Lab Protocols
BLOCK 1
Membrane Potential, model
Hemocoagulation tests
Blood groups
Hemoglobinometry, Hematocrit, S.E.
Blood Cells Counting
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physiology.lf1.cuni.cz
Labs aim: Explore biology in context through brain and hands
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LAB: Membrane Potential
Computational Model (Metaneuron)
WHAT
1. Membrane potentials definition:
o PubMed MeSH: Ratio of inside versus outside concentration of potassium,
sodium, chloride and other ions in diffusible tissues or cells. Also called
transmembrane and resting potentials, they are measured by recording
electrophysiologic responses in voltage-dependent ionic channels of (e.g.)
nerve, muscle and blood cells as well as artificial membranes.
o Easy words: MP is a difference in charges across biological (e.g. cellular)
membrane
AIM of the lab
• Understand core principles of membrane potential generation, its maintenance and
changes
• Help to explain the effect of certain drugs interfering with MP (e.g. anesthetics)
• See the mechanisms influencing MP through interactive simulation
REQUIRED KNOWLEDGE (major topics)
• Cellular membrane, selective permeability, concentration gradients (Na, K, Ca, Cl),
resting membrane potential, action potential, pos-synaptic potential, equilibrium
potential (Nernst eq.), ionic channels, channel gating, all or none law
TASKS - overview
1. Resting membrane potential
1.1. Basics
2. Action potential
2.1. Threshold stimulus, summation
3. Effect of changes in conductivity – channel blockers (Na, K)
4. Effect of changes in extracellular ionic concentrations (Na, K)
WHY …
1. … do cells maintain membrane potental?
a. Homeostasis
b. Transmembrane processes
c. Energy store
d. Signal processing/conduction
2. … do we perform the lab
a. Clinical relevance:
i. Demonstrate effect of ionic dysbalances
ii. Drugs action explanation - Some common drugs are channel-blockers
(e.g. anesthetics, antiarrhytmics, Ca antagonists…)
b. Physiology – principle of
i. Neural activity, receptor sensing,
ii. Muscle control
iii. Cardiac rhythm generation and propagation
iv. Many others
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HOW does that work?
1. potential implies unequal distribution of ions and charges, thus it must be powered
and maintained in quite sophisticated manner
2. membrane potential (of biological membranes, e.g. cells) depends chiefly on:
o concentration (chemical) gradient across the membrane for different
ions
force that makes ions to move across the membrane. Otherwise no
ions (charges) flux would occur thus no potential would be
generated.
o membrane selective permeability for ions
though forced, the ions must be allowed to cross the membrane.
Otherwise no charge can be generated
if all the ions could pass through at the same time, all charges would
equilibrate since concentration
2
1.
o
3
what is equilibrium potential?
if ion follows its concentration (chemical –CH) gradient, it generates
electrical gradient (by movement of its charge).
The more ions move, the greater the developed charge is
Electrical (E) and chemical (CH) gradients are opposite direction
Once electrical gradient (force) equals chemical one, ionic flow stops
(more accurately ie equal both directions. THIS IS EQUILIBRIUM
POTENTIAL Simply we can say:
E = CH
This is Nernst equation - in principle ☺
E is electrical force (determined by potential)
CH is chemical force determined by conc. gradient which depends on
the (logarithm of the) ratio between concentration outside (OUT) and
inside (IN) the cell, co:
E = k ⋅ log
k stands for some constants (
con[OUT ]
con[ IN ]
R ⋅T
).
z⋅F
Thus electrical (equilibrium) potential for given ion depends just on
its concentration gradient.
Surprisingly, concentration almost does not change !!!!
Each ion, if allowed (i.e. if having channel open) tends to reach its
equilibrium.
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SETUP
1. Simualtion software – Metaneuron (google for it)
a. simulates membrane processes based on Hodgkin-Huxley model (Nobel Prize
1963).
b. Intuitive interface allowing control of ionic concentration and permeabilities
(conductances) and more
c. The graph to show the results
2. The screenshot:
TASKS - detailed
1. Resting membrane potential
1.1. Basics – read the graph, notice RMP, EK, ENa
1.2. explore the effect of changes in ECF ions concentrations
1.2.1. change the values in a meaningful way
1.2.2. explain how the finding is different for each ion and why?
1.3. see the effect of changed conductivity
2. Action potential
2.1. basics
2.1.1. read the graph, notice stimulus trace, MP trace, conductivities (turn them
on)
2.1.2. find threshold stimulus
2.1.3. explore summation of two stimuli
2.2. Effect of changes in conductivity - demonstration of effects of channel blockers
(Na, K)
2.3. Effect of changes in extracellular ionic concentrations (Na, K)
MORE QUESTIONS:
Q1: what is the effect of increasing amplitude of a stimulus?
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Blood Coagulation tests
BRIEFLY:
• Hemocoagulation is one of (three major) hemostatic (stopping bleeding) mechanisms.
• During coagulation blood becomes solid - principally due to the transformation of
plasma protein fibrinogen into polymerized insoluble fibrin.
• Blood Coagulation Tests determine the speed of coagulation. They are necessary
before starting surgery and during anticoagulation therapy (e.g. of thrombosis)
AIM of the lab
• Understand coagulation and anticoagulation mechanisms
• Know coagulation tests and usage
• Know anticoagulation procedures
REQUIRED KNOWLEDGE (major topics)
Hemostasis, hemocoagulation, clotting factors (principle), coagulation pathways
(principles), vitamin K, anticoagulation, Heparin, thrombus, thrombosis, plasma
calcium
TASKS
1. Anticoagulation tests
1.1. INR
1.2. APTT
1.3. capillary fragility test
WHAT
• Blood Coagulation:
o PubMed: The process of the interaction of BLOOD COAGULATION
FACTORS that results in an insoluble FIBRIN clot. (MeSH tree#
o Wikipedia: The coagulation of blood is a complex process during which
blood forms solid clots. It is an important part of hemostasis (the cessation of
blood loss from a damaged vessel) whereby a damaged blood vessel wall is
covered by a fibrin clot to stop hemorrhage and aid repair of the damaged
vessel. Disorders in coagulation can lead to increased hemorrhage and/or
thrombosis and embolism.
WHY
• Why do we perform the lab:
o Understand common coagulation tests
o Know why and when to test coagulation
• Surgery. Why?
• Delivery. Why?
• Anticoagulation therapy. Why?
• …
o Discuss and see anticoagulation methods.
HOW does that work?
1. COAGULATION
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o
o
o
Hemocoagulation has one endpoint - formation of fibrin polymer.
It has two distinct starting points:
• Stimulation from inside a vessel. Ttypically endothelial damage and
exposition of blood to collagen (intrinsinc pathway)
• Stimulation from outside a vessel. Contact of blood with tissue
substance Tissue thromboplastin –TT (extrinsinc pathway)
Between the starting and end points, a cascade of proteolytic reactions
occurs. The actors of the reactions are called clotting factors. Most factors are
plasma proteins (others represent: Ca++ and phospholipids)
2. ANTICOAGULATION
o Physiology
• At any time, blood contains everything necessary for clotting
• Thus it might clot even in vessels, that could be fatal.
• Thus spontaneous unwanted coagulation must be prevented by
effective and reliable mechanisms, such as:
• Blood flow – reduces the chance of interaction of factors
• Intact endothelium – prevents from activation of factors
• Anticoagulation factors (endogenous)– inhibit ongoing
coagulation process. E.g. Heparin – Antithrombin, …
o Pharmacology
• Anticoagulation is an important therapeutical strategy in some
diseases where thrombosis (intravascular coagulation) appears. E.g.:
stroke, coronary heart disease, deep venous thrombosis, implants, etc.
short
• Major anticoagulation procedures:
• Heparin – with Antithrombin (AT) deactivates some factors
(mainly thrombin and factor X) already activated during
coagulation.
• Anti-vitamin K (AVK) e.g. coumarin (Warfarin, Macumar)
interfere with the production of certain coagulation factors
(II, VII, IX, X) Consequently factors are less effective and
coagulation times are prolonged.
• “de-calcification” Ca++ (ionized, free) is necessary for many
steps of coagulation cascade. If Ca++ is unavailable, clotting
does not occur. The easiest way to let Ca++ interact with
substances that form insoluble molecules/complexes. E.g.:
o EDTA, Oxalate, citrate, EGTA
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3. COAGULATION TESTS
o There are many tests, just two most common are mentioned.
o The ones we perform (APTT, INR) evaluate the time of coagulation after
specific activation (i.e. “how long it takes to clot”)
o The simplest test would be just to wait until blood spontaneously clots in a
test tube (cca 5-10 minutes)
o “Our” tests are just slightly smarter (more sophisticted)
• Use just plasma. Why?
• Use specific activator:
Activator Test
name
TT
Quick
INR
PT
•
•
extrinsinc
Normal
result
15 s
0.9 – 1.1
APTT
intrinsinc
40 – 50 s
TT – tissue Thromboplastin
INR - international normalized ratio
INR =
•
pathway
Quick ( patient )
Quick (norm)
PT – partial thromboplastin
o PT = diluted thromboplastin + clay
o “pretends” endothelial damage
o also called KK = kefalin + kaolin
SETUP:
Test tubes, water-bath, little hook to pull the fiber, stopwatch (on your phone;-) Really
so simple ☺
Procedure
1. Get ready
a. Chck that all reagents are available and heated to 37 °C. Why?
b. Chck that test tubes are clean. Preheat them. Why?
c. Make sure to use proper pipettes and fresh tips
2. Add reagents (100 µL of each, but always fresh tips!)
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a. Decalcified plasma. Why decalcified?
b. Activator (TT or PT)
3. Start timing once CaCl2 was added. Still keep in the bath. Why?
4. Use hook to pull the fiber
5. Stop timing once the first fiber is seen
TASKS:
• APTT and Quick (INR) test for provided plasma(s) – marked P1, P2, …
Perform as many tests (equal number of APTT and INR) as possible from
given plasma.
RESULTS:
Sample
APTT [s]
P1
P1
P1
P2
P2
P2
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Quick [s]
Rem.
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Blood Groups
AIM of the lab
• Understand blood group evaluation
• Understand importance of blood group testing
REQUIRED KNOWLEDGE
• Blood, blood groups, antigen, antibody, antibody class, agglutination, cross-match,
fetal erythroblastosis,
WHAT?
Blood group is antigen determination of erythrocytes
There are many antigens on each ery,
thus there are many systems of blood groups.
o Most important are: AB0, Rhesus, MN, …
WHY?
… is it important to know the group?
1. because of potentially fatal complications in case of incompatibility, typically:
a. during transfusion and organ transplantations
b. during certain pregnancies (of some Rh-neg. mothers)
Knowledge of groups can completely prevent the complications
HOW?
… does that work?
Erythrocytes have antigens on their surface
Only sometimes plasma contains antibodies against non-self group. Why?
system
group
Antigen on RBC
Antibody in plasma
ABO
A
A
Anti B
AB0
B
B
Anti A
ABO
AB
A and B
None of above
AB0
0
None of above
Anti A and Anti B
Rhesus
POS
D
usually none
Rhesus
NEG
D
usually none
MN
M
M
usually none
MN
N
N
usually none
…
usually none
The problem is if erythrocytes and antibodies against them (e.g. A and anti-A) meet in
bloodstream. Why? RBC – AB form complexes, stimulate immune system, and
systemic shock can develop. This can be fatal in tens of minutes. Also hemolysis,
renal failure and other complications occur.
HOW? … do we test the blood group?
By mixing blood with specific monoclonal antibodies against RBC group antigens
For AB0 group we need:
o 2 drops of blood (e.g. from disinfected!!!!! finger)
o 2 types of antibodies (Anti-A and Anti-B)
o Something to work on and mix with
Agglutination (clumping) occurs if RBCs are mixed with specific antibodies targeted
against them.
AGGLUTINATION IS NOT CLOTTING! ☺
WHICH GROUP IT IS?
o In the picture below showing the procedure, try to estimate which group it is
(step 6)! Write your result.
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LAB: Hemoglobinometry, Hematocrit,
Sedimentation rate
AIM of the lab
• See and understand basic blood test
• See the different aspects of red blood cells evaluation
• Basics of oxygen transportation
REQUIRED KNOWLEDGE
• Blood, plasma, blood composition, RBCs, hemoglobin, methemoglobin, oxygenation
and oxidation of hemoglobin, spectrophotometry, sample, blank, suspension stability
Hemoglobinometry
WHAT
Estimation of hemoglobin concentration in blood.
Physiologically cca: women 150 g/L, men 160 g/L (+/- 10 g/L)
→ Tells us about O2 transporting capacity of blood
TASKS
•
•
In three blood samples that are provided perform hemoglobin concentration
measurement (at least three tests per each sample per class)
Calculate MCHC once other necessary results are available. Which?
WHY …
… do we measure it?
i. Amount of hemoglobin belongs among elementary tests of body’s
homeostasis. It crucially determines O2 transporting capacity and thus
O2 delivery to the tissues.
ii. Pathologically, amount of hemoglobin can change, typically in
anemias. Hb content can change independently of erythrocyte count
and size (reflected by hematocrit). Knowledge of both hemoglobin
content, number of RBCs and their size (volume) can help identify
ethiology (reason) of anemia and thus suggest suitable treatment
iii. Critically low hemoglobin concentration will indicate, that
transfusion might be necessary. More on transfusion e.g.:
www.guideline.gov: Indications for and techniques of red cell
transfusion
HOW
1. Colorimetry (visual assessment)
o In principle, amount of Hb is proportional to colour of blood
o Thus it is (theoretically) possible just to look and see ☺. Colorimetry is
almost that simple ☺
o Practically this method would be inaccurate, mainly due to:
Colur assessment Why?
Oxygenation of hemoglobin. Why?
o Thus the procedure contains
Hb oxidation by HCl
Blood dilution
Comparison to standard colour (available in the set)
2. Photometry
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a. The principle remains the same as for colorimetry but (spectro)photometer is
used for evaluation. Thus subjective assessment is excluded
Procedure:
i. Fill 10 cuvettes with Drabkin solution (3 ml). Why ten?
1. contains K-cyanate (Why?)
2. it is toxic (Why?)
ii. Take blood from samples 20 µl and transfer to cuvettes
1. How would you obtain such amount of blood?
iii. mix properly!!! (use pipette or glass stick)
iv. incubate 10 mins
v. mix again if sedimentation occurs (why?)
vi. perform photometry using Vernier photometer
1. 565 nm
2. use drabkin sol for blank
vii. calculate hemoglobin concentration
viii. what info is needed
3. gasometry (not performed)
a. Principle:
i. blood is fully saturated by O2 (how?)
ii. blood is fully desaturated
iii. amount of released O2 during desaturation is measured
RESULTS:
Sample
1
2
3
test 1
extinction
test 2
test 3
avg
HB conc
[g/l]
QUESTIONS:
1. What could be the sources major errors during the procedure?
2. Which of the methods would NOT be suitable in methemoglobinamemia?
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Hematocrit (HCT)
WHAT
1.
medline:
The (relative) volume of packed RED BLOOD CELLS in a blood specimen. The
volume is measured by centrifugation in a tube with graduated markings, or with
automated blood cell counters. It is an indicator of erythrocyte status in disease. For
example, ANEMIA shows a low value; POLYCYTHEMIA, a high value.
2. easy words:
Percentage of volume of erythrocytes out of total volume of blood.
Physiologically cca 45% (+/- 5 %)
→ Tells us about blood fluidity (viscosity) and more
Use A B C from the picture to fill the equation: HCT = –––––
TASKS
In three blood samples that are provided estimate hematocrit
(Perform at least three tests per each sample - per class)
WHY …
•
… do we check hematocrit? this all?
i. Generally, these belong among elementary tests of body’s
homeostasis. Most in-door patients would have it done on admission.
ii. Specifically HCT, Hb and RBCs count allow us to distinguish among
different anemias.
iii. Proportion between RBCs and plasma determines blood fluidity.
Lower hematocrit means better fluidity and thus sometimes better
tissue oxygen delivery.
HOW?
1. Microhematrocrit (just from “a drop “of blood)
o Due to higher specific gravity of cells these tend to separate from plasma.
o Spontaneous separation would be very slow if possible.
o Thus centrifugation is used to speed the separation
o Also, blood must be anticoagulated (Why?)
o The anticoagulation must not interfere with the measurement.
How could it interfere?
• …
• …
• …
Which anticoagulant would be suitable?
Procedure:
i. Find the samples 1, 2 and 3 and shake properly (why?)
ii. For each sample fill at least 3 capillaries with blood
1. Fill about 2/3 of capillary (who not much more / less?)
2. How?
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iii. Seal one end of capillaries in flame. Which end?
iv. Sealed capillaries place into the stand. Notice the positions!
v. Ask lab assistant to perform centrifugation.
NOTE: the capillaries will be returned in the same places as when
handed to the assistant.
vi. Estimate hematocrit either using a ruler or the device in the lab.
RESULTS
Sample
1
2
3
test 1
Hematocrit [%]
test 2
test 3
avg
HB
[g/L]
MCHC
[%]
Calculated values for RBCs
•
•
RBCs parameters such as count, Hb and HCT are somehow independent (RBC can be
big but have low amount of Hb)
In order to see whether the parametersch change proportionally or not some
calculated values are used. They allow for “one-look” assessment of two params:
o
MCV = mean corpuscular volume = avg. volume of one erythrocyte
MCV =
o
MCH = mean corpuscular hemoglobin = avg. Hb content in one ery.
MCH =
o
HCT
, norm cca 100 fl/ery
RBCcount
Hb
, norm cca 30 pg/ery
RBCcount
MCHC = mean corpuscular hemoglobin concentration = avg. Hb
concentration in erythrocytes
MCHC =
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Hb
,
HCT
norm cca 35%
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Blood sedimentation
(or Ery. sedimentation rate (ESR), or FW - Farheus Westergreen method)
WHAT
1. Medline: Measurement of rate of settling of erythrocytes in anticoagulated blood.
2. Medline Plus: ESR (erythrocyte sedimentation rate) is a nonspecific screening test for
various diseases. This 1-hour test measures the distance (in millimeters) that red
blood cells settle in unclotted blood toward the bottom of a specially marked test
tube.
Physiologically cca 3-12 mm/h
WHY?
… do erythrocytes sediment?
… the test is performed?
• The erythrocyte sedimentation rate (ESR) can be used to monitor (progress
of) inflammatory or malignant disease. Although it is a screening, nonspecific
test (cannot be used to diagnose a specific disorder), it is useful in detecting
and monitoring tuberculosis, tissue necrosis, rheumatologic disorders, or an
otherwise unsuspected disease in which symptoms are vague or physical
findings are minimal.
HOW?
… is ESR influenced?
• By many factors,
o Elevated fibrinogen: helps formation of stacked erythrocytes called
rolleaux that are relatively heavier and settle fastest.
o Erythrocytes: number (anemia/polycytemia) size
(macrocytosis/microcytosis, spherocytosis)
o
… is Sedimentation measured?
• Blood withdrawn (How much? How?)
• Diluted and anticoagulated (anticoagulant prefilled in test-tube/syringe)
o Which anticoagulant will be suitable?
• The sedimentation tube filled
• After one hour evaluate
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LAB: Blood Cells Counting
WHAT
1. Medline (MeSH ID D00177)
Blood cells count: The number of LEUKOCYTES and ERYTHROCYTES per unit
volume in a sample of venous BLOOD.
A complete blood count (CBC): also includes measurement of the HEMOGLOBIN;
HEMATOCRIT; and ERYTHROCYTE INDICES.
2. easy words:
Number of cells per volume of blood (not plasma! ;-)
REQUIRED KNOWLEDGE:
Composition of blood, Erythrocytes (number, volume, composition, function),
Leucocytes (types, differential count, composition, funcrion), Plasma, Osmolarity,
more;-)
PRACTICAL TASKS
In three blood samples that are provided cont RBCs and WBCs
(each student counts both RBCs and WBCs)
WHY …
•
•
•
… do we count blood cells?
i. Generally, these also belong among elementary tests of body’s
homeostasis. Many in-door patients would have it done on
admission.
ii. Helps to diagnose many diseases, monitor their progress
… do we count RBCs?
i. Specifically RBCs count (with HCT, Hb) allows us to discover and
distinguish different anemias.
Anemias are quite common, often unidentified, may accompany
chronical diseases and may significantly affect prognosis
ii. To help identify other hematological diseases (e.g. polycytaemia)
… do we count WBCs
i. WBC count (and differential count) is non-specific or semi-specific
marker of inflammatory processes such as
1. Infection (bacterial, viral, parazital)
2. Allergy
3. Autoimmunity
4. Shock
5. neoplasms
ii. WBC count (and differential count) may be a specific sign of
hematological neoplasms.
HOW?
1. Counting chamber (e.g. Hemocytometer , Buerker chamber) and microscope
o Just a drop of blood is sufficient (Why only drop?)
o Blood is diluted (Why?), anticoagulated (if necessary)
o Blood cells stained and fixed if necessary (which cells need staining?)
o Cells are counted visually under the microscope
o The volume in which the cells are counted needs to be known. Thus, specific
slide called counting chamber or hemocytometer is used. (see the picture)
Tiny grid is engraved into the slide and is seen under microscope only. Grid
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o
determines area, space between slide and cover slip determines height. Area
times height gives volume.
final blood count needs to be calculated (from # of cells, dilution and volue)
count =
number _ of _ cells
× dilution
volume
2. Automated machine (Flow cytometer) [NOT AVAILABLE IN THE LAB]
a. Blood sample is processed in machine (diluted, stained, etc.)
b. Sample is forced through a tiny capillary where cells travel one – by – one.
(What is the diameter of such capillary?)
c. Each cell passing thru the capillary is detected and counted. (optically or
electrically).
d. Thus cells count can be calculated from: known volume of sample, dilution
of blood,and passed cells.
Notes:
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TASK
Estimatate blood cels count (both RBCs and WBCs) in given samples (Hemocytometer)
Each working group (students at one microscope) is supposed to process one sample.
Record and DISCUSS the results.
Procedure smmary:
Cells
Solution
RBCs
Heym’s sol.
- cells fixation
- hypertonic
- no staining
WBCs
Turck’s sol.
- stains WBCs nuclei
- hemolyzes all cells
(Why?)
dilution
200x
Area for counting
Small squares
1/400 mm2
Count in
80 squares
20x
Large squares
1/25 mm2
50 squares
.STEP-BY-STEP Procedure (Hemocytometer):
i. Find the blood samples 1, 2 and 3 and shake properly (why?)
ii. Transfer 25 µl of blood into pre-filled vile with respective working solution. (Heym
solu. for RBCs, Turck solu. for WBCs).
Use automatic pipette to transfer blood/.
iii. Shake in the shaker for 10 minutes. (why?)
iv. Apply one drop of processed sample at the edge of cover slip as shown in the picture.
One edge for RBCs, opposite one for WBCs.
v. Allow for sedimentation for 5 minutes (why???)
vi. Count cells under the microscope
1. Recommended magnification 1:100
2. Count RBCs above 80 smallest squares
3. Cont WBCs above 50 large squares
ii. From data obtained calculate numbers of cells in original blood samples. The
calculation must include:
1. Number of cells obtained
2. Volume in which counting occurred
3. Dilution of the sample
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RESULTS
Team
Blood
(microscope) sample ID
1
RBC count
[x106/µl]
WBC count
[x103/µl]
interpretation
2
3
4
5
6
7
8
SUMMARY:
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