1 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
Red Blood Cell Count and
Haemoglobin Determination
Prepared by;
Hakami, Hana A
Viewed by;
Dr.Naseem Siddiqui
2 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
The Blood
The blood consists of a pale yellow fluid called plasma in which one
finds mixed:
a. Red blood cells called erythrocytes (RBC)
b. White blood cells called leucocytes (WBC)
c. Platelets called thrombocytes (PLT)
The blood remains in motion continuously flowing through arteries
carrying oxygenated blood from heart to various parts of the body by
the pumping action of heart. Blood also flows through veins carrying
deoxygenated blood (dark red) from different parts of body to heart and
to lungs. The arteries divide into smaller blood vessels called capillaries
which supply blood to the various tissues. The capillaries then rejoin to
form veins.
Red blood cells (Erthrocytes)
Shape:
Round cells filled with haemoglobin
When seen from their side they look like biconcave.
Do not contain nuclei
Number: In general is 5,000,000 RBCs per cu mm of blood (cu=cubic)
3 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
Hemocytometry
Improved Neubauer Hemacytometer
Hemacytometry means the use of the hemacytometer counting
chamber to count blood cells (to count WBC, RBC, and Platelets, as will
as, counting cells in other body fluids, e.g. CSF and semen analysis).
Hemacytometer is a counting chamber device made of heavy glass
with strict specifications,
it
resemble
hemacytometer have a special glass slide
a glass
slide. Also, the
manufactured to strict
specifications, it is very thick and non-flexible. There are many types of
hemacytometers,
in
which
they
differ
in
rulings,
but
the
commonest and the easiest one is the Improved Neubauer Chamber,
bright line type. When viewing the hemacytometer from the top
4 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
(figure below), it has 2 raised platforms surrounded by depressions on
three sides, each raised platform has a ruled counting area marked off
by precise lines etched into the glass. The raised areas and depression
form H letter, this “H” has two coverglass supports on each side which
are exactly 0.1 mm higher than the raised platforms. The coverglass is
placed on top of the coverglass supports so it covers both ruled areas.
The depth between the bottom of the ruled area and the coverglass
is exactly 0.1 mm. So, coverglass function is to confines the fluid and
regulates the depth of the fluid to be applied.
PLATFORM WITH
RULED AREA
H-SHAPED DEPRESSION
AL AZHAR
COVERGLASS
COUNTING
COUNTING
CHAMBER
CHAMBER
COVERGLASS SUPPORTS
Figure1: Top view of the hemacytometer
COVERGLASS
COVERGLASS
SUPPORT
CENTER PLATFORM
Figure 2: Coverglass position on the hemacytometer
5 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
Hemacytometer Counting Areas
Hemacytometer has 2
composed of etched area
identical
consists
ruled
of
counting areas, each
a large
square, with a
diameter of 3 mm. This large square is subdivided to 9 small squares,
each
with
a diameter
of
1 mm. So, each 1mm square can
accommodate a volume of 1 mm x 1mm x 0.1 mm (depth) = 0.1
mm³ (cubic millimeter). WBC cells are counted in the entire 9
squares.
The central square is further subdivided into 25 smaller
squares
each
with
a
diameter
of
0.2
mm, so the volume
accommodated within this square will be 0.2 mm x 0.2 mm x 0.1
mm(depth) = 0.004 mm³
(cubic millimeter). Red blood cells are
counted in the large central square (1 from 9 squares), in which only the
four corner squares and the center square (look figure 3 , in which “R”
denotes for red blood cells). Platelets are counted in the entire large
center squares (the 25 small squares).
Figure 3 - Red Blood Cells Counting Area
6 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
Using The Hemacytometer
1- Position a clean, dust free, coverslip so it covers the ruled
counting areas of a clean hemacytometer.
2- Fill the hemacytometer with the fluid containing cells to be
counted, by touching the tip of the capillary tube or
micropipette tip to the point where the coverslip and raised
platform meet on one side, the fluid will drawn under the
coverslip and over the counting area by capillary action, this
requires about 10 l.
3- Repeat on opposite side of the chamber.
4- The chamber must not be overfilled or underfilled, if accurate
results are needed!.
5- Place the hemacytometer on the microscope stage, so one of
the ruled counting areas is aligned directly above the light
source (condenser); rotate the low power objective (x10) into
place; using the coarse focus knob, move the low power
objective very near the coverslip; rotate coarse focus knob to
increase the distance between the low power objective (X10)
and the hemacytometer until etched/ruled lines come into
focus; all nine large squares must be viewable; very carefully,
rotate the high power objective (X40) into place, with the aid
of fine focus knob, adjust the focus until the etched lines come
into focus, you can now carefully move the hemacytometer by
using the mechanical stage, so that the ruled area on the other
side can be viewed.
7 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
The Counting Pattern
Either left to right or right to left counting pattern can be used (
fig.4); but with the insurance that each cell is counted only once, to
accomplish this, cells that touch the right boundary lines or the
bottom boundary lines are not counted, because they will be counted
with the other squares (look figure). After cells are counted on one side,
the hemacytometer is moved and the cells are counted on the other
side. Results for each side are recorded, then are totaled and the
average is calculateed.
Begin
Figure 4 Counting Pattern
Cells
touching
right, and
bottom
boundaries
are not
counted!
Figure 5: Cells touching the right and bottom boundaries are not counted
8 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
Calculating The Cell Counts
1st.
The total number of cells per cubic millimeter of sample can be
calculated from:
1. The average number of cells counted.
2. The ruled areas contain an exact volume of diluted sample.
3. The dilution of the sample.
2nd. The hemacytometer Formula:
N x D (mm) x DF
= c/mm3
A (mm2 )
Where:
C/mm³ = number of cells/ mm³
N= Total number of cells counted in the counting chamber.
D (mm) = Depth factor in mm
DF
= Dilution Factor
A (mm²) = Area counted (mm²)
1. The dilution factor is determined by the blood dilution
made by you as a laboratory technologist..
2. The depth factor is always = 10 (1/0.1).
3. The area counted will vary for each type of cell count and is
calculated using the dimensions of the ruled area.
Comments
Although some specialists still considers hemacytometry is the
standard method of cell counting, but its C.V. is high, which indicates
impression and sometimes inaccuracy, especially when counting red
blood cells . In cases of leukopenia (low WBC count, below normal
ranges ), still hemacytometry the method of choice for cell counting.
9 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
10 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
RBC Manual Count
Principle:
A specified volume of blood is diluted with a specified volume of
isotonic fluid. This isotonic diluting fluid will not lyse RBC’s, and will
facilitate counting with the aid of the hemacytometer.
Sample:
EDTA anticoagulated whole venous blood.
Diluting Fluid:
Isotonic saline:0.85% sodium chloride (NaCl) in distilled water.
OR
10 ml of 40% Formalin made up to 1 liter with 32 g/l Trisodium Citrate.
OR
6.25 g of crystalline Sodium Sulfate. Transfer to 100 cc
volumetric flask, and add approximately 50 cc distilled
water. Then add 16.7 ml of Glacial Acetic Acid. Finally add
distilled water up to the mark.
Apparatus and Equipment:
1- Micropipette – 20 l is the desired volume.
2- Serological Pipette, 5ml.
3- Handy Tally counter.
4- Improved Neubauer counting chamber with the cover
slips.
5- Conventional light microscope.
Procedure
11 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
In clean aseptic work area :
1- Pipette 4.0 ml of diluting fluid into a tube.
2- Pipette 20 l of will mixed anticoagulated whole blood to
the tube.
3- Mix continuously for 2-3 minutes.
4- Load the cleaned hemacytometer.
5- Place the hemacytometer on the microscope stage, lower
the condenser.
6- Focus with x10 objective lens on the large central
square. This square is ruled into 25 small squares, each
of which is further divided into 16 smaller squares, of the
25 squares, only the four corner squares, and one
middle square are used to count RBC’s.
7- Switch to x40 objective lens, and start counting in the five
designated squares.
Calculations:
Total RBC count =
N x Dilution Factor x Depth Factor
Area Counted (mm2 )
= c/mm3
Where:
N= Total number of red cells counted in the counting chamber.
Dil. Factor = 0.02 : 4 = 2 : 400 = 1:200, Dilution Factor = 200.
Depth Factor = 10
Area Counted = 0.2 x 0.2 x 5 = 0.2 mm²
So,
Total RBC count =
N x 200 x 10
0.2
= N x 10.000 c/mm3
Results :
……………………………………………………………………………
12 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
Hemoglobin ( Hb ) Determination
Introduction :
Hemoglobin (also spelled haemoglobin
and abbreviated Hb or Hgb) is the ironcontaining oxygen-transport metalloprotein in
the red blood cells of vertebrates, and the
tissues of some invertebrates.
A
substance
contained
within
erythrocytes (red blood cells) that is responsible for their color and their
remarkably high oxygen-carrying capacity. Hemoglobin is the most
efficient oxygen-carrier known. Oxyhemoglobin is scarlet in color;
reduced hemoglobin is of a purplish color.
In mammals, the protein makes up about 97% of the red blood
cell's dry content, and around 35% of the total content (including water).
Hemoglobin transports oxygen from the lungs or gills to the rest of
the body (i.e. the tissues) where it releases the oxygen for cell use.
Hemoglobin is also found in outside red blood cells and their
progenitor lines. Other cells that contain hemoglobin include the A9
dopaminergic neurons in the substantia nigra, macrophages, alveolar
cells, and mesangial cells in the kidney. In these tissues, hemoglobin has
a non-oxygen carrying function as an antioxidant and a regulator of iron
metabolism.
In most humans, the hemoglobin molecule
is an assembly of four globular protein subunits.
Each subunit is composed of a protein chain
tightly associated with a non-protein heme
group. Each protein chain arranges into a set of
alpha-helix structural segments connected
together in a globin fold arrangement, so called because this
arrangement is the same folding motif used in other heme/globin
13 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
proteins such as myoglobin. This folding pattern contains a pocket which
strongly binds the heme group.
Myoglobin is an oxygen-binding protein found in the muscle
tissue of vertebrates in general and in almost all mammals.
Myoglobin (abbreviated Mb) is a single-chain globular protein of
153 or 154 amino acids, containing a haem (iron-containing porphyrin)
prosthetic group in the center around which the remaining apoprotein
folds.
Methods for hemoglobin determination are many and varied.
The most widely used automated method is the cyanmethemoglobin
method.
To perform this method, blood is mixed with Drabkin’s
solution, a solution that contains ferricyanide and cyanide.
The ferricyanide oxidizes the iron in the hemoglobin, thereby
changing hemoglobin to methemoglobin. Methemoglobin then unites
with the cyanide to form cyanmethemoglobin. Cyanmethemoglobin
produces a color which is measured in a
colorimeter,
spectrophotometer, or automated instrument. The color relates to the
concentration of hemoglobin in the blood.
Manual methods for determining blood
hemoglobin include the Haden-Hausse and SahliHellige methods. In both methods, blood is mixed with
dilute hydrochloric acid. This process hemolyzes the red
cells, disrupting the integrity of the red cells’
membrane and causing the release of hemoglobin,
which, in turn, is converted to a brownish-colored
solution of acid hematin. The acid hematin solution
is then compared with a color standard.
Hemoglobinometer is a laboratory instrument for colorimetric
determination of the hemoglobin content of the blood; abbreviated Hbmeter.
14 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
 Sahli h. — is based on the separation of globin from hemoglobin
by treatment with hydrochloric acid to produce acid hematin
which is measured by colorimetry.
 Spencer h. — measures oxyhemoglobin by light absorption using a
green filter.
The normal values for hemoglobin determinations are as follows:
Hemoglobin Normal Values
Grams per 100 ml blood
Men
14 to 17
Women
12.5 to 15
Newborn infants
17 to 23
Determination of
Hemoglobinometer:
Hemoglobin
Percent
97 to 124
83 to 110
97 to 138
concentration
with
Spencer
Purpose :
To determine your Hemoglobin concentration by use Spencer
Hemoglobinometer.
Materials :
Alcohol swab , Lancet , Gloves , Spencer Hemoglobinometer , Saponin
coated applicator , Chamber slide , cover slide , clip , Sharp container
Procedure :
In clean aseptic work area :
1. Sterilize your finger with alcohol swab and prick
it by lancet .
2. Withdraw blood by place enough quantity of it
in a chamber slide.
3. stir blood with the end of a hemolysis applicator
until the blood appears as a transparent red
rather than a cloudy liquid.
15 Human Physiology-I (PHSL 205) Lab
1st Lab
Hakami,Hana A- 2010/2011
4. Cover chumber slide top and place both are slid
into the metal clip.
5. transport blood chamber slid into the slot on the
side of the hemoglobinometer.
6. Read and determine your hemoglobin that are
located on the reader.
7. Wash the slide then Sterilize it , and discard from
wastes by use Sharp container
Results :
Write result of sample and study it :
Sample Name
Grams per 100 ml blood
Percent
Note