198
PART TWO (2)
Diagnostic Hematology
Clinical Laboratory Diagnosis:
Diagnostic Hematology
For
General Clinical Laboratory Scientists
By;
Kourosh Teymourian, CLS
199
General Contents
Page
Chapter One:
203
Orientation and Safety Measures;
203
1.
2.
3.
4.
203
203
213
213
Instrumentation and Automation
Safety in Hematology Laboratory
Quality Control and Quality Assessment
Specimen Collection, handling and manipulations
Chapter Two:
220
Fundamentals in Hematology;
220
1.
2.
3.
4.
220
220
220
224
Historical Perspectives in Hematology
Basic Concepts
Origin of Hematopoietic Cells
Different Cell Lines
Chapter Three:
Diagnostic Hematology;
227
230
200
1. Approach to identification of Red cells, White cells and Platelet anomalies/alterations
230
2. Different Diagnostic and Identification Indications of Hematopoietic Disorders; 230
3. Testing Methods (Methodology)
238
Pathological Disorders/Hematopathology
243
Anemia/Definition
243
1. Megaloblastic Anemia
2. Vitamin B-12 Deficiency Anemia
3. Pernicious Anemia
4. Microcytic, Hypochromic Anemia
5. Iron Deficiency Anemia
6. Sideroblastic Anemia
7. Anemia of Alcoholism
8. Anemia Associated with Hepatocytes/Liver
9. Normocytic, Normochromic Anemia
10. Aplastic Anemia
11. Pure Red Cell Aplasia & Anemia of Chronic disease (ACD)
Chapter Four:
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252
253
254
Hemaglobinopathies
254
1. Sickle Cell Disease
2. Pathophysiology of Thalassemia
3. α-Thalassemia and β-Thalassemia
Abnormal and Unstable Hemoglobin
254
256
256
258
1.
258
Hemoglobin- H, C etc. as unstable Hgbs.
Chapter Five:
Hemolytic Diseases (HD)
1. Intravascular
2. Acute Alloimmune Hemolytic Transfusion Reaction (AAIHTR), etc.
3. Extravascular;
4. Reticuloendothelial Destruction of RBCs
5. Autoimmune Hemolytic Anemias (AIHAs)
6. Cold AIHA
7. Warm AIHA
8. Extrinsic HDs
9. AIHA
10. Hemolytic Disease of the Newborn (HDN)
11. Paroxysmal Cold Hemoglobinuria (PCH)
12. Intrinsic HDs
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201
13. Drug-induced Hemolytic Anemia (DIHA)
14. Erythrocytes membrane Defects and Associated Disorders
15. Spherocytosis, Elliptocytosis and Stomatocytosis, etc.
16. RBC Enzymes and Defect in ancillary Erythrocytes Resident Enzymes:
Enzymopathy
1. G6PDHase Deficiency
2. AK/Adenylate-Kinase Deficiency
Chapter Six:
265
266
266
267
267
267
268
268
1.
2.
3.
5.
6.
Non-Immune Hemolytic Anemia(s)
268
Microangiopathic Hemolytic Anemia
268
Thrombotic Thrombocytopenic Purpura (TTP)
268
Hemolytic Uremic Syndrome (HUS)
269
Acute and Chronic Disseminated Intravascular Degradation/Destruction/Coagulation
(DIC)
270
7. Macroangiopathic Hemolytic Anemia
271
8. Artificial Valves
271
9. Thermal and Physical Destruction of RBCs, etc.
272
10. HD due to Parasites and Intracellular Infections
272
Chapter Seven:
275
1. Myeloproliferative Disorders (MPDs)
2. Chronic Myelogenous/Myeloid Leukemia (CML)
3. Idiopathic Myelofibrosis (IMF); Fibrosis with Myeloid Metaplasia (FMMP)
275
276
277
4.
5.
6.
7.
279
280
281
283
Polycythemia Rubra Vera (PRV)
Essential Thrombocythemia (ET)
Acute Myelogenous/Myeloid Leukemia (AML)
Myelodysplastic Syndrome (MDS)
Chapter Eight:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Lymphoproliferative Disorders (LPDs)
Acute Lymphoid Leukemia (ALL)
Chronic Lymphoid Leukemia (CLL)
Hodgkin’s and Non-Hodgkin’s Lymphoma (HD) & (NHL)
Hairy Cell Leukemia (HCL)
Sezary Syndrome (SS)/Cutaneous T-Cell Lymphoma
Monoclonal Gammapathies
Multiple Myeloma (MM)
Waldenstrom Macroglobulinonemia (WMN)
284
284
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286
288
290
290-291
292
293
294
202
10. Infectious Mononucleosis/(IMN) with Reactive Lymphocytes
Chapter Nine:
1. Thrombosis and Coagulation
2. Instrumentations/Automations
3. Components of Coagulation
4. Basics Concepts
5. Role of vessels
6. Role of Platelets
7. Blood Factors & Relevant Pathways
8. Intrinsic
9. Extrinsic
10. Common
11. Role of Plasma Procoagulants
12. Inhibitors
13. Related Coagulopathies and Other Bleeding Disorders
14. Congenital
15. Acquired
16. Testing Regiments/Protocols/Panels
17. aPTT (Activated Partial Thromboplastin Time)
18. PT (Prothrombin Time)
19. Fibrinogen estimation/Assay (FA)
20. FDP (Fibribnogen Degraded Products) & D-dimer
21. Reptilase Time (RT)
22. Platelet Aggregometry with Rich and Poor Plasma/Platelet Function Test
23. Bleeding Time (BT)
24. Monitoring Thrombolytic and AnticoagulationTherapy
25. Heparin
26. Warfarin/Comadin/Comarin
27. Antibodies to Coagulation Factors.
28. References
29. Normal values
Chapter One (1)
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203
Orientation
Measures
and
Instrumentation
Automations;(KC4
Safety
and
Delta products
info & Rodak et al.)
he era of instrumentation and
automation commenced during early
40 and 50s, in all aspect of Medical
T
Laboratory
Technology/Science,
especially in the field of Clinical Chemistry
and
subsequently
spread
to
the
Hematology Department and later
within
other
Departments
(interdepartmentals) as the leading force to ease
the operation and the accuracy of the test
procedures.
These had not only increased the batch load
but also improved and enhanced test
volume, precision and accuracy of the result
with concomitant augmentation of the
Laboratory revenues. Thus Laboratory
became a business oriented entity and a
Medical Facility in which diagnostic
procedures occurred and intermingled with
less avail of Technical Personnel.
In this era the idea of robotics utility
although was in the mind of Health Care
Systems and especially Laboratory and
Medical Technology, but it did not
configured until its blossom in the 70s, thus
a period of flourishing of Technologies and
Methodologies emerged into the Medical
Market. In this respect, Hematology was not
exempted from these improvements and
innovations in the aforesaid Laboratory
field.
By
60s
and
70s,
Computer
attaché/adjunct to the main diagnostic
instruments/device become popular but due
to high costs and low revenues, Government
and private laboratories possessed a lower
number of automation and this obstacle were
gradually eliminated later and many types of
machines conjoined with robotic systems
have become main stream and hallmark of
technological advancement in the particular
field of Laboratory Medicine.
These Instruments such as, Flow
cytometerincorporating
hydrodynamics focusing and laser
scanning systems, advanced Light
Microscopes, compound Polarizing,
Dark-field, Inversion and others as
Scanning (SEM) and Transmission
Electron Microscopes (TEM) benefited
Hematology in the era of automations. Also
Coulter System- embracing laser and
hydrodynamics and Coulter principle are
part of a continuum of state of the art
technologies. These cutting edge automated
instruments systems technology have almost
fulfilled the promise of Science for a better
and a healthier life of the patients by
accurately and precisely diagnosing
disorders/illnesses and management of the
patient’s aliment with a better therapeutic
choice and prognosis.
Other automated devices and instruments
included in the hematology laboratories
encompass coagulation machines such as,
AMAX-400® by Trinity Biotech Inc.; or
MDA-2® by the same Incorporation,
ACL-Elite System® by Coulter
Diagnostics, with other automation systems
that are either Mechanical, Immunoassay
systems, Photo optical, Chromogenic or
Nephelometric Systems approach to test for
the coagulation profiles and factors. In
addition,
Photo
optical
and
Nephelometric assays possess the ability
204
of Turbidometric analysis of coagulation
test panels. Elisa (Enzyme Linked
Immunosorbent Assay) has a versatility
and adaptability to major substance
detection and identification, e.g. in
Coagulation factor Assays and antigen and
antibody identification and estimations,
(ACL, Operators Manual, 2003 and
Bernadette, F. R, et al) fig. 1-1. (Thomas
LC. Et al.)
Fig. 1-1: A Flow Cytometer System for
Identification of Cell Lines and Cluster of
differentiation (CD) in lymphocytes and
leukocytes surface markers
Turbidometric,
immunoassays
and
Nephelometric analyses and instruments’
ultrastructure have been discuss in the
clinical chemistry section of this text and
will not be covered longer in here. Other
devices
embracing
the
hematology
department
are
erythrocyte
sedimentation rate apparatus (ESR).
Enzyme liked Immunoassay system is one
of the most versatile detection systems that
has been utilized in hematology, clinical
chemistry, transfusion medicine,
microbiology and most likely in
histotechnology and histopathology
departments. These systems are used in
protein
detection
and
quantitation,
antigen/antibody estimation, coagulation
factor assays, RBC (erythrocytes) antibody
quantitation and WBC’s (leukocytes/white
cells) CD antigen and antibody evaluation
by which electrophoresis and Southern blot
analysis can be followed as a diagnostic
sequel.
In these arrays of diagnostic machines, the
most applicable and versatile instrument in
hematology is the flow cytometer, which
can be used for detection of a spectrum of
normal
and
abnormal
blood
cell
lines/precursors and progeny lines, such as
WBCs (e.g. leukocyte, lymphocyte,
monocyte, eosinophil and basophil)
cell lines, as well as RBCs (erythrocyte),
marrow cells and other likely cellular lines
in other body fluids and even DNA analysis.
Few detection systems are visible in
hematology such as Coulter Counter and
Flow cytometers in hematology and thus for
the this reason, Flowcytometry and Coulter
Principle, which both have a significant
outcome in diagnostic hematology, will
be shortly described and their ultrastructure
as a couple of diagnostic systems within
their scope of functions will be explored, so
that a general understanding of their
function and interpretation of results may be
elucidated in brief (vide infra).
Flow cytometer is based on hydrodynamic
focusing and laser principles. In this a
linearly focused stream of isotonic fluid
(isotone) mixed with citrated/EDTA
(ethylenediamintetraacetic
acid)
anticoagulated blood is produced and
consequently a beam of narrow single line
of blood cell focuses at several junction
where laser system (light amplification
by stimulated emission of the
radiation) detect and scan the external and
205
the internal structures of the cell(s) through
certain degree of excitation and or priming.
These angles of radiation strikes the cell(s)
at 0 degree scatter where indicates
cellular surface composition and granularity,
90 degree scatter (Side Scatter) which
scans the structural composition of the cell’s
internal structure and 180° scatter (or 180
degree scatter) known to scan the same
analogous structures as in 90 degree scatter
on the Scattergram. To detect the external
surface structures of the CD molecules
(Cluster of Differentiation of surface
markers, such as glycoproteins making
receptors for different and variety of
molecules), these glycoproteins are stained
with fluorescent dyes such as Acridine
divided
Orange, Auramine, Rhodamin Orange
G and Fluorescent Isothiocyanide
(FITC) Green so that may detect surface
Fig. 1-2, shows a Scattergram or a
Scatterplot on the coordinates with
boundaries indicating cell population
processed by a Flow cytometer; Cell
population may be normal or abnormal.
markers of CD structures, once conjugated
with corresponding antibody to the
particular molecule(s). The source of laser
can be of Argon, Krypton or glass filled
tubes of Neon and Helium. Figure 1-2 shows
an example of a Scattergram. In this typical
“Scattergram”, the eosinophils, basophils,
monocytes and neutrophils are shown on the
scatter gram. The ghost cells, which are
RBC cells with lost hemoglobin as internal
content washed out and can be observed as
well.
Scattergram is a plot window on which the
number of cells and intensity of fluorescence
are plotted on specific boundaries, which is
on
the
plot.
Are the colored-coded/enhanced scattered
areas of the plot indicates the intensities of
the normal or abnormal cell populations as
antibodies conjugated to specific cell
marker(s) traced by fluorescent dyes as
mentioned above. Onboard sensors detect
the signal generated fluorescent dyes and
laser/photo optical scan differentiates
between the internal and external
complexities and characteristics of the
target cell(s) or any surface molecules in
the cell(s).
Fluorescent and laser scanning applications
can identify and use for DNA and RNA
quantifications and estimations, this
application of flow cytometer is confined to
Clinical Genetic specialty in the field
of Clinical Laboratory Science. This text is
206
rather a general approach to five general
specialty fields in Medical Laboratory
Division as in Clinical Chemistry,
Diagnostic
Hematology,
Diagnostic
Microbiology, Transfusion Medicine and
Histotechnology
and
Histopathology
(Histology).
Other instrument in the hand of Clinical
Laboratory Scientist and Technicians
are the powerful diagnostic tool as Coulter
Counter, some Coulter Counters applies the
laser application and/or the fluorescent
dye staining of the particles and cells, but
a genuine Coulter Counter is the one within
which the Coulter Principle is applied to
this device. Coulter Principle states that,
if a particle or cell passes through an
aperture of an electrical conductance in an
isotonic medium, during its transition it
will
produce
an
electrical
resistance/conduction signal proportional to
the particle or the cell size and ultimately
its structure. Thus a cell could be
numerated according to their size and
nuclearity as in RBC, WBC or Platelet,
etc. The nucleated RBCs will be classified
as WBCs, however, these categories of
cells in a normal situation will be counted
as WBC thus this erroneous count could
not effect/overlooked due to rarity of the
nucleated RBCs (as in Erythroblastosis
Fetalis/Polycytemia Rubra Vera, or
some
Hematological
Cancers/Leukemia and/or HDN. In a
situation where the abnormal and nucleated
RBC numbers indicate (as examples
above/vide supra), otherwise in higher
counts, it mandates to do a WBC
differential count including nucleated
RBCs either manually or by the Coulter
Counter and estimates for the correct count
of nucleated RBCs in a WBCs differential
count in a blood smear/film or
automatically by the instrument as per se.
The following Figure (Fig.1-3) is a
schematic representation of Coulter
Principle.
Fig. 1-3: The Coulter Principle of a Coulter
Counter (e.g. Beckman Coulter® Machines
for
Multichannel
Pulse
Sensitive/Resistance Particle Counter).
The
normal
size
ranges
(Anisocytosis/changes in cell size) of
RBCs lapse between 11-15 units and for
Platelets/ Thrombocytes/megakaryocytes
varies between 2- 20 units on the
Histogram Plot (e.g. RDW & MPV).
Platelet may be counted accordingly and
sized alternates between the normal ranges
cited. Specimen source may be whole
citrated or EDTA Blood or Body
Fluids in some cases and CSF
(Cerebrospinal Fluid) in other instances,
for applicability of specimen application
consult the device manual instructions. Fig.
1-4 shows a sample of a Histogram Plot of
a Coulter Counter.
207
Fig. 1-4: A Histogram Plot of a particle
sizes/Anisocytosis e.g. RBC (RDW/mean
corpuscular distribution width), WBC or
Thrombocytes
(MVP/mean
platelet
volume).
The most Modern Coulter Counter is
currently from Technicon Inc. The model
STKS® instrument uses robotic arms to
handle the sample/specimens and within
the system finds photo-optical detection
onboard, laser scanning, hydrodynamic
focusing comprising a unifluidic sample
streaming and fluorescent detection
systems and sensors.
Under instrumentation as mentioned we
have the most popular instruments for
coagulation profiling systems such as;
ACL Elite system® (ACL 8000
operator manual Brea) by Coulter
Diagnostics and AMAX-400® by Trinity
Biotech. Coagulation Profiling systems are
based
on
photo-optical
detection,
mechanical systems, immunoassays
detection
and
electrochemical/electromagnetic
systems and chromogenic assays. In
photo-optical types, the fibrin clot
formation leads to optical density
changes/variation and thus estimation of
the aforesaid parameter. In the mechanical
systems, the changes in location and the
speed of the oscillation of a magnetic ball
in the reaction vessel is detected by the
formation of the fibrin clot and the slowing
down of the oscillator ball due to fibrin
formation indicates clot formation. In the
electromagnetic system stirring of a
metallic bar enhances the formation of
the clot (fibrin) and the final
quantification of the amount of fibrin. All
results are plotted and the amount estimated
by computer attaché algorithm software
application. Figure 1-5 shows ACL Elite
System®
by
Coulter
Diagnostics.
Fig.1-5: ACL Elite System® by Coulter
Diagnostics Inc., Coagulation Profiling
Instruments
(courtesy
of
Coulter
Diagnostics®).
Coagulation systems are capable of
coagulation factor assays with quantitative
and qualitative analysis. The above various
types of operations and analysis totally
depend on the manufacturer and producer
of the instrumentations’ technological
scheme/design and functional capabilities
and performance criteria of the machine
itself. This is unique to the manufacturers
and companies producing these types of
208
machines. The most important instrument
in hematology department is the light
microscope and is efficient in the hand of a
hematologist/technologist, which will be
explained briefly in here (vide infra).
An average light compound microscope
(Turgeon et al.) is composed of binocular
optical lenses (10x), a body tube, a
carousel/or revolving/rotating nose piece,
the stage and illumination system (a lamp
or mirror, etc.), other details onboard are,
objectives (lenses); 40, 50 and 100xs; with
a
maximum
of
1000x
total
magnification, vernier scale, stands,
coarse adjustment, and fine adjustment
knobs, condenser, off and on switch, and
sub-stage light adjustment aperture added
with light adjustment aperture located in
the stand. This apparatus needs subtle care
and cleaning and that should be done with
70% ethyl alcohol. Formalin and 2%
gluteraldehyde vapors are corrosive to
the body and the lenses and these should be
eliminated; lenses must be cleaned by a
lens paper. As for use in hematology and
other purposes, Köhler illumination
should be followed for light path
adjustment; for this method, objectives and
optical lens adjustment with condenser, and
sub-stage adjustment aperture with light
adjustment aperture located in the stand,
must be brought to interplay, so that
illumination path is correct and maximal
and the optical and light path
alignment are in place. The following
figure (Fig. 1-6) is an example of light
compound microscope (Polarizing also see
Histology part 5).
Fig. 1-6: Components of a compound
polarized microscope.
Safety
in
Hematology
Laboratory: (Rodak et al.)
As safety in all aspects of clinical
laboratory,
safety
in
Hematology
Laboratory is of prime importance, in order
to visualize the significance of safety in
Hematology is to consider open wounds,
cuts or scratches come in contact to blood,
body fluids and or other blood/tissue
products. Such careless omission of safety
protocol in this atmosphere is inviting to
most sever of infections, for instance in
acquisition of AIDS (HIV infection),
Hepatitis B, and C or other sort of
infectious etiology in the clinical and
nosocomial settings.
209
One of the valuable insignia in the
laboratory area is the biohazard sign, as
shown in figure 1-7.
fluid for cell counting and other bodily
products as in CSF (Cerebrospinal Fluid),
plasma, serum or other somatic
secretions and excretions/discharges
manipulations must be considered and
avoid opening the centrifuges while
spinning (this can add to droplet and
aerosol formation) as warranted.
During blood smear/film preparation and
blood manipulation/handling in this
department care should be exercised to
prevent aerosolization of particles within
the droplets of infectious origin and later
contraction of the infecting agent causing
laboratory and nosocomial infections.
Fig. 1-7: Biohazard symbol indicating
possibility of contracting infectious agents.
As in this biohazard signage is posted in all
laboratory sections that deal with blood and
blood
products
and
bodily
tissues/biological fluids, being in process,
especially
in
Microbiology/Surgical
Pathology areas/departments. Subsequent
to observation of such hazard sign,
laboratory personnel must use PPEs
(Personal Protective Equipment) and follow
the institution’s safety rules/regulations as
in Standard Precaution (previous
Universal Precaution) set by CLSI (Clinical
and Laboratory Standard Institute/ previous
NCCLS/National Committee on Clinical
Laboratory Standards). Aseptic procedures
such as no mouth pipetting, caution in
removing caps of blood vials/tubes and
needles (do not bend or recap) and syringes
in preventing aerosols generation are inplace. Caution with containers of body
After utility of hemocytometer for
manual cell counts it is essential to
disinfect the piece of equipment with
proper disinfectant of 75% ethanol so that it
is germ free for the subsequent usage. Also
at the age of automated machines, there is
much less need of pipetting for dilutions of
RBCs, WBCs and platelet counts. In any
case if there is a need to use RBC and
WBC pipettes the performer should
utilize different available safety bulbs or
aspirators to safely pipette the desired
dilutions. Relatively, for setting up the
Wintrobe or Westergren tube methods
for erythrocyte sedimentation (ESR) care
should be exercised not to pipette by mouth
and use aspirator or safety bulbs to fill the
tubes.
In using the Unopette system for blood
dilutions it must be handled with caution
and care, therefore contamination and
infectivity could not occur and kept to the
minimum, this is used for blood dilutions in
a safer manner. CLSI requires all blood and
body fluids must be handled within the
210
domain of Standard Precaution along with
CLIA’
88
recommendations,
after
processing, all contaminated equipment,
tissue remains and blood and body fluid,
must be sterilized by either autoclaving
or other means of sterilizing (as Physical or
thermal) and be discarded by a waste
disposal
management
system
contractor to the hospital or the lab
facility. Shields, masks, goggles, gloves,
gowns, lab coats (including scrapes) and
other barrier protection such as respirators
should be used appropriately, the latter
must be in place at the time of specimen
collection from a T.B. (Tubercle
bacilli/tuberculosis patients, etc.) patient
contaminated with this pathogen; as in
cases of respiratory lavage/aspirate or
blood sampling/collection from the said
patients.
Scrapes, lab coats and gowns should be
laundered by the hospital or lab facility,
therefore, must not be taken out of the
premise of the laboratory. Personal
belongings and text books or other items
have to be left in the locker or they should
be kept to minimum: current to the lab in
use, so that they would not be the item of
public infectivity during outdoor activity
or indirectly contaminating the other public
areas such as hospital canteen or restaurants
or vicinity gift shops.
Pen should not be chewed on and jewelries
should be kept to the minimum, nails
should be trimmed short and hairs tied back
due to the danger of contamination and
getting caught and strangulate during
centrifugation or other operations, etc. ties
should have a warning of getting caught
into and strangulation with centrifuges
operated by an open lid and with rotor.
There should be no cuts or abrasion on
the skin, as the skin should be intact; in
case of injuries to the personnel, an
incidence report should be made in less
than 24 hours and the victim must be sent
to the Employee Health Office for further
evaluation and follow ups. Cuts and broken
skin (dermis) mandates attention by
covering the inflicted area by plaster/ or
band aid and covering it with two gloves
(Latex or Nitrile). Rubber gloves are
utilized in the chemical works with strong
acids and bases, which gives stronger
protection and resistance to these
corrosives actions.
In this respect, Chemical Hygiene Plan
should be in place as well in hematology
department in order to handle chemical
agents dangerous to health and lives of
Technologists
and
Technicians,
storage requirement of chemicals should
follow
CLSI
and
OHSA/OSHA
(Occupational
Safety
and
Health
Act/Association). It means they must not be
arranged alphabetically, but in category and
classified arrangement, example, strong
bases away from strong acids and reactive
agents, etc.
In hematology lab the most dangerous
chemical
used
is
the
hemoglobinocyanide
or
cyanomethemaglobin for determination
of total hemoglobin by Unopette system or
other traditional approaches. However, this
methodology is proven to be hazardous due
to containment of cynical element in the
reagent and thus is highly hemotoxic,
with very minute amount of cyanide being
fatal. Use of other reagent and staining
solutions can be a health hazard as
Isothiocyanide Green or Rhodamin
211
Orang G and Periodic Acid Schiff
(PAS) reagent for manual cellular staining,
others as esterases; direct and indirect, can
be toxic and irritant once exposed to the
reagent, care should be exercised in safe
handling of these chemicals,
Lastly concentrated acid and basic
solutions rarely used in hematology lab
although examples are present; one of
example is the 2N hydrochloric acid in
HAM’s test for confirmation of
Proxysmal
Nacturnal
Hemoglobinuria or commonly known
as,” PNH”. This is an acidified serum lysis
test. Personal safety is always first in doing
things in the lab atmosphere.
In collecting blood for cell counts,
hemoglobin determination, ESR, sCRP
(sensitivity C-reactive protein) and
screening, the possibility of needle stick
injuries always exist. In case of needle stick
injury to the own person, the same mandate
of reporting an incident case is remarkably
essential and similar policy applies. A 24
hour report should be installed and
followed by initial testing for HIV and
Hepatitis screening. It follows in 2
months, and a 6 months retesting protocol.
Standard protocols for aseptic performance
are indispensable and any infectious case
occurrences must be reported to the
infection control committee of the
diagnostic/therapeutic facility in which the
worker works.
During phlebotomy with vacutainers
(tubes) and micro-collection needles,
such as butterfly infusion set(s) with
micro-containers warrants a standard
operation procedure be instituted so that it
does not harm the patient and the
technician
and/or
the
technologist
him/herself, this may be in the form of
needle stick injuries or cuts and for this
tech should wear gloves.
Some procedures such as preparing smears
or handling fresh specimen and/or tissues
or working with infectious materials (e.g.
sputum count, seminal fluid cell count,
body fluid cell count and other analysis for
pleurocentesis materials, paracentesis
materials, arthrocentesis and CSF cell
and protein count and measure, must be
manipulated under biohazard safety
hood/cabinet with HEPA installations
and chemical preparation and handling
under Chemical Fume Hood.
The first line of defense against infection is
proper hand washing, this should be before
entering and after exiting the lab area,
before and after starting and finishing a
procedure, before and after using the toilet,
and must follow the protocol for hand
washing. Contaminated sharps and
needles, scalpels and others such as
contaminated and broken glass must be
discarded in the safety sharp container
with red color coding and yellow
containers for bandaids and contaminated
and soiled cotton-balls, etc. In addition to
the above safety tips, fire drills must be
periodically installed and performed and in
this relation, fire extinguishers for each
type of fire have to be trained and followed
with internal and external quality
audits. Glassware must be arranged
according to the standards available; such
as taller/larger cylinders/beakers, etc. has to
be stored behind the smaller vessels in the
cabinet and to the reach of the handler.
212
Also, for chemical safety plan, while
preparing chemical solution, diluents and
standards, the safety aspect of the chemical
in preparation should be consulted with
MSDS, which means Material Safety
Data Sheets. This group of chemical
filing system is universal and warn the
operator or the preparer of the danger of
each chemical, plus the emergency
treatment in case of physical injuries due to
the chemical itself. This systematic type of
filing has been introduced by the
manufacturer of the products and is utilized
in the laboratory for awareness of the
laboratory personnel and the action
preformed at the emergency time.
Product information encompass physical
properties of the substance, chemical
properties, thermal properties, fire and
explosion hazard, hazardous ingredients,
reactivity data, toxicological properties,
preventative measures, first aid measures
and
preparation
information
with
manufacturer’s contact information.
In addition to the above MSDS, in cases the
product label provided by the manufacturer,
importer or distributer and/or the
producer, has been lost or faded, it is the
responsibility of the consumer lab to supply
the work place label within specified time,
or if the chemical being manufactured or
produced in the workplace and/or the
original container being decanted to a new
container, therefore
a new label
(Workplace label) must be supplied by
the workplace.
The following figure (Fig. 1-8, A-B), is the
example of MSDS and workplace label,
used by the manufacturers and suppliers
(A)
(B)
Fig. 1-8: examples of (A) workplace label
and (B) MSDS label.
In respect to MSDS, the hazard sign of this
hazard warning system are depicted in the
Fig. 1-9 in form of WHIMS. It means
Workplace Hazardous Information Systems
and it has been classified as Class A hazard
types, Class B, Class C, Class D (with
subdivisions), Class E and Class F
forwarded by Canadian health authorities.
These are warning signs that each use of
dangerous chemicals carry and the
technician is alerted to the precaution cited
by the warning signs. Although not totally
comprehensible but the classification
213
covers
most
parts
of
the
manufacturing/manufactured chemicals.
lab and as we know how important these
topics in Clinical/Laboratory Medicine are
for today’s lab operation.
Quality Control and Quality Assessment,
particularly in today’s Clinical Laboratories
are indispensable procedures that had
commenced while before than the robotic
automation and mechanization of modern
clinical laboratory conforms, it is a core to
properly functioning of the laboratory in
yesterday and today’s era/labs.
In respect to the above, Quality Control,
starts within the department procedural
controls and observation of the test
performance, so that the productivity and
the reproducibility of the result are
maintained through a precise statistical
performance
and
algorithms.
This
guarantees the results are in consistent with
Standard Operation Procedures (SOP)
and that emphasizes all aspect of
controlling and useful monitoring of the
test procedure is operational and the results
are valid.
This starts with calibration of the
instruments with standards, controls and
calibrators and considered prior to the test
procedure. The mean and the Standard
Deviation (SD/reference interval(s))
and the Coefficient of variation (CV)
are implemented to have a meaningful
interval range in the hematology labs.
Methodologies must have calculated
interval ranges for all hematology
parameters and consistent results have to
be established before reporting. To attain
such consistent, accurate and precise result
need application of statistical tools, (see
Clinical Chemistry section of the text) and
other algorithms to establish such ideal
result meaningful to the physician. These
could be the statistical mean, mode and
median used in constructing CV and SD
and establishing and application of
Quality Control and later its overall
observation
embrace
Quality
Assurance/Assessment implementations
consecutively.
Fig. 1-9: WHIMS hazard warning classes
of chemicals in symbolic form.
Quality Control and Quality
Assessment: (Rodak et al.)
and total quality improvement (TQI) in the
Westgard-mutlirule
procedures
(Watson and Shwarts) with Levey
Jenning’s control chart monitoring
214
Also Quality Control points to the details in
the procedures such as right amount of
stain in blood smear/film preparation or
the right PH of the buffer, right amount of
preservative/anticoagulant used and its
effect on stain precipitation or color of the
stain etc. This is exemplified by using
heparin as an anticoagulant and staining
with Wright’s stain, which results in blue
precipitated stain on the slide rather than
expected/conventional stain colors for
Wright-Geimsa stain without precipitation
with EDTA anticoagulant as an optimal
hematology anticoagulant for this method
of staining. As cited, heparin is also used in
this department with the disadvantage of
leaving blue precipitate/smudges on the
slide with Wright or Geimsa staining
procedure (vide supra).
Use of correct procedure in smearing the
blood film including proper angle of
application of the spreader on the glass
slide for thick or thin smears, or the right
amount of the blood drop on the edge of the
smear or how old is the reagents such as
isotone, and stain (whether filtered before
use) are significant quality applications.
Other related solutions utilize in operation
of the Coulter Counter or the Flow
Cytometer with other likely method of
calibration of the instrument(s) for other
automated or manual methodologies are
distinct and vivid points in Quality Controls
rules.
All Quality Control essentials are classified
into three levels, known as, Pre analytic
or frontend, analytic and post analytic
or backend aspects. In addition to
frontend, specimen collection should
optimize control errors in this phase, the
analytic phase, which is the domain of
Quality Control mentioned earlier, includes
testing controls, standards calibration,
and plus trouble -shooting/problem solving
of
the
instrument
or
the
procedure/methodology are the most
important aspects of this phase and at last
clerical aspects optimization and freedom
of erroneous reporting are the ideals of
backend systems and a manifesto of the
post analytic phase as in quality assessment
and TQI.
The frontend (i.e. right amount of
anticoagulant to blood or transportation
requirements, etc.) and the backend
categories are the domains of Quality
Assurance/Assessment and analytic aspect
are the limits of Quality Controls, these all
together with total optimization are arched
over the title of Quality Assessment
(QA) and Total Quality Improvements
(TQI) (Stewart CE. et al.).
Quality Assessment in Clinical and
Diagnostic Hematology embraces, all the
triad of Quality measures as some part of
which was cited above, so that results are
up to the desired and expectations. As with
Quality Controls procedures of such tests as
Hemoglobin (Hb) determination and
Hematocrit (Hct) estimation need proper
functioning of micro-centrifuges known as
Hematocrit Centrifuges, this has to be
periodically upgraded and checked with
strobes like equipment similar to other
centrifuges. Tissue centrifuges called
“Cytospin” should be occasionally
checked for the assemblage and the
centrifuge’s speed with a tachometer as
well. In addition, Unopette systems have
to be preform according to the
manufacturer’s direction with a proper
assembly. For smear preparation Quality
215
staining is a must in these procedures so
that it is sustained with a good quality
staining for elemental, cellular, diagnostic
clues and other inclusion body detection.
As in malarial parasites detection with
the use of thick smear, staining properties
such as color, appearance, controlled
thickness of the smear, accumulation of the
leukocytes and red cells in the center of the
smear or in the edges are determinants to
optical or visual detection/elemental
identification. Along with this, magnetic
beans are used to stir the vial of blood
suspected to have malarial parasite in thick
smear test, this is so as to rupture the cells
(RBCs) to release the parasite for easier
visualization and need extra attention to the
procedure,
timing
and
preparation
procedures and qualities. In case of
reticulocytes (Retic.) estimation/count for
the diagnoses of absolute and relative
erythropoiesis (medullary or extramedullary or ineffective or effective
erythropoiesis) a good blood film is
mandated. This is to count the number of
reticulocytes among 1000 erythrocytes
being counted and calculated in percentage
after stained with supravital staining with
methylene blue. Thus proper staining of the
reticulocytes is of such an importance.
The result may be reported according to a
good Quality Control protocol practices for
the tests and covered by Quality Assurance
and Assessment then subsequently the
results will be optimally released to the
hospital wards or posted online in
LIS/HIS (Laboratory and Hospital
Information Systems) systems or in
hard copy backup files. Internal and
external quality audits/controls are
significant part of the hematology labs,
these include tests procedural review,
quality checks, delta checks, review of the
control values, use of positive and negative
controls, viability/validity of standard
solutions, standard curves and erection of
histogram and nomograms, calibration
criteria, clerical checks, proper specimen
collection and transport, optimal storage
temperatures requirements, professional
reviews and training/testing and at last
assessment of total procedures and
methodologies, all and all are few to
consider for quality checks..
Specimen
collection,
Handling and Manipulation
Once the requisition from(s) arrives at the
Specimen Collection Center (SCC)
from the requesting physician or if the
specimen have to be collected within the
ward in hospital facility, the technologist or
the technician preforms and collects all the
requested samples and specimens. These
are in form of (for the hematology
department purposes) whole blood and
anticoagulated blood or serum/plasma for
testing purposes.
For
the
purpose
of
venipuncture/venisection,
the
most
important/valid vacutainer tube for
hematology purposes is the lavender
color coded top/rubber cap with or
without screw cap/Hemogard. This
vacutainer
contains
EDTA/ethylenediaminetetraacidic
acid
anticoagulant and along with this, heparin
216
anticoagulant [green cap vacotainer(s)] also
is useable for some hematology tests, e.g.
ESR, sCRP or blood smears, for this latter,
care/caution must be applied for staining
with Wright/Geimsa stains to avoid blue
precipitates of heparin with these
Romanovsky stains. This staining
procedure is optimal with EDTA
anticoagulant. As for coagulation purposes
and study light blue stopper/cap with citrate
anticoagulant warrents.
In short blood extraction or phlebotomy
(Garza et al.) consists of the following short
procedure; once the patient is in the ward,
prepare
the
patient
for
venipuncture/venisection, and set up the
phlebotomy tray and the arm makes ready
to piercing. (McCall RE, et al.)
Locate the vein in the bend of the arm
[either in antecubital fossa/vein, or
cephalic vein (and rarely basilic veindue to its disadvantage of to being too
elastic
and
rubbery)],
never
use
saphenous vein of the leg, in the patient
whom
may
have
cardiac
insufficiency/failure or other heart problem
predisposing the patient to clot formation,
or thrombotic condition. This should be
done with the consent of the attending
physician.
The next step is to tie around the arm a
tourniquet, search the vein once located,
use an 75% alcohol pad or rub (ethanol/for
routine puncture and beta-dine or povidon
iodine/cholerhexdine for culture purposes)
to sterilize the area, with an outward
circular motion cleanse the tending piercing
area with the alc. rub. Let for few seconds
to evaporate the alcohol rub residues to
prevent stinging sensation to the patient and
hemolysis, and then perform the
venipuncture. At this stage, before piercing,
anchor/pull down the below area of the
intended skin for the piercing/venipuncture
while holding the vacuatiner cylinder
with attached needle to its hub with the left
hand (the hub at the other end, inside the
cylinder, is protected by a rubber sleeve to
prevent leakage of the blood drawn inside
the cylinder) and with right hand slowly but
deliberately penetrate the skin at 25-30
degree angle deep enough to be in the vein
at about 2 inches below the tourniquet line.
Subsequently,
feed
the
intended
vacutainer with the right hand into the
cylinder cautiously and shift hand and draw
blood by the order of draw; this is as
followings; black blotched cap first for
culture purposes, then yellow cap tube
(for chemistry works), third red caps (for
trace element and chemistry works), fourth
light blue cap (for coagulation purposes),
royal blue next for some toxicology
purposes, sixth lavender top tube (for
hematology) and the last for glucose, etc.
the grey cap vacutainer tubes should be
utilized (consult with Clinical Chemistry
section)
with
their
appropriate
anticoagulants.
Likely there is often need of blood
collection either from the neonates/infants,
old/debilitated patients or in situation
where there is difficulty in obtaining from
the causal locations/sites, therefore, microcollection will be performed.
In this procedure either sole of their feet
(external and medial aspects of the
sole) may be tried by doing microcollection with lancets or autolets® (new
products are laser application, which
produces heath rather than piercing
sensation). Following figure (Fig. 1-10)
shows the anticipated areas to micro-
217
collecting from the sole of feet. (Turgeon et
al)
avoided, as these can cause nerve
compression/damage and discomfort to the
patient. In serious cases mal-extraction can
cause arm amputation and should be
exercise with caution.
Under this topic we have handling and
manipulation of the specimen; if Whole
blood is anticoagulated for blood counts,
hemoglobin estimation, and ESR has to be
kept in 4° C (fridge) temperature when not
tested immediately. It can be kept up to 24
hours prior to testing.
Fig.1-10: The area where micro-collection
can be performed in green.
The different approach to collecting blood
from the infant and the old/debilitated
patient is by collection through a butterfly
infusion set or by microtainers and
lancets. This (butterfly) set is used almost
with the same procedure and precautions
with vacutainer system venipuncture (this
is also a venipuncture method) with the
advantage
of
seeing/observing
the
flashback of the blood in the tube near the
vicinity of the needle indicating penetration
of the needle correctly into the vein, except
with an addition of the leur adopter. This
connects the needle along stubing to the
vacutainer’s cylinder. The size of tubes and
needle matters and should be reviewed
carefully while venipuncture is current.
The areas involved in butterfly specimen
collection, is from the back of the hand in
prone position. In addition, trusting the
needle must be deliberate and continuous
between angles of 20-35 degree.
Hematomas and bruises must be
Smears are made by preparing a blood drop
to the frosted end of the slide and
spreading it according to CLSI
recommendations. The size of the blood
drop from an EDTA anticoagulant and
thinness and the thickness of the smear are
important where a first part of the film must
be thick and the preceding has to be thinned
out. Angle of spread/application must
be 45 degree and in cases of too thick an
application
results
into
an
over
accumulation of heavy
elements
(WBCs/large cells) at the edges of the slide
in thick preparation. As in thin preparation
the distribution of cells; neutrophils,
basophils, etc. would be even throughout
the smear. In this connection, if the angle of
application is too large (>45°) it will
appear too thin the reverse is (<45°) true
when it will be too thick. Therefore, the
speed of application and or the spread is
critical as well. This means a slow
deliberate motion will ensue to a thick
film and a speedy one cause a thin smear.
Excess or too little amount use of buffers,
stains concentration, plus (color index of
the satins) the timing all rule over the
optimal preparation of the wedge smear
(such as too red, too blue, etc.).
218
Still with handling and manipulations, the
blood drop size, if too large causes a thick
and when a smaller size used causes a
thinner wedge smear. A proper
application of the wedge smear is the one
not too thick with a length of 2-3
centimeters applied from the frosted end of
the slide in feather like shape/appearance,
WBCs and platelets/thrombocytes
should be well distributed among and/or
along body of the slide other than the edges
too. Figure 1-11 shows a properly prepared
wedge blood film/smear. Reading of cell
morphology/inclusions and detection starts
at the thinner end of the slide with red cells
not overlapping, in a described zigzag
manner, along the length of the smear with
cross-wise (crisscross manner) progression.
Fig. 1-11: A properly made wedge blood
smear/film.
To stain a properly made smear need to use
“dip method” with Romanovsky stains
such as, Wright or Geimsa stain. For
this method after making the smear with
anticoagulated blood, fix the slide with
either a 70% ethanol/or 85% methyl
alc. and/or heat fixation/or air dry (less
recommended), subsequently dry and use
the intended satins and solutions as in dip
technique. In this technique, dip the slide
into Wright stain container, then into
Wright/Geimsa stain one, then into Wright
stain mixed with phosphate buffer and
rinse with deionized distilled water for
three times, all these have separate
containers. Next and at last, smear is ready
to screen and to read cells’ morphology and
properties as in morphoanalysis. Scan the
10X magnification to locate the cells then
utilize 40-50X magnification to detect
morphology/morphogenesis and read the
slide. 100X is the magnification by which
the details such as parasites as in malarial,
Schistosomas, Leishmonias and other
miniature elements such as red cell
clumping/agglutination,
rouleaux
formation, bar bodies, Howell Jolly
bodies or other detailed morphological
entities and pathomorphology indications
etc. can be differentiated.
After wedge smear, we have to set up ESR
tube, using the Westergren technique,
ESR tubes are filled with anticoagulated
whole blood from the lavender top tubes,
with the assistance of safety bulbs or
aspirators attached to the ESR tubes, then
place them in the Westergren rack. The
ESR tubes plugged with cotton tips so that
tip absorbs excess blood. Subsequently read
the tubes after 1 hour in millimeters. The
normal range for males is 0-15 mm and for
female subjects is 0-20 mm. The
Wintrobe ESR tubes are different from
Westergren in the length and size of tubes.
Automated
ESR
(erythrocyte
sedimentation rate) is also available, e.g.
Ves Tec® or Vacu Tec® these systems
measure the opacity of the blood by photooptical systems onboard. The ESR is not so
informative except in multiple myeloma
(MM) presumptive/tentative diagnosis.
ESR by the time will be removed from
hematology diagnostic tests. An ESR of
219
100 mm or over may be indicative of MM.
(Quirt I et al.)
The
next
chapter
two
discusses
fundamental in Hematology, as historical
perspective and basic concepts.
In regard to Hgb. the classical hemoglobin
estimation needs Drabkin’s solution
consists of anticoagulated whole blood, and
potassium ferricyanide, potassium
cyanide and dihydrogen phosphate.
Addition of these solutions to each other
produces cyanomethhemoglobin. The test
reads spectrophotometrically along with the
standards curve that will be instituted.
Unopette system can be used for dilution
and mixtures of these solutions. The
automated systems had made the procedure
easy with the same chemical principles and
are available in market with automated
instrumentation. HemoCue® is a popular
manual methodology with an ease of
performance. Standards and test will be
read at an absorbance of 540 nm
spectrophotometrically.
Hb/Hgb
(hemoglobin) ranges between 14-17.5 g/dl
for males and 12.3-15.3 g/dl for females.
CLIA
’88
(Clinical Laboratory
Improvement Act, 1988) recommends
use of HemoCue® in the event of POCT
(Point of Care Testing). In regard to
Hematocrit/Hct.
determination,
microhematocrit tubes/capillary tubes (such
as Natelson/Natelson®) are available,
which are filled by whole heparinized
blood (some capillary tubes has coat of
heparin inside them and color coded). The
capillary tube once sealed with clay (or
self-sealant, etc.) with specimen inside will
be centrifuged and reads by a reader or
caliber. This type of centrifuge used for
hematocrits is called, ”hematocrit
centrifuge”.
Chapter Two (2)
Fundamentals
Hematology
in
Historical Perspective
T
he first advancement in Hematology
commenced by the discovery of
microscope and red cells by
Anthony van Leeuwenhoek, in 1642
and worms observed by Athanasius Kircher
in 1657 followed by platelet discovery in
1800s. Wright stain was developed
during 1902 by the developer James H.
Wright. This discovery years later
preceded by the developments of many
automated instruments in hematology,
smear preparation and blood screening for
hematological disorders and Oncology
through morphology as these remains the
core and central pivot of Clinical
Hematology/Oncology labs.
These discoveries in Hematology are cited
in short by timeline as followings;
In 460-377 BC., Hippocrates teaches the
humoral theory, which describes excess
or deficiency equals illness this was as a
hypothetical theory, in humoral, which this
term later coined to be “blood”, its factors
and other constituents.
In 1616 William Harvey introduces the
concept of circulation. Afterward in 1642
as mentioned earlier, van Leeuwenhoek
discovered microscope and discoveries of
red blood cells followed in the same years.
220
In 1842 Alexander Donne identifies
platelets and during later years Rudolph
Virchow expresses the importance of
fibrin and coagulation process.
Discoveries
continue to 1925 when
Thomas Cooley defines and describes
Mediterranean
Hematologic
Syndrome
of
Anemia,
Erythroblastosis
fetalis
and
Splenomegaly, which later named as
Cooley’s anemia and now as
Thalassemia.
In 1938 Louis Diamond along with Dr.
Kenneth Blackfan described a type of
Anemia known as, “Diamond-Blackfan
Anemia”.
In 1990s G-CSF and GM-CSF becomes
clinically
available
along
with
recombinant factor replacement
products. By 2000 research continues the
endless discoveries and new
scientific research emerged.
era
of
Hematology is an essential discipline
among other pathology subjects that mainly
deals with blood constituents, diseases and
coagulation factors along with coagulation
system and disorders.
Basic Concepts: (Dyssypris EN, et
al. & Rodak et al.)
The Origin of Hematopoietic
Cells
Average Human Blood is about 6-7 liters in
a 70 kilogram individual with 45% consists
of elemental cells (WBC, RBC etc.)
constituents and the other 55% is liquid
plasma (Clinical Chemistry, Fig. 5-4).
90% of plasma contains water and the left
10% as soluble constituents such as
carbohydrate,
lipids,
amino
acids,
vitamins, glycoproteins, proteins and
electrolytes etc.
The hematopoiesis (blood formation)
starts at embryo as an embryonic
development at Yolk Sac. This is the
commencing of the generation of all cell
lines. As we know, the development
originates primarily by the appearance of
pluripotential
(pluripotent),
multifunctional hematopoietic stem cell.
The stem cell is a primordial cell whose
function is to generate and produce variety
of cell lines that have definitive function to
the somatic tissue cells; they proliferate
and differentiate to become precursor cells.
These cells are classified accordingly to
White
Blood
Cell/WBC
(granulocytes/leukocyte
and
agranulocytes), Red Blood Cell/RBC
(erythrocyte)
and
Platelet/Plt
(or
thrombocytes in peripheral blood and
megakaryocytes in bone marrow).
CD 34+ pluripotential stem cell rises to
myeloid and lymphoid progenitor
cells.
Uncommitted stem cell (CFU-S and
CFU-M/ colony forming unit-stem cell and
the macrophages) divides to become the
committed lymphoid stem cell, which later
produces lymphoid series (CFS-L/colony
forming stem cell-lymphoid), while from
the CSU-S/CSU-M merges all cell lines
and precursors mentioned above,
221
including,
Monocytes,
Neutrophils/Basophil/Eosinophil,
erythrocytes and platelets: Lymphocytes
are agranulocytes as with Monocytes.
Blood formation at early stages is confined
to liver, spleen and thymus during
embryonic development and the later years,
bone marrow remains the active blood
forming sites during adulthood.
In regard to site of production, red
marrow is the only active site of blood
formation in most of the flat bones or
trabecular bone however are not actively
forming in the canaliculated (Canaliculi)
bones. This red marrow transformed to
yellow marrow at later years in diaphysis
bones/long bones, which is rich in adipose
tissues. After the age of 18-20 red marrow
remains the only active site for blood
formation. Yellow marrow consists of fat
(adipose tissue), and bone elements
(Calcium appetite, sulfate, carbonate and
phosphates, etc.). The following picture
depicts the cell lineages of the
originating cells from the primary stem cell
(Fig. 2-1)
.
Fig.2-1: Hematopoietic cell progeny line or
cell pedigree.
Hematopoiesis is the formation of blood
cells and cellularity in the bone marrow,
bone structure consists of an outer layer of
extravascular compartment and a
vascular
compartment.
Vascular
compartment branches out to form bone
marrow longitudinal artery into sinuses
or sinusoids and finally leads to formation
of veins and its branches and ramifications.
It is in extravascular compartment that
hematopoiesis actually takes place. This is
the place of genesis of erythroid series,
myeloid
(granulocytes),
platelet
(thrombocytes/megakaryocytes),
monocytes and lymphoid cells. The inner
layer next to outer layer is the fatty or lipid
layer. The trabecular projections are
located in between the islands of
hematopoietic tissues, in this respect
erythropoietin and thrombopoietin are
synthesized
in
the
kidneys
mesenchymal cells that affect the
222
production of the red cells and platelets in
the blood forming tissues, especially bone
marrow. The ratio of cells to fat in a normal
subject is about 3:1 in the bone marrow,
which in pathologic conditions changes
in numerator and denominator are
apparent. This represents the ratio of fats to
cells; in other word it is the bone marrow
cellularity, which would be in question in
conditions such as bone marrow
dyscracias or defects in erythropoiesis,
leucopoiesis,
megakaryocytopoiesis
(thrombocytopoiesis). This may reflect as a
decrement, or an increment of cells and/or a
partial
or
total/absolute
aplasia/dysplasia of several residence
cells in marrow and or other hematopoietic
organs, e.g. aplastic anemia or refractory
endothelium
(Rodak
et
al.)
(A)
anemia or Essential Thrombocytemia
(ET). In normal conditions of effective
bone marrow (b.m.) erythropoiesis in bone
marrow can increase 8 times than the
production
of
normal
erythrocytes
generations. Bone marrow cellularity to
adipose tissue (islands of fat cells) is
indicative of the effective and ineffective
erythrocytes production (-poiesis),
leucopoiesis, and platelet generations.
Macrophages as histocytes, in this
scenario, can be loaded/laden with
hemosiderin/iron
pigments/deposition in a condition
known as hemosiderosis. Bone marrow
also is involved in another likely condition
in which the fibroid tissue (Fibrosis)
distort the architecture of the trabecular
and adipose tissues (and hematopoietic
islands) leading to bone marrow failure or
aplasia. Figure 2-2 represents bone marrow
structures and constituents between
extravascular
and
vascular
(B)
Fig.2-2:
AMicrostructure/microenvironment of Bone
Marrow, B- Normal ratio of Cellularity and
adipose tissue
Also bone marrow contains cells such as
osteoblasts, which are building blocks of
bone trabeculae and osteoclasts that are
merely destructive cells of the bone marrow
by which resorption takes place, there is a
subtle hormonal relationship in bone
maintenance as have been discussed in
223
Clinical Chemistry
calcium metabolism.
section/chapter
of
In regard to lymphoid series such as T-cells
and B-cells, (small and large lymphocytes),
these cells are fostered and developed in
the bone marrow, thymus, spleen, lymph
nodes, lymphatic tissues and peyer’s
patches of intestine (GI) among which
are the mesothelial/epithelial layer of
intestines.
Primary lymphoid tissues consist of
bone marrow, liver, spleen and thymus.
The secondary lymphoid tissues consist of
lymph nodes and GALT/MALT associated
lymphoid tissues, e.g. gut associated
lymphoid tissues (GALT) equivalent to
Bursa in chickens and mucosal
associated lymphoid tissues (MALT).
Other immunologically derived cells
encompass NK cells (Natural killer) and
Null cells. These cells act to kill and
eliminate offender/intruder cells (non-self)
or microbes (microorganisms) by secretion
of perforin causes pores or holes into the
target cells, the mechanism of destruction is
similar to complement activation
subsequent to porous formation; there will
be ion exchanges of electrolyte to the outer
environment and final destruction and burst
of the target cell.
All hematopoietic cells except red cells (as
they contain red cell antigens or blood
group systems) contains on their surface
glycoproteins molecules in nature; as in
these, B-cells possess immunoglobulin
and leukocytes consist of Human
Leucocytes Antigen as cell surface
markers (Cell identity antigens/markers). In
this series platelet also absorbs HLA
antigens
(histocompatibility antigens)
from the circulation, as the leukocytes.
These cells (platelets) should be matched
and transfused to patients through a long
term/chronic
platelet
rich
products
transfusion therapy strategies (consult
transfusion chapter).
As we will notice in subsequent chapter the
cell lines; such as progeny and precursors
cells to all erythroid, myeloid and
thrombocytes series, and additionally to
monocytes/
macrophage
(M-theta
cells/Mθ), and histocytes, (etc.) are active
hematopoietic cells that are generated to
maintain homeostasis in our somatic and
circulatory
system
and
avoid
hematopathologic insults. All morphology,
functions, cell specifications, inclusions in
health and diseases will be reviewed in
later chapters.
Different Cell Lines
NE et al./ Rodak et al.)
(Dyssypris
The progenitor cell lines of all types of
blood cells starts with primary stem cells.
In fact at this stage of discussion I will
focus on the white cells, red cells and
platelets progenitors and precursors.
White blood cells or leukocytes are derived
from the primary stem cells (Pluripotent
stem cell) through committed myeloid stem
cell as CFU-GM to myeloblast.
Myeloblast
gives
rise
to
polymorphonuclear leukocytes; these
cell lines include the precursors such as,
Neutrophils, Eosinophils, Basophils, and
macrophages
(types
of
cellular
monocytes/histocytes). The early cell
bursts to these cell line is Progenitor cells
224
in myelopoiesis, this is named as
Pluripotential stem cell, these cells also
derives
to
Lymphocytes through
Lymphoid series. The following figure
(Fig. 2-3) indicates normal mature myeloid
series, as Polymorphonuclear Neutrophils,
Leukocytes and Lymphocytes.
Fig. 2-3: Mature Myeloid Blood Cells
Leukocytes, blood film.
The first line after myeloid series come
Myeloblast. This cell is 10-18 um in overall
size and oval to round in the shape of
nucleus, N/C ratio of 4:1 (nuclear to
cytoplasmic ratio) reflecting an oval
nuclear shape with 1-5 nucleoli containing
Auer Rods ( a type of purplish red
degranulated neutrophilic or azurophilic
granules –primary or non-specific
granules- and lysosomal materials) with
no granules might be present.
Next is Promyelocytes after Myeloblast.
This cell has some of the morphological
and functional characteristics of Meyloblast
in which the size is larger than Myeloblast
(14-20 um) with heavy granulation of
auzorophilic granules and N/C ratio of 3:1
with an oval nucleus. The third series
would be Myelocyte with a size of 12-13
um in diameter and N/C of 2:1 or 1:1 with
indentations and a round nucleus. This cell
possesses some blue granulation and has
characteristics of its previous series.
Subsequent
to
myelocyte
is
Metamyelocyte, this cell has a nuclear
indentation and with blue granulations
mostly primary granules or known as
granulocytic granules (neutrophilic or
secondary granules). Its size equals to 1018 um with a C/N ratio of 1:1.
Band Cells are the next is in catergory
that has an N/C ratio of 1:1 and with blue
granules and an elongated, single lobe and
curved nucleus almost Reni shape, it has a
very typical nuclear morphology, it should
be present in peripheral circulation by 5%
as still indicating a normal indicator. This
cell has remnant of blue granules from
Metamyelocytes.
The last series are Neutrophilic
leukocyte/or segmented neutrophil,
with purplish pink cytoplasmic staining with
Wright and Geimsa stain, contains 2-5
nuclear bands or lobes, size equal to 10-16
um and with specific blue pink granules,
with an N/C of 1:1. The leukocytes have
inflammatory action potential and increases
in number at the site of inflammation
when bacteria invade tissue. They release
febrile active cytokines (such as leukotriene,
cytokines and interleukins), this is when
inflammation response be instituted. Heavy
azurophilic
granules
or
secondary
granulation
in
Leukocytes
indicates
intoxication with heavy elements such as
copper, zinc or mercurial toxicities,
vacuole formation in these cells. They have
the same connotations as these two
predicators (vacuole and granulations) of
intoxication can also indicate infection.
Defect in granulation manifest in an
225
exemplary of condition such as, Chёdiak
Hegashi Syndrome, a genetic disorder of
From the same Promyelocyte comes forth
basophilic myelocytes and then
basophil. Mature Basophil is 10-16 um in
diameter and has a distinct nuclear lobes and
a dark-blue overall granular staining; it
contains
heparin like-substance
and
peroxidases. It contributes to inflammation
and chemotaxis, the priming of the target
and offender cell with IgE and chemotactic
factors that bring about phagocytosis,
which is the engulfment of bacteria and
foreign/non-self, substances/organisms and
subsequent killing and elimination of the
said organisms, all leukocytes have
inflammatory properties.
Basophils participate in graft versus host
reaction (GvHR) and immediate
hypersensitivity if the response is to
Allergens. Eosinophil derives from
progenitor cell such as the same as
leukocyte evolutionary line; it is from
Myeloblast to Promyelocyte and then to
Eosinophilic myeloctye, this follows
with Eosinophilic Metamyelocyte forms
and then to Eosinophilic band cell to
mature Eosinophil. Figure 2-4 shows some
of immature myeloid series.
granules.
Fig.2-4: showing, myeloid series, immature
and mature, left shift in peripheral blood.
Mature Eosinophil contains orange granule,
size is 10-16 um in diameter with distinct
nucleus with a C/N ratio of 1:1. These cells
have
locomotion
and
phagocytosis
properties and participate in immunologic
reaction/response; it is an effective
participant against viruses, fungi and
parasitic infections and organisms.
The function of this cell is not well
distinguished (Quirt I et al. Dyssypris NE et
al.).
The next series are erythroid series. The
occurrence of erythroid progenitor cells has
come to existence primarily through
Pluripotential stem cell; the subsequent
stages as myeloid stem cell, to Blast
Forming Unit-Erythroid (BFU-F) and
Colony Forming Unit- Eryhtroid
226
(CFU-E),
Pronormoblast,
Basophilic
Normoblast, Polychromatic Normoblast,
Orthochromatic
Normoblast,
Polychromatic
Erythrocyte
(or
Reticulocyte)
and
finally
mature
Erythrocyte (red cell). This is the European
nomenclature for red cell’s evolving stages.
For
American version
we have;
Rubriblast which occupies 1% of the
marrow with a size of 12-20 um, N/C ratio
of 8:1 and it has round nucleus with fine
clumped chromatin and 1-2 nucleoli.
Cytoplasmic color is intensely blue. The
other stage next to Rubriblast is
Prorubricyte, this cell has inheritance
from the primary stages, Rubricyte with
different self-properties, these include 14% of the bone marrow, a bit smaller in
size as 10-15 um, N/C ratio of 6:1. It has 1
or no nucleoli with some chromatin
clumping and a rich deeper blue color more
than blast. The third stage is Rubricyte with
N/C ratio of 4:1, with 10-20% containment
in bone marrow and clumpy chromatin,
and it is Murkey (dark) grey blue
cytoplasmic color. The penultimate stage is
the Metarubricyte, with pink to slightly
bluish cytoplasmic color. It has an N/C
ratio of 1:2 and bone marrow contains 510% of them. With almost or totally
Pyknotic chromosome the cytoplasmic
color is pink-organ to slightly bluish hue.
The last stage consists of Reticulocyte,
which can have I nucleus or without
nucleus, it has 1% of the bone marrow and
basophilic stippling with cytoplasmic color
of bluish hue. Figure 2-5 indicates red cells
morphologic classifications (Turgeon et al.
Rodak et al).
Red cells are responsible for oxygen
saturation and carries oxygen (O2) to the
tissue cells and vice versa it takes back
carbon dioxide (CO2) to the lungs. It plays
a crucial role in different kinds of Anemia
and
its
reduction
in
size
(Anisocytosis/size variation), number and
shape
variation/morphology
(Poikilocytosis) affects the intensity of
the particular Anemia.
Effective and ineffective erythropoiesis
depends on the balance of Reticulocytes
and mature red cells either in bone marrow
or peripheral blood. Normal erythropoiesis
consists of 1-2% Reticulocyte in blood
stream/circulation in adults and 5-6% in
neonates. These percentages indicate
normal marrow functionality and variation
from these normal ranges show effective or
ineffective
erythropoiesis,
dysplastic/aplastic/fibrotic conditions
of marrow and bone marrow failure with its
lack of productivity.
Fig.
2-5:
The
appearance
and
morphology/morphogenesis of erythroid
series progenitors, with some normal red
cells; source; bone marrow aspirate, 1000x
magnification.
227
Next categories would be Thrombocytes
or
Megakaryocytes
and
Macrophages/histocytes.
Megkaryocytes
are
derived
from,
pluripotential stem cell and progenitor cell,
which subsequently transforms into
Megakaryoblast and Megakaryocyte and
finally to Thrombocytes/platelets. Their
size varies from as little as 4 um in
diameter to a much bigger sizes (as 30-35
um) and is multinucleated. They are
produced and reside in bone marrow and
are responsible for coagulation and
thrombosis, they contain primary Platelet
factors (PF) and mature to normal
platelet/Thrombocyte as big as 1-4 um in
size. The process encompasses chipping of
part of the cytoplasm into platelet
formation. This is when a large young
Megkaryocytes transforms into a normal
platelet. It will divide into many
margination boundaries and then
cytoplasmic division takes place. Platelets
contain
Platelet
factors
(Glycoprotein/Gps, Plt.F1, Plt.F II, Plt F
III & IV) with specific functions in
coagulation
and
thrombogenesis
(clot/thrombus formation).
The last category of cells are Macrophages
these are the same as Monocytes; when
these cells are active they become large and
with many pseudopods (false feet), that
engulf any foreign or self-damaged
particles or organisms. It is an APC or
antigen processing cell active in
adaptive immunity. It is also called Mtheta and is a variety of Monocytes; in
tissues it is called Histocytes. All these
types or morphotypes have the same
function. It has different names in different
organ tissues, such as microglial cells of
the nervous system, kupffer cells of the
liver (Hepatocytes), dust cells of alveolar
cells/tissues,
adipose
tissue
macrophages in adipose tissues,
sinusoidal or dendritic cells of spleen,
peritoneal macrophages in Peritoneum,
histocytes/giant cells of connective
tissue, Hofbauer cells of Placental
tissues, and osteoblast of bone marrow
plus epithelioid cells of granulomas.
These are all part of reticuloendothelial
system.
The term shift cells (Left shift) in regards
to the cell linage applies to immature cell
lines especially during the time they transit
to the peripheral circulation in pathologic
conditions is from bone marrow or blood
cell producing organs toward peripheral
blood, these manifest in pathological
conditions
such as Leukemia,
lymphomas or metastatic carcinomas.
It definitely applies to blood cells series,
but not to the solid-organ-tissue
malignancies. These pathological solid
carcinomas are secondary to immature
blood formation in some of these
conditions.
Other situations in which there may shift
cells apply are such as different anemia.
These can show and present with the
picture of left shit (e.g. Thalassemia, or
Megaloblastic and pernicious Anemia,
Hairy T-cell lymphoma, or Sёzary
Syndrome (SS), Hodgkin and nonHodgkin’s lymphoma (HL, NHL), etc.).
For the cells of Lymphoid categories, they
originates from pluripotential stem cells to
progenitor cells, then they successively
228
reside within the primary lymphoid organs
such as liver, spleen, bone marrow and
thymus to become pre T-Cells and pre
B-Cells, subsequently the transit to
secondary lymphoid organs such as MALT
and GALT to transform into T-Cell, B-Cell,
lymphoblast/plasma-blast,
and
plasmacyte//plasma cell.
participate in parasitic infection along with
basophil such as, Trichinella spiralis
(Trichinosis), hook worms/round worms,
as Diphyllobothrium latum and Ascaris
lumbericoides
(Ascariasis)
and
(Helminthes and Nematodes) infection,
fungal and like bacterial infective insults,
induced allergies.
The appearance of lymphoblast has a size
of 15-20 um, N/C 4:1 with round to oval
nucleus containing 1-2 nucleoli with
cytoplasmic medium-blue staining.
Also in this series we have Prolymphocyte, with N/C ratio of 4:1 to 3:1
and a size compared to the size of a
Lymphoblast but smaller equals to 15-18
with oval to slightly indented nucleus and a
color of medium-blue cytoplasm with few
azurophilic
granules.
Mature
lymphocytes embraces; small lymphocyte
with a 6-9 um in diameter and with an N/C
ratio equals to 4:1-3:1 it has a round to oval
nucleus. It has medium-blue cytoplasm
with few azurophilic granules, the other
mature lymphocyte is large lymphocyte
with a size equals to 17-20 um in diameter,
N/C ratio of 2:1 and a medium-blue
cytoplasm and few auzrophilic granules.
Their nuclei have a dense chromatin with
dense
euchromatin/heterochromatin
and a less parachromatin.
Lymphocytes as explained in chemistry
section and partly prior to here are cells of
innate and acquired immunity. It consists of
humoral and cellular arms of immune
systems
that
control
HLA
and
Immunoglobulin (Igs) production. The
most common types of T-cells are helper Tcells
or
CD4+
cells
and
Tsuppressor/cytotoxic cell or CD 8+ cells,
they participate in immune reaction to
either protect the individual from acquiring
infection or conversely damaging organ
system by their action in autoimmunity
(such
as
in
Good
Pasture
Other immune related cells are Plasma cells
and Mast cells, which have a certain role in
immune apparatus. Plasma cells are active
in immunoglobulin production while Mast
Cells are active in cellular immunity upon
stimulation by allergens and production of
IgE and it contains bradykinin, heparin,
interleukins
and
other
cytokines,
chemokines and serotonin (neural
transmitters) respectively. It is actively
Syndrome/Autoimmune
Glomerulonephritis) and in Henoch
Schönlein Autoimmune Allergic
Purpura or phlebitis/vasculitis). These
are examples of pathology of immune
system that do not recognize the self and
due to changes in molecular integrity
(homeostasis) of the tissues they arose into
an autoimmune response to self as a nonself.
Recognition of self from non-self, will
elaborate a pathologic response toward
the damaged organ systems. In doing so
there is a cytotoxic antibodies or
immunoglobulin reaction against the
offending antigen either auto-antigen or
foreign antigen e.g. in Rheumatic fever
(RF) and Rheumatic Heart Disease (RHD)
in which the cytotoxic antibodies attacks
229
the common “M” receptor on the heart
muscle leading to valvular defect and
thus fibrosis and calcification/damage to
the heart valves due to the offending
Streptococcus pyogenes; this is a βhemolytic strep and has antigen
homologous to M antigen in the cardiac
muscle. This can pursue to destruction of
this tissue through complement activation.
Increased total number or differential
percentages of mature lymphocytes are
indicators of viral and fungal infections,
with no purulent or pyogenic infections and
lack fever in the patient except in febrile
reaction due to some cytokines. A blood
smear with lymphocytosis is likely an
indication of viremia and viral meningitis
especially if found in CSF (Cerebrospinal
fluid) spinal tap.
In summary, blood linages derive from
primary stem cell progenitor, and they
include neutrophils, basophils, eosinophils,
monocytes, lymphocytes and macrophages
and plasma cells/plasmacytes.
The
key
to
understanding
the
morphological
determination
or
differentiation of these cells particularly by
relaying on observation of their size
variation, nuclear to cytoplasmic ratio,
shape of the nucleus and the cytoplasm,
granulations, their staining properties,
nuclear
and
cytoplasmic
staining
characteristics and functional assays, over
all are tools in identification of alteration in
these normal morphogenetic evolution.
Diagnostic
Hematology/Oncology: (Quirt I
et al. Rodal et al.)
Chapter Three (3)
Differentiation
and
Identification of red cells,
white cells and platelet
anomalies/alterations: (Rodak et
al.)
D
ifferentiation and diagnosis of
different erythrocyte, leukocytes
and thrombocyte’s pathologic
alterations in hematology laboratory solely
depends on morphology in manual
methodology or by other procedure
explained
before
by
sophisticated
instruments such as flowcytometry. As I
have had some discussion about the
methods earlier by which a flowcytometer
analyzes and detects morphological
changes, differences and sorts information
with a subsequent data analysis of result,
then these successively will be reported,
there would be no need of citation of this
procedure (mentioned in clinical chemistry
section), and we adhere to manual
interpretation of blood smear/or film, which
today still it is a mandate to hematology
laboratory.
Leukocytes, Lymphocytes and
Thrombocytes: (Glader B et al.)
Most of the changes in cell structure are
due to abnormalities or anomalies of the
cell function or physiology due to disease
process. These structural changes can be
specific and reflect the disorder in which
the cell has been afflicted from.
In most cases the changes may be typical
predicator of the particular diseases such
as LE cells for Systemic Lupus
230
Erytheomatosus (SLE) or Sickle Cell
disease (SCD and trait). These pathologies
are also reflected in Alder Reilly
syndrome with Alder Reilly cells
(specific heavy granulation predicator of
affected Neutrophils) as a distinctive
disorder. Other cellular defects are
manifested in Pegler Huёt cell, which
has a pence-nez appearance of nucleus,
similar example typically associated with
pseudo-Pelger Huёt cells represent
drug toxicity, infections or typically is
significant
in
Chronic
Myelogenous/Myeloid Leukemia (CML) or
acute leukemia. Figure 3-1 shows some of
the hematologic cell alterations in
peripheral blood.
(B)
Toxic Granulation
Howell Jolly Body
(A)
(C)
Vacuolization in Monocytes
231
bone marrow macrophages laden within a
heavy loads of iron are typical for
hemosiderosis.
(D)
Fig. 3-1: some of inclusion bodies in
Leukocytes, A) Howell Jolly body, B) May
Hegglin anomaly, note the giant
thrombocyte next to it, C) Heavy toxic
granulation,
D)
Vacuolization
of
monocytes indicating infection and heavy
metal poisoning as lead toxicity. With total
Magnification 1000x; oil immersion lens.
Nevertheless changes may be common and
universal for such examples we have
Howell Jolly bodies (remnant of DNA
in RBCs) present abnormality in red cells
for a variety of anemia. Other conditions
such as hyper-granulation of neutrophils
represent heavy metal toxicity or infection
with viruses, bacteria or fungi. These
insults result into abnormal granulation and
vacuolization.
WBCs’
include
alteration
pathologically
also
of
vacuolization
Polymorphonuclear
Neutrophils
leukocytes (PMN) and formation of
phygolysosome, which is an indicative of
toxicity, drug poisoning, bacterial and
fungal infection (e.g. Aspergillosis and
pneumonia by Aspergillus fumigatus, etc).
Also other inclusion bodies is the presence
of siderosomes; this is significant in iron
overload and refractory anemia with
sideroblastosis and ring cells, in these,
In regards to cell abnormalities, May
Hegglin anomaly and May Hegglin cell
are differentiated along with Döhle
bodies; these Döhle bodies are denatured
RNA (Ribonucleic Acid) remnants and are
manifestation of genetic disorder such as
storage pool disease, burns, infection and
drug abuse as well. These two pathologies
(May Hegglin and storage pool disease)
are distinct and identical/unique disease
entities. In order to visualize May
Hegglin’s cell inclusion bodies, an electron
photomicrograph is essential.
Increased leukocyte alterations in size is
called megalocytes (the same name
applies for erythrocytes/megalocytosis),
variation in color of the said cells is called,
Hyper- or Hypochromasia (the same
for erythrocytes series/precursors). Increase
in total number is known as Leukocytosis
and the decrease reflects Leukopenia.
Basophils increment is Basophilia and
decrease rarely named as Basopenia these
types of phenomena will be discussed in
later chapter as the correlation in disease
conditions.
Eosinophilia and Eosinopenia are
terms coined for increase and reduce levels
of eosinophil respectively. Additionally,
monocytes alteration in numbers includes
Monocytosis for increase and for
decrease, we have Monocytopenia.
Furthermore, monocyte vacuolization is
seen in mercurial/cyanic and other heavy
metal poisoning/intoxication, burn and
232
infection.
For
erythrocytes
we
lymphocytes
and
use
the
terms
Lymphocytosis (for increase numbers)
and Lymphocytopenia (for decrease
numbers), for augmented numbers in
erythrocytes collateral terms applies as
Polycythemia (Rubra Vera) and for
decrease Erythrocytopenia.
With regards to platelets, thrombocytosis
is for increment of platelets total number
and
decrement
includes
thrombocytopenia. Abnormally looking
platelets
(morphopathology)
are
Megathrombocytes or giant platelets and
bizarre thrombocytes. MPV (mean platelet
volume) is an average of all/total platelets
volumes. Its range spans between 2-20
units and is significant in platelets disorders
such as ET, TTP, ITP, and in Chronic
Myelofibrosis
with
extramedullary
hematopoiesis (CMF).
All these
conditions have indication in many anemia,
leukemia, lymphomas, and in most of
myeloprolifertative
and
lymphoproliferative disorders, and
hemoglobinopathies, as well.
Still under the said rubric, we have
basophilic stippling, monocytic dust
granules, bar bodies (chromosome-X
bodies- in females), and smudge cells
(these
are
increased
in
Chronic
Lymphocytic Leukemia/CLL), increase in
size of all cells expresses as in
macrocytosis and decrease as in
microcytosis. These cellular phenomena
represent aberration and diseased states.
Erythrocytes
abnormality
anisocytosis (variation
consists of
in
size),
poikilocytosis (variation in shape),
chromaticity/chromasia
(staining
properties variation) and inclusions, these
are exemplified as Heinz bodies, which
reflects hemoglobin denaturation and are
indications of unstable hemoglobin M,
G6PD deficiency and drug induced with
such medications as dapsone, quinine
and sulfasalazine. Other inclusions such
as Pappenheimer bodies, which are the
denatured hemoglobin (hemosiderin), are
seen in some conditions such as
sideroblastosis, lead poisoning, anemia
and hepatosplenomegally and some
iron loaded states with hepatocellular
degeneration, are significant in
identification of the related disorders.
Erythrocyte Series
Normal erythrocyte is discoid and
biconcave and appears as a shape of a
doughnut with 7 μm in diameter with
depression at the center. Other alternate red
cells abnormalities include, spherocytes
in which red blood cell loses discoid shape
and becomes rounded/oval with no central
pallor , it is a common sign in some
hemolytic anemia and in Warm
Autoimmune Hemolytic Anemia
(WAIHA) or Warm Agglutinin Disease
(WAD/ In these RBCs agglutinate in blood
smears and are significant indicator of this
condition), in this disorder red cell changes
shape as from discoid appearance and then
once passed through the spleen, parts of it
breaks up/off due to complement activation
in sinusoids, it is during this time that
afflicted red cell sustain an oval shape
representing spherocyte. It is common in
post splenectomy by few numbers, also
in neonates and newborns; these two
233
conditions clinical implication is not
significant; nor is significant to report less
than 1% of these cells in adult
spherocytosis. If the count is more than
normal, it is predicative of WAIHA and
hereditary
spherocytosis
(HS).
Laboratory Scientist should report presence
of normal 1% spherocytes in adult patient
while a normal 3% count on blood smears
should be reported in neonates and children
for
hereditary
spherocytosis,
microspherocytes are very common or
better to say are indication of a burn case.
Stomatocytosis and Elliptocytosis are
the other types of anisocytosis and
poikilocytosis in morphological and clinical
finding on blood smear. Stomatocytes are
common in hereditary Stomatocytosis,
acute alcoholism, in neoplasm and in
hepatobiliary/obstructive
diseases.
They look like open mouth or a slit like
opening.
In Elliptocytosis, cells (RBCs) appear
elliptical and are commons in hereditary
Elliptocytosis, if they appear more than
25% of time in all red cells indicated
morphology they have to be reported and
are
significant
for
this
genetic
disorder/hereditary condition; these cells
are common in disorders such as
microcytic
anemia
and
iron
deficiency anemia (IDA).
Next we have ovalocytes in the states of
ovalocytosis, this is almost the same
appearance as Elliptocytes but much round
and a bit smaller, they may appear ovoid or
elliptic.
They
are
significant
in
megaloblastic anemia and other conditions
related to Elliptocytosis.
In addition to the above less significant and
less clinically apparent conditions are RBC
dyscrasia and abnormalities, encompass
Sickle Cell (in Sickle Cell anemia),
Malarial Cells and Burr cells (which have
spaced blunt edges), Burr Cells are
significant in MAHA (microangiopathic
hemolytic anemia), ITP (idiopathic
hemolytic anemia), liver disease, uremia
and carcinomas.
Target Cells or codocytes appear like
bulls-eye with more central concavity. In
this
hemoglobin
is
less
dense
(hypochromia) and are found in liver
disease, thalassemia, post-splenectomy and
iron deficiency.
With tear drop cells or dacryocytes, there
are shaped like a tear (quasi-tear). They are
found in cases of especially in
myelofibrosis, subsequently in ineffective
erythropoiesis, myelophthastic anemia
and in megaloblastic anemia and
thalassemia.
Crenated Cells or Echinocytes are cells
with equally shaped short projections with
a number of projections equal to 10-30.
They can be artifacts or found in blood
smear of patients with kidney disease in
vivo or they can be in vitro artifacts due to
PH changes and shrinkage of RBCs due to
faulty smear preparation.
Helmet Cells are particularly found in TTP
(thrombotic
thrombocytopenic
purpura),
DIC
(disseminated
intravascular
coagulation),
HUS
(hemolytic uremic syndrome) and
MAHA, these cells
keratocytes.
are also called,
234
Schistocytes or fragmented cells with
many sharp-like projections must be
distinguished and differentiated from
created cells, are particularly indicative of
MAHA, TTP and DIC, also it is common in
macroangiopathic-hemolytic-anemia such
as heart valve hemolysis, in carcinomas,
uremia and severe burns and in thermal
insults.
Also other cells will be mentioned here is
Blister
Cells
with
a
single
bulging/projection and Bite Cells are found
in hereditary defect of G6PD deficiency
(enzyme defects) and are shaped according
to oxidative degeneration damage of the
cell because of the lack of glutathione and
NADPH reduction, they are shown as
partly gone as an impression of bitten
apple, etc. this is an oxidative damage to
the cell.
Lastly Acanthocytes with spicules, thorn
shape appearance, therefore they are
predicative sign of MAHA, alcoholic liver
disease, hereditary acanthocytosis and
abetalipoproteinemia, known as Spur
Cell as well.
Microcytosis have correlates with a low
MCV (mean Corpuscular/cell volume)
and RDW (red cell distribution widths,
which is an average of total red cell
volume) are indication of hypochromic
microcytic anemia.
Macrocytosis
indicates
increase
reticulocytes formation (reticulocytosis)
with high MCV, and round macrocytes
designates liver disease, hyperthyroidism
and alcoholism.
Finally with regards to the platelets
morphopathology
and
abnormalities,
thrombocyte morphological abnormality is
only mentioned once and at this point
shortly states that this form a giant platelets
(thrombocyte) that are seen in some
hematological and pathological condition
an example is May Hegglin and Essential
Thrombocytosis (ET) and bone marrow
aplasia, in addition, some coagulation and
thrombotic
disorders,
they
display
abnormal giant or microplatelets or other
bizarre types according to the type of
anomaly.
The above Poikilocytic and Anisocytic
criteria including macrocytic/microcytic
morphology are the most encountered in
clinical laboratory hematology practice and
they essentially need a thorough review of
the blood smears in which certain disorder
has been suspected and these blood
screening determinants should be in the
mind of the lab scientist when
examining/reviewing the blood film/smear.
In this association, (alterations) we need to
add in vivo and in vitro rouleaux formation.
This is when red blood cells can pile up on
each other as a stack of coins it is the cause
of either in vitro PH of the medium changes
or for the reason of reduction in the Zeta
Potential zone of RBC’s surface negative
potential. The following figure (Fig. 3-2)
shows some of the blood cells
abnormalities and alterations.
235
(A)
(D)
Fig. 3-2: Some microphotographs of Red
Blood Cell Alteration, (A) Elliptical and
oval erythrocytes (ovalocytes) with
Hypochromasia, (B) some of tear drop
cells, Spherocytes, few crenated cells and
Schistocyes with Hyperchromasia, (C)
Hypochromasia
with
anisoand
poikilocytosis,
(D)
Sickle
cells,
Schistocytes
and
Target
cells,
magnification 1000x (courtesy of Adam).
(B)
(C)
Other abnormal constituents of
hematopoietic cells
Under this topic we have hemoglobin
(Hgb.), which decreases significantly in
anemic conditions either in microcytic
hypochromic anemia, or in bleedings
and external or internal hemorrhages,
however, at the current active stage of
bleeding anemia is normochromic, whereas
subsequently it converts to hypochromic
anemia due to the chronicity of the
underlying bleeding or hemorrhagic
condition, different types of bleeding
consist of petechiae, mucocutaneous or
subcutaneous
hemorrhages,
ecchymosis, purpura and freckles, etc.
236
Hemoglobin may increases as well as in a
variety of conditions (respectively MCHC
does not increase in intensity of the color
due to definitive size and the limited
amount of hemoglobin present in the
RBC/MCHC.) it is increased in MCHC
(Mean
Corpuscular Hemoglobin
Concentration) artifactually, so relatively
that is evidenced in Spherocytosis where
we
have
macrocytic
hyperchromic
(hyperchromia) anemia; in practice
hyperchromia does not exist due the said
explanation,
Hemoglobin increases, moderately increase
or decreases in many hematopoietic
disorders (refer to the later chapters) and
anemia, e.g. include, Iron Deficiency
Anemia (IDA), vitamin B 12 deficiency,
megaloblastic anemia, leukemia and
lymphomas.
Moreover, hematocrit (Hct.) has a direct
relationship with Hgb. Increase or
reduction in Hct. correlates with Hgb. In
these
ESR
(Erythrocytes
Sedimentation Rate) is similar to Hct.
and Hgb., but distinctively different. It is
the rate of sedimentation rather than the
pack cell volume (Hct.) or the contents of
RBCs (Hgb.). Increases in ESR are
particularly seen in Multiple Myeloma
(MM) or other leukemia. Decrease/increase
production due to
fluctuation in
biosynthesis (Hgb.) result in low/high Hgb.
and Hct. (vice versa).
Other parameters that oscillates in
hematologic conditions are MCV (Mean
Corpuscular Volume), MCH (Mean
Corpuscular Hemoglobin) and MCHC
(long forms mentioned before), These are
RBC indices and are significant red cell
profiles in such disorders as Iron
Deficiency
Anemia,
(hereditary)
Spherocytosis,
G6PD
deficiency,
megaloblastic anemia, liver associated
anemia with its pathology, alcoholism
associated anemia, HUS, TTP, MAHA,
intrinsic and extrinsic anemia, and
other hemolytic anemia, etc.
Reference interval ranges include; MCV in
femtolitter (fl), MCH, is in pico-gram per
litter (pg) and lastly MCHC is in gram per
litter (g/l) or percentage (%) units. Normal
indices reference rages are respectively as
following: 80-90 fl., 26-32 pg, and 320-360
g/l.
In vis-á-vis reticulocytes have been
described in this topic, thus I will escape
explaining it in this section.
Reticulocyte Production Index has
clinical implication in, effective and
ineffective erythropoiesis. It is formulated
as; RPI= corrected reticulocyte count/
maturation time. It is increased in anemia
by greater than two (more in method
section). Anemia can ensue by short life
span or reduced survival rates of these cells
(RBCs), which is equal to 120 days.
(Rodak et al)._
Anemia (Glader D. et al.) may be due to
bleeding or short red cell survival as
mentioned, this can be either secondary to
infection or super-infection, and or due to
leukemia and lymphoma, or extendedly due
to the reason of different types of
autoimmunity, bone marrow defects/or
failures, GvHR and post transplantation and
acute and chronic hemolytic transfusion
reactions (A-CHTRs) as well. It may be
additionally due to drug reactivity/or solely
to RBC(s) components (blood grouping
antigens) and therefore due to their
different
types
of
autoimmune
reactions/responses.
237
All these hematological parameters for
RBC and other cells pathologies may be
used to evaluate type of anemia,
Lymphproliferative or Myeloproliferative
Disorders
(LPDs
& MPDs)
and
medical/hematological status quo of the
patient. A good studied clinical history of
the patient medical record is a key
approach to diagnosis and identification of
the underlying disorder(s)/conditions. In
fact a thorough examination of smear and
other flowcytometric information must
be reviewed and analyzed deliberately in
the light of clinical history so that a
complete picture of the condition is
elucidated and thus a definitive diagnosis
may be instituted. To this end, CBC counts,
Hgb determination, Hct evaluation, RI, RPI
and the result of total RBC, WBC and
Platelets count with morphological studies
and histocytochemistry (such as
peroxidases, Periodic Acid Schiff (PAS)
application, Pearl’s Prussian Blue (PPB)
dyes and different kind of esterase all can
point to a presumptive and thus a
definitive diagnosis of the patient’s
condition.
Most anemia have a better physiological
adaptation to the condition thus delude the
Scientists attention, therefore a subtle and
elaborate examination of all testing
modalities and evidences must be called
to
faculty
for
an
tentative
(provisional/presumptive)
or
definitive diagnosis. Following table
is taken from Cornell College 1995, for
classification of anemia (Table 3-1).
Table 3-1; an abridged
classification
chart
of
anemia; courtesy of Cornell
University Medical College,
1995.
Different Diagnostic and
Identification Indications of
Hematopoietic Disorder
Testing
(Methodology)
Method
Although, ESR, CBC
and
other
hemoglobin/hematocrit and reticulocyte
counts and estimations/procedures are
common in hematology labs however, these
are not limited and other methods in
hematology exist. These applications and
other methodologies as ESR test, Ham test
(known as acidified serum lysis test),
238
malarial smears (thick and thin smear),
anemia blood smear screening test, Hgb F
quantitation (alkaline acid denaturation
test), acid elution test, and others as
electrophoresis of serum, iron profiling
tests such as TIBC (Total Iron Binding
Capacity), transferrin, Hgb A (and so on)
quantitation, are several numerous tests
performed in these labs.
In this section we focus on manual CBC,
and hemoglobin/hematocrit and ESR (this
is mentioned before) estimation, which are
the most common and relevant procedure in
hematology.
Routine Testing:
(Turgeon et al
and Dyssypris NE et al.)
CBC
For CBC (or Complete Blood Count) we
use hemacytometer. This is an
apparatus by which the cell counts such as
red cell count, white cell count and plate let
count, will be performed. The figure below
is an example of a hemacytometer (Fig. 33).
(A)
(B)
Fig. 3-3: (A) example of a hemacytometer
with Levy chamber, (B) detail strufcture of
a hemacytometer.
The most common hemacytometer is Levy
chamber with improved Neubauer
ruling (above fig. 2-9, (B)), This chamber
consists of two moats with two raised
surfaces, which have 3-mm X 3-mm with a
dimension of 9mm squared surface area
(preimeter). This is separated by a Hshape moats and on the top of these
surfaces the Neubauer ruling is etched.
Each of these two rulings are composed of
a squar shape etches and as stated total
dimensions are 9-mm 2 . This 9-mm square
surface (for each 2 surfaces at each side of
the hemacytometer) is further divided down
into 3-mm square by 3mm squares,
therehence these further divide down into
one square with 1-mm X 1-mm square
dimension.
These pattern of each 1-mm X 1-mm
square has further reduced to 16 squares for
all of the 1-mm squared boxes, except for
the the middle or central box or the
square where it is divided to 25 squares
with 0.04-mm 2 for the smallest squares in
these (1/25). The largest four corner
squares are for WBC and platelet counts
and central square with 25 squares uses for
RBC count. Fig. 3-4 shows the structure of
239
Levy chamber with Neubauer rulings.
There are identical ruling in each side of
hemacytometer. The depth between the
cover glass and the two surfaces is 0.1-mm,
therefore all squares have 0.1-mm depth
from the cover glass placed on the surface
for counting.
(A)
(B) area R at the fig. A for red cell counts
in a Neubauer ruling.
This chamber not only uses for Leukocyte,
Erythrocytes, and Platelet counts also
utilized in Sperm count with the same
principle counting with different diluting
agent.The counting of each cell is done by
a counter such as Clay Adams® or
Biovation® differential counter.
Using a normal counter we count the cells
in the hemacytometer ruling areas, while
differential counting is done on the
peripheral blood/bone marrow or body
fluids’ smear/film by said former counters.
Previosly pipetts were used in diluting the
blood so that with the provided dimension
we would have fairly accurate estimation of
the cells. RBC count is no longer done by
the manual method for having a large
differences with automated systems due
to much lesser counts in manual
methodologies and high numbers of RBCs.
However still WBC counts, plarelet and
sperm counts are done with manual
procedures.
(B)
Fig. 3-4: (A) Areas W for counting sperm,
and white blood cells and platelet counts,
In preparing the dilution as mentioned in
early part of this chapter, it can be done
with Unopette® system without using
WBC and RBC pipettes. Once the dilution
has been made the blood coagulated with
EDTA or heaprin, dispenses from the tip
of the Unoppette® dispensing tip onto
the v- shape slit at the center of the outer
side of the two Neubauer ruling and then
cover slipped and what we may have in
inspection would be elements under the
light or bright field photomicrograph, some
thing as RBCs in Fig. 3-4 in image (B).
When we are reading and counting cells in
areas W (white cells) for white cell, sperm
240
cells and platelets the cells on the outer
lines should be counted while the cells on
the inner lines of these squares should not
be counted.
It merits to point that the chamber should
not be overflowed with the dispensing
anticoagulated blood and always there
must be two counts in both side rulings and
obtain the average of the two counts. The
following general equation is used to count
cells.
Eq. 3-1
Total leukocyte count is important in
leukemias, anemia, autoimmune disorders,
and infections.
Total platelet counts are significant in
coagulation disorders, coagulopathies,
thrombolytic therapy, thronmbosis and
some other inherited, familial and genetic
abnormalities. Toatl RBC count will be
used to intrepret conditions such as
Ploycytemia vera, thalassemias, malarias,
leukemia
and
lymphomas
and
autoimmune diseases, in bone marrow
dyscrasias and many other conditions.
Total cell counts = cell counted X factor
2
dilution/ area counted (mm ) X depth
Example WBC total = 98 X 20/ 4 X 0.1 = 4900/mm
9
squared or 4.9 x 10 //L
The same principle equation applies to
platelets and sperm and RBC counts with
different dilution; for WBCs dilution is
about 1:20 and for RBC is 1:100 depending
what ever intended dilution used. For blood
of children or neonates or patients with
hemocncentration there may be higher
dilution and vice versa; patients with
anemia may have a lower dilutions.
In counting WBCs if nucleated RBCs
(NRBC) are present there may be a
correction formula be used. In this equation;
uncorrected WBC count will be timed to
100 and the result will be divided by the
number of nucleated RBCs per 100 WBC
plus 100.
Hemaglobin measurement
Under this topic we have hemoglibin
(Hgb/Hb) estimation, as it still uses
Unopptete
sytsem, while using
cyanomethhemaglobin method. In this test
by the addition of potassiun ferricyanide (K
3 Fe(CN) 6 ) and poatssiun cyanide (KCN) to
anticoagulated
whole
blood the
hemoglubin oxydizes to hemoglubin
ferricyanide to methhemaglubinocyanide.
This is where its name is derived from. For
such test there is a need of stanadard curve
on a log-log and semi-log paper/sheets. The
result reads spectrophotometrically at 540
nm. In addition, sulfhemaglobin will not
be measure by this method for it does not
bind
or
react
with
cyanomethhemaglobin. There are only
5% sulfhemaglobin available in blood,
which does not count and estimate with
other hemoglobins. This method are also
called, “methhemoglobinocyanide”.
In measuring Hgb. It is crucial to consider
the source of errors in estimation, such as
wrong stroage of the potassium ferricyanide
(it shoud be in brown bottle for being
241
light/photo sensitive) also high leukocyte
count may cause turbidity and also other
types of Hemoglobin (e.g. Hgb. S, Hgb. C)
may cause turbidity and erring in
mearurements, in addition there are several
other point of error to the procedures. One
example is, precipitation of globulins in
dieases
as
Waldenström
Macroglobulinemia (WM) or Multiple
Myeloma (MM), which can intefer with
the result. Also along this topic we have
smoker who have higher levels of
Carboxyhemoglobins where it can takes
one
hour
to
convert
to
methhemoglobinocyanide. To have a
quick check of the correctness of Hct and
Hgb estimation one may do a rule of three.
In this the value of Hct. should be three
times more or less than Hgb. Value.
Hematocrit estimation (Hct./
or packed cell volume/PCV)
Using the Capillary tube and sealings, the
blood obtained from the patients in vials or
microtainers will be filled and dispensed in
these special capillary tubes and sealed with
clay or sealant and subsequently placed in
the hematocrit centrifuges. Some of this
type has caliber onboard. Therefore the
column of red cells after spinning will be
measured by a measure/ruler or caliber./
ESR
(Erythrocyte
Sedimentation Rate)
This has been cited already, and therefore a
short citation of the past statements will be
included in here. ESR has a lower value in
clinical hematology these days for not being
very informative except in case of Multiple
Myeloma and gamma globulinopathies
as Waldeströn’s. The Westergren or
Wintrobe
tubes
are
filled
with
anticoagulated blood (EDTA, heparin or
citrate) and set up in the special racks and
after one hour the sediment will be read in
millimeter as already said the normal
expected values for female is 0-20mm and
for male is 0-15mm/hr. The exmple of this
types is Sediplast® by Polymedco
incorporation.
The ESR automated system is an exmple of
Sedimat® 15 automation sedimentation
system by Polymedco. With almost the same
principles of maual application including an
automatic reading device.
Reticulocyte count
The reticulocytes count are significant in
estimation of effective and or ineffective
erythropoiesis. These cells are immature
form of RBCs and the last stage of
denucleation
of
nucleated
RBCs.
Reticulocytes possess remnant of RNA
stained with supravital stains. Other RBC
inclusion bodies that stain with these dyes
include Heinz bodies, Howell Jolly and
Pappenheimer bodies. In addition, one
example of supravital stains are brilliant
creysl blue, cryestal vioet and
methylene blue. Pappenheimer bodies
are also another inclusion with siderosomes
granules and stains particularly with iron
stains such as Prussian Blue. Figure 3-5
shows an example of reticylocyte in
peripheral blood.
242
erythropoiesis. In conditions such as
polycytemia rubra vera, leukemia, some
myeloproliferative,
lymphoproliferative states and some othe
type of anemia, reticulocytes estimation
varies. this will be disscussed later.
Fig.
3-5:
Presentation
of
typical
reticulocytes with RNA and mitochondrial
inclusions in the peripheral blood (arrows)
under oil immersion lens, total 1000x
magnification.
Reticulocytes are counted in an EDTA
anticoaglated whole blood, while
counting 1000 RBCs, then along with these
cells count the number of retics under high
or oil immersion magnification lens of a
light photomicrograph. Counting and
estimation of reticulocytes are for assessing
the bone marrow activity and eryhtropoiesis.
The cells spend 2-3 days in the bone marrow
and then appear in the peripheral blood
afterward. Its count should be equal to 26% for neonates and very young children
and about 1-2% in adults.
Counting reticulocytes with lower count
of 1% or 5% in adult and neonates
respectively present with the clinical picture
of ineffective erythropoiesis and up or
below the reference range is an indication of
hypercellularity or hypocellularity of
the bone marrow erythrocytes respectively.
Normal
counts
indicates
effective
Principles of counting such cells composed
of preparation of wedge smear/film and
fixing and subsequently staining the sample
with methylene blue supravital stain and
scanning with oil immersion lens or
objective. There should be 1000 cells
(RBCs) in an area of the smear where red
cells are not touching each other in
otherwise are close to one another. Thence,
the retic count is included with the total red
cell counts. Then retic counts is divided by
1000 red cells with result multiply with 100
(percent) which gives the total number
precentage of reticulocytes. Equation 3-2
represent this relationship.
Eq. 3-2
number of reticulocytes/1000 red
cells X 100 = reticulocytes precent.
Example. % = 15/1000 X 100 = 1.5 %
reticulocyte percent
For calculation of Absolute Reticulocyte
Count (ARC) we may follow the next
equation:
Eq. 3-3 ARC = reticulocyte precent (%) X
RBC count (10 12 )/ 100
Reference range for ARC is between 25 X
10 9 /L to 75 X 10 9 /L
Other measures in retic counting have the
following equations for correct retic
count (CRC), which is :
243
Eq. 3-4
CRC = retics % X ( Hct %/ 45),
when Hct. is low, the precentage of retics is
factually higher than whole blood
reticulocyte count. Retic count of whole
blood has a lower number of retics than
Hct. with a higher retic counts. Thus the
preceding equation applies for corrrection.
There is also Reticulocyte production
index (RPI) that has its own use and
implications. This equation has not been
mentioned here (refer to fundamentals of
hematology texts.
Pathological
Disorders/Hematopathology
Anemia definition:
(Glader B et
al.)
It is defined as low hemoglobin contents and
the effect is oxygen carrying capacity.
Anemia may come in differeing forms and
is the result of numerous disease conditions.
It can be due to reduction in number of
circulating red cells (erythrocytes).
They are also classified according to relative
amount of RBCs and this anemia is due to
amount over hydration and thus a blood
volume depletion as in physiological
anemia (as in pregnancy) and the second
one is an absolute anemia in which the
number of RBC reduces (in hemorrhage,
myelophthastic anemia, etc.) and effects the
oxygen carring capacity of the blood and
tissue oxygenation. This reflects some
degree of tissue hypoxia.
For understanding the oxygenation and
oxygen carrying capacity of blood refer to
clinical chemistry chapter as in oxygen
saturation curve.
Hemodilution of blood in physiologic or
relative anemia may be renal in origin
(renal pathology). This is a reduction or lack
of hematopoietin/erythropoietin made
by kidneys and or other condition such as
cardiac states; e.g. congestive heart failure
or cardiaovascular shock.
Hemoglobin reduction or anemia may be
mild, moderate or sever depending upon the
amount of hemoglobin present in the blood.
Whereas the clinical
signs and
symptoms involve reduction in oxygen
capacity and blood level saturation, the
reduced blood volume and hemodilution.
The third sign will be augmented cardic
output or over load.
Symptoms of anemia in effect depends on
heart status, severity of the anemia itself and
speed by which the anemia developes
(speed of onset). Reduced oxygen capacity
manifests itself as syncope, and dyspea,
blood volume reduction results into
hemoconcentration.
In
this
regards
hypotension and increased cardiac
load and therefore output ensue to
tachycardia.
There are numerous types of anemia. The
most clinically significant anemia,
compose of current calssification based
on morphological and physiological
groupings/classification. Example of
some of them include, megaloblastic
anemia, pernicoius anemia, prophyrias,
sickle cell anemia, malaria, G6PD anemia,
intrinsic and extrinsic anemias, anemia
244
due to RBC membrane defects
myelodysplastic syndromes, etc..
and
Anemia’s etiology and causation are
erythropoietin dependant in form of
reduction as in; defects in myeloid stem
cells, defects in erythropoietin and reduce
cell maturation, also hemolysis and acute
and chronic bleeding (hemorrhage) decrease
survival rate of RBC and so on.
Once
the
haemeostasis
disrupt,
hematological disorders sequel would ensue.
The most clinically relevant anemias will be
considered in following section.
Megaloblastic Anemia
As the name applies these type of anemia
accompanies by giant RBC or macrocytes
with a definitive size (>110 fl.),
Megaloblastic anemia is a macrocytic
normochromic type anemia and bone
marrow
contains
macrocytic
megaloblastic RBCs. Usually various
pancytopenia
is
accompanied
by
megaloblastic cells. Etiology and causation
is due to DNA slowing down or arrest in the
bone marrow and develops in situ and other
hematological
sites.
Thymidine
phospahtes arrest and reduction in velocity
production ensues to this type of anemia. Its
calssification includes, folic acid, intrinsic
factor and vitamion B-12 deficiency and
others.
Clincial symptoms cosists of, indigestion,
glossitis (beefy red tongue) diarrhia and
different degree of mild to moderate
juandice. In this regards bone marrow is
hypercellular and
with myeloid to
erythroid ratio (M:E) reduction from 3:1 to
1:1. Blood smear study shows erythroid and
nuclear retardation and nuclear and
cytoplasmic
dysnchrony,
meaning,
younger nucleaus to more mature cytoplasm.
Obvious macrocytosis, with numerous
immature RBCs (nucleated) in left shift with
several
Howell
Jolly
bodies:
Karyorrhexsis or breakdown of nucleus is
a manifestation of Howll Jolly bodies
formation.
Also
giant
myelocytes
and
hypersegmentation of the nucleus are the
significance finding on the blood film for
myeloid series. Platelets in bone marrow
(megakarocytes) slightly increased and
sometimes
acompanied
by
thromboctopenia reflecting state of
ineffective
thrombocytopoiesis. In
Macrocytic anemia the red cell indces of
MCV reaches to more then 100 fl (>100 fl).
Ineffective RBCs are due to marrow death
and premature destruction and reduced
survival in the bone marrow these also
covers
granulopoiesis
and
thrombopoiesis leading to pancytopenia.
Marrow turn over and contents can be
estimated by enzymatic tests such as LDH 1
anad LDH 2 and bilirubing level. (for
estimation of these enzymes refer to clinical
chemistry chapter).
Morphological
identification
approaches with ovalocytes and presence of
macrocytes,
giant
band
cells,
hypersegmented cells with 6 nuclear lobes,
atypical granulocytes and pleomorphic
thrombocytes (-penia). MCV often more
then 100 fl and hemoglubin less than 0.20
(L/L) there are cabot rings present,
schistocytes and ansiocytosis with
ovalocytes. Additionally reticulocytes are
reduced, whereas, RBC dimorphism
245
(presence of normal and reticytocytes
synchronuosly)
may
contribute
to
ansiocytosis and mixing of Vit. B-12
deficiency anemia. The cause and or
etiology of meglaoblastic anemia is
usually due to defects in absorption of Vit.
B-12, and folate (Folic acid), it may be due
to preganacy, neoplasms, hemolytic
anemias, alochol abuse, drugs such as folate
antagonist as in methotrixate and or inborne
error of metabolism plus deficient diet for
folic acid source (nuritive source). Diagnosis
is by estimation of folate and others as
decficiency of the transporter molecules for
monoglutamate essential for folate transpot
to the tissues and liver.
In these type of anemia Vit. B-12 deficiency
also could be due to the reduction in
transporter molecule known as
transcobalamin II for intrinsic factor
and can entail to these classification of
anemia. Both vit. and intrinsic factor are
present in the stomach fundus region and in
ileum portion of the small intestine.
The treatment modalities consists of 1 mg
/day of Vit. B-12.
Vitamin
Anemia
B-12
deficiency
As mentioned is due to lack of transporter
transcobalamin II (deficiecncy) and this
can be congenital or acquired: some
substances such as nitrogen oxide and
anesthesia can ensue to Vit. dificiency.
There are several causes of Vit. B-12
shortage due to parasite such as D. latum,
and other hookworms in the instine also
sprue, Crohn’s diesease, inflamatory
bowl syndrome, bacterial consumption,
jejunal diverticlum, blind loop syndrome
, gastric potentials as, carcinoma/sarcoma,
gastrectomy,
pernicious
anemia,
infiltration and methastasis of sarcoma and
carcinoma in situ (stomach) or other causes
as infiltration of leukemic and lymphoma
cells, etc. food sources and just
vegeterianism, can presue to its deficiency.
Pernicious Anemia (PA)
This is some what an autoimmune in order
due to antibodies to intrinsic and parietal
cells of the stomach fundus. Blocking
antibodies prevents binding of the vit. B12 and intrinsic factors, In addition, nonbloking antobodies with less implication on
binding of the said fator and Vit. play a role
in this scenario. It is found in 75% of
patients with prenicous anemia, in their
serum, saliva and GI juices. In these
category their polyclonal types antibodies
incompass IgG and IgA immunoglobulin
types, whereas to the above, gastric
infiltration by immunoglubolins (Igs)
points to an inflammatory or autoimmune
condition as in alchorhydria.. there are
hypersegmentation in neutrophils with
megaloblastic marrow. Schilling’s test
is diagnostic for this type of anemia with
acompanying low level of intrinsic factor.
Clinical signs and symptoms are peripheral
neuropathy, association to Hashimoto’s
thyroditis
(autoimmune
thyroditis),
megaloblastic psychosis and optic nerve
atrophy. Figue 3-6 represent Schilling’s
test principle for Pernicous anemia.
246
some
conditions
Schilling’s test stages
Fig. 3-6: presents principle of Schilling’s
test for diagnosis of pernicious anemia,
courtesy of Adam.
Above figure indicates IM (intramascular)
injection of unlabeled vitamin B-12 and
collectively ingesting radioactively labeled
vit B-12. Liver unsaturated transcobalamin
II normally binds to the vit. B-12 substrate
through intrinsic factor mediation and there
must be a least more than 10%
radioactivity in the collected urine, any
changes in the said amount indicates gastric
parietal cell damage therefore low or absent
intrinsic
factor
deficient
to
bind
transcobalamin. This is a diagnostic test
for prenicious anemia.
Laboratory findings include macrocytosis,
hypersegmented neutrophils low serum vit.
B-12 antibodies to both parital cells and
intrinsic factor with an abnormal schilling’s.
Table 3-2 indicates differential diagnosis of
some conditions by Schiling’s test in
different stages of the test.
Table
3-2
indicates
differential diagnosis of
pernicious anemia among
by
Microcytic and hypochromic
anemia
In determining microcytic hypochromic
anemia, normacytic normochromic anemia
amd macrocytic hyperchromic anemia, the
utiliy of red cell indices comes handy and
these are in form of three special formulas
such as in the equation 4-6 for red cells
status.
Eq. 4, 5 & 6:
Normal Range: 80-100 fl.
Normal Range: 27-31 pg/l.
Normal Range: 32-36 g/dl.
As
it
shows
the
MCV
(mean
corposcular/cell volume) is used for
microcytic
hypochromic
anemias
correltated with MCHC (mean corpocular
hemoglobin concentration. If MCHC and
MCV both are low the microcytic and
hypochromic condition indicates in
patients with this type of anemia.
247
These anemic parameters are observed in a
variety of anemia such as chronic bleedings
and hemorrhages, or some types of
thalassemias. Indices are used to type and
classify anemia according to anisocytic and
chromacity of the red blood cells such as
microcytic hypochromatic or marcrocytic
hyperchromatic and finally normcytic
normochromatic anemia, this latter
condition is seen often in cases of
neoplasms, in chronic and acute illnesses
and also in early stages of different
hemorrhages before shifting to microcytic
hypochromic type.
Microcytes have commonly reduced volume
and content/concentration in disorders such
as
thalasemias,
lead
poisoning,
sideroblastic anemia and iron defficiency
anemia.
These cells are smaller than 6-um
(micrometer) with MCVs lower than 80 fl.
(MCV<80 fl.).
On the contrary MCV in Macrocytes are
more than 80 fl. (>80 fl.) and with a size
greater than 8- um. Normocytic and
normochromic cells are normal in
parameters, however these may have
indications of aggresive and sever
neoplasms or other debilitating conditions.
Iron Defficiency
(IDA)
Anemia
Iron defficiency anemia is a microcytic
hypochromic anemia, where erythrocyte
suffer a metabolic iron sub-loads (or
overloads) a decrease (or increase) iron
storage respectively. This has been reflected
in bone marrow iron stores and peripheral
microcytosis and hypochromasia (reduced
Hgb. content in the red cells). Clinical signs
and symptoms indicates atrophic tongue and
spoon shaped nails known as koilonychia.
Also, the cells manifest an iron defficient
overall signs and mucosal atrophic
symptoms with pica (chewing on
artifactual objects, such as ice, soil, etc.).
These abnormalities are caused by reduction
in hemoglobin in the normal red cells as
well as their bone marrow counterparts
nucleated and anucleated (retics) cells. In
normal cells iron forms and incorporates
into portoporphrins and subsequently into
hem for the purpose of combining to oxygen
in reduced state. These overall process lead
to oxygen transport by hem to the tissues.
The implement takes place in the erythroid
precursor and progeny cells in their
mitochondria. Iron is included in the hem
and in its identical counterpart in muscles
(in form of myoglobin) in cells interior
milieu, and in cytochromes oxidase (
cyclooxyginase and catlase, oxidase etc.).
In pregnancy, the mother-to-be is short of
iron and thus hem for the reasons that the
fetus consumes and uses iron as a part of
their metabolic needs, and these decrease
must be supplied by mother and mother has
to take a 20-25 mg of iron during
pregnancy on a daily bases.
Iron is contained in hemaglobin and in
storage locations as in liver, b.m. (bone
marrow), myglobin and in transporters
molecules in plasma in form of
ferrihemoglobin and ceruloplasmin.
The following chart indicates normal
differential lab findings in iron
defficiency anemia in table 3-3.
Absorption of iron takes place in the gastric
mucosal cells and hydrochloric acid
248
whereas, this facilitates absorption through
Vitamin-C presence, to be absorbed it needs
to be in the ferrous states (Fe ++) and
once it enters the mucosal cells of the
stomach and intestinal epithelia it turns into
ferric states (/Fe+++ for more information
refer some clinical chemistry texts in trace
elements section).
Table 3-3. Some normal
laboratory fingdings in Iron
Defficiency Anemia (IDA)
Gender/Age
Hematocrit<%
Females
(yrs)
Hemoglobin<g/dL
12-14.9 11.8 35.7
15-17.9 12.0 35.9
18+ 12.0 35.9
Males
12-14.9 12.5 37.3
15-17.9 13.3 39.7
18+ 13.5 39.9
Laboratory Test Value
Ferritin <15 ug/L
Serum transferrin receptor concentration (TfR)
>8.5 mg/
Transferrin saturation <16%
Mean cell volume (MCV) <82/85 fL*
Red cell distribution width (RDW) >14%
Erythrocyte protoporphyrin (FEP) >70 ug/dL
<15 yrs/>15 yrs of age
There are two main iron storage types,
one called ferritin, which is a main store of
iron in form of ferric hydroxide and
apoferritin and is negative for Pearl’s
Prussian Blue (PPB/specific stain for iron
stores) and the second one is the
hemosiderin, which is clumps and
aggregates of ferric hydroxide and stains
with the said dye and with a rapid positive
reaction.
Laboratory findings encompass of a
decreased MCV, MCH and MCHC with a
higher RDW, microcytic anemia and
hypochromasia. These follows with
normal leukocytes, normal or elevated
thrombocytes and retics are either normal
or decreased in total counts. For iron
stores and other lab parameters in IDA,
refer to table 3-3 above.
.
Sideroblast Anemia
This is a microcytic hypochromic
anemia likey the same as iron defficiecncy
anemia with iron metabolism defect.
Overloads of iron is appearant in bone
marrow deposits and manifests in
normoblast and in the cells interior as we
can find aggregates of iron depositis
along the vicinity of the neucleus.
This type of anemia can be hereditary or
acquired. Siderosomes and sideroblast
rings of iron percipitates in the cell’s around
the neucleus and are found in refractory
anemia and myelodysplastic syndroms as
well.
Iron overload can be definitely being
obsereved in bone marrow cells (erythroid)
and peripheral circulation by Pearl’s
Prussian Blue stain for iron deposits.
Macrosphages also conatin residual
amount of iron overload that seems to be
ingested and processed by this cells.
Pappenheimer
bodies
(siderosomes/hemosiderin pigments) may be
found in these cells and are reactive with the
said stains.
The iron loading seems to be a problem
associated with iron metabolism where
249
pyrodoxine treatment of these patients can
help in synthesis of the hem portion of the
molecule responsible for iron metabolism.
Some drugs and chemical elements can
cause the aquired forms of the condition
such
as
lead,
alcohol
and
chloramphenicol; the genetic form
consists of x-linked and autosomal varieties.
It is present is myeloma, myelosclerosis,
AML
(acute
myeloid
leukemia),
polycytemia vera and myelodysplastic
syndromes. Lab findings dimorphic blood
picture, elevated RDW (red cell
distribution width) some degrees of
anemia with ansiopoikilocytosis in blood
film. Anemia usually present in form of
microcytic hypochromic due to enzyme
defects (ALA synthase-aminolevulonic acid
synthase).
There
is
acquired
and
congenital form of disorder.
Iron studies shows, reduced serum iron,
increased
ferritin, decreased free
erythrocyte protoporpherin (FEP) and
elevated serum iron stores. In this there is an
increased of 15% of ringed sideroblasts.
Anemia of Alcoholism
Ethyl alcohol is one of the substances that
affects the vitality of the red cells and other
body tissues. Red blood cells and it
precursors are affected by alcohol in the
form of vacuolization, macrocytosis and
megaloblastic forms of megaloblastic
anemia. Bone marrow and erythrocytes and
cellularity in these compartments are
severly affected. Alcohol is toxic to all
cellular series and its chronic abuse
leading finally to cirrhosis of the liver, one
of the essential organ for blood cells
production and synthesis in the early
stages of individual development..
There are decrease cellular production in the
bone marrow. Vacuolization occurs in
the erythroblasts and subsequent progenitor
cells. This substance interfer with folate
metabolism in the stomach and affects and
leads to appearance of meglaoblastic
anemia as
mentioned along with
macrocytosis. There are dimorphic blood
pictures in the blood film due to presence of
reticulocytes and red cells with coexisting
iron defficiency due to folate interruption
synthesis in gastric mucosal epithelium.
Macrophages
in
the
spleen as in
reticuloendothelial system interfer with
red cells functional and morphological
intergrity in the said organ with cells where
they are high in cholesterol and
phospholipid contents. This mechanism of
red cells destruction follows with
acanthocytes anemia or spur cell anemia
due to the said high contents of the red cells
cholesterol and lipids contents within the
cells mosaic bilayer . therefore increasing
blood
viscosity
and
red
cell
deformability. These series of blow to the
cells in spleen sinosoids result into
formation of acanthocytes. The resultant
fatty liver
(fatty streaks) ensues to
hemolytic episodes and therefore to Zieve’s
syndrome.
Normocytic
and
normochromic anemia are common in
patient’s with inflammation (anemia) and
other hepatic involvement.
such as alcoholic
thrombocytopenia is the result of chronic
and long term alcoholic abuse and
ingestion. The resultant reduction in
Other
conditions
250
megakarypoiesis is the direct result of
alcohol toxicity. Platelets are affected by an
unknown
mechanism.
Mild
thrombocytopenia is shown in patients with
mild ingestion of ethanol and therefore a
normal thrombocytes number can be
resumed after cessation of the consumption.
Usually
there
is
a
rebound
of
thrombocytosis after stopage of alcohol.
The ultimate damage of ethanol ingestion is
cirrhosis and cancer of the liver known as
hepatic carcinoma or hepatolenticular
carcinoma.
Hepatic Disease Associated
Anemia
There
are
associated
numerous
hematological
diseases and anemia
correlated with liver diseases and pathology.
The causes of the anemia is classified
according to the following processes: Portal
hypertension and hemorrhages related
cirrhosis of the liver and thus esophageal
varices with subsequent hemorrhage, as
well as peptic ulcers, gastric bleedings,
hemorrhoids can ensure to the types of
anemia associated with these consequences
of liver pathology. These cause
normocytic normochromic anemia or
subsequently with concurrent bleeding
leading to microcytic hypochromic anemia.
As mentioned alcoholic liver cirrhosis is
another type of liver pathology that ensues
to concomitant liver disease associated
anemia as mentioned in the relevant section,
whereas, aspirin and some other medications
such as some NSAID (Non-Steroid AntiInflammatory Drugs) can as well cause
anemia of hepatic diseases associated
anemia.
This is the same as with alcoholic anemia
with thrombocytopenia. Liver diseases
associated anemia has a component of
platelet
dysfunction
and
abnormal
morphology.
Thrombocytopenia
and
thrombocytosis are seen in this category
of patients.
Other laboratory findings are due to
membrane cholesterol and phospholipid
contents affected by liver and splenic
metabolism, the blood film findings
encompass target cells, macrocytes,
acanthocytes (spur cells), whose
morphology been changed by red cell
membrane properties in the spleen.
Macrocytes
are
round
nonpolychromatic with increased volume to
100-110 fl. Precursors in bone marrow are
likely macronormoblastic in appearance
without any megaloblastic changes, this
include hypersegmented neutrophils in bone
marrow.
Nutritional
folate
deficiency
also
affects the clinical laboratory findings;
alcohol affects the secretion of folate, vit.B12 and other gastric secretive contents.
In patients with liver disease and hepatic
cirrhosis there will be hypersplenism and
anemia accompanying with leukopenia and
thrombocytopenia. Hemolytic processes and
episodes such as hemolytic anemia can be
seen along with the said cellularity (spur
cells, etc.) due to increase cholesterol and
phospholipids in their membrane, these cells
are rigid and lose their permeability, with
concurrent
splenic
sequestration.
Coomb’s test and DAT (Direct
Antiglobulin Test) is positive in chronic
251
active hepatitis due to autoimmunity, such
as autoimmune hemolytic anemia. The
anemia of the liver-associated-diseases
is a mixture of variety of disorders that lead
to some anemia with certain clinical
outcome.
exist in conditions mentioned under the said
topic
(Microcytic,
hypochromic
anemia).
Aplastic Anemia
pancytopenia and
hypoplastic marrow in which there is
This
Normocytic
Anemia
Normochromic
As mentioned before these type of anemia
are manifestation of a spectrum of diseases
and pathologies that have sever and grief
clinical outcomes. Pathology such as
leukemia, lymphomas, myelophthistic
anemia, viral infections, drugs, bone
marrow
associated
anemia
and
dyserythropoietic anemia.
Under bone marrow response an increased
retics counts, due to acute blood loss and
hemolytic anemia are marked. In defective
bone marrow, a low or normal retics cell
(reticulocytes) consists of intrinsic bone
abnormalities as in idiopathic and secondary
causes, with acquired and congenital
syndromes consequent impact.
These hereditary syndromes include
Fanconi’s anemia (Cooley’s anemia)
and aplastic (anaplastic) and dysplastic
anemia, with multiple chromosomal
defects and mutations in DNA. In
dyserythropoietic
anemia,
there
is
depression of erythropoietin production with
concomitant kidney pathology (renal
insufficiency, failure and neoplasms,
etc.). With myelophthistic anemia, nucleated
red cells (NRBC) and left shift cells, as in
leukocytes/myeloid series, are prominent.
Chronic diseases are 70% normocytic
normochromic and with others (30%), with
microcytic and hypochromic anemia do
consists
of
reduction of total spectrum of hematopoietic
cells with depletion of hematopoietic stem
cells. This condition is rare however, it is
potentially fatal. It is a bone marrow
failure syndrome whereas Pancytopenia
covers
neutrophils,
erythrocytes,
thrombocytes and other leukocytes (myeloid
and erythroids/megakaryocytic cell lines).,
the yellow bone marrow (fatty marrow) is
dysplastic and shows reduce cellularity or
acellular. This is in forms of either
hereditary or acquired. Hereditary forms are
due to congenital malformations and
genetic mutations. Hereditary types include
Fanconi’s
syndrome
in
which
pancytopenia is predominant figure and the
second type is dyskeratosis congenital.
Acquired forms are either idiopathic or
secondary forms. Neonates and children
being affected consist of 70% of acquired
cases and the rest of 30% are hereditary.
Initially the anemia is in form of
normocytic, normochromic anemia, this is
accompanied by sever pancytopenia and
marked thrombocytopenia. Secondary
types are caused by various type of infection
either viral, bacteria or do to sever radiation
exposure and other mutagenic, teratogens
and chemical toxicity/insult.
Hypocellular mechanisms involve and
caused by stem cells depletion/depression
at first and second etiology is the placement
252
of total cells by yellow marrow as fats.
Few cells such as plasma cells, lymphocytes
and histocytes may be observed in bone
marrow. In this condition the ratio between
cells to fat islets changes dramatically to a
total presence of fat globules/islets, with
very few islet cells left to defend and
participate in their metabolic destiny. Stem
cell transplant/transplantation is the
treatment of choice in these categories of
patients
The following figure (Fig. 3-7) depicts
cellularity of bone marrow in aplastic
anemia.
Fig. 3-7: Aplastic anemia with hypoplastic
bone marrow with cells replacement by
fatty/adipose islets/tissues.
Pure Red Cell Aplasia
(Diamond-Blackfan Anemia)
and Anemia of Chronic
Disease (ACD)
Pure red cell aplasia (PRCA) is an anemia
with depletion in red cell progenitor cells
and it is different from that of aplastic
anemia in that hypoplastic leukopoiesis
and thrombopoiesis are absent.
It is caused by parvovirus B-19 infection
and subsequent rapid fall in the erythroid
series and its bone marrow counterparts.
The stem cells depletion does not exist and
differentiation of other myeloid and
megakaryocytic cells are normal. Acute
forms of the condition warranted etiology in
other associated diseases such as sickle cell
disease/anemia
and
hereditary
spherocytosis in which there may be a
rapid fall of erythroid series. This virus is
cytotoxic to eryhtroid progenitor cells and
there are drug linked to acute red cell aplasia
incidence. Example of drug insult includes
chloramphenicol and phenytoin. There is
severe anemia and reticulocytopenia in
this respect. Chronic form exists as well, for
such overall marrow cellularity is normal
with increased retics cells and anemia
however, erythroid precursors are
dramatically depleted. Treatment includes
treating the underlying causes. Figure 3-8, is
the representation of pure red cell aplasia in
a child.
253
Fig. 3-8: the presentation of pure red cell
aplasia in a child; the bone marrow cells, oil
immersion magnification, 500 X.
In respects to ACD or anemia of chronic
disease, is due to inflammations whether in
chronic infectious diseases inflammations or
chronic noninfectious inflammatory
conditions. In the former, iron sequesters in
the macrophages and erythropoietin does
not produce appropriately as well. The red
cell survival is slightly decreases causing
mild anemia. in the latter, conditions such as
tuberculosis,
rheumatic
fever
(RF),
rheumatic heart disease (RHD),
pneumonia and acute and chronic and subacute bacterial endocarditis can be the
causative and etiologies of this type of
anemia.
Neoplasms, carcinomas and
lymphomas/sarcomas are involved in the
pathogenesis of ACD. Lab findings have
normocytic normochromic anemia, with
accompanying normal reticulocytes and
thrombocytes and leukocytes. In some cases
there may be increases in leukocytes and
platelets. On cell morphology we have
anisocytosis and poikilocytosis with
hypochromasia. Iron stores in serum
reduced and decreased in TIBC (total iron
binding capacity; this is elevated in IDA
/iron deficiency anemia). Stores of iron
shows elevated stainable iron store in
macrophages in spite of decreased iron
stores in sideroblasts; iron deposits show
increased iron staining in macrophages,
(In IDA hemosiderin is absent). Indeed
treatment of this kind of anemia is directed
toward the treatment of the underlying
conditions/disorders.
Chapter Four (4)
Hemoglobinopathies
Sickle Cell Disease (SD):
(Glader et al, Quirt et al. and Kocmarek et
al.)
his hemoglobinopathy was
discovered by a local physician in
Chicago during 1910. As the name
applies, the red cell is sickled shape
and hemoglobin chain is being affected by
changes in the amino acid sequences
accordingly. The defect is a genetic one with
amino acids substitution. The hemoglobin
that represents sickle cells disease and
sickle cell trait has a genetic mutation
between GAG to become GUG, this
mutation in the codon encodes amino acid
glutamic (glutamate) for valine. The
chromosomes involved for globulin chain
are chromosome 11 and 16. For more
information of globulin chain refer to
clinical chemistry section, but however, the
phenomenon is typed to homozygous and
heterozygous variety.
Hemoglobin SS is the homozygous
form of the Hgb., while AS is the
heterozygous form. Hemoglobin S or
sickle cell disease individuals are affected
by Hgb. S. This type of Hgb. is normal when
oxygenated whereas under reduced
oxygen
tension
resumes
sickle
morphology. The disease is quite prevalent
in central Africa and other part of the USA
in black population in form of sickle cell
traits. About 90-100 % of the hemoglobin S
is homozygous and 20-40% are AS or
heterozygous.
T
254
Its pathophysiology consists of four crisis
types, the infarctive crisis, where there is
vasocculsive events after stasis of the
vessels by the sickled red cells, these factors
are responsible for such phenomenon as
reduced oxygen tension, PH of the blood,
stasis of the vessels and with a clinical signs
including, pain, circulatory failure and
dehydration as the other forms of risk
factors. Organs such as eyes, kidneys and
so on may be affected by the vasocculsive
and vascular stasis
Next stage is aplastic crisis, bone marrow
failure and viral etiology of type
parvovirus infection can be the triggering
agents in this stage.
The third crisis is the hemolytic crisis,
whereas there is hemolysis of red cells due
to rupture and membrane defects. Infection
can be a predisposing factor correlated with
G6PD deficiency.
The last crisis is the sequestration crisis,
which happen usually in children and
neonates
commences
by
hepatosplenomegaly
and
frank
hemolysis. Sickle cells are shown in the
following figure (Fig. 4-1).
Fig. 4-1: The appearance of sickle cells in
the peripheral blood.
Red cells with certain blood group such as
Duffy (fy) type are resistant to
Plasmodium falciparum, also mutation
in S hemoglobin can produce resistance to
the same malarial parasite. When P.
falciparum infects red cells with sickling
reaction progresses the parasite dies, this
may induce resistance to the parasite rather
than immunity.
Laboratory
findings
include,
Hgb.
concentration of 50-90 g/L, with hematocrit
value of .150-.300 L/L. there is
polychromasia and target cells. The
pathogonomic sign is presence of sickle
cells along with Howell Jolly bodies.
Neutropenia is common and there are
increased thrombocytes. In addition, ESR
is low and serum bilirubin increased to
25-80 umol/L (normal is 3-20umol/L).
Finally the red cells life span is much
reduced. Sickle cell solubility test
employs sodium metabisulfide and
sodium dithionate, these are reducing
agent to reduce S hemoglobin. These agents
255
once added to the blood it changes and turns
turbid. Turbidity indicates presence of sickle
cells. They can be done in tube or on slide
for presumptive diagnosis of sickle cell
disease. Sickle cell trait (AS) is the same
however, there is 40 % hemoglobin A and
60% hemoglobin S; the prognosis is less
severe than sickle cell disease with less
sickling of cells. There are combination of
hemoglobin C (a different type of
hemoglobin) and Hemoglobin S making
Hgb. SC with less consequence than Hgb. S
disease. Hemoglobin S-thal is also another
variety of sickle cell with thalassemia traits
as well.
Hemoglobin C homozygous is Hgb. CC
with more sever onset than Hgb. SC
heterozygousity. Hgb. C has substitution
of valine for glutamate at position 6 of βglobin chain.
Although, there is hemoglobin AC
heterozygous type which is less severe in
clinical presentation and is a combination
of hemoglobin A and hemoglobin C,
hemoglobin AC has more sickling than
Hgb. SS and the infarction and
vasocculsion is more severe than Hgb.
SS, it is proven fatal during pregnancy and
nephropathy is more common in Hgb. SC
than SS. There are also some forms of
Sickle cell, thalassemia trait as
mentioned that can be reviewed in the
reference clinical hematology texts.
Pathophysiology
Thalassemia: (Rodak et al.)
of
Alpha and Beta or α-β
Thalassemia
Usually thalassemia is divided into two
categories, quantitative and qualitative,
quantitative is indicated by structural
decrease production in alpha, beta and
gamma globulin chains and it is mostly
microcytic hypochromic with Hgb.
reduction in RBCs, while the qualitative,
which is a variant thalassemia is decreased
amount of one of peptides chains. This is a
genetic disorder.
There are two types of globulin chains, 1)
alpha (α) or 2) non-alpha (β-like). In this
respect fetal hemoglobin is composed of
two quasi-alpha chains [as theta (θ)] and
zeta (ζ) chains) with combination of beta
like hemoglobin (epsilon (ε)) these are
formed in the hemoglobin as Gower I,
Gower II and Portland. Later in fetal life
the alpha chain changes to gamma chain to
produce fetal Hgb. and it remains the
major hemoglobin until 6 months of age,
when hemoglobin A (adult Hgb.) becomes
the major hemoglobin (96%) that is α2β2
type. Hemoglobin A2 would be 0.1%
during neonatal period and 3% during adult
life. Hemoglobin F would be 1% in adult
life and 96% at fetal and neonatal periods.
Chromosome 16 controls alpha globulin
chains and chromosome 11 controls beta
chains. There are two types of
thalassemia, alpha and beta.
Alpha types have divisions such as minima,
minor, intermediate and major (beta type)
and hydrops fetalis. The latter possesses
no alpha genes, the same categories goes
with beta thalassemia, except the last types
256
hydrops fetalis does not exist in β-type.
Gene deletions happen majority of times
in both types (alpha and beta) causing αchain deletions and β-chain deletion in the
chromosomes 16 and 11 respectively.
manifestations
of
alpha
thalassemia minima include, normal
parameters except MCV is equal to 85 fl.
Alpha thalassemia minor is slightly low
in Hgb. with microcytic hypochromic
character and an MCV of less than 80 fl.
The intermediate variety is more severe
and Hgb. is between 60-100 g/L. Anemia is
moderate and iron may be overloaded.
There is splenomegaly and transfusion
may be needed.
Thalassemia
major
or
beta
thalassemia, Hgb. is less than 60 g/L with
severe anemia, hepatosplenomegaly and
iron overload.
In hydrops fetalis, an intrauterine
anemia is obvious and this type of anemia
is incompatible with life. Figure 4-2, is the
bone/skull changes in Thalassemia
major, known, as “hair-on-end” with
frontal bossing, due to extreme bone
marrow
and
skeletal
hypererythropoiesis, with bone marrow
erythroid to myeloid ratio 20:1 (normal
is (3:1 or 1.5:1).
Clinical
Fig. 4-2: radiographic picture of skull/bone
of a thalassemia major patient with the
phenomenon known as “hair-on-end”.
The following table (table: 4-1) indicates
differences between thalassemia minor
and major. In major thalassemia, there is
microcytic hypochromic anemia,
while minor type has mild anemia. In major
there is evidence of severe dysplasia with
marked variation in hemoglobin A and
erythroblastosis. In minor type we have
microcytic hypochromic anemia without
iron deficiency, and Hgb. A fraction has
been raised as well.
Table
4-1:
indicates
differences
between
Thalassemia minor and
major.
257
Abnormal and
Hemoglobins
unstable
Hemoglobin- H, C
unstable Hemoglobin
Hemoglobin H
Clinical laboratory tests for all alpha
and
beta
thalassemia include, Hgb.
electrophoresis, CBC, serum ferritin,
alkali
denaturation,
(Hgb.
F
quantitation), hemoglobin A quantitation,
reticulocyte estimation, H-inclusion
bodies and RPI (reticulocyte production
index).
For beta thalassemia, thalassemia major, in
addition to anemia of microcytic
hypochromic type we have as well target
cells NRBCs, slight leukocytosis and
thrombocytosis, along with plasma Hgb.
and serum iron increased with bone marrow
hyperplasia and increased iron stores.
Lab findings for thalassemia minor are
milder in comparison with the mentioned
major type, in this NRBCs are not usually
seen and there is relative decrease in
quantity of target cells.
Indeed there are other Hgb. types that are
less significant in clinical presentation,
these are rare such as hemoglobin H
disease
and
other
stable
hemoglobin(s), will be covered in next
section.
and
As it was cited at the end of the previous
section, hemoglobin H and C and
unstable Hgb. will be briefly pointed here.
Hemoglobin H is due to three α-globulin
gene deletion out of four. There are
heterozygous
and
homozygous
varieties and it is common in Southeast
Asia, Middle East, and in Mediterranean
population.
The defect is due to reduction in the
synthesis of alpha chain formation of H
type hemoglobin.
Hemoglobin H does precipitate in the
senescent red cells and cause removal
prematurely by spleen. There is consistent
chronic hemolytic anemia. Anemia
develops over years since infancy. As a
result Hgb. reaches to 90-120 g/L in this
respect red cell indices changed and
reduced in amount. Despite of the fact that
retics (reticulocytes) are 5% in adults and
unlikely to that, RPI shows inefficient
erythropoiesis.
Blood
film
shows
anisoctyosis and poilkilocytosis and
augmented RDW. In the early years of
development Hgb. H is 5% and Hgb. A
andA2 is 30 %.
Hemoglobin H can be shown by 1%
brilliant cresyl blue indicating multiple
inclusions (the precipitated Hgb. H). The
258
diagnosis is based of serum electrophoresis.
Indirect (unconjugated) bilirubin would
be increased. Finally defective oxygen
transportation, ineffective erythropoiesis
and defective Hgb. synthesis are other risk
factors. Hgb. H can be occasionally seen in
chronic granulocytic leukemia and
sideroblastosis
along
other
myeloproliferative disorders.
Hemoglobin C
This is another amino acid substitution
resulting into valine substitution for
glutamine at the sixth position on the
globulin chain.
There are homozygous and heterozygous
(or hemoglobin C trait) form in which
the homozygous (Hgb. CC) is more serious
in nature, consisting of hemoglobin C
disease. These are commonly and
exclusively found in black population,
particularly in Northern Ghana.
Hgb. C trait is only of interest and
significant in genetic studies. C disease
clinical findings include hemolytic
anemia, splenomegaly and abdominal
discomfort with less severity than Hgb. SS;
microcirculatory complication occurs
when there is dehydration and subsequent
hemolysis. The affected red cells show
hemoglobin C crystals, in form of either
hexagonal or rod shape.
Other type of this hemoglobinopathy is
found in Hgb. SC patients with less severe
than SS type. The sickle cell concentration
is higher in SC than in SS. Severe
infarction
during
pregnancy
and
puerperium happens and has proven fatal,
also proliferative retinopathy in more
common in SC trait than Hgb. SS disease.
Lab finding compose of hemolytic
anemia; mild, large target cells, some
intracellular crystals and sickle cells, the
sickle cell preparation is positive with a
peculiar electrophoresis pattern.
Unstable Hemoglobin
Examples are such as Kölin, Zurich and
M type.
There are also genetic mutations in this
hemoglobin
changing
amino
acid
sequences and resulting into altered
hemoglobin solubility and or binding of
hem moiety.
Beta chain mutations are common in
comparison with alpha chain. Hemoglobin
aggregates known as Heinz bodies are
precipitated hemoglobin in these red cells.
Clinical signs indicate childhood
splenomegaly and jaundice, cyanosis
and frequent episodes of hemoglobin
precipitation caused by infection and
oxidant drugs. Lab findings consist of
hypochromia,
anisocytosis,
poikilocytosis
and
reticulocytosis.
Supravital stain (1% methylene blue)
indicates Heinz body. Electrophoresis
does not usually help. The best lab test for
instance is detection of unstable
hemoglobin with isopropyl alcohol
precipitation and denaturation test. In this
test unstable hemoglobin precipitates on
addition of isopropanol alcohol and
indicates
presence
of
unstable
hemoglobin(s).
259
Chapter Five (5)
Hemolytic Diseases
Intravascular
Hemolytic
Diseases/Hemolysis
W
hen red blood cells rupture
inside the circulation the
intravascular hemolysis and
anemia incurs. Hemoglobin releases and its
metabolic products will be trapped by
spleen and liver and partly clears by the
kidneys. Example of these are complement
mediated destruction by trauma and
physical injuries such as , chemical
poisoning of the cells with shock causing
RBCs damages, immunologic injuries in
nature such as immune hemolytic
anemia (IHA), and finally hemolytic
transfusion reaction or hemolysis. The
products mentioned include but not limited
to
hemoglobinemia
and
hemoglubinouria,
methemoglobinemia
hemosiderinemia, accordingly
and
the
hemoglobinemia is the presence of
hemoglobin in the blood stream. In this,
free Hgb. binds to haptoglobin, and then
the complex is cleared by the
reticuloendothelial system. In regards
to hemoglobinuria, the hemoglobin appears
on the urine through kidneys filtration, and
due to the size of the Hgb. molecule, the
GFR (glomerular filtration rate) may
hamper, this is when haptoglobin saturates
and free Hgb. filters through this organ. In
this renal thresholds overwhelms and the
product of the destruction appears in urine.
The threshold for Hgb. is 1.35 g/L. Over
saturation of haptoglobin only happens
when
there
is
severe,
massive
intravascular destruction, and so much
Hgb. released into the circulation. The next
terminology is methemoglobinemia, it is
when there is free Hgb. in plasma oxidized
into methemoglobin, which further
dissociated
to
hem
and
globin
chains/molecules. Hem makes complex
with
plasma
hemopexin
(methemoglobinemia); a plasma protein
and will be cleared subsequently by
reticuloendothelial
system
especially
hepatic sinusoids. It may also combine with
plasma albumin forming a brown color of
methemogloninemia. It remains in the
blood for few days and is an indication of
massive and severe intravascular
hemolysis. The followings are example of
intravascular destruction of red cells (Quirt
et al.).
Acute
Alloimmune
Hemolytic
Transfusion
Reaction (AAHTR)
This will be explained in short, for the topic
can be found in transfusion medicine
chapter. The AAHTR is the consequence of
sensitization of the recipient of blood
through transfusion of red cells or red cell
products (whole blood or cell
concentrates).
incompatible
RBCs
are
transfused or infused to the Patient (as a
recipient of blood) with mismatched
When
products there may be a reaction to the
foreign erythrocytes therefore patient
might suffer a an acute and sometime a
fatal transfusion reaction. Alloantigen
260
(foreign antigens but from the same
species) can cause these type of reactions,
the antibodies formed in the recipient to the
donors
mismatched
blood
causes
destruction of the foreign red cells releasing
massive amount of hemoglobin in the
recipient circulation, which is morbid
and can cause kidney and shock in the
patient as a recipient. This phenomenon
will be discussed further in the blood
bank
and
transfusion
medicine
section/.part four (4).
Fig. 5-1: Extravascular destruction of RBC.
gWhiz©
Extravascular
Hemolytic
Diseases/Hemolysis
This type of hemolysis is due to RBCs
destruction in the reticuloendothelials
system (RE cells) and out of
intravascular compartments in the
tissues. The liver (hepatocytes) sinusoids
and splenic sinuses handle the removal of
released hemoglobin and cells matrix. The
product of destruction and hemolysis is in
from of bilirubin products, first in this
form unconjugated bilirubin bound with
albumin and then clears by the liver
through direct conjugation with liver
enzyme glucuronide transferase (refer
to clinical chemistry chapter). The
formation of urobilinogen and urobilin
in the urine and feces (stercobilinogen
and stercobilin) are the final product of
this type of process. The following
schematic diagram represents extravascular
destruction scheme in vivo (Fig. 5-1) the
followings are examples of extravascular
hemolytic destruction (vide infra). This
type of destruction is the most common
type in hemolytic anemia(s).
Reticuloendothelial
Destruction of RBCs
As mentioned under extravascular
section in the preceding part this is caused
by destruction of erythrocytes in the RES
(reticuloendothelial system) and the
product of destructions such as by the
oxidases in RES system releases free Hgb.
into the circulation in combination with
albumin to form indirect bilirubin and
pursuing to hepatic sinusoids to further
conjugates with glucuronides enzyme to
form conjugated bilirubin or direct
bilirubin. The rest is same as explained in
the extravascular hemolytic disease.
Autoimmune
Hemolytic
Anemia (AIHA)
The individual immune response is
responsible
for
production
of
autoantibodies to self-red cell
antigens that conceive them as foreign
causing immune destruction of the patient
261
red cells. This premature removal of red
cells causes hemolysis of erythrocytes and
this destruction may be mediated by
complement or without complement (one
of proteins of immune system).
syndrome is idiopathic in nature. Anti- I
Antibodies involve could be of type IgG or
IgM
with
activation
of
classic
complement system pathway. In here
the spleen and liver RES system takes
over by phagocytic engulfment of
damaged cells and hemolysis; this may
cause hepatosplenomegaly, if it resists
for longer time. In this type of reaction, the
immune system response is toward the
autoantigen resulting to production of
autoantibodies. These are classified into
two types, cold and warm autoimmune
hemolytic anemia (AIHA).
The drop in temperature in these afflicted
patients may result in hemolysis and
hemoglobinemia and hemoglobinuria. Lab
findings consists of a blood films showing
agglutination of RBCs and spherocytes
(spherocytosis) .The anticoagulant EDTA
should be warm at 37° C. this should be
done in the light of the fact that
agglutination of red cells due to cold
agglutinins is disruptive, causing false
positive in vitro test When there is an
increased titer of the cold agglutinins the
CBC test should be prepared with saline
replacement to reduce the titer and thus a
better smear preparation and antibody
titration. For healthy individual the titer is
64 at 4° C whereas at the same temperature
the affected patients have a 1000
times/folds of titer. During temperature
drops, the hemolysis is great and the
hemoglobin drops as well, while at warm
temperature there is no hemolytic
episode.
Cold
Autoimmune
Hemolytic Anemia (CAIHA)
The destruction process is the same as cited
above with production of cold antibodies
that react at 0-10° C by binding to the red
cells surface sensitized with IgM
antibodies in vivo and in vitro. It is a cold
reacting antibody with a cause and etiology
of disease due to cold autoantibodies to red
cell antigens such as anti-I, anti-i, anti-H
and anti-HI. Therefore it is pursuing a
hemolytic process. The Raynaud’s
phenomenon/syndrome is due to
exposure of the patient to cold and thus
occurring of acrocyanosis. This is also
called
cold
hemoaglutinin/or
cold
agglutinin disease. These causative are
causation of secondary type agglutinin
disease while the primary agglutinin
is caused by infectious mononucleosis,
lymphoproliferative diseases and PCH,
also anti-I caused by mycoplasma
pneumonia, etc in secondary types.
Warm
Hemolytic
(WAIHA)
Autoimmune
Anemia
This type of anemia is autoimmune in
nature and it is IgG and IgA derived, it
consists of 70% of AIHAs, and it is
directed towards certain red cell antigens as
Rh antigens and others blood groups such
as anti-U, anti-W etc. There are primary
and secondary types. The primary type is
262
idiopathic and the secondary type happens
to other autoimmune diseases such as
lymphoma
and
lymphproliferative
disorders,
systemic
lupus
erythromatosus
(SLE),
chronic
lymphocytic leukemia (CLL) and
rheumatic arthritis (RA).
Viral
infection
and
other
immunodeficiency
diseases/syndrome are etiologic agents
and processes responsible for autoimmunity
in this disorder.
Clinical manifestation and signs include
jaundice, dyspnea, anemia, fever,
hemoglobinuria, hepatosplenomegaly and
lymphoadenopathy are common. Lab
findings consist of macrocytosis, variable
anemia, marked retics count, showing
likely bone marrow compensation,
agglutination of red cells and prominent
spherocytosis.
Also
there
are
poikilocytosis and anisocytosis along with
erythrophagia/erythrophagocytosis by
neutrophils and monocytes. WBC count
may be variable.
There are products of hemoglobin
hemolysis
such
as
unconjugated
bilirubin, in severe cases there may be
hemoglobinemia, hemoglobinuria and
decrease haptoglobin with LDH (lactic
dehydrogenase) increases. The antibody
reacts at 37° C and evidence of hemolysis
at the said temperature, therefore, the so
called warm types. DAT (direct
antiglobulin test) is positive, as the same
for cold antibody.
An increased mortality and morbidity
rate due to uncommon signs such as
reduced reticulocytes (reticulocytopenia)
that is a manifestation of marrow release
and subsequent destruction are likely
uncommon clinical signs of this rare mortal
condition.
The cells are coated with IgG and taken to
the spleen and liver. They pose surface Fc
receptor
for
macrophages
and
monocytes these cells engulf portion of
the affected red cells so that erythrocytes
look as bite cells, the red cells subsequently
after releasing to circulation repair the
removed
portion
and
sustain
as
spherocytes integration.
Extrinsic
Diseases
Hemolytic
These hemolytic diseases are classified
as following and they embrace conditions
that involve injuries external to RBCs and
the RBCs are normal in overall but could
damage by extrinsic sources and factors
such as antibodies in AIHA, or membrane
defects as in hereditary forms of
spherocytosis and elliptocytosis and
enzyme defects or as in sickle cell disease
and finally due to paroxysmal cold
hemoglobinuria (PCH).
AIHA
This
has
been
discussed
under
autoimmune hemolytic anemia.
Hemolytic Disease of the
Newborn
(HDN)
or
Erythroblastosis fetalis
263
intrauterine/in utero
hemolytic process that occurs due to
HDN
is
an
RBC antigen incompatibility between
mother and the fetus. During the time of
pregnancy, the child possess an antigen
from the father unlike the mother red cell
antigens,
thus
provoking
mother’s
immune system to produce antibodies to
the child foreign red cell as inherited
from the father. If this has not been
controlled can cause severe hemolysis
and in vivo mother compensate with her
liver the excess hemoglobin turning it into
bilirubin and thus excreting into urine,
however after delivery neonates increased
red cells destruction continues more
dramatically if it is not control either by
phototherapy and or exchanged blood
transfusion due to immature fetal
liver/hepatocytes. If it has not been
controlled the accumulated bilirubin passes
the blood-brain barrier and causes lysis
of the neurons and microglial cells
resulting into so called, “Kernicterus”; in
this condition, the patient can die due to
brain damage and excess bilirubin or
luckily survives it would have brain
damage.
The antibody responsible for such
phenomenon
is
IgG
capable
of
transferring through placenta and
harming the fetus. The red cell antigens
responsible for such disorder are Rh
antigens, ABO types or Kell, Kidd and
Duffy systems. 65% of cases are
emerged from ABO incompatibility and
33% from Rh mismatches and 2% from
other blood types mentioned.
Clinical
signs
encompass
massive
generalized edema with variable anemia
along with jaundice at first day and
hepatosplenomegaly and finally in
severe cases Kernicterus. Lab findings
consist of mild anemia to severe forms
<80g/L, leukocytosis, erythroblastosis
(with NRBCs), neutrophilia. We may
have marked spherocytosis in ABO
incompatibility and mild or non in RH
incompatibilities.,
severe
hyperbilirubinemia indicating exchange
transfusion treatment and prevention
include, 300 ug of RHIgG, or D
immunoglobulin to the mother at 28 weeks
of gestation, provided the mother has not
developed D antibodies.
Paroxysmal
Cold
Hemoglobinuria (PCH)
The condition is rare and occurs after
exposure to cold temperature. Patients
may have congenital syphilis or now in
children suffering from mumps, measles
and chickenpox, and other likely viral
infections inclusively.
Autoantibodies are cold types to RBCs and
are so called autohemolysin causing
severe hemolysis. This autoantibodies
bind to red cells and fixes complement at
cold temperature. However, the hemolysis
happens at 37° C, once hemolysin binds
to RBCs. The destruction of RBCs is
intravascular
and
classical
complement pathway accordingly
activated. The antibody is IgG type
(different from the one in CAIHA). These
antibodies are also known as DonathLandsteiner antibody (DLA), this is a
264
biphasic antibody (hemolysin) which
will be explained in transfusion chapter in
depth. The specificity of the antibody is
towards the P-antigen (P-antibody).
Hemoglobin drops to low when exposed
to heat (at the body temperature), and there
is severe hemolysis, this is an acute from of
the illness with clinical signs of chills and
abdominal distress and vomiting and
flank pain and malaise.
Lab findings include, spherocytosis,
leukopenia followed by leukocytosis,
schistocytosis,
erythrophagia/phagocytosis
and
presence of biphasic anitbody (DLA), and
DAT (direct antiglobulin test) is positive
in cold climates or temperatures.
Haptoglobin is overwhelmed and
saturated by hemoglobin due to massive
hemolysis. The treatment may require
corticosteroids and transfusion in
severe and disabling situations. Following
figure (Fig. 5-2) shows a typical hemolysis
in tubes with different degrees of
hemolysis. This is the most common
findings in hemolytic anemia.
Fig.5-2: indicates degree of hemolysis from
mild to severe in the subsequent tubes from
left to right. The first tube on the left is a
normal serum color.
Intrinsic Hemolytic Diseases
(IHDs)
Hemolytic diseases are when destruction
of RBCs enhanced and within an
accelerated pace; the bone marrow
erythropoiesis is unimpaired. Examples
include intrinsic hemolytic diseases or
processes. The increased red cells
production may be apparent as in hemolysis
and
with
marrow
increased
cellularity/cytopoieses while there may
be no hemolysis as well depending on the
marrow response without any hemolysis.
The following description is in respects to
examples of
intrinsic hemolytic
diseases. Subsequent section explains the
sorts of intrinsic classifications.
Drug Induced Hemolytic
Anemia (DIHA)
265
There are four types of drug induced
hemolytic anemia the first to explore is
the Immune complex or commonly
known
as
innocent
bystander
mechanism or drug dependent
antibody mechanism. In this mechanism
the red cells are affected by the production
of the antibodies toward the complex of
drug as in quinidine and phenacetin and
red cells. This is a complement nonimmune mediated attachment to these
immunogenic complexes. Antibodies
are of IgG and IgM types, these antibody
types can bind complement. The term
applies for accidental affection of the red
cells by the complement.
The hemolysis is severe and can cause
renal failure or insufficiency. In some cases
spherocytes, leukocytes and thrombocytosis
are evident. PTT (partial thromboplastin
time) are elevated and fibrinogen
increases with low factor VIII are
manifestation
of
intravascular
involvement. Upon removal of the
offending medication the process halts and
hemolysis disappears and returns to normal.
The second type of drug induced anemia is
drug absorption mechanism or hapten
carrier mechanism this is when the drugs
(as penicillin) combine with plasma
proteins and as carriers with subsequent
non-specific attachment to red cells. The
antibody production is IgG class, once this
coats the erythrocytes the warm reactive
antibody bind to target red cells and noncomplement mediated red cell lysis occurs.
The DAT is positive.
The third type of DIHA, include αmethyldopa
or
autoimmune
mechanism (Idiopathic in nature), in
such situation the antibody is an
autoimmune antibody towards the drug
with specificities to red cell antigen. The
drug methyldopa causes hemolysis and red
cell destruction. DAT is prominently and
strongly positive with IgG sensitization.
The fourth kind of drug AIHA is membrane
modification or non-immunologic protein
absorption. In this there is absorption of
drug as cephalosporin to the red cell
surface and changing the surface antigen
integrity thus provoking antibody response
to cell membrane.
Erythrocyte
Membrane
Defect
and
Associated
Disorders: (Blood Review, 2013)
Hereditary
Spherocytosis,
Elliptocytosis
and
Stomatocytosis: (Baillieres Clin
Haematol. 1993)
The red cell membrane is a bilayer of
phospholipids
and
cholesterol
molecules anchor within this mosaic
membrane. Among these molecules there
exist molecules that give cohesion and
integrity (integrins) to the membrane such
as ankyrin, spectrin and protein band 3
and protein band 4.1. These proteins
give flexibility, permeability and lateral
stability and elastisity. When these
molecules are deficient and there are
genetic mutations, naturally the spectrin,
ankarin and other proteins are not available
and there will be a defect in the membrane
causing phenomenon as spherocytosis.
266
In spherocytosis the membrane spectrin
and protein band 4.1 is lacking and these
linking protein to cell membrane
skeletal framework, leading to loss of
surface tension and area and integrity,
forming a spherocytes.
In elliptocytes and stomatocytes (-osis)
the same principle protein are involved.
Particularly the elliptocytes the lateral
spectrin based skeletal defects cause
membrane fragmentation and loss of
stability forming elliptoid cell. While the
stomatocytes experience these defects in
these proteins in form of the abnormal
membrane proteins that correlates with
anomalous univalent cations across the
plasma membrane. The defect is not
concern with membrane transporter but
likely the increase in permeability to
cations, which is quite evident. The recent
research indicates the
membrane
integrity skeletal proteins as mentioned
above may play some role in the univalent
cations deficiency.
Laboratory
diagnosis
is
by
electrophoresis of membrane proteins,
red blood cell cytology and
flowcytometry with genetic studies of
membrane proteins. These types of red cell
defects may be found in variety of anemia
such as, megaloblastic anemia, hemolytic
anemia, membrane defects anemia, and
intrinsic and extrinsic anemia, and so on.
The figure 5-3 shows the complexity of the
red cell membrane skeletal proteins.
Fig. 5-3: Schematic presentation of the
structure of red cell membrane skeletal
proteins including RBC antigens/blood
grouping systems.
RBC Enzymes and Defects
in Ancillary RBC Resident
Enzymes/RBC Enzymopathy
Glucose
6-phosphate
Dehydrogenase Deficiency
(G6PDase) (American Association
for
Clinical
Chemistry,
G6PDase
deficiency 2001-2013)
G6PDase deficiency is an autosomal
dominant trait with both homozygous
and heterozygous individuals. The
genetic defects are prevalent in regions of
Africa, Mediterranean countries, and as
well in Southeast region of Asia. The defect
is due to mutations in the said genes and
manifests as an acute hemolytic
anemia. The enzyme deficiency causes
reduction in G6PD and low level
production of NADP from NADPH causing
267
disruptive oxidation and reduction
reaction, which can jeopardize red cell to
oxidant drugs or chemical or dietary
substances such as fava beans.
The condition is known as fauvism. The
hemolysis is due to exposure of the
molecules to oxidative agents in fava beans
and other chemical similar in action to fava
beans, i.e. phenacetine, naphthalene,
sulfa drugs, viral and bacterial infection,
metabolic acidosis and malarial
medication such as quinidine/quinine.
Clinical signs are jaundice, severe acute
hemolytic anemia, dark urine, fatigue,
pallor, fainting, tachycardia, increased
pulse, splenomegaly and shortness of
breath or dyspnea. These are some of the
signs and symptoms present in the disease.
Women are carriers and possess one
defective gene and one healthy gene as in
heterozygous, while the homozygous have
both abnormal genes causing G6PDase
deficiency, severe enough to ensue to
massive hemolytic anemia during the
acute reaction/phase. Therapy includes
transfusion of either whole blood or
red cell concentrates, and supportive
therapy.
Lab findings include, blood smear showing
bite cells, hypochromasia, microcytic
hypochromic anemia, Heinz bodies,
increased reticulocytosis with bone
marrow compensation, erythropoiesis,
hematuria,
hemoglobinemia
and
hemoglobinuria, and finally there may be
hemosiderosis and presence of
hyperbilirubinemia due to jaundice and
icterus. The hemoglobin electrophoresis is
the definitive identifying test for diagnosis.
The next enzyme defect most common to
RBC ancillary enzyme deficiency is
Adenylate Kinase.
Andenylate
Deficiency (AK):
Kinase
(NA Lachant et
al.)
AK is an enzyme that mediates the
interconversion of ADP to ATP and is
responsible for red cells energy resources
and survival. This is a genetically
inherited disorder of red cells and it is
an enzymopathy as G6PD, and
Pyruvate kinase deficiency. The
chemical agents and mutations and
dietary substances can add to the deficiency
through an inherited or acquired pattern.
Most of the lab findings are the same as
other enzymopathies as well as in G6PD
deficiency. It includes significant increase
in
LDH
(LD),
anisocytosis,
poikiocytoisis,
hemoglobinopathies,
such as decreased Hgb. reduced MCV
(<90 fl. ), reticulocytes significantly
augmented indicating marrow erythroid
hyperplasia, white blood cells count
(CBC) variable and with bilirubin
significantly elevated.
Clinical signs and symptoms are
consistent
with
other
red
cells
enzymopathies including dark urine, sever
chronic or acute hemolytic anemia, pallor,
dyspnea, tachycardia and increased pulse.
Other findings include microcytic
hypochromic anemia, this clinical
268
condition
warrants transfusion,
corticosteroid treatment along
supportive therapy.
and
with
Chapter Six (6)
Non-immune
Hemolytic
Anemia
Microangiopathic Hemolytic
Anemia(s)
Thrombotic
Thrombocytopenic Purpura
(TTP):(Sadler JE, et al/ Marie Scully et
al.)
he PTT syndrome is of
significance in hematology due to
it relation and similarity to HUS
(Hemolytic
Uremic
Syndrome). Although there are subtle
differences between these spectrums and
arrays of these similar disorders, however
there are coagulopathies with regards to
both of these syndromes. There are
microcirculatory thrombi formation and
small vessels clogging by minutes thrombi.
T
PTT is essentially a microangiopathic
hemolytic anemia, where there is
significant
thrombocytopenia.
The
intravascular thrombosis is less in TTP than
in HUS and more likely in DIC.
Fibrinogenemia is not involved or
observed in TTP, hemoglobin is severely
decreased and there are evidence of
subcutaneous purpura and ecchymosis
under the dermis. Due to thrombotic
episode and severe hemolysis, leading to
very low hemoglobin, low MCV, raised
LDH
due
to
hemolysis
and
schistocytosis,
anisocytosis
and
poikilocytosis, Red cell fragmentation
is seen along with reduced haptoglobin
due to increased red cell and platelet
destruction and subsequent binding to
haptoglobin. Other hematology lab finding
includes a negative DAT with cardiac
irregularities as one of the clinical signs.
Blood smear shows schistocytes, RBC
fragmentation and target cells. One of the
distinctions of TTP is the lack of kidney
involvement in spite of the circulatory
disturbances. Recently there have been
evidences that molecules such as
ADAMTS13 responsible for thrombotic
thrombocytopenic
events/complications,
this molecule is the cleaving factor of the
VWF (von willebrand factor), its assay
is available in diagnostic reagents
manufacturers
and
pharmaceutical
companies. The patient with TTP should
not ever be treated with the platelet
concentrates
or
with
thrombocytopheresis because these
product only aggravate and exacerbate the
condition and is course turn into
dramatically mortal.
Hemolytic
Uremic
Syndrome (HUS): (Gillespie,
Robert S et al.)
This condition is also a microagiopathic
hemolytic anemia (MAHA), which is
quite common in children with typical
uremic episodes followed by E. coli
infection causing enterohemorrhagic E. coli
enteropathy resulting into severe hemolysis
and uremia with eventual kidney failure.
269
The strain of E. coli responsible is O157:
H7, in this infection WBCs are increased.
Clinically uremia is typical in this
thrombotic condition as mentioned this
would be similar and of the same spectrum
of PTT variety. Figure 6-1: shows a typical
HUS blood picture with thrombocytopenia,
WBC and helmet cells are identified.
Fig. 6-1: Blood smear of a typical HUS
individual showing, thrombocytopenia,
WBC, and helmet cells and schistocytes,
total magnification; 500x.
The most obvious clinical signs and
syndrome is marked with variable
fibrogenemia; a distinction from PTT.
Other blood smear findings are helmet
cells (blood smear predicator of the
disorder),
schistocytes
and
RBCs
fragmentation, other findings involved
coagulation panel such as variable
fibrinogenemia, reduced haptoglobin due
to augmented hemoglobin because of
increased red cells lysis and destruction
with low survival rate, reduced amount of
fibrin degradation products increased
(FDP), along with elevated reticulocytes
indicating bone marrow compensation and
hyperplasia with increased RPI
(reticulocyte production index), bilirubin
may be increased. WBCs differential shows
left shifts usually of myeloid series. The
differential diagnosis of HUS and DIC
(disseminated
intravascular
coagulation) consists of increased fibrin
degradation products and increased
fibrinogen, which both are pathognomonic
for DIC while variable fibrinogen and
increased FDP are predicators of HUS,
helmet cells are in the blood film is
predicator of HUS events. One of the
clinical chemistry tests that are typical for
this condition is urea/creatinine is high
and there may be higher uric acid levels
found.
The patient may be treated for the
underlying condition and supportive
therapy such as electrolytes monitoring,
transfusion of platelet in case there is an
active hemorrhage, and dialysis.
Transfusion of other causes of thrombosis
is controversial. The prognosis may be poor
if treatment would not be substituted
promptly.
Acute
and
Chronic
Disseminated Intravascular
Coagulopathies: (Yu M, et al/ Ferri
et al.)
This is
kind of consumptive
coagulopathy in which coagulation
system progresses to complete derange and
chaotic events. Microthrombi, microand macrovascular thrombi are formed
and coagulation factors are consumed in
a
270
the process. The FDP and other degradation
products of coagulation are seen,
Fibrinogenemia and fibrinogen/fibrin
degradation
products
are
pathognomonic to laboratory findings in
DIC.
This is a life threating condition with 75%
mortality.
Immediately
underlying
condition must be identifies and treated
accordingly.
Bleeding,
fever
and
thrombosis are cardinal signs and patient
circulatory hypervolemia should be
increased so that prevents shock and death
of the patient. Cardiac signs include
tachycardia, dysrhythmias and circulatory
failure.
Hematology laboratory findings are typical
schistocytosis, fragmented red cells
and
thrombocytopenia
along
with
prolonged PT, PTT and thrombin time.
Clinical presentation shows hemorrhages,
ecchymosis, and purpura as this is a severe
and radical form of microangiopathic
hemolytic anemia, with poor prognosis.
Chronic form is milder in clinical
presentation while acute DIC is invariably
may be fatal. The treatment should be
focused in the underlying condition and
therapy may be substituted according to
the therapeutic scheme. Kidney and liver
failure are targets once the thrombotic
clots prevent and block and clog these and
other organs circulatory apparatus leading
to ischemia, infarction and organ
failure.
Electrolytes may also be disturbed and
general acidosis may ensue in form of
metabolic and respiratory acidemia. Prompt
attention and recognition of the underlying
etiology are crucial. Etiologic condition
such as carcinoma in situ, leukemia (s),
lymphomas, debilitating conditions, and
also eclampsia and preeclampsia as so
called, “fever of pregnancy”, are some
of the causes of DIC and should be
considered once the condition recognized
and responded adequately to prevent
clinical disaster. LDH is invariably high,
due to Hepatic insufficiency and
failure and should be paid attention to it
diligently.
Macroangiopathic Hemolytic
Anemia
Anemia due to prosthetic
valve: (Williams Hematology)
The practice of valve replacement
during 1950s, started with patients
experiencing with some mild to severe
macroangiopathic hemolytic anemia.
Today even though by the advance in
cardiac valve replacements, there still
hemolysis of red cells exists among the
patient having their valve changed, this is
either by natural or synthetic valve
prostheses.
Our point of study in this section is to have
an overview of the hematology lab
findings in cardiac prostheses and we
will keep the scope of this section to the
said topic. Although natural and normal
heart
valve
when
sick
retains
classification fibrillation and fibrosis
and though becoming hard in natural
consistency thus causing a frank or overt
hemolysis; this phenomenon is worse in
artificial valves.
271
Temperature above 37°C can cause mild
Usually the hemolysis consists of red
cells rupture due to turbulence of the
regurgitation and backflow jet causing
torque to the erythrocytes and then rupture
of the cells releasing hemoglobin into the
circulation therefore we have low HGb.
hemoglobinemia, hemosiderinuria and
a low MCV due to microcytic hypochromic
anemia for red cell lysis and elevated LDH.
Other lab findings may include increased
WBCs, decreased haptogloblin, presence of
schistocyte, helmet cell, fragmented
red cells with sharp angles and spikes,
thrombosis due to platelet adhesion to
the
surface and triggering
thrombogenesis and active bone marrow
due to thrombocytopenia, eryhtroid
hyperplasia
with
reticulotcytes
augmentation. Bone marrow spends in
compensation for most of the dysplasia.
A patient with chronic over hemolysis and
dark urine would be an example of
valvular hemolysis in patients with
artificial heart valves. Treatment usually is
the replacement of a proper less
immunogenic and hematogenic valves
in nature and in some cases transfusion to
replace red cell mass and other factors. At
the end it worth to remind that the main
reason of hemolysis is shear force applied
to the cells during regurgitation or leakage
of the blood to in either backflow
(regurgitation) or forward contact force
within the parts of valve.
destruction and lysis if erythrocytes,
however if it reaches to above >40°C can
be quite destructive and cause severe frank
hemolysis. This would be the same as
traumatic hemolysis and as too cold a
temperature, if the traumatic blow is too
hard, there will be bleeding during
trauma, and hemolysis would be in form of
microcytic hypochromic anemia,
hemoglobin increases and haptoglobin
decreases. WBCs augment if there are signs
of infection or release of immunogenic
and cellular mediators. Treatment
consists of monitoring normal temperature
and treatment of injuries against trauma.
valve
Thermal
and
Physical
Destruction of RBCs
Hemolytic Disease due to
Parasites and Intracellular
infections
There are several parasitic diseases that
cause red cell destruction and then
hemolysis. Examples of these are
babesiosis, malarial parasites as
Plasmodium spp. and others such as
Leshmamiasis (Leshmonia donavani,
braziliensis and gombiansie) and
Trypanosomiasis (Trypanomosomia
burcei, rheodesienses, and cruzi)
causing typical hemolysis and anemia in
general. These anemia(s) are mostly
identical to one another therefore we will
consider here the most morbid one among
others; malarial parasites, Antibodies are
formed in malarial infection however they
are not immune and protective. For
Malarial
parasites
there
are
exoeryhtrocytic and erythrocytic
272
stages. The mosquito stage is in the
spirogony and erythrocytic pigmented
stage is schizogony.
Malaria parasites or Plasmodium spp. is
divided and classified into four species
accordingly; first and the most morbid one
is Plasmodium falciparum, then
Plasmodium
malariae,
third
Plasmodium
ovale
and
fourth
Plasmodium vivax.
The most endemic regions in the worlds
of malarias are subtropical and tropical
areas in all geographical regions except
Australia which is considered free of
malarial parasites. Here we will review
shortly some aspects of malarial life
cycle. The first time when the parasites
invade the epithelial cells of the vertebrates
by the bite of Anopheles mosquito, the
cycle starts by meroziotes penetrating the
red cells therefore changing from
trophoziotes to schizonts, as in
schizogony when it invades red cells
cytoplasm and turning into zygots, the
cell disrupts with anemia and destruction of
the cells involved, it subsequently enters
into the new red cell and the process
reinitiates. Again when the mosquito bites
the patient; the vector cycle starts and
with another morphogenesis by spiroziotes
and oogony (schizogony) in sexual and
asexual reproduction phases respectively.
The following figure is the picture of
Plasmodium falciparum; schizonts
stage in the blood film (Fig. 6-2).
Fig. 6-2: The Plasmodium falciparum,
schizonts in blood smear
The malaria studies starts through thick
and
thin
smears,
as
technologist/technician prepares the thick
smear, the anticoagulated blood with
EDTA will be defibrinated with magnetic
balls to let release the ruptured RBCs
content permitting observation of the
parasite stage of infectivity. For thin blood
smear the preparation is the same as normal
blood films with Romanowsky stains
such as Wright’s and Geimsa.
In case of P. falciparum (DPDx,
laboratory et al.) there are ring forms and
they are very delicate with one or two small
dots, although there are often appliqué
forms (rings in the periphery of the red
cell) can be seen.
Multiple red cells infections are common in
P. falciparum infection than other malarial
species. In the older smears Maurer’s
clefts may be observed and rings are
common in the trophozoites stages. The
273
classical ‘headphone” and “appliqué”
ring forms are quite typical. The following
figure (Fig. 6-3) is an example of
headphone rings.
surrounding milieu. In this type of infection
(P. falciparum) RBCs are small in
comparison with other types.
For Plasmodium
vivax, (DPDX
Laboratory et al) blood collection is by
fingers
sticks/puncture
and
microcollection and delay in processing
can distort parasites morphology, which is
the same for other species in this respect
the staining characteristic may change due
to
malpractice
in
quality
assurance/assessment.
Fig. 6-3: representation of Headphone rings
in the peripheral blood. Total magnification
1000x.
Plasmodium falciparum’s gametocytes
(Gardner, Malcolm J. et al.) are crescent
and sausage shape with chromatin either
large (microgametocyte) or diffuse type
(microgametocytes),
P.
falciparum
trophozoites are seldom seen in the
peripheral blood and if found it’s in
schizonts stage with 8-28 merozoites and
a compact form in the thin smear, with red
cells meroziotes having a dark pigment
and a clumping mass.
For detection of malarial parasites thick
smear give a better detecting of the
parasites
within
identification
parameters than thin smear, because of
the density of the blood and defibrination
and release of RBC contents in the
The RBCs are large due to infectivity of
this microorganism (P.vivax) and under
optimal condition Schuffner’s dots are
obvious with large chromatin dots.
Schuffner’s dots appear fine in
comparison
with
P.
ovale.
The
trophozoites are large and ameboid and
in other cases they are band shape forms of
trophozoites. This can be distinguished
from P. falciparum by the large sizes of the
trophoziotes bands (large cells and
bands). Gametocytes are round and oval
with large schizonts with 12-24 merozoites
that may fill the RBCs. Schizonts
looks/appears either mature or immature in
the thick or thin blood smear.
In P. malariae, the infected red cells are
smaller, with sturdy cytoplasm and
chromatin dots, trophoziotes have
compact cytoplasm with large chromatin
dots and occasional bands and basket forms
are seen in either thick or thin blood film.
In these cells dark brown pigment can be
observed. Figure 6-3 indicates band form
of P. malariae.
274
Fig. 6-4: Schizonts of Plasmodium
malariae in form of rosette. Total
magnification 1000x.
Lastly for P. ovale, the cytoplasm rings is
sturdy with large chromatin dots.
Schuffenr’s dots may be present, and
cells look as round and oval and are slightly
large.
Fig. 6-3: indicates band form of P.
malariae.
Additionally, merozoites of schizonts
are sometimes in rosette shape in form of
dark brown pigments. Merozoites are 612 in number with large nuclei with coarse
and massive clustered pigments around the
nucleus. Figure 6-4 represents a rosette
of P. malariae schizonts. The
gametocytes are round and ovale as they
fill almost the whole cell.
Trophosoides are slightly large with sturdy
cytoplasm and chromatin dots are large and
irregular in shape. Trophozoites could
be fimbriated (bordering fringe) and
gametocytes are round and oval with
compact appearance. Pigments are brown
and much coarser, which discriminates
between P. ovale and vivax. Schuffner’s
dots are present and gametocytes divides
into macrogametocytes or a diffused
form
as
in
microgametocytes.
Schizonts are 6-14 in number and large
nuclei clustered around a mass of dark
brown pigment.
In general with all species of malaria,
there is lactic acidosis and electrolyte
changes, ions changes are due to
pulmonary and metabolic lactic
acidosis, white blood cells may be
variable with often -cytopenias and cytosis.
The
increased
WBC
count/neutrophilia is an indication of
current infections and release of other
inflammatory mediators; anemia is an
indication of red cell lysis and
transformation of Hgb to amino acids.
Thrombocytopenia is variable and
usually
observed.
In
addition,
Microvascular thrombosis and ozzing
275
from
injection
sites or other likely
mucosal orifices are likely and should be
tackled efficiently to reduce morbidity and
mortality; the microvascular occlusion
and hyperviscosity syndrome can cause
organs reperfusion failure and organ
failure or insufficiency with tissue
ischemia due to lack of sufficient oxygen
tension, oxygen supply and red cells
damage. Treatments include transfusion
or exchanged transfusion for the
neonates and children, and chemotherapy
with such cliché as primaquine, quinine
and quinidine and some other modern and
new chemotherapeutic agents (David J.
Weatherall, et al).
Chapter Seven (7)
Myeloproliferative Disorders
(MPDs)
M
yeloproliferative
disorders are a group of
disorders
for many years after the initial diagnosis. In
addition, the disorders under proliferation
have their own specific diagnosis and
treatment modalities. Clinical signs
include, throbbing and burning
sensation
on the skin, headache,
stomach ache, bleeding, breathing
difficulties and malaise with general pain
and lethargy. This class of disorder is as
following:
1) Chronic Myelogenous/Myeloid Leukemia
(CML)
2) Chronic Idiopathic Myelofibrosis (IMF);
Fibrosis with Myeloid Metaplasia (FMMP)
3) Polycythemia
Rubra
Vera
(PRV)
4) Essential
Thrombocythemia
(ET)
a. Acute
Myelogenous/Myeloid
Leukemia (AML)
b. Myelodysplastic
Syndrome (MDS)
I will explain and cover the preceding
accordingly.
involving
proliferation of myeloid
precursors and progenitors cells. These
cells
are
leukocytes,
erythrocytes,
thrombocytes or megakaryocytes and
other blood cell lineage that grow
abnormally in the bone marrow and
sometimes will release in peripheral
circulation.
The conditions under this category are
serious disorders and patients may live
Chronic
Myelogenous/Myeloid
Leukemia
(CML):
(Koyamangalath Krishnan et al.)
CML is a proliferation of all myeloid series
including polymorphs, erythrocytes, and
platelets, the disease stages divides into
chronic phase, accelerated phase and a
blast phase. This disease clinical signs are
276
low grade fever due to hyper metabolism
with bleeding from mucosal membrane
and other orifices, bone pain, headache, left
upper quadrant abdominal pain as in
stomach pain due to splenomegaly: as an
encroachment of spleen into stomach, along
with splenic infarction, anemia and
hepatomegaly, shortness of breath and
lethargy and malaise. Bleeding in acute
phase may be ecchymosis, petechiae and
mucocutaneous hemorrhages.
It is more common in older patients with an
age range of middle ages, or sometimes in
20s or older people.
In hematology laboratory finding, we have
hepatosplenomegaly, due to infiltration
of increase hyperplastic myeloid cells,
leukoerythroblastosis, erythroid and
myeloid dysplasia, basophilia (could
be variable), eosinophilia and increased
megakaryocytes
and
thrombocytes
(thrombocytosis), there are thrombotic
tendencies and augmented inflammatory
mediators, the leukocyte alkaline
phosphates (LAP) is low or absent
whereas LAP in leukemoid reaction
increased, leukemoid reaction (there are
more toxic granules, more toxic vacuoles
frequent döhle bodies and Lap as said is
high with no Philadelphia chromosome) and
late polycytemia vera and IMF(for IMF
there are many NRBCs stress reticulocytes,
means very basophilic appearance, with
fibrosis in b.m.), must be differentiated
from CML.
There
may
be
bone
marrow
hypercellularity with myeloid series as,
myeloblast,
promyeloblast,
myelocytes, metamyelocytes and
bands, also erythroid progenitors such
as nucleated red cells ( as erythroblast,
prorubriblast, rubriblast and metachromatic
rubriblast) can be find in marrow and
peripheral cells.
The cause of the condition is the aberrant
Philadelphia chromosome with bcr and
abl translocation between chromosome 9
and 22 may be obvious in the bone marrow
cells.
Bone marrow white cell count correlates
with the peripheral cells, in which there may
be increased to < from 50-600 x10 9 /L as
well, reticulocytes are increased, as the
compensation is disturbed due to
hyperplasia and hyper cellularity. Both
myeloid to eryhtroid ratio in b.m. would be
significantly increased (10:1 where normal
is 3:1) and adipose tissues and fatty islands
are decreased while there is a mild fibrosis
by reticulin stain.
Recovery and remission depends on the
diagnosis and installment of the therapy.
The therapy may be in the form of
transfusion of blood products and
coagulation factors, interferon alpha, or
the most advanced and newly passed drug
by FDA as tryosine kinase inhibitors
such as Imatinib mesylate, and other
cliché medications as busulfan, and
hydroxyl urea. The following is the
peripheral blood smear of a CML case (Fig.
7-1).
277
to anemia and
thrombocytosis
red cell lysis with
LAP
(neutrophil
alkaline phosphatase) raised due to
tissue breakdown and cytolysis. The
hematologic triad of findings includes:
 Fibrosis of marrow
 Extramedullary hematopoiesis or
myelois metaplasia of spleen and
liver
 Leukoerythroblastosis and teardrop
poikilocytosis of the peripheral
blood.
Fig. 7-1: Photomicrograph of CML with
myeloid series, progenitor cells such as
myeloblasts, metamyelocytes, myelocytes
and bands (left shift) with some normal cell
lines, so on. Total magnification 500x.
Chronic
Idiopathic
Myelofibrosis (IM)/Fibrosis
with Myeloid Metaplasia
(FMMP)
The IM is a
disorder
clonal proliferation
that affects stem cell CFUGEMM phenotypes, it affects ages > 70
years and clinically manifested by
hepatosplenomegaly,
MPD
(myeloproliferative disorder), and low
fever, bone pain, anemia with malaise and
lassitude/lethargy,
also
episodic
hemorrhage.
Common laboratory and
hematologic findings are as of increased
to marked thrombocytes (in pre -fibrotic
state, but variable in fibrotic state),
elevated, normal or decreased WBC count
(variable in fibrotic state, but moderately
increased in pre-fibrotic state), slightly
reduced erythrocytes, LDH increased due
Reticulocytes are decreased and there is
reduced compensation in bone marrow.
In this organ, there are significant increased
myeloid
hyperplasia with severe
leukoerythroblastosis
and
megakaryocytopenia (variable in this
stage) with resulting increment in
peripheral eukocytosis/erythrocytosis
and thrombocytopenia in fibrotic stage.
Bleeding episodes/diatheses are due to
increased platelets and slightly decreased
erythrocytes with NRBCs in the peripheral
blood. In blood film there are evidence of
low platelet count, increased WBCs and the
pathognomonic signs as teardrop red
cells
(dacrocytes)
and
anisopoikilocytosis.
Figure
7-2,
indicates an example of blood film for IMF.
Myeloid metaplasia is usually seen in
blood
and
phenomenons
proliferation of
system (RES)
bone marrow.
bone
marrow.
These
are indicated by the
the reticuloendothelial
such as liver, spleen and
There is evidence of
myelomatosis and ESR is raised due to
inflammation, and folic acid can be
elevated because of tissue breakdown and
hyper -metabolism. IMF (or fibrosis with
278
myeloid metaplasia), is a precancerous
states in which it can ensue to CML and
other types of sever Myeloproliferative
and lymphoproliferative conditions. As
the name applies it may occur by an
unknown origin/or etiology, it may happen
by some chemicals, genetics or infectious
nature. Bone marrow indicates fibrosis with
increased fibroblasts with signs of
neovascularization with reticulin stain.
There is also splenic fibrosis.
Fig. 7-2: A) Teardrop red cells, B) Giant
thrombocytes, C) NRBC, D) Myeloid left
shift cell as Promyeloblast, and E & F)
Giant thrombocytes and teardrop cells.
These are indications of CIMF (chronic
idiopathic
myelofibrosis).
Total
magnification 400x.
Polycytemia
(PRV)
Rubra
Vera
This is also a hematopoietic stem cell
disease, where there is accelerated
erythropoiesis and to some degrees of
increased
granulopoiesis
and
thrombopoiesis in bone marrow. The
distinction may be made between relative
and secondary types of PRV. The
secondary type is caused by cardiovascular
diseases, or heavy smoking and poor
erythropoietin production. The relative
type is due to dehydration and plasma loss
due to burns, severe diarrhea, etc. causing
concentration of RBCs mass.
Clinical signs and symptoms include
headache, weight loss and malaise
(weakness), there are mucocutaneous
signs such as hot bath pruritis,
ecchymosis and petechiae due to platelet
malfunction, also cardiovascular signs as
arteriovenous thrombosis and hyperviscosity syndrome. Indeed there would
be also gasterointestinal signs as ulcers
hemorrhages and 75 % of affected have
splenomegaly and 50% hepatomegaly.
Hematologic findings consist of 2-3 times
increased than the normal erythrocyte
counts, hemoglobin and hematocrit
increased proportionately. MCV, MCH and
MCHC are normal but may be augmented
along
with
hyperchromasia and
additionally red cell indices are normal
however may be variable or increased.
WBCs significantly increased
(15-50x10 9 /L), whereas there is often
neutrophilia with increased LAP score,
neutrophilia exists along with left shift and
basophilia. Platelets moderately increased
in 2/3rd of patients occasionally may shoot
up to 1000x10 9 /L. Megathrombocytes
are the main platelet morphology.
Bone marrow is hypercellular with
increased
ratio
in
cell
to
fat,
megakaryocytes clusters are increased and
this can differentiate between PRV and
other polycytemias. In this regards, iron
279
deposits are absent and shows utility of
storage iron for increased HGb. synthesis.
RBC mass studies are increased, however
plasma volume is normal. There is
increased vitamin B 12 and 10% raise in
Hgb. F. Treatment approach consists of
phlebotomy or venisection to let blood
and thus reducing red cell mass and
maintain normal Hct.
Busulfan an
immunosuppressive agent may be used in
conjunction. Prognosis may be poor if
transforms
into
some
malignant
disorders such as AML in 10% of patients
also may change to
IMF
and
thrombohemorrhagic complications.
The following figure indicates blood
picture of PRV (Fig. 7-3).
Fig. 7-3: Blood film of a case of
Polycytemia Rubra Vera; note the number
of RBCs in the peripheral circulation 400x.
Essential Thrombocythemia
(ET)
This is an overproduction of platelets in the
absence of recognizable stimulus, the
platelet/thrombocytes shoots to 400x10
9
/L. This is a rare condition, and as
mentioned
there
is
marked
thrombocytosis, but their functions are
normal; it must be differentiated from
reactive
thrombocytosis
such
as
malignancy, iron deficiency and acute
hemorrhage. It is seen primarily in older
adults after the fifth decade.
Clinical signs and symptoms are bleedings,
although thrombocytes are too many but
are helpless in closing the bleeding. There
is
subsequent
thrombosis
and
splenomegaly, with rapid cell turnover
such as CML and IMF. Patients are usually
asymptomatic and when symptomatic
they represent with clinical picture of
hemorrhage, vasoclusive states, along
with skin and mucocutaneous bleedings
such as ecchymosis, epistaxis and stroke
may happen when there are large clogs of
thrombi and clotting.
Hematology lab findings consist of platelet
functional defects, Phosphates, potassium
and calcium release from platelets into the
circulation and change in electrolytes.
Platelet
count
always
elevated,
thrombocytes morphology include giant
platelets,
megathrombocytes,
microthrombocytes and anemia is in
form of normocytic normochromic
anemia in 20% of the patients, 33.3% of
the patients show leukocytosis but counts
mostly are confined to and not more than
50x10 9 /L and occasionally there may be
eosinophilia. LAP may be variable
however it is usually normal. Treatment
280
could be placed with hydroxyl urea
administration and radioactive phosphorous
32
P may warrant, also transfusion of
plateletpheresis/thrombopheresis or
concentrates
may
be
indicated.
Transformation
to
leukemia and
myelofibrosis with myeloid metaplasia is
feasible and likely.
With regards to bone marrow, it is
trilineage hypercellular or hyperplasia sort,
therefore, increase in megakaryocytes and
no fibrosis and absence of iron stores may
be apparent. Bone marrow karyotype is
normal in which there is a deletion of long
arm of chromosome 21 (21-q). Platelet
aggregation is often decreased with
epinephrine and there may be different
responses and reaction to ADP and
collagen. Platelet factor 3 and adhesion is
reduced with low protein S, bleeding time
has been reported most variably with
prolongation or normal response. Figure
7-4 is indication of ET case blood smear,
with increases thrombocytes has been
shown.
description refer to
magnification 1000x
the
text.
Total
Acute Myelogenous/Myeloid
Leukemia (AML)(Kocmarek et
al.)
In this disorder the myeloid cells fail to
differentiate beyond the blast stage.
Immature hematopoietic cell lines
proliferate clonally; this is a malignant
state.
Incidence increases by age. The blast cells
take over the bone marrow and
peripheral architecture of normal myeloid
cells. The malignant myeloblasts proliferate
and mature but they do not die. This leads
to thrombocytopenia and anemia. The
diagnosis is based on FAB system
(French, American and British system
of classification). The cytogenetic,
cytochemistry and other molecular and
chemical approaches can have great
implication in establishing a diagnosis and
therefore treatment.
Table 7-1 shows
classification of AML according to
histochemical stains.
Table
7-1:
Stain
Characteristic of MLA
Stain/Leukemia MLA MLA MLA MLA MLA MLA MLA
M1 M2 M3 M4 M5 M6 M7
Peroxiase
Fig. 7-4: Essential thrombocythemia with
platelet polymorphism and dysplasia. For
+
1-3+ 3+
2-3+ 0
0
0
Spec. Esterase +
2+
3+
1-3+ 0
0
0
Non-Spec. Est. 0
0
0
2+
3+
0
~+
PSA
0
0
0
0
3+
2+
0
281
Spec. Esterase = Specific Esterase NonSpec. Est. =Non-Specific Esterase
PSA: Periodic Acid Schiff
The causes are idiopathic, however agents
such as alkylating agents as in drugs,
chemical as benzene or viruses and genetic
abnormality may be involved: example this
is Down’s syndrome can be the culprit
agents. FAB classification uses Wright’s
May-Grunwald stain for distinction and
morphological
classification.
FAB
classifies AML into several classes,
including M1 to M7; in this M3 has M3m
subclass and M4 with M4Eo. M5 have to
subdivision M5a and M5b. These are
define shortly as,




AML M1) There is no maturation
and these are myeloid leukemia,
cells are essentially myeloblasts and
no azurophilic granules.
AML M2) There is maturation and
beyond that. The maturation could
be at or beyond promyeloblast.
AML
M3)
Hypergranular
promyelocytic
leukemia.
Granulations are anomalous. There
are predominantly promyelocytes
with distinctive Auer rods.
M3m consists of hypogranulation,
with
microgranular
promyelocytic leukemia, DIC
is common in this type of leukemia
along with an often reniform nuclei.
AML
M4)
This
is
myelomonocytic
leukemia
there are multilineage cell line such
as granulocytes and monocytes are
present, for
M4Eo, is the
myelomonocytic leukemia with
presence of eosinophils.



M5a-M5b)
monocytic
leukemia, where there is
predominantly monoblasts in F5b
there is primarily promonovytes in
blood film.
AML
M6)
is
the
erythroleukemia,
with
erythrocytes predominates. More
than 50%% of marrow cells are
bizarre cell lines such as
megaloblstoids.
AML M7) is mainly thrombocytes
and
megakaryocytes/megakaryocytic
leukemia, there may be dry tap.
AML
The histochemical staining strategy to
classify AML are divided into 3 category,
these are peroxidases and Sudan black;
specific for M1 (1+), M2 (1-3+), M3 (3+),
M4 (2-3+), M5, M6 and M7 (0+).
The second stain under the said category is
Esterase (specific and non-specific), the
specific esterase is positive for M1 (+), M2
(2+), M3 (3+), and M4 (1-3+) the rests are
0+.
Non-specific esterase enzyme is
positive for M4 (1-3+), M5 (3+) and M7
(almost ~1+) and there in AMLs are 0+. For
the third stains PAS as we have 3+ for M6
and 2+ for M7, ALL (acute lymphoid
leukemia) shows block reactivity with PAS
(Periodic acid Schiff).
Clinical findings include anemia in form of
normocytic normochromic, dyspnea
upon exertion, pallor and weakness,
thrombocytopenia in association with DIC,
bleeding in mucocutaneous, purpura,
neutropenia due to infection; this is in from
of septicemia, pneumonitis, epithelial and
mucosal infections. There are metabolic
282
consequences with large tumor mass,
overwhelming blasts occupy bone marrow,
peripheral blood and other sites in other
organs, etc.
Bony tenderness especially in the sternum is
seen along with lymphoadenopathy and
hepatosplenomegaly. Hemoglobin is
decreased, hematocrit correlates with Hgb
and lysosomes are present and may be one
of the causes of DIC, once released in the
circulation it can affect plasma proteins and
blood factors. Bone
marrow is
occasionally hypercellular with blast as
the main motif, but myelopoiesis,
erythropoiesis
and
megakaryocytopoiesis are normal. INR
(PT), PTT, FDP and fibrinogen are altered
in cases of DIC also there is increased LDH
and LFT (liver function test) with reduced
calcium ion.
Treatment is supportive with transfusion and
antibiotic for infection and treatment of
metabolic abnormality, bone marrow
transplant may be an option when conjoin
with other therapies. The figure below is a
blood film that shows hypercellularity of
peripheral with leukemic multilineage cells
in M2 category (Fig. 7-5).
Fig. 7-6: AML with multilineage cell lines,
classified as M2. Total magnification 1000x
Myelodysplastic Syndromes
(MDS): (Attilio Orazi, et al.)
The MDS is a clonal disorder, which is
characterized by anemia and proliferation
of one or more cell lineages. It is a
heterogeneous group of disorders related to
clonal hematopoietic stem cell disease.
Most of the cellular lineage experience
cytopenias. These disorders are either
primary idiopathic or a secondary type,
which follows some cause as either a
radiation or chemical exposure such as
benzene.
There is chromosomal abnormality in
chromosome 5 (5q-) has been found in 4050% of patients, except in chronic
myelomonocytic leukemia (CMML).
Affected individuals are above 60 years of
age and some of them evolve to CML (preleukemic state) with insidious onset.
There are also ineffective hematopoiesis in
spite of presence of adequate numbers of
283
progenitor cells in the marrow (marrow is
hyper-cellular).
Clinical presentation encompasses lethargy,
fatigue, malaise and weakness, infections,
bruising, weight lost (rarely), fever and
hepatosplenomegaly. There is a triad of
findings as anemia ± thrombocytopenia
± neutropenia, bone marrow is either
hypercellular or normocellular and
dysmyelopoiesis is mostly found in bone
marrow precursors.
Hematological findings include RBCs are
variable in count and there is decrease in
reticulocytes showing failure of bone
marrow to compensate, also WBCs as
granulocytes are decreased and their
functions are abnormal. Platelets are too
large
and
too
small
with
thrombocytopenia.
FAB classification composed of:


Refractory Anemia, (RA)
nuclear cytoplasmic dyssynchrony, and
dysplastic pictures such as , binuclearity,
multinuclearity, fragmentation and increased
Howell Jolly bodies, and the staining of
cytoplasm is uneven. The leukocytes
abnormality consists of neutropenia, which
is
common,
hypogranulation,
hyposegmentation along with abnormal
chromatin pattern as pseudo-Pelger-Huet,
hypersegmentation with bizarre shapes
abnormal monocytosis with granulocytic
hyperplasia or granulocytic hypoplasia.
Platelets show thrombocytopenia (common),
thrombocytosis
(uncommon),
dual
population of platelets with blood picture of,
hypergranulation
or
agranulation
(variable)
bizarre
platelet,
megathrombocytes
and
micromegakaryocytes with mono- or
binucleated forms.
Treatment consists of sympatomatic
approach; transfusion, antibiotics and bone
marrow transplant. Figure 7-7 represents
RA class of MDS in blood smear.
Refractory Anemia with ring
sideroblast (RARS)
 Refractory Anemia with
excess blast (RAEB)
 Refractory Anemia with
excess
blast
in
transformation (RAEB-T)
 Chronic
Myelomonocytic
leukemia (CMML)
Each classification has its own definition. In
RAs there are erythrocytes abnormality as
mentioned; hematologic findings include
dimorphic
blood
film,
anisopoikilocytosis such as tear drop
(acrocytes, schistocytes, ovalocytes
etc.), megalobalstic changes consists of
Fig. 7-7: Represent erythroid and myeloid
granulocytic dysplasia, in RA with multiple
lineage dysplasia; A courtesy of G. Bhgat©.
284
Cytogenetic study may be taken to
diagnosis in conjunction with other lab
tests to diagnose
C this condition. Some
percentage (5-10%) of newly diagnosed
h
patients have poor prognosis.
CSF
a
(cerebrospinal
fluid) shows protein
increasedp along with elevated cells, there
may be metastases in CSF by malignant
t
cells infusion. Patient with this condition as
e nervous system involvement
with central
have poor
r prognosis.
Lab findings include WBC elevated in most
cases with
E hyperuricemia and hyperphosphotemia
along
with
i
hypocalcaemia, there is moderate to
g
severe thrombocytosis.
Kidneys may be
damagedhdue to increased uric acid in the
blood.
t
Lymphoproliferative
Disorders (LPDs): (Rodak et al.)
Acute Lymphoid Leukemia
(ALL)
his is a childhood cancer, and
accounts for 30% of cancers in this
age group. This develops from a
lymphoid cell line (block) at certain
stage of development. Clinical figures are
the same as AML. There is no specific
morphological and cytochemical features
in lymophoblastic leukemia stage.
T
There is a need of bone marrow aspiration
for morphological differentiation. These
(
studies include immunophentotyping,
8
karyotyping
and
cytochemical
examination
and
staining.
Hematologic
lab
)
findings show increase in WBC, which
comprise >50% of lymphoblasts. For
cytochemistry myeloperoxidase and
Sudan black are negative while > 50% of
cells is in lymphoblast blocks, with PAS
(Periodic Acid Schiff stain) staining
positivity. Bone marrow morphological
features of lymophoblast’s nucleus are
folding (there may be several folds it is
observed more in ALL than myeloblast),
the nucleus is reddish purple in color, and
number of nucleoli may be 0-1 and
indistinct, chromatin is more dense than that
myeloblasts, and evenly distributed. The
nuclear to cytoplasmic ratio or N/C ratio is
increased in comparison with myeloblasts,
285
with intense blue cytoplasm and granules are
usually absent with more vacuolization.
The FAB classification includes 1) FAB
L1, 2) FAB L2 and 3) FAB L3;
FAB L1 features consists of 25 % have Tantigens and mostly are null cells, it is
common in children. The lymphoblasts
are small with much reduced cytoplasm
there is homogenous chromatin pattern
however it varies from patient to patient.
Nuclear clefts are regular and cytoplasm is
less
basophilic
with
variable
vacuolization.
maturation detect beyond myeloblast or
promyelocyte while in ALL it is beyond
lymphoblast.
Treatment is the same as AML along with
elimination of abnormal clone. This is a
condition today with remission depends on
the initial induction and childhood
prognosis with following relapse. First
remission 60-90% and childhood ALL
80% with long term remission (>5 years),
adults have 30-40 % chance of remission
with > 5 years of survival. Figure 8-1, shows
peripheral blood cells of ALL case.
FAB L2 features composed of large
lymphoblasts (twice the size of small
ones) varies within the patient, along with
variable chromatin pattern, nucleus with
frequent folds, nucleoli vary in number and
are large and prominent. The cytoplasm
varies in amount but usually abundant. The
basophilia varies accordingly.
FAB L3; also called Burkitt’s lymphoma
with
large
lumphoblasts
and
are
hemogenous. Fine in nuclear chromatin and
stippled. Prominent nucleoli may be one
to two in number; cytoplasm is deeply
basophilic
with
noticeable
vacuolization. Some of the differentiated
features between AML and ALL are: AML
involves adults whereas ALL patients are
children, in AML blasts are big whereas in
ALL are small, AML has lots of cytoplasm
while ALL do not, and AML has lots of
nucleoli (3-5), where ALL only 1-3, AML
has lots of granules and
Auer rods whereas in ALL there are no
granules, AML treatment sustain toxicity as
ALL do not, stain for AML are
myeloperoxidase and Sudan black
while for ALL are PSA and lastly AML
Fig. 8-1: Acute lymphoblastic leukemia,
note distinctive vacuolization in the
cytoplasm. Total magnification 1000x.
Chronic Lymphoid Leukemia
(CLL)
This is a clonal lymphoid disease as
well with indolent characterization. The Blymphocytes have poor function with
accumulation of neoplastic cells such as
lymphocytes in the blood, bone marrow and
tissues as lymph nodes, spleen and liver.
There is small number of T-cell
population in this regard. This is the most
286
common type of lymphoid leukemia in the
western countries. The population
demographic involves older patients with
up to 60% asymptomatic. Almost 9 years
median survival and sometimes this
varies with the course and length of illness.
Causes include chromosomal trisomy or
translocation, genetic predisposition and
retroviruses.
Clinical signs encompass of 25%
asymptomatic at diagnosis, adenopathy,
hepatosplenomegaly, fatigue and weight
loss are other clinical signs and symptoms.
Laboratory findings are lymphocytosis
between 5-10 x10 9 /L the lymphocytes are
small and mature. A lot of smudge cells,
lymphocytosis, and infiltration of marrow
with the malignant cells (lymphocytes)
(more than 30% at 6 months) are dominant
features, there are λ and κ monoclonality
with
lymphocytes
immunoglobulin
specificity. The normal ratio of these lambda
and kappa chains is 50-50 however, the
malignant lymphocytes proliferate and
differentiate producing only one type of
heavy chain specificities as the kappa (or
likewise lambda) increase the ratio changes
from 50-50 to 80-20, which is the diagnostic
for this monoclonal neoplasm. We can
use T-cell receptor genes for identifying
the T-cell types.
There is
shooting
lymphocytosis with WBC
50-250
x
10 9 /L.
Thrombocytopenia occurs as well with
autoimmune hemolytic anemia and
spherocytosis in 25 % of cases.
Morphology of these cells are consistent
with small mature lymphocytes, there
to
may be abnormal lymphocytes with
different morphological features. These cells
(small and mature) may transform into prolymphocytes found in many prolymphocytic leukemia patients.
Bone marrow shows infiltration of the
neoplastic cells, which replaces normal
eryhtroid
and
myeloid
and
megakaryocytic cells, there would be
increase in number of immature
lymphocytes as the disease progresses.
Bone marrow aspiration and sampling
is not essential to establish a diagnosis but
could give out excellent clues to the disease
process.
Lymphocytes are well differentiated with
small size and non-cleaved in CLL. The
aspirate and bone marrow print shows
increased immature lymph and blasts.
Coomb’s test is positive in 1/3rd of the
patients and there would be autoimmune
complications. Also 5% of patients might
have T-cell leukemia that may invade
skin with lytic bone lesions and
hypercalicaemia with differentiation
difficult between B and T-cells. For
diagnosis of T-cells Southern blot is
indicated.
mentioned
pro-lymphocytic
leukemia might progress from CLL and
has rapid clinical course with poor
prognosis. The morphology consists of
large lymphocytes and prominent nuclei,
with mostly blasts from.
As
Complications of CLL might turn into,
bone
marrow
failure,
287
thrombocytopenia,
massive
lymphoadenopathy,
hypogammaglobulinemia,
and
transformation to histiocytic lymphoma.
Treatment of CLL consists of corticosteroid
therapy, radiotherapy and chemotherapy,
etc. pictures below uses Romanowsky
stains as in Fig. 8-2 is the peripheral blood
of CLL, with abnormal lymphocytic
distribution.
Fig. 8-3: Smudge cells and small
lymphocytes
features
of
chronic
lymphocytic leukemia. Total magnification
under 450x.
The next section covers Hodgkin’s and
non-Hodgkin’s lymphoma.
Fig. 8-2: Blood film indicates chronic
lymphocytic leukemia with predominant
small and mature lymphocytes. Total
magnification 1000x.
Figure 8-3, indicates presence of small
lymphocytes and lots of Smudge cells in
CLL with Romanowsky staining option.
Hodgkin’s
and
nonHodgkin’s Lymphoma (HD)
& (NHL)
Hodgkin’s
lymphoma (HD) is
practically a
monoclonal B-cell
(lymphocytes) disorder. Its demographic
population peak and age of onset is at 20
years of age or more than 50 years of age.
It is more common in males than females.
Clinical
features
consist
as
lymphoadenopathies as in neck and
axilla; B-cell proliferation and classical
symptoms consist of pruritus and painful
nodes subsequent to alcohol consumption.
Diagnosis is based on lymph nodes
biopsy and bone marrow biopsy having
obvious cell types known as “Reed-
288
Sternberg cells” and poly-nucleated cells
derived
from
B-cells.
Histological
examination includes nodular sclerosis
that must be distinguished from other types
of sclerosis as in NHL. Lymph nodes
overwhelms by malignant cells and thus
forming sclerotic lesions.
Peripheral blood shows variability in
results. The anemia is in type of
normocytic
normochromic
with
occasional hemolysis, there may be
increase to marked leukocytosis (occur
in 1/3rd of patients), leukopenia or
normal. These cloud be classified as
variable, eosinophilia is seen in 20 % of the
affected patients. The lymphocytosis may
be relative or absolute, there may be
neutrophilia
and
monocytosis
discovered. Thrombocytosis and or
thrombocytopenia may occur in
instances of time and about 5-15 % of the
patients may experience lymphocytosis
with
predominant
malignant
lymphocytes.
Bone marrow biopsy and sampling
indicates infiltration of neoplastic
cells at the time of diagnosis, bone marrow
biopsy and lymph-nodes aspiration (as
FNAB) is essential to establish diagnosis,
there are IV stages in leukemic process.
Stage 1, is involvement of a single lymph
node region and stage 4, consists of diffuse
involvement of one or more extralymphatic organ including bone marrow,
with stage 2 & 3 in intermediate stages and
IV stage as malignant advanced stage.
Immune defense decrement especially in
cell mediated immunity may result into all
from of infections as bacterial, viral,
fungal, etc. Following figure is the typical
Reed-Strenberg cells in HL.
Fig 8-5: shows one typical Reed-Sternberg
cells/lymphocytes in lymph node aspirate
or biopsy. Total magnification 1000x with
oil-immersion lens.
Liver function tests (LFT) may rules out
liver involvement and there are increased
Ca++ and ALP and phosphates when there
is metastasis to other organs, other tests
may be essential as, CT scan (computed
tomography),
radiographic
examination and abdominal and pelvis
scan.
Treatment may be indicated by radiation,
chemotherapy and focal field
radiation for stage I and II (there is a less
risk of malignancy), stage III, and IV may
be combined chemotherapy such as
ABVD, etc.
Treatment may lead to acute and chronic
toxicity and secondary malignancy and
infertility, in this latter oophoropexy and
sperm banking may indicate however,
there is high rate of cure.
289
Non-Hodgkin’s
(NHL)
Lymphoma
This is a lymphocyte malignancy
affecting lymph nodes unlike CLL is more
common in adults than children. Incidence
increases by progressive increase from
childhood to adulthood up to 80 years of
age.
It is almost idiopathic and the causes are
not clear, they may be viral, especially
retroviruses, drugs, chemicals and
organic solvents as benzene, along with
ionizing radiation come as culprit to
affection of the disorder.
Clinical pictures composed of painless
superficial
lymphoadenopathy, the
disorder may be presented as a widespread
disease. Constitutional symptoms may
indicate fever, weight loss, night sweats;
these are not as common as in Hodgkin’s
type of lymphoma. Cytopenia with
anemia and with neutropenia and
thrombocytopenia are variable (+/_) as if
the bone marrow fails. Abdominal and
mesenteric symptoms points to lymphoma
involvement and infiltration with these
organs and the probability of other organ
being affected. The oropharyngeal
tissues consist of 5-10% of patients with
sore throat and obstructive apnea.
Diagnosis
that are
concerned
to
hematology; are finding histological
changes in lymph nodes, cell morphology
as in type of lymphoma; these cells are
hardest
to
identify.
Using
flowcytometery can be performed and
make a solid detection and can
distinguished between lymphoma and
leukemia. 15% of patients show
malignant cells (lymphocytes) in PB
(peripheral blood). In addition 50 % of
them have small cleaved lymphoma and
70-80 % of the said patient population
shows mixed types such as follicular and
small cleaved lymphoma cells.
The condition can aggravate to a term
coined as
“lymphosarcoma
cell
leukemia”, this is the leukemic phase of
lymphoma. There also exist two main
morphological
cell
(lymphoma)
classifications;
one
is
the
well
differentiated and the other 2) is the poorly
differentiated
blastoid
cells
(lymphocytes). For further differentiation
of these cells refer to hematology/oncology
texts. Figure 8-6 is the blood film of a case
of NHL.
Fig. 8-6: shows cohesive sheets of lacunar
cells in lymphoid nodules, the nodular
sclerosis of nodule (whiter areas in the
germinal centers) is known as a “syncytial
variant”; a distinction from HL a classical
NHL. The figure indicates histochemistry
290
of lymph node. Total magnification, 4050x.
Hairy Cell leukemia (HCL)
This is a type of chronic lymphocytic
leukemia; the most predominant
characteristic of HCL is the “wispy or
hair like tail or projection” in
cytoplasm” of the mononuclear cells. This
phenomenon would be seen in the
peripheral circulation or blood and bone
marrow core biopsies. Clinical features
consist of thrombocytopenia and
hypersplenism, with recurrent infection
due to leukocytopenia can be the cause
of death in this patient’s population. The
immune system, due overwhelming
infections, may be helpless. The
populations demography includes male
are affected more than females and age
ranges 22-84 with the median of 55 years.
Hematologic lab findings are anemia with
somehow low hemoglobin, and hematocrit,
anemia
is
usually
normocytic
normochromic type and there is
leukopenia
and
monocytopenia,
9
Leukopenia is <4.0 x 10 /L. in 50-60 % of
the patients also there is pancytopenia
due to filtration of bone marrow with the
leukemic cells (Hairy’ cells) and splenic
pooling of damaged cells and therefore
their destruction may occur, and finally
there may be expansion of plasma due to
splenomegaly.
The morphology of the cells includes wispy
appearance with abundant cytoplasm with
blue-gray color. Their nuclei are round and
oval with a homogenous chromatin
pattern with fewer clumps (chromatin).
The nuclei have a lighter staining than
normal
lymphocytes;
nucleoli
are
prominent and variable in number. Bone
marrow shows hyperplasia and high
cellularity, the affected lymphocytes appear
as “fried eggs” along with infiltration of
bone marrow. The following is a peripheral
blood showing these typical cells (fried
eggs), Figure 8-7. In addition, bone
marrow aspiration is unproductive due
to infiltration of hairy cells (dry tap) and
core biopsy show hairy cells pattern with
nucleus separated by an abundant
cytoplasm. In a fine fibrillar network, the
nucleus appears as mentioned like fried egg
pattern. (Refer to the following figure).
Fig. 8-7: is the presentation of Wispy cells
(lymphocytes) known as “fried eggs” cells,
are indications of Hairy Cell Leukemia,
peripheral blood smear with total
magnification of 1000x.
The diagnostic test for hairy cell
leukemia
is
the
cytochemical
investigation of HCL in form of tartrate
resistant acid phosphatase (TRAP)
staining, majority of the neoplastic cells
are of B-lymphocyte types with some
291
possessing
T-cell
markers,
morphological studies with different
cytochemical stains are in common use in
diagnosis of hairy cell leukemia and other
leukemias. A positive TRAP staining with
conjunction to bone marrow core biopsy is
diagnostic of HCL. In this regards bone
marrow filtration and hyperplasia causes
dry tap and
reticulin fibrosis.
Immunophenotyping of lymphocytes
are definitive identification of this type of
lymphocytic leukemia.
Sézary
(SS)/Cutaneous
Lymphoma
Syndrome
T-Cell
Sézary syndrome is some forms of
cutaneous lymphoma (s) that involve skin
with affected cells are T-cell class and the
syndrome has been described by Albert
Sézary. In the T-cells there are increased
quantities
of
pathological
mucopolysaccharide.
Once
these
pathological malignant cells reach the skin
would
be
named
as
“mycoides
fungiodes”, with lymphoadentopathy.
Epidermothropism of neoplastic CD4+
T-cells for the epidermis are found
mainly in the dermis and thus in the
mycoides fungoides syndrome. The
course of the illness may take years to
develop fully.
Clinical
signs
involve
generalized
erythroderma,
lymphoadenopathy,
atypical Sézary cells in the peripheral
blood and hepatosplenomegaly.
Hematological lab findings are not helpful
but there are Sézary cells infiltration into
epidermis and peripheral blood, there is
absolute lymphocytosis, with 15% Sézary
cells. The cytoplasm is gray to blue and
these cells have large folded grooves and
nuclei are rare and indistinct the chromatin
is coarse and cytoplasm of the cells is
agranular. Immnophenotyping may be
of some utility. The following figure is
indicative of Sézary cells in peripheral blood
film (Fig. 8-8).
Therefore Sézary syndrome occurs when the
disease would be systemic and spread to
different organs.
Initially, the skin is involved and at later
events it turns to other systemic
involvement. Both conditions are
originated
as
cutaneous
T-cell
lymphoma and these cells/lymphocytes are
inclined towards such a marker as CD 4+
cells helper cells and some with CD8+
and CD4- suppersor or cytotoxic cells.
Fig.8-8: arrows indicate Sézary cells in
peripheral blood, note atypical lymphocytes
in this figure along with some creneated
292
cells/burr cells or echinocytes may be
artifactual or pathological.
Bone
marrow
infiltration
usually
happens late in the disease process.
Treatment of all cases are problematic,
however, sometimes Zolinza as a second
line drug for the condition in combination
with phototherapy and chemotherapy
may be utilized.
Monoclonal Gammapathies
Monoclonal gammapathies of unknown
significance or commonly known as benign
monoclonal gammapathy (BMG) has an
incidence of .15% in the general
population, and in 3% of people more than
70 years of age. It is characterized by large
amount of homogenous immunogobulins.
This is observed on electrophoresis (SEP)
of serum proteins and other fractionation
techniques, this is seen in light of the
absence of clinical or laboratory
evidence of plasma cell dyscrasias
(multiple myeloma and so on). There
have been some etiologic arguments of the
origin of the disorder. It seems that T-cell
lymphocytes’ function deficiency most
likely play a role in the pathologic process.
There are typical hematologic findings that
indicate the diagnosis by lab approach,
these are plasma cells increased in bone
marrow, but in about 1/3rd of the patients
have normal bone marrow. Plasma cells has
a prominent Golgi apparatus with rare
multinucleated cells may be observed.
The monoclonal gammapathies include IgG
immunoglobulins class and the amount
would be near or less than 30 g/L. All
proteins on electrophoresis are normal
except gammaglobulins.
In addition to these there is another type of
gammapathies in which the episode is
transient,
called
transient benign
monoclonal gammapathy (TBMG), with
better prognosis and the immunoglobulins
are not associated with lymphoreticular
disease, and do not normally contribute to
the disease process, this is with much less
benign forms. It may be linked to different
condition such as immunodeficiency
syndrome, or autoimmune disorders
and drug allergies. The cause as
mentioned may be the T-cell function
deficiency however infections of any kinds
as, viral, bacterial and cytomegalovirus,
mycoplasma and tumors are likely play a
crucial role in the pathogenesis and
etiology of the disorder. BMG must be
differentiated from other plasma cell
dyscrasias.
Absence of osteolysis, urinary light chain
(Bence
Jones
Protein/BJP),
lymphoprioliferative diseases with IgG
>30g/L. and marrow plasma cells be less
than 15% are deferential investigation in
the BMG diagnosis and identifications.
BMG state may be a preleukemic state,
exposing the propositus/proband to
multiple
myeloma
and
other
lymphoproliferative states.
Multiple Myeloma (MM)
This is a malignancy of plasma cells
and is engaged in production of
paraproteins and it occurs in individual
over 60 and the bone marrow content
replaces by malignant plasma cells as they
293
infiltrate the bone marrow with subsequent
bone marrow architecture destruction.
Multiple myeloma is rare under 40 years of
age. The chromosomal defect has been
detected in the stem cell and the proteins
produced are monoclonal in origin as in one
class of heavy chain and one type of light
chain (M paraprotein). The light chain
disease is about 15% common in this
population. Karyotyping shows the
chromosomes affected are t(11:14) (q13:
q32).
In
addition
immunoglobulin
phenotypes IgD and IgE are rare. Causes are
at present unknown but it is associated with
malignancies as, autoimmune diseases
such as rheumatoid arthritis, Sjögren
syndrome, etc. radiation and constant
stimulation
of
reticuloendothelial
system may play significant role in the
pathogenesis of the disease.
Clinical signs and symptoms are; the onset of the disease is between 40-70 years of
age, bone pain, bone tenderness and
deformity, lethargy and malaise due to
anemia, weight loss, night sweats with
advanced
disease,
also
abdominal
hemorrhage as in epistaxis and purpura
and petechiae, infections, renal failure
and on the exam there are pallor deformity
and pathological fracture and tenderness,
this
are
accompanied
by
hepatosplenomegaly. Excess protein may
cause
hyperviscocity
syndrome,
amyloidosis, activation of coagulation
factors, with acquired von Willebrand’s
factor (vWF) reduction unavailable for
coagulation, and lastly there will be renal
failure or disease due to large molecular
sizes and excess Bence Jones Protein (BJP).
Laboratory diagnosis and hematologic
findings are based on serum protein analysis
(elevated
proteins
with
decreased
albumin/globulin
ratio),
and
immunoelectrophoresis
of
urine
paraproteins in 98% of patients. Bone
marrow has diffuse or focal increase in
plasma cells, in this respect plasma cells are
primitive and with anemia with 60% of
patients
experience
normocytic
normochromic anemia in morphology.
In peripheral smear background rouleaux
and staining occur. The WBCs and Platelets
are usually normal there might be increase
or decrease of these cells in 20% of the
patients,
with
extensive
marrow
replacement.
ESR
(erythrocyte
sedimentation rate) is very high reaching
to more than 100. mm/hr. This test is quite
rare in other conditions with such an
increased scale and level. The end stage
disease manifests by MM cells in the
peripheral blood.
Due to immune derange there is recurrent
infections with Diplococcus pneumonia
(Streptococcus
pneumonia),
Staphylococcus
aureus
and
Escherichia coli. Serum calcium shoots
up very high and there are signs of
osteolytic bone lesions and bone pain,
serum ALP is either normal or slightly high,
with hypercalciuria causing dehydration
and further damage to the kidney such as
hyperglycosouria and aminoaciduria
along
with
other
complications.
Respectively there may be reduced serum
uric acid and renal potassium wasting and
renal loss of phosphates,
Urinalysis consists of albumin and casts
and there may be cryoglobulinemia
294
observed. The staging of disease/tumor has
three stages and the 2nd stage is the
intermediary between the first stage (low
tumor mass) and the third stage (high
tumor mass) (for staging refer to
hematology texts). Treatment monitoring
depends on Bence Jones Protein serial
measurement and serum globulins
estimations.
Other similar condition as plasma cell
leukemia is predominantly identifies by
finding plasma cells in the peripheral blood.
It is indistinct from MM. WBCs are slightly
high and with
> 20% plasma cells for this condition and
other
monoclonal gammapathies,
cytochemical
staining
and
immunophenotyping and markers
detection are useful. The image below
(Fig. 8-9) shows MM cells in bone marrow
biopsy smear.
Waldenstrom’s
Macroglobulinonemia
In this condition there is proliferation of
lymphoplasmocytoids cells as hybirds
of plasma cells and lymphocytes. This is
also a monoclonal
gammapathy
however the immunoglobulin involved are
IgM class. In this immunoglobulin
paraproteins are produced. It differs from
multiple myeloma in that there are no
osteolytic events and there is also absence
of hypercalicemia.
Patient’s demographics include affected
male more than females with occurrence in
later years of life (elderlies), as it is cited
lymphocytes and plasma cells are the
cells that produced IgM immunoglobulin.
Clinical signs and symptoms considered as
weakness and lethargy, fatigue, malaise,
hemorrhage in oronasal, recurrent
infections, dyspnea, CHF, weight loss and
with concurrent neurological symptoms
such as peripheral neuropathy and
cerebral dysfunction, signs are pallor,
hepatosplenomegaly,
lymphoadenopathy and retinal lesions.
Fig. 8-9: Bone marrow biopsy indicates the
presence of plasma cells with eccentric
nucleus and halo around the nuclear
cytoplasm and large in size. Total
magnification 1000x.
IgM is ≥ 30g/L. while excess IgM may
cause cryoglobulinemia with hyper
viscosity and thrombocytopenia.
Bone
marrow
aspirates
show
plasmocytoids lymphocytes and lesions
are not normally present. In addition there is
possibility of cold hemagglutinin syndrome
that may occur. Anemia is normocytic and
normochromic with rouleaux formation,
high ESR, this is when there is no
295
hyperviscosity syndrome
present.
There may be hyperviscosity syndrome and
must be watched and monitored
predominant atypical lymphocytes or known
as reactive lymphocytes or Downey
cells in the peripheral blood.
Infectious
Mononucleosis/
(IMN)
with
Reactive
Lymphocytes: (Wikipedia, the free
encyclopedia, et al)
IMN is usually an acute infection caused by
Epstein Bar virus (EBV) that is often
transmitted by salivary contamination of the
infected individual to the new host. This
can happen in a form when there is
exchange of saliva between these
individuals by drinking of the same glass or
cup of drinks or by kissing (also it is known
as kissing disease). The virus can
occasionally be transmitted by blood
transfusion.
Clinical signs include malaise, fever and
lymphoadenopathy
(lymph
node
enlargement), fatigue, sweating and
orolaryngitis (sore throat), also headache
and nausea are all constitutional signs
and symptoms.
Peak incidence reaches in North America
in adolescence and young adults and
between the ages of 17-25 and commonly
occurs in young children however it may
occur in over the age 40 rarely.
It is generally accepted and reported to be a
self-limit disease and the symptoms
recovers after a short time: there may be no
need of specific treatment.
Hematology lab findings consist of 50%
lymphocytes with at least 10% of that are
atypical lymphocytes. Fig. 8-10, shows
Fig. 8-10: shows blood smear of two
atypical lymphocytes also known as reactive
lymphocytes/or
downy
cells.
Total
magnification is 1000x.
The atypical lymphocytes at first
discovered looked like monocyte that is why
it is called mononucleosis. The monotest is
a latex agglutination test with specificities
for the antigen, or antibody depends on the
test format, this is a heterophil antibody
test for identification of IMN. Other lab
findings are, neutropenia, leukopenia
occur during the first week of infection and
WBC might reach to 30. x 10 9 /L. False
positive serology test may occur during
infections or co-infections with syphilis,
rheumatic arthritis with transient
antinuclear
antibody
positivity.
Lymphocytes are of T-cell suppressor or
cytotoxic CD8+ phenotypes. T-cells
suppress
the
growth
of
B-cells
(lymphocytes) by attempting to control the
viral infectivity in the B-lymphocytes.
Thrombocytosis is seen in cases of IMN.
Differentiation must be made between
Toxoplasma gondii infection and
296
cytomegalovirus
infectivity.
The
reactive lymphocytes are called as well as
downey
cells,
virocytes,
or
transformed lymphocytes however, only
reactive or atypical lymphocyte terms
should be used for IMN.
.
Lastly reactive lymphocytes are T- or Blymphocytes in origin and once provoked
they transform into blastoid like cells.
These cells have fine euchromatin and
develop nucleoli; the cytoplasm RNA and
endoplasmic reticulum are prominent.
Morphology of these cells they are large
lymphocytes with cytoplasm flows toward
adjacent cells (Fig. 8-10) and may have
peripheral basophilia. The cytoplasmic
contour and color is like, “Dutch skirt”
known as; ”Ballerina”.
There may be few azurophilic granules
and some vacuolization. The nucleus may
have central folds, or be round or oval
with fine chromatin as mentioned along with
clumped euchromatin. The following
examples are infections that causes
formation of reactive lymphocytosis; IMN,
infectious
lymphocytosis,
cytomegalovirus
infection,
viral
infection such as mumps, German
measles (rubella), chicken pox
(varicella), brucellosis, pertussis and viral
pneumonia and infectious hepatitis are
lest in row to count to causing reactive
lymphocytosis.
The next section is coagulation studies
and coagulation lab, which is the last
chapter of this hematology part. Focus will
be in coagulation mechanics, factors and
other ancillary mechanisms and elements
playing critical role in coagulopathy with
related disorders.
Chapter Nine (9): (Rodak et
al. & Marie Scully et al.)
Thrombosis and coagulation
Instrumentation
and
automation
Please refer to the Chapter one (1) under the
same topic.
Component of Coagulation
and Hemostasis
Basic Concepts
C
oagulation and hemostasis
system includes blood factors and
platelet and vessels walls to
interplay in the clotting process
and hemostasis so that the bleeding and
injury to the vessels are repaired and
patented.
Primary Hemostasis
When platelets and blood factors and vessel
walls come into action the blood clot forms,
in this scenario collagen with tissue
factors in the vessels walls when injured
activates platelets and clotting factors
(such as F VII, F II or FIX) to patent and
repair the injured site. Blood is normally a
fluid. Once the injury sustained, blood with
adherence factors occurs and subsequent
to repaired vessel, the bleedings will be
controlled. Basement membrane factors
as collagen and tissue factor exposes and
then platelets accumulates in the injured
site by forming a platelet plug and
297
containing the site; this process initiates by
collagen and ends by fibrinolysis through
the action of inhibitors of hemostasis.
Coagulation normally consists of three
aspects of hemostasis; these are 1) blood
vessel, 2) platelets and 3) clotting factors.
Starting with blood vessel when the
injury is not there, the vessel is intact, once
the damage has been sustained collagen with
other tissue factors replenish and releases.
Thrombocytes are responsible for
maintaining epithelial cells lining in the
vascular lamina. Once the injury happens,
the vessel contracts due to platelet releases
of thromboxane A2 and its natural
response to injury,
Thromboxane A2 is a strong vasoconstrictor
to cause vessel constriction. Thrombin
affects this constriction process through
prostaglandin released by endothelial
cells, which last almost one minutes
(constriction). There are some vascular
disorders that cause somehow provocation
of coagulation. These disorders are
divided into two categories as acquired and
inherited/congenital. Clinical signs of which
include,
easy
bruising,
bleeding
subcutaneously and also as ecchymosis
and petechiae. To diagnose this vessel
disorders one may exclude the plasma
factors deficiency (as von Willebrand’s
factor) and other qualitative and
quantitative platelet dysfunctions and
defects.
Inherited defects of blood vessels are
collagen abnormality. Elastic fibers and
fibronectin deficiency are the inherited
vascular diseases. Two of these disorders
will be covered in here, one is HHT (or
hereditary hemorrhagic telangiectasia) and
the
other
one
is
Ehlers
Donlos
syndrome.
HHT is a genetic autosomal dominant trait
leads to abnormal blood vessel in
epidermis,
mucocutaneous
tissue/membranes and sometime in brain,
liver and lungs.
The defect is located at peculiar places as
subendothelial section of vessel wall
tissue with bleeding in form of epistaxis and
GI bleeding; the bleeding may occur in
form of petechiae, ecchymosis on finger
tips, lips, tongue and cheeks. In addition the
loss of iron can occur during chronic or
acute bleedings, thus the patient is becoming
anemic.
The other disorder is Ehlers Donlos
Syndrome, which is a rare condition as
HHT is an autosomal dominant genetic
trait, in this vessel wall connective tissue is
affected and defective (collagen). The
bleeding may be mild to severe and there is
anemia.
The acquired condition of vascular
endothelium consists of the following
disorders:



Simple bruising (or easy bruising),
this is due to vessel fragility the
condition is benign and may occur
during child bearing age.
Senile purpura, due to old ages and is
due to connective tissue loss
(collagen). This is a benign
condition.
Scurvy, lack of vitamin C causing
loss of connective tissue, the
tourniquet test is abnormal. Full
recovery by vitamin C administration
occurs.
298
 Cushing’s disease. This is an
atrophy of connective tissue.
 Allergic
purpura
including
Henoch Schönlein syndrome. It
is a systemic inflammation of
vascular bed. It may be a
hypersensitivity reaction.
 Autoimmune vascular diseases. Such
as
Systemic
Lupus
Erythematosus (SLE). Antigen
antibody complex tissue damage.
Laboratory tests for vessel integrity
include PT, PTT, tourniquet test, bleeding
time (BT) and tissue biopsy.
Platelets maintain the endothelium of the
vessels and participate in the clotting by
adherence to the surfaces and vessel
subendothelial cells, these releases of
vasoactive amines and its granular contents;
as in activation and aggregation and
retraction.
Adherence is the attaching or adhesion of
platelets in area of high shear rate in the
microvasculature. The platelet factor 9 or
PF Ib-IX facilitates the attachment to the
cell membrane receptors also initially
von Willebrand factor (vWF) participate
in the adhesion, this is released by platelets
primarily.
The second properties of platelets as
mentioned is the activation in this process,
upon adhesion to the collagen surface
(phospholipid surface) in subendothelial
membrane, platelets will be activated and
receptors on the collagen such as
glycoprotein PF Ib- IIIa, which is an
integrin molecule releases and the
reaction take place from the platelets by the
formation of alpha and dense granules.
Subsequently, thromboxane A2 releases
from thrombocytes releases and then
release of platelet
activation protein or factor (PAF) from
the platelets follows, this is a potent PAF
agonist and vasoconstrictor. This will
complete the task in activation process.
In aggregation, after production of
thromboxane A2 and APF and ADP (with
serotonin), which are platelet agonists to the
phospholipid surface with other platelet
factor releases from platelets and thrombin
from coagulation cascade forms so further
aggregation commences by primarily the
presence of fibrinogen which completes
the coagulation process by forming the
platelet plug(s)
subsequently
the
The last sequence is the retraction; this
stabilizes the plug and there is microtubule
formation and cytoskeleton compacts
and adds to the plug stability.
Collagen activates release of phosphatase
then manufacturing of phosphatase causes
production of archidonic acid and then
endoperoxidase and thromboxane A2 and
lastly ADP release from platelets, which
these ultimately affects adhesion and
aggregation and retraction.
Platelet disorders consist of qualitative
and quantitative types. Clinical signs of
platelet dysfunction and defect include
epistaxis, subcutaneous bleeding, oozing,
petechiae, bleeding in mucous membranes
as in dental extraction, etc.
In short, platelets are produced from
pluripotential stem cell. These are
although differentiate to all myeloid,
erythroid and megakaryocytic progenitors,
such as megakaryoblast, subsequent to
formation of megakaryoblasts the cell
undergoes nuclear division accordingly and
finally
the
normal
platelets
or
299
thrombocytes lodges in the circulation.
Kinetically thrombocytes are stimulated to
produce more platelets by the action of
thrombopoietin, which can be made by
kidney. The healthy platelets live (life span)
is about 7-10 days in the circulation and then
die where collected and filtered by the
spleen. Naturally as it is happens to other
cell
lineage,
platelets
undergo
maturation and this brings about smaller
sizes. When the cells are young there are
large and immature, after nuclear division
the cells pinches off from cytoplasm and a
new platelets is formed. Normally increased
platelet MPV is seen when there are
immature and with reduced parameter
(decreased in size) when mature.
Thrombocytosis
and
thrombocytopenia are the increased and
decreased spectrum in numbers of platelets
in
the
circulation
respectively.
Thrombocytopenia and anemia and other
fragmented cells (RBC) are typically
associated with DIC, TTP and HUS in blood
smear.
Thrombocytopenia may be acquired or
inherited and there are always tendency to
bleed when platelets are low in number.
In acquired form it could be due to
aplastic anemia, myelofibrosis, TTP,
HUS or DIC, in these productions of platelet
is secondary to the illness itself. Also sepsis
can cause anemia and thrombocytopenia due
to their toxins. Other infections as AIDS,
infectious mononucleosis, can cause
platelet destruction, immune destruction
such
as
immune
complexes
and
autoimmunity can eventually destroy
platelets
with
their
antibodies
or
autoantibodies/cytotoxic antibodies. Drug
induced hemolytic anemia can ensue to
thrombocytopenia, as penicillin or
cephalosporin. In addition to these we
may have HIT (heparin induced
thrombocytopenia) syndrome in which
the heparin induction causes antibody
complex between heparin and the immune
system. The complex activates complement
system leading to platelet destruction and
reduction in number. ITP (Idiopathic
thrombocytopenic purpura) also, is a
cause of thrombocytopenia. As we have
several other examples by which there is a
decreased in the number of these cells which
are essential for life. Respectively DIC
(disseminated
intravascular
coagulation),
lymphoproliferative
disorders are other examples may lead to
thrombocytopenia. Hypersplenism either
dilutional thrombocytopenia or post
transfusion can be the cause as well. There
are certain cerebrovascular diseases such as
cerebral-aneurism and hemangiomas
can also induce platelet reduction due to
platelets endothelial interaction and
subsequent adhesion and their destruction.
The inherited forms are, Bernard
Soulier Syndrome, TAR and May Hegglin anomaly. In the first disorder
there are megathrombocytes, mild
thrombocytopenia and bleeding time
markedly decreased also there will be
decreased
ristocetin
induced
aggregation. This does not correct by
mixing test or VWF. In May-Hegglin there
is reduced platelet numbers and finally,
TAR is known as thrombocytopenia
absent radius which is a type of
thrombocytopenic condition. It is
genetically induced. There are artifactual
(spurious) thrombocytopenias, as EDTA,
300
this can induce reduction of platelets and
other
causes
of
aretifactual
thrombocytopenias consists of platelet
satellitism, and platelet agglutinins
along with giant platelets.
Qualitative platelets disorders are
characterized by excessive bleeding time
which reflects platelet adhesion, activation,
aggregation and medication. This does not
differentiate between which function is
defective but can indicate whether it is
hereditary or acquired.
Inherited disorders include BernardSoulier syndrome, von Wellibrand
disease and Glanzmann’s thrombasthenia,
with decreased platelet aggregation with
ADP, epinephrine or collagen in the latter.
The
platelets
are
normally
morphologically are normal with normal
number. The last disorder is the storage
pool disease which is a type of disorder in
which defective release of platelet granules
are evidence that causes reduce aggregation.
Acquired type of quantitative platelet
defects are several types, as in drugs,
hypergammaglobulinemia, dexran or
FDPs (may coat the platelets and preventing
adhesion/ as in fibrin degradation
products),
Quantitative platelet defect consists of
production, destruction, sequestration, and
dilutional defects. At first, there is
production; either reduced or ineffective;
in reduced we have examples as infiltration
as in tumors, and in aplasia as in
chemically induced or congenital. The
ineffective production may be in form of
megaloblastic
anemia
and
myelodysplasia. In destruction defects we
have immune induced and consumptive
destruction; examples of immune are
autoantibodies as in TTP and alloantibodies
such as NAIT, and HIT. The examples of
the latter are as DIC, HUS and TTP and
mechanical.
Also there are sequestration as in
splenomegaly, and in hemodilution;
examples
are
excessive
platelet
concentrates
or
whole
blood
transfusion and at last could be real defects
or spurious. Table 9-2 & 9-1 indicate the
quantitative and qualitative defects or
disorders of platelets.
Table
9-1:
Quantitative
platelet disorders
Quantitative disorders
1. Immune thrombocytopenic purpura
(ITP)
2. Thrombotic microangiopathies
3. Post-transfusion purpura (PTP)
4. Heparin-induced thrombocytopenia
(HIT)
5. Disseminated
intravascular
coagulation (DIC)
Table
9-2:
Qualitative
Platelet Disorders
Qualitative Platelet Disorders
1. Drug induced (e.g. Aminotryptyline &
Asprin)
2. Uremia (mechanism of induction not
understood)
3. Liver Disease (Acute and Chronic)
4. Acquired von Wellibrand Disease (vWD)
5. Paraneoplastic Platelet Dysfunction (e.g.
myelodysplastic and myeloproliferative)
301
Blood Factors & Relevant
Coagulation Pathways
Secondary Hemostasis
The coagulation factors are a series of
chemicals and zymogens that trigger
coagulation namely as clotting. The purpose
of clotting is to prevent blood loss from
injurious tissue or from the blood vessel.
These factors in combination with blood
vessel tissue factors and plate let
interplay a system that eventually stops
bleeding. There are 13 blood factors and few
substances that participate in
the
coagulation cascade, which is a system
of triggering in a flow-through manner of
different activation factors and electrolytes
such as calcium ion to accumulate the
bleeding factors at the site off injury. The
blood factors are known also as,
“Procoagulant”. These factors are namely
mentioned with their properties in the
following table (Table 9-3).
Table
9-3:
Factors.
Coagulation
Factor number/Descriptive name
form
Active
I Fibrinogen
subunit
II Prothrombin
protease
III
Tissue
Receptor/cofactor*
V Labile factor
VII Proconvertin
protease
VIII Antihaemophilic factor
IX Christmas factor
protease
X Stuart–Prower factor
protease
Fibrin
XI Plasma thromboplastin antecedent
protease
XII Hageman (contact) factor
protease
XIII
Fibrin-stabilizing
Transglutaminase
Prekallikrein/ (Fletcher factor)
protease
HMWK
(Fitzgerald
Cofactor*
HMWK, high molecular weight kininogen.
* Active without proteolytic modification.
Serine
Serine
factor
Serine
factor)
All of the forementioned factors are
manufactured in the liver except Factors
von Willebrand and F VIII. These made
in the endothelium of the vascular beds
and megakaryocytes/platelets.
For initial activation of the coagulation
pathways and fibrinogen transformation to
fibrin with intermediary steps, presence of
contact factors such as XII, XI and HMWK
is essential. This group does not consume in
the coagulation process and they follow
upon contact to activation of XII converts to
XII a then preKallikrein to Kallikrein,
which in turn the HMWK will be converted
to Bradykinins. This latter system
subsequently
initiates
complement
activation and plasminogen. There three
kinds of Pathways under the coagulation
cascade:
Serine
factor
Cofactor
Serine
Cofactor
Serine
Serine



Intrinsic Pathway
Extrinsic Pathway
Common Pathway.
In intrinsic the coagulation initiates by
preKallikeirin and high molecular
weight kininogen or HMWK. Then it
follows the flow of cascade in form of
302
activation and conversion and mentioned
above. This is depicted in the figure 9-1.
that are residents in tissues such as
collagen, sneak venom etc. The tissue
factor activates factor III to covert and
participate in conversion of factor VII →
VIII a , at this time factor VIII activated
(subordinate “a” after the Romance number
for that factor, means the molecule is
activated) by the participation of ca++ ion
changes activates factor X, which is the
kick start of common pathway (Fig. 9-1).
Next is the activation of the last common
pathway from activation of factor X to V
then II and fibrinogen to fibrin by the cross
linked, fibrin will be stable by the action of
XIII.
Cofactors in the play of the cascade are
Fig. 9-1: Coagulation cascade and intrinsic,
extrinsic and common pathways.
In this pathway ca++ ion plays crucial role
on conversion of one factor to another
especially in conversion of factor IX → IX a
and VIII → VIII a and from VIII a to X a
series of complex factors and elements play
role such as PF (platelet factors) + VIII a +
IX a until these activation reach at the
and common
pathways in here, after activation of
common pathway coagulation precedes
toward thrombus formation/clot by the
action of fibrinogen to fibrin in common
pathway.
junction
of
intrinsic
In extrinsic factor as can be seen in figure
9-1, it starts by activation of tissue factors
coagulation checking points in the
coagulation, which correct and make the
process progress and adjusting the process
e.g. such as V a that controls and
accelerate X a
and the same for factor
VIII a .
Role
of
Procoagulants
Plasma
The procoagulants (coagulation factors)
are serine protease and glycoproteins,
which acts by cleaving downstream proteins.
Some of these factors are not serine protease
but transglutaminase, such as factor VIII
and V. These coagulants circulate as active
zymogens. And as mentioned they activate
at each step of the process from the
preceding factor and generation of the next
procoagulant in the cascade to end
formation of thrombosis, which is
indispensable for hemostasis and the
cessation of the bleeding.
303
The
Inhibitors
The formation of clotting is important for
the
purpose
of
cessation
of
hemorrhage/bleeding diathesis but
however once these clotting process goes on
and on without physiological checks and
monitoring can be morbid and ultimately
fatal. The substance that enforce the
checking processes are called coagulation
inhibitors, these as antithrombin III,
protein C [inhibits plasminogen activator
inhibitor (PAI)]and its cofactor protein S
(this is made in the liver, vitamin K
dependence and affects by the liver
diseases).
The formation of clot may be prevented by
other means in therapeutic approach as these
are
called
anticoagulants
and
thrombolytic
substances;
examples
include heparin, heparin sulfate and EDTA,
other anticoagulant such as drugs; ticlid,
warfarin are used therapeutically to prevent
cardiac or other organ infarction upon their
administration,
others
are
used
diagnostically as citrates and oxalates for
the purpose of lab testing. But the concern
for here is the coagulation inhibitors cited
earlier, not the anticoagulant. Natural
coagulation inhibitors include also
heparin as mentioned, it actually is a
cofactor that accelerates the action of
antithrombin III and with increased APTT
formation time. The contact system can
effectively be controlled by the action of
α1-antitrypsin and α2 macroglolbulin.
As the name conveys the antithrombin
inactivates thrombin and factor X a as well
as factors XII, XI and IX.
following
9-3 shows
coagulation inhibitor interplays.
Table
figure
the
24.2
Fig. 9-3: The action (inactivation) of Protein
antithrombin III, Protein C and its cofactor
protein S.
Related Coagulopathies and
Other Bleeding Disorders
Congenital
Hereditary bleeding disorders merely
consists
of
Hemophilia A and
Hemophilia B, which are reflected in
certain coagulation factor deficiency as
for hemophilia A is the deficiency of factor
VIII and for Hemophilia B is factor IX
deficiency or Christmas factor. .Other
hereditary
coagulation
factor
deficiencies exist that can be detected by
laboratory
means. In hemophilia A,
bleeding time may be prolonged but PT is
normal, PTT is increased and TCT normal
and its incidence occur in as low as .01 %.
This is a sex-linked recessive disorder.
For hemophilia B, PT and PTT with TCT
are the same as factor VIII deficiency. This
304
is a sex-linked recessive disease with and
incidence rate of 0.002% which is quite
low. Bleeding in F VIII deficiency is severe
with severity depending on bleeding
according to the level of factor, this is
similar to F IX deficiency.
For von
Wellibrand factor deficiency the
incidence is similar to F VIII deficiency
with PT normal, PTT prolonged and TCT
(thrombin clotting time) normal. Table
9-4 shows coagulation factor deficiencies.
Table 9-4: Shows some major
coagulation
factor
deficiencies.
Defects
Inheritan PT
ce
PTT
TCT
Bleedin Inciden
g
ce
IX:
hemophil SR
ia A
Normal Increas Normal Severe 0.002%
ed
*
VIII:
hemophil SR
ia B
Normal Increas Normal Severe 0.01%
ed
*
VIII:
Von
AD
Willebra
nd
Reports
Normal Increas Normal Severe vary
ed
*
I:
AR
Fibrinoge
n
Increas Increas Increas Severe Very
ed
ed
ed
*
rare
SR: sex-linked recessive
AD: Autosomal Dominant
AR: Autosomal recessive
Acquired
Bleeding
Disorders
these are more frequent in comparison to
the hereditary forms, usually they manifest
in form of destruction of blood factors
by interfering substances, and also
increased consumption. There is no family
history or clinical history of the bleeding
diathesis in the patient. Examples of these
types of defective coagulation disorders are
liver diseases; this is because all blood
factors are manufactured in the liver except
vWF and factor VIII. Any liver pathology
can
eventually
result
to
factor
deficiencies. Factor VIII is normal and
may be increased in hepatopathology as
this fact can differentiate between DIC and
liver pathology.
In mild liver disease, factor VII may be
deficient due to short half-life and PT may
rise. If the intrinsic and extrinsic factors
be affected and deficient, aPTT and PT
respectively will increase. In moderate to
severe liver disease the common
pathway’s blood factors may elevate the
APTT. TCT (thrombin clotting factor) will
increase if fibrinogen is affected by the
liver disorders, its impairment and decrease
synthesis affects an increase in TCT.
Other conditions that affects factor
deficiency in liver pathology are, decrease
in antithrombin III and antiplasmin in
liver disease may affect the blood factors.
These
cause
increase
thrombin
neutralization and increased plasmin
generation. Also, HDN (hemorrhagic
disease of the newborn due to immature
liver) and Vitamin K deficiency can
result into vitamin K dependent factors
deficiency. This decrease in vitamin K
factor may be from altered gut flora as
well. Likely as in different intestinal
disorders, such as biliary obstruction in
GI (gasterointestinal) disorders can ensue
to vitamin K dependent factors (as II,
VII, and IX and X) deficiency. Massive
transfusion can induce factor deficiency
as well along with DIC. And finally
305
thrombolytic drug therapy such as
warfarin and/or coumarin can lead to
factor deficiency.
Etiology of DIC could be intravascular
platelet aggregation, tissue injury, and
activation of coagulation factors; the
pathophysiology of DIC include thrombin
formation due to lost compensatory
mechanism leading to a perpetuating
cycle of events as mentioned above, then
plasmin is generated due to tPA (tissue
plasminogen activator) and factor XII,
fibrin is generated as well. Once
plasminogen digest fibrin, FDP (fibrin
degradation products) is formed. The
last cause of DIC is primary
fibrinogenolysis.
Plasmin
digests
fibrinogen. In this regards, neoplasm can
cause fibrinogenolysis as well by
secreting plasmin-like substances therefore
complicating the condition to DIC.
Testing
Regiments/Protocols/Panels;
aPTT (Activated Partial
Thromboplastin Time)
PT, aPTT and TCT are clot based
procoagulant screen. Activated partial
thromboplastin time as mentioned briefly
before is useful for checking/monitoring
the integrity of the intrinsic pathway
coagulation factors and monitor the
unfractionated heparin for thrombolytic
therapy; also it is useful in detecting lupus
anticoagulants. It detects congenital and
acquired factor XIII and VII deficiency; all
factors in the intrinsic pathway can be
easily estimated including factors in the
common pathway (as II, V, IX and X plus
fibrinogen).The
procedure
involves
application of PPP (plasma poor
proteins) plasma, activator such as
kaolin (or Celite) and phospholipid
(previously called activated thromboplastin),
the previous two are supplied in the
diagnostic
reagents
forwarded
by
manufacturer. The regent is pre-warmed
to 37° C and subsequently the mixture with
PPP (plasma) is incubated at the same
temperature for certain time (e.g. 3 minutes).
At the end of the incubation there the
calcium ion is forcibly added by a pipette to
the mixture and a timer runs, upon
observation of coagulation or clotting of
the plasma the timer stops the unit will be
read and records the level of activity time.
This can be reads elecromechanically or
photo-optically
[Refer
to
the
instrumentation chapter (1)]. The test should
be run in duplicate for quality control
purposes and must be within 10%
difference.
PT (Prothrombin Time)
The purpose of measuring PT is to monitor
and check the integrity of extrinsic
pathway of coagulation such as factor VII,
and common pathway’s blood factors such
as factors, II, V and IX, including
fibrinogen as they are detected as well and
it can measure the efficacy of thrombolytic
drug such as warfarin and coumadin
(coumarin). In the procedure the test
components
are
thromboplastin,
phospholipid and chloride reagent are prewarmed to 37°C and PPP aliquot is
incubate separately in another reaction
vessel for almost 3 minutes (according to
manufacturer) and once the incubation
306
finishes, PPP is forcibly added to the reagent
and elapsed time is recorded or measured
the activity time. The unit is in activity
time scale.
testing protocol for monitoring the course of
DIC and relevant pathways derange. Figure
9-2 indicates fibrinogen reference curve.
Fibrinogen estimation/Assay
(FA)
This test measures the integrity of the
fibrinogen molecule and its functional
level and some parts of the common
pathway. The following figure is schematic
presentation of fibrinogen molecule (Fig. 91).
Fig. 9-2: showing fibrinogen reference
curve.
FDP (Fibrinogen Degradation
Products) and D-dimer
Practically during the coagulation, fibrin
polymers bind/cross linked by factor XIII a
Fig. 9-1: schematic representation of
microstructure of fibrinogen molecule.
The procedure and methodology uses the
PPP, which is added to bovine thrombin and
catalyzing the formation of fibrinogen to
fibrin. PPP in this test is diluted 1:10 times
and a control such as Owen’s buffer can
be run along the test, then bovine
thrombin is added to the incubated PPP
(for 3 minutes) and the time scale will be
recorded for the activity. We can use ELISA
(Enzyme
Linked Immunosorbent
Assay) for quantitation of the fibrinogen
assay. This test fibrinogen assay is an ideal
and binds to plasma plasminogen and
tissue plasminogen activator (TPA).
Subsequently several hours later the TPA
bound to plasminogen to form plasmin
thus bound plasmin cleaves fibrin and
results into formation of FDP and other
products of fibrin degradation such as
D, E, X and Y and lastly D-dimer. These
FDPs are original fibrinogen domains. And
these are especially FDP and D-dimer is
essential to testing fibrinogen as in DIC
and other thrombotic episodes and
coagulopathies where fibrinogen and its
products are sought.
Testing the D-dimer for FDPs are commonly
performed by automated and semi-
307
automated and immunoassays systems,
the immunoassays methodology in vitro
systems make use of latex particles
agglutination with D-dimer products and
antigen, in an antigen antibody reaction.
Reptilase is an enzyme obtained from
the venom of the sneak Bothrops atrox.
This is used along with thrombin to measure
hypofibrinogenemia
or
dysfibrinogenemia in especially in
patients with heparin anticoagulants. The
enzyme cleaves fibrinogen to fibrin
polymers fibrinopeptide A in contrast to
thrombin which does it by producing
fibrinopeptide A & B. This test indicates
fibrinogen function in plasma. Atroxin
is available by Trinity Bioteck Inc.®.
Platelet Aggregometry with
Rich
and
Poor
Plasma/Platelet Function Test
One of the
functions
of platelets is
aggregation. Aggregometry is an
instrument or an in vitro test to estimate the
degree of aggregation and adhesion of
platelets to the inert surfaces. This
measures platelet functions in terms of
their α-granules, dense bodies and
lysosomes. The automated test is design
to produce curves and charts of the
aggregation process. Lumiaggregometry
or optical-aggregomery is advanced
instrumentations in coagulation lab for
platelet
aggregation
and
adhesion
functional assays. In this procedure PRP
(Platelet rich plasma) and be used by PPP
(Platelet poor plasma) adjustment and an
aggregometer, which is a photometer
detects the changes in the density of the
PRP. In this procedure cuvette is filled
with PRP adjusted by PPP with a bar in it
(warmed at 37°C) and then stirred while an
agonist is added while time elapse, the PRP
sticks/adhere to the bar and the density
changes. The first reading is “zero
transmittance”. The actual run of the test
will measure the aggregation in time as
recoded. The advance aggregometer have
computers attaché that quantitates with
graphs, histograms and algorithm.
Bleeding Time (BT)
The test is simply used to estimate the
aggregation and adhesion of the
platelets. This is done by piercing the volar
surface of the forearm by a standard
lancet with standard length and depth of the
puncture (5 mm width and 1 mm deep).
This is done under pressure of a cuff
attached to arm (40 mm Hg.) then
subsequent to puncture the bleeding is
blotted by filter paper until stopped and
the elapsed time is recorded. The lancet is
standard such as Simplate® by Organon
Teknika Corp. or by Surgicutt® by
International Technidyne Corp. in
Durham N.C., and Edison in N.J.
respectively. At the end of the procedure,
laboratory scientist should follow standard
protocol
for
blood
extraction/phlebotomy to prevent injury
to the patient and self. Blotting with filter
paper should be done every 30 seconds.
308
Then when the bleeding stops the time will
be recorded as said.
Monitoring Thrombolytic and
AnticoagulationTherapy
This is used to prevent thrombosis and clot
formation in the patients predispose to them.
Unfractionated
Heparin
(UFH) and Heparin
UFH is of porcine mucosa and its
catalytic and anticoagulant actions are
measured indirectly. It inactivates serine
protease II (thrombin) in coagulation
pathways with conformational change in
its structure that exposes anticoagulant site
through steric changes.
It supports thrombin- antithrombin
reaction through bridging mechanism. Its
(heparin) activity after administration can be
detected within minutes. Heparin therapy is
monitored through the APTT hemostatic
assay and it is initially (heparin) given to
the patient before coumarin or warfarin
commences. Once the patient is stable with
heparin coumarin is given. Sometimes
heparin is jammed in the muscles after
operation to prevent clot formation. After
initial does coumarin is given orally. It
naturally bind antithrombin III (AT III)
thus make acceleration of thrombin
inactivation, activated factor X and IX. It
major effect naturally is on thrombin.
The major action potential of thrombin is
aggregation, adhesion and activation of
thrombocytes and other factors in extrinsic
pathway.
The
monitoring
heparin
administration is through measurement of its
activity. If too much bolus of heparin is
given it may result in hemorrhage and if too
low can result in effective thrombolysis.
Thus the dose and the bolus should be
adjusted by the laboratory monitoring of
PTT. The therapeutic range is about 1.52.5 (level of normal). If too much is given,
test PTT and stop infusion and if too less is
administered or it is below the range a bolus
given and test PTT again so that in this way
monitoring is accomplished with measuring
the PTT. As mentioned low molecular
weight heparin (LMWH) is available
(named as fractionated), with much smaller
the size in comparison to heparin. Because
action of LMWH is stronger on factor X a
than on thrombin, it anticoagulation power
should be monitored by factor X assay
rather than PTT as PTT can only monitor
thrombin (factors in intrinsic pathway). It
can be administered once a day due to its
longer action potential and predictable
clinical response. (Kocmarek et al).
Antiplatelets also play a significant role
in
thrombolysis
and
thrombolytic
therapy. They have different modes of
action. Some block irreversibly platelet
enzymes as cyclooxygenase this further
affects platelet life span, the block of this
enzyme prevent the release of ADP needed
for aggregation and this mode can be used
in atherosclerotic and cardiac infarction.
Examples
are
ticlid™
and
dipyridamole™.
Coumarin
Once the initial anticoagulation with heparin
instituted we follow the thrombolytic
therapy with coumarin. This is monitored
through PT estimation of the extrinsic
pathway for factor VII and X + V and other
309
common pathway factors. To standardized
the procedure and reporting system of
testing PTs between laboratories, there have
been an equation set forward by the World
Health Organization (WHO) so that there be
uniformity between every lab results for PT
it is called International Sensitivity Index
(ISI and INR or International Normalization
Ratio); the equation is as following;
Eq. 9-1
INR = [patient’s thrombin time
(PT) /means of PT reference interval] ISI
As you can see from the formula, the ISI is
the international sensitivity index as it
is the measure of prolongation of PT or
responsiveness of PT compared to
international reference preparations, it is
usually equal to one (ISI =1). High ISI is
when there is a loss of precision or a narrow
therapeutic index and low ISI is a wider
therapeutic window, which are a more
precise monitoring and a close correlation
to ISI. The normal range of PT is 2.0-3.0. It
is a prophylactic measure in high risk
surgeries and cardiac status, thrombosis,
pulmonary emboli and so on. Above the
therapeutic range can predispose patient to
hemorrhage and below that can be
ineffective.
Antibodies to Coagulation
Factors
I only mention here the factor VIII
antibodies in these patients with severe
hemophilia develop these kinds of
antibodies. These people are likely
transfused with massive amount of
transfusion (multiple), and hemorrhage is
severe with APTT increases indicating
neutralization of VIII in intrinsic
pathway. Other tests as PT and TCT are
normal. Antibodies to factors IX and V are
similar to VIII with some variance.
References
1) KC4 Delta product information, Bay,
County Wicklow, Ireland, Trinity Biotech,
PLc, 2001. 2) Thomas LC., Sochynsky CL:
Multiple measuring mode of coagulation
instruments, Clin Hemost Rev. 1999.
3) ACL 8000 Operator’s manual Brea, CA:
Instrumentation Laboratory/Beckman Colter
Inc., 2003.
4) Garza D. Becan-Mcbride K: Phlebotomy
hand book, 7th ed. upper Saddle River, N.J.
Pearson Prentice Hall, Appleton and Lange,
2005.
5) Stewart CE, Koepke JA: Basic Quality
Assurance Practices for clinical laboratories.
Philadelphia: JB, Lippincott 1987.
6) McCall RE, Tankersle CM: phlebotomy
essentials, 3rd ed. Philadelphia; Lippincott
Williams & Wilkins, 2003.
7)
Dyssypris EN, Sawyer ST:
Erythropoiesis. In Greer JP, Foerster J,
Lukens et al (ed): Wintrobe’s clinical
hematology.
11th
ed.
Philadelphia,
Lippincott Williams and Wilkins, 2004.
8) Glader B: Anemia: General consideration.
In Greer JP, Foerster J, Luken’s JN,et al(ed)
Wintrobe’s clinical hematology. 11th ed.
Philadelphia, Lippincott Williams and
Wilkins, 2004.
9) Quirt I et al, Hematology, Review notes,
2002.
10) Hereditary spherocytosis, elliptocytosis,
and other red cell membrane disorders.
1
Blood
Rev.
2013 May 9. pii: S0268-960X(13)00019-2.
doi: 10.1016/j.blre.2013.04.003.
310
11) The membrane defect in hereditary
stomatocytosis. Baillieres Clin Haematol.
1993 Jun;6(2):371-99.
12) ©2001 - 2013 by American Association
for Clinical Chemistry, G6PDase deficiency,
http://labtestsonline.org/understanding/analy
tes/g6pd/tab/test
13) NA Lachant, CR Zerez, J Barredo, DW
Lee, SM Savely and KR Tanaka: multiple
phosphotransferases? Hereditary erythrocyte
adenylate kinase deficiency: a defect of
Blood, Vol77, No 12 (June 15), 1991: pp
2774-278.
14) Sadler JE, Moake JL, Miyata T, George
JN. Recent advances in thrombotic
thrombocytopenic purpura. Hematology Am
Soc Hematol Educ Program. 2004:407-23.
15) Marie Scully,1 Beverley J. Hunt,2
Sylvia Benjamin,3 Ri Liesner,4 Peter Rose,5
Flora Peyvandi,6 Betty Cheung,7 Samuel J.
Machin8 and on behalf of British Committee
for Standards in Haematology Guidelines
on the diagnosis and management of
thrombotic thrombocytopenic purpura and
other
thromboticmicroangiopathies,
doi:10.1111/j.1365-2141.2012.09167.x.
16)
Pediatric
Hemolytic
Uremic
Syndrome Clinical Presentation Author:
Robert S Gillespie, MD, MPH; Chief Editor:
Craig
B
Langman,
MD,
http://emedicine.medscape.com/article/9820
25-clinical.
17) Screening tests of disseminated
intravascular coagulation: guidelines for
rapid and specific laboratory diagnosis. Yu
M, Nardella A, Pechet L. Crit Care Med.
2000 Jun; 28(6):1777-80.
18) Ferri: Ferri's Clinical Advisor 2013, 1st
ed./disseminated intravascular coagulopathy,
Singh MM - Clin Liver Dis - 01-FEB-2011;
15(1): 69-87/ hematologic and oncologic
diseases of the liver, Coagulation response
in sepsis, Levi M - Clin Chest Med December, 2008; 29(4); 627-642.
19) Chapter 50 traumatic cardiac hemolytic
anemia
Williams
Hematology,
http://medtextfree.wordpress.com/2011/12/3
0/chapter-50-traumatic-cardiac-hemolyticanemia/
20) Gardner, Malcolm J. et al. Genome
sequence of the human malaria parasite
plasmodium falciparum. Nature (419) 03
October 2002. 498-511.
21)DPDx, laboratory identifiaction of
parasite of public interest.P. falciparum, ,
http://www.dpd.cdc.gov/dpdx/HTMLPDF_F
iles/Pfalciparum_benchaidV2.pdf
22)DPDX Laboratory identification of
parasites of public health concern, P. vivax
http://www.dpd.cdc.gov/dpdx/HTML/PDF_
Files/Pvivax_benchaidV2.pdf/
23)
http://www.dpd.cdc.gov/dpdx/HTML/PDF_
Files/Povale_benchaidV2.pdf , by David J.
Weatherall, et al., Treatment of Malaria, doi:
10.1182/asheducation-2002.1.35
ASH
Education Book January 1, 2002 vol. 2002
no. 1 35-57.
24) Chronic Myelogenous Leukemia,
Author: Emmanuel C Besa, MD; Chief
Editor: Koyamangalath Krishnan, MD,
FRCP,
FACP,
http://emedicine.medscape.com/article/1994
25-overview.
25) Myelodysplastic Syndromes, Attilio
Orazi, MD, FRCPath1 and Magdalena B.
Czader,
MD,
PhD.,
doi:
10.1309/AJCPRCXX4R0YHKWV (2009)
311
American Journal of Clinical Pathology,
132, 290-305.
26)
Infectious
mononucleosis,
http://en.wikipedia.org/wiki/Infectious_mon
onucleosis, Wikipedia, the free encyclopedia
27) Bernadette F. Rodak et al, Hematology,
clincial principle and application, 3rd ed.,
2007, Sunders publication.
28) Mary L Turgeon, Clinical Laboratory
Science, the basic and routine techniques, 5th
ed. 2007, Mosby Elsiveir Publication.
29) Kocmarek B et al. refresher course in
hematology, CSMLS, Sep. 1999.
312