1335687089.75523 Lab PracticalHematologyManual PDF

Practical Hematology Dr.
Alaa-eldin
Colleges of Dentistry
1st SEMESTER 1432/1433 H
Practical Hematology
For 1ST Year Student
Dr. Alaa-Eldin Salah-Eldin
Associated Professor of Molecular Biology and

Physiology
Zolfi College of Science
Department of Medical Laboratories
1
Practical Hematology Dr. Alaa-eldin
Practical
Hematology
Manual
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ANTICOAGULANTS
Used in Hematology
Laboratory
Anticoagulants Used In The Hematology Laboratory

Anticoagulants are defined as substances which prevent blood clotting /
coagulation, and allow separation of the blood into cellular and liquid (plasma)
components. Generally plasma contains coagulation factors. The three
anticoagulants commonly used in hematology laboratory are:
1] Ethylene Di-Amine Tetra-Acetic Acid (EDTA):

EDTA can be found in three salt forms:
1- Tri-Potassium EDTA
2- Di-Sodium EDTA
3- Di-Lithium EDTA
Also, EDTA can be crystalline or liquid. Liquid EDTA tubes requires
specific filling volume to avoid dilution effect. So, blood: anticoagulant ratio must
be maintained (this is applicable to all anticoagulants). EDTA is also known as
Versene or Sequestrene. EDTA acts by chelating/removing ionized calcium
(calcium is required for blood to clot, so when it is removed blood will not clot).
Generally tri-Potassium EDTA is better than di-Sodium EDTA and di-Lithium
EDTA.
Always, be sure to mix blood with anticoagulant in a manner that guarantee proper
complete mixing, by gentle repeated inversion of the tube, in figure of 8 inversion for at
least 20 times, do not shake or use vigorous inversion, since this may cause hemolysis, and
disintegration of cells, and the final effect will be erroneous low results for cellular
components of blood
EDTA is the most commonly used anticoagulant in the hematology laboratory,

and is the anticoagulant of choice for the CBC.
Excess EDTA (i.e. more EDTA, you fill less blood volume, so EDTA is in
excess), causes shrinkage of RBC’s, causing falsely/erroneously reduced
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hematocrit (HCT), and subsequent increase in MCHC and decrease in MCV

(MCV and MCHC are RBC indices that will be studied later). Platelets are also
affected, they will swell and subsequently disintegrate, causing erroneously high
platelet count, since platelets will be disintegrated into more than one fragment,
each fragment will be counted as one platelet (for example if one platelet will be
disintegrated into 4 fragments, the 4 fragments will be counted as 4 platelets, but
actually they represent one platelet, causing erroneously high platelet count).
2] Sodium Citrate
Is the anticoagulant of choice for coagulation and platelet function tests,
also is used for ESR (erythrocyte sedimentation rate test). It acts by
precipitating calcium, thus it will not be available for clotting process. It came
in a liquid form, as 3.8% tri-sodium citrate. For coagulation testing, the ratio
of 9 volumes of blood to one volume of anticoagulant (9 volumes blood : 1
volume anticoagulant) is very critical (very important), as variation from this
ratio may cause errors.
For ESR (4) volumes of blood to one volume of anticoagulant is used (4 :
1).Generally, this anticoagulant is not suitable for routine hematology testing.
From this we conclude that sodium citrate acts as anticoagulant and as diluent
(as in the case of ESR). Because of its dilution effect it can’t be used for CBC.
3] Heparin
Heparin is an acid mucopolysaccharide, it acts by complexing with anti-
thrombin to prevent blood clotting (antithrombin is one of the
natural/physiological inhibitors of blood coagulation). It is not suitable for
blood films staining, since it gives too blue coloration to the background, when
films are stained with Romanovsky stains, also, heparin may cause leukocyte
and platelet clumping, this is why heparin is not suitable for routine hematology
tests. It is the preferred anticoagulant for osmotic fragility test. Heparin also is
used in capillary tubes for spun hematocrit (HCT) (heparin cover the entire
capillary tube glass), these capillary tubes are also called microhematocrit
capillary tubes. Heparin is also used for L.E. cell preparation (L.E.= Lupus
Erythromatosus).
• Heparin is found in basophil and mast cell granules.
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• Heparin is used therapeutically as an in vivo anticoagulant.

Anticoagulants commonly Used in the Hematology Laboratory and their use:
No. Anticoagulant Hematology Laboratory Use Universal Color Code

1 EDTA Routine Hematology Procedures. Lavender, Pink
2 Sodium citrate Coagulation , Platelets Tests, ESR. Blue
3 Heparin Osmotic Fragility, Spun Hematocrit Green, Brown
I am a tube containing
specified volume of
anticoagulant, please only
fill me with the correct
required volume of blood
from the patient, do not
attempt to overfill or
under fill me, your results
may be negatively
affected, so help me and
help your self.
5
HEMOCYTOMETRY
Improved Neubauer Hemacytometer

Hemacytometry
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 a glass slide. Also, the
hemacytometer have a special glass slide manufactured to strict
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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 (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: Cover glass position on the hemacytometer
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Hemacytometer Counting Areas

Hemacytometer has 2 identical ruled counting areas, each
composed of etched area consists of 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
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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 µl.
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.
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
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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
calculated.
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
Calculating The Cell Counts

1st. The total number of cells per cubic millimeter of sample can be
calculated from:
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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 (mm ²)
Where:
A- C/mm³ = number of cells/ mm³
B- N= Total number of cells counted in the counting chamber.
C- D (mm) = Depth factor in mm
D- DF = Dilution Factor
E- 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.
Hematology, also spelled haematology, is the branch of internal medicine,

physiology, pathology, clinical laboratory work, and pediatrics that is concerned
with the study of blood, the blood-forming organs, and blood diseases.
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Hematology includes the study of etiology, diagnosis, treatment, prognosis, and

prevention of blood diseases.
The laboratology work that goes into the study of blood is frequently
performed by a medical technologist. Hematologists physicians also very
frequently do further study in oncology - the medical treatment of cancer.
In a clinical laboratory the hematology department performs numerous

different tests on blood. The most commonly performed test is the complete blood
count (CBC) also called full blood count (FBC). Studies of blood coagulation is a
sub-specialty of hematology; basic general coagulation tests are the prothrombin
time (PT) and partial thromboplastin time (PTT). Another common hematology
test in the erythrocyte sedimentation rate (ESR).
Hematology Tests
Changes in time of day and fasting state may alter some values.
Blood consists of red cells (erythrocytes), white cells (leukocytes), and platelets
(thrombocytes), suspended in a liquid called plasma.
A CBC usually includes white blood cell count (WBC), red blood cell count
(RBC), hemoglobin, hematocrit, red cell indices (MCV, MCH, MCHC), and
platelet count. Some others tests listed under the CBC include red cell distribution
width (RDW), mean platelet volume (MPV) and a differential examination of the
quality and quantity of various white cells reported either in percent or absolute
terms.
Reference values for the different parts of the CBC are difficult to list as some vary
by age, sex, and altitude. Also different instruments will have different principles
of measurement that can impact on the final values.
Blood Tests
BMP: electrolytes (Na+/K+, Cl-/HCO3-) · renal function,
BUN-to-creatinine ratio (BUN/Creatinine) · Glucose ·
Clinical Ca
Metabolic panel
biochemistry
CMP: BMP + protein tests (Human serum albumin,
Serum total protein) · liver function tests (ALP, ALT,
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AST, Bilirubin)
derived values: Plasma osmolality · Serum osmolal gap

Acid-base Arterial blood gas · Base excess · Anion gap · CO2
homeostasis content
Transferrin saturation = Serum iron / Total iron-binding
Iron tests capacity
Ferritin · Transferrin · Transferrin receptor
Glucose test · Glucose tolerance test · Glycemia ·
Blood sugar Noninvasive glucose · C-peptide · Fructosamine ·
Random glucose test · Glycosylated hemoglobin
Endocrine ACTH stimulation test · Thyroid function tests
Troponin test · CPK-MB test · Glycogen phosphorylase
Cardiac marker
isoenzyme BB
Other Beutler test · Blood lipids · Tumor marker
vWF: Ristocetin induced platelet agglutination
clotting factors: Prothrombin time · Partial

thromboplastin time · Thrombin time
Clotting other/general coagulation: Bleeding time · animal
enzyme (Reptilase time, Ecarin clotting time, Dilute
Russell's viper venom time) · Thromboelastography
Hematology/ fibrinolysis: Euglobulin lysis time · D-dimer

CBC Hematocrit · Hemoglobin · RBC count
ratios: Mean corpuscular hemoglobin · Mean corpuscular

hemoglobin concentration · Mean corpuscular volume
Red blood cell indices
Fetal hemoglobin: Apt-Downey test · Kleihauer-Betke
test · Red blood cell distribution width
Reticulocyte index · Haptoglobin
Other Blood film · Blood viscosity · Absolute neutrophil count
viral infection: HIV (HIV test, BDNA test) · Epstein-Barr

virus (Monospot test)
Immunology Infections
bacterial infection: syphilis (VDRL, Rapid plasma reagin,
Wassermann test, FTA-ABS) · rickettsia (Weil-Felix test) ·
helicobacter (HelicoCARE direct) · streptococcus
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(Antistreptolysin O titre)
protozoan infection: toxoplasmosis (Sabin-Feldman dye

test)
C-reactive protein · Erythrocyte sedimentation rate ·
Inflammation
MELISA · RAST test
A complete blood count (CBC), also known as full blood count (FBC) or
full blood exam (FBE) or blood panel, is a test requested by a doctor or other
medical professional that gives information about the cells in a patient's blood. A
scientist or lab technician performs the requested testing and provides the
requesting medical professional with the results of the CBC.
The cells that circulate in the bloodstream are generally divided into three types:
white blood cells (leukocytes), red blood cells (erythrocytes), and platelets
(thrombocytes). Abnormally high or low counts may indicate the presence of
many forms of disease, and hence blood counts are amongst the most commonly
performed blood tests in medicine, as they can provide an overview of a patient's
general health status. A CBC is routinely performed during annual physical
examinations in some jurisdictions.
Manual blood count
Counting chambers that hold a specified volume of diluted blood (as there are
far too many cells if it is not diluted) are used to calculate the number of red and
white cells per litre of blood.
To identify the numbers of different white cells, a blood film is made, and a large
number of white cells (at least 100) are counted. This gives the percentage of cells
that are of each type. By multiplying the percentage with the total number of white
blood cells, the absolute number of each type of white cell can be obtained.
30% of CBCs have medical scientists manually looking at a blood film down the
microscope, not only to find abnormal white cells, but also because variation in the
shape of red cells is an important diagnostic tool. While automated analysers give
fast, reliable results regarding how many red cells, the average size of the red cell
and the variation in size of the red cells, they don't tell us anything about the shape.
Also, a percentage of normal patient's platelets will clump in EDTA
anticoagulated blood. In these cases the automatic analysers will give a falsely
lower platelet count. On looking manually at the slide in these cases, clumps of
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platelets will be visible, and the scientist will estimate if there are low, normal or
high numbers of platelets but an absolute number cannot be reported.
A complete blood count will normally include:
Red cells
• Total red blood cells (RBC)- The number of red cells is given as an
absolute number per litre.
• Hemoglobin (Hb)- The amount of hemoglobin in the blood, expressed in
grams per decilitre. (Low hemoglobin is called anemia.)
• Hematocrit or packed cell volume (PCV) - This is the fraction of whole
blood volume that consists of red blood cells.
• Red blood cell indices
1. Mean corpuscular volume (MCV) - the average volume of the red
cells, measured in femtolitres. Anemia is classified as microcytic or
macrocytic based on whether this value is above or below the
expected normal range. Other conditions that can affect MCV include
thalassemia and reticulocytosis.
2. Mean corpuscular hemoglobin (MCH) - the average amount of
hemoglobin per red blood cell, in picograms.
3. Mean corpuscular hemoglobin concentration (MCHC) - the
average concentration of hemoglobin in the cells.
• Red blood cell distribution width (RDW) - a measure of the variation of
the RBC population
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RED BLOOD CELL (RBC’s) COUNT
According to the Merck Manual, normal values of RBC/cmm for males is 5.4 +
0.8 million, and 4.8 + 0.6 for females. Anemic levels for adult males are below 4.5
million, for females below 4.0 million. We will perform red and white blood cell
counts on your blood in the lab using a hemocytometer and appropriately diluted
blood.
Blood cell counts can be performed using the hemacytometer. This precision
instrument possesses a platform with microscopic grid scoring. Rails on either side
hold up a cover slip so that a specified quantity of fluid is held. By properly
diluting blood, counting all cells in specified squares, and multiplying by the
proper conversion factor, the number of cells per cubic millimeter can be
determined.
Set up all equipment on your desk so that you are sure to have everything at
your finger tips for the procedure. Prepare an autolet with a sterile lance and
platform.
1. Swab towards the side of the tip of a little-used finger with 70% EtOH.
Ø Hemacytometer
Ø Coverslip
Ø Rbc pipette with hose and Mouthpiece
Ø Autolet
Ø Lancet needless (for drawing blood)
Ø Platforms (for drawing blood)
Ø Microscope
Ø Squirt bottle with 70% ethanol
Ø Kimwipes (to be soaked in etoh)
Ø Bottle ringer's solution (a clear diluent for rbc)
Ø Paper towel
2. Lance by placing the platform of the autolet against the finger tip and pressing
the trigger.
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3. Using the dilution pipette with RED mixer from hemacytometer kit, draw blood
up to the 0.5 mark. (Do not allow air to be drawn into the pipette or it will
not draw the correct volume of blood. Do not allow blood to congeal in
pipette! Immediately proceed to the next step:
4. Continuing to hold the pipette as horizontal as possible, draw Ringer's solution

diluent up to the 101 mark. (Dilution of 1 to 200.)
5. Seal the tip with your finger and shake well to mix.
6. Empty ~1/2 of pipette into waste container
add a small amount of the diluted blood to one chamber of the

hemacytometer to just fill the chamber of the hemacytometer. (Do not
over fill).
7. Let the preparation sit for a minute (for cells to settle).
8. Center the grid at 100x, switch to 400x and count and record the RBCs in each
of five fields (each with 16 smallest squares) with a clicker (fields: top R
& L, bottom R & L, center). Include in the count all cells touching left and
bottom sides, ignore cells touching top and right sides.
Calculate the RBCs/cmm by adding the cells in the 5 groups and

multiplying by 10,000 (i.e., add four zeros). Enter your RBCs/mm in the
class data table.
9. Wash out the pipette thoroughly with soap and water, rinse well, finish with
distilled H2O rinse, replace in case.
Calculation:
(1) Routinely, blood is drawn to the 0.5 mark and diluted to the 101 mark with
RBC diluting fluid. All the blood is washed into the bulb of the pipette (which has
a volume of 100). Therefore, 0.5 volumes of blood are contained in 100 volumes of
diluting fluid. The resulting dilution is 1:200. (These figures are arbitrary and refer
strictly to dilution and not to specific volumetric measurements.)
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Figure 5-1. Hemacytometer counting chamber (RBCs). Central areas are used to count red blood
cells.
Ringer's Solution, per 100 mL:
860 mg NaCl
30mg KCl
35mg CaCl2
dissolve in dH2O and q.s. to 100 mL.
18
19
No. of RBC’s counted/

80 small square 1 1
3
RBC’s Count/mm Blood = -------------------------- X ----------------- X --------------
No. of small square Volume of Dilution
(80) small square
• No. of RBC’s counted 80 small square = ??? /

• No. of small square = 80
• Volume = Length X Width X Height = 1/20 X 1/20 X 1/10 = 1/4000
• Dilution = 1:200 = 1/200
i.e. Factor = 10000
RBC’s Count/mm3 Blood = ??? X 10000
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HEMOCYTOMETRY
Improved Neubauer Hemacytometer
PLATFORM WITH
RULED AREA H-SHAPED DEPRESSION
COVERGLASS
COVERGLASS AL AZHAR
SUPPORT
CENTER PLATFORM
COVERGLASS
COUNTING
COUNTING
CHAMBER
CHAMBER COVERGLASS SUPPORTS
21
Hemacytometer Counting Areas
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 calculated.
Begin
Cells
touching
right, and
bottom
boundaries
are not
counted!
Sample:
EDTA anticoagulated whole venous blood.
Diluting Fluid:
§ Isotonic saline:0.85% sodium chloride (NaCl) in distilled water.
Apparatus and Equipment:
1- Micropipette – 20 µl 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.
Normal Reference Range:
Males : 4.6 – 6.2 x 1012/L
Females : 4.2 – 5.4 x 1012/L
Children: 4.5 – 5.1 x 1012/L
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Hemoglobinometry
(Hemoglobin Determination)
Decrease in hemoglobin concentration beyond established normal ranges for

age and sex is called “ anemia”, whereas increase in hemoglobin concentration
beyond established normal ranges for age , sex, and geographical distribution is
called “polycythemia”. So that, for correct diagnosis it is important to determine
accurately and precisely hemoglobin concentration.
Many methods are available for the determination of hemoglobin, but among
them the relevant, and the recommended one is the Modified Drabkin’s Method.
ICSH (International Committee for Standardization in Hematology) consider this
method as the reference method for hemoglobin determination.
Drabkin’s solution contains the following:-

1- Potassium Ferricyanide
2- Potassium Cyanide.
3- Non- ionic Detergent
4- Dihydrogen Potassium Phosphate.
Well mixed EDTA anticoagulated blood is diluted in Drabkin’s solution; non-
ionic detergent will lyse the red cells to (1) liberate hemoglobin, and to (2)
decrease the turbidity caused by red cell membrane fragments by dissolving them.
Then, hemoglobin is oxidized and converted to methemoglobin (Hi) by
potassium ferricyanide, this step is accelerated by the dihydrogen potassium
phosphate, and requires approximately 3 minutes for total conversion. Potassium
cyanide will provide cyanide ions to form cyanomethemoglobin (HiCN), which
have a broad spectrum of absorption at 540 nm. The absorption can then be
compared with a hemoglobin standard with a known hemoglobin concentration,
and by applying Beer’s law extract the hemoglobin concentration of the unknown
(i.e. the patient).
Hemoglobin + Potassium Ferricyanide Methemoglobin (Hi)
Methemoglobin + Potassium Cyanide Cyanomethemoglobin
23
Hemoglobin Concentration Determination

Modified Drabkin’s Method
Principle:
Whole blood is diluted in a solution containing Potassium Ferricyanide and

Potassium Cyanide. Hemoglobin will be oxidized by the action of Potassium
Ferricyanide to form Methemoglobin (Hemiglobin, Hi). Potassium Cyanide will
provide Cyanide ions to form Cyanomethemoglobin (HiCN). This solution can be
measured spectrophotometrically and compared to known hemoglobin standards.
This procedure is applicable in diagnosing and monitoring therapy in cases of
hemoglobin deficiency anemia’s.
v Sample: EDTA anticoagulated venous whole blood.
v Apparatus:
1- Brown bottle- 1 liter
2- Volumetric flask 1 liter
3- Balance
4- Spectrophotometer adjusted at 540 nm
5- Spectrophotometer Cuvettes
6- Glass or plastic centrifuge tubes
7- Micropipette (adjusted at 20 µl)
8- 5 ml volumetric pipette or graduated pipette
v Reagents:
1- Potassium Ferricyanide K3Fe (CN)6 0.200 g (200 mg)
2- Potassium Cyanide (KCN) 0.050 g (50 mg)
3- Dihydrogen Potassium Phosphate KH2PO4 0.140 g (140 mg)
4- Distilled Water (laboratory grade 1)
5- Non-Ionic Detergent:
- Sterox S.E. 0.5 ml
or - Trinton X-100 1.0 ml
or - Quolac Nic 218 1.0 ml
6- Standard Cyanomethemoglobin solution/ solutions.
v Working Drabkin’s Solution Preparation:

Add the reagents to the volumetric flask and add distilled water up to the mark
(avoid bubble formation), with continuous mixing as you are adding the distilled
water. Transfer to a stoppered brown bottle, and label with name, date of
preparation, and the name of the technologist who prepared the solution.
24
Ready made commercial preparations are available in the market, just dilution with
distilled water is required, such that preparations are for example available from
Randox company.
v Procedure:
1- Add 20 µl of whole anticoagulated blood to 5 ml of Drabkin’s solution.
2- Mix well.
3- Allow the mixture to stand at room temperature for at least 3 minutes.
4- Measure the absorbance at 540 nm against a diluent Drabkin’s solution
(blank).
5- Measure the absorbance of the standard HiCN solution in the same manner.
6- Extract the unknown hemoglobin concentration, using the following
equation:
Abs. of Unknown
Unknown Hb concentration = x Conc. Of STD
Abs. of STD
Or read directly from the hemoglobin standard curve. See below, how to
prepare the hemoglobin standard curve.
v Hemoglobin Standard Curve Preparation:

A standard curve must be made each time new Drabkin’s solution is prepared.
A commercially prepared standard kit with hemoglobin concentrations of 20, 15,
10, 5 g/dl is available, read each corresponding absorbance, and plot the results on
a linear graph paper (absorbance versus Hb concentration). Or, you can use a stock
standard solution of 20 g/d, and dilute it to various Hb concentrations with
Drabkin’s solution.
Absorbance
0.3
0.2
0.1
5 10 15 20 Hb-Concentration- g/dl
Hemoglobin Standard Curve
25
Notes:
1- Drabkin’s method is the recommended method by the ICSH.
2- Drabkin’s solution should be clear and have a pH of 7.0 to 7.4, discard if
turbid.
3- The Drabkin’s solution is the blank, and should read zero (0) absorption.
4- Take care when preparing the solution, as cyanide is fatal, and toxic,
although the amount of cyanide in the prepared solution is less than the
human lethal dose.
5- Do not expose the solution to acids, because cyanide will be released.
6- Keep the solution in a dark bottle at room temperature, but discard after a
month.
7- All types of hemoglobin which include hemoglobin, oxyhemoglobin,
carboxy hemoglobin, methemoglobin are measured by this method, only
Sulfhemoglobin (S-Hb) is not measured by Drabkin’s method..
8- Sulfhemoglobin is found at increased amounts in cases of Clostridium
septicemia, as a result of drug intake, and in severe cases of constipation.
At increased amounts blood sample may color as lavender to green.
9- Increased methemoglobin concentration occurs as a result of inherited
conditions or more commonly as acquired as a result of drug intake or
exposure to chemicals.
10-Turbidity due to leukocytosis , lipemia, or high protein levels as seen in
para proteinemias ( e. g. multiple myeloma ) may interfere and cause
erroneously high hemoglobin concentration.
Prepare sample blank to overcome erroneous readings).
11-Try as possible as you can to obtain venous whole blood.
12- Use automatic micropipettes, but not Sahli pipettes, to reduce technical
errors.
13- Panic values for hemoglobin is less than 6.0 g/dl.
14- Hemoglobin concentration is unrelated to patient eating status.
15- Blood obtained from heavy smoker’s requires 3 minutes more incubation
time for full conversion of hemoglobin to methemoglobin.
16- Do not expose Drabkin’s solution and standards to light, and sunlight.
17- People living at high attitudes tend to have polycythemic hemoglobin
concentrations, because of the low oxygen tension at high attitudes,
causing tissue hypoxia, so that body compensate and adapt for this by
increasing hemoglobin.
18- Pregnant females tend to have lower hemoglobin concentrations than
normal for their age, because their fetuses compete with them for iron,
vitamins and other essential substances for hemoglobin synthesis, this is
26
why pregnant females are of great needs for iron and vitamin
supplements during their pregnancies.
Preparation Of Sample Blank

Turbidity can cause erroneously increased hemoglobin concentration due to
increased light scattering, to overcome this you can prepare a sample blank. To
prepare a sample blank use the formula:
5 ml
N=
(1- Hct)
Where N = ml of Drabkin’s to be added to 20 µl of patient plasma.
So, when you have a turbid sample, take a portion from it, and centrifuge it,
take 20 µl of it’s turbid plasma and add it to the calculated Drabkin’s solution
amount according to the above formula. This is considered the zero blank, adjust
the spectrophotometer absorbance reading to zero, then read the absorbance of the
patient hemoglobin according to the procedure above. The sample blank will
correct the erroneous hemoglobin result caused by turbidity.
Example:
If a patient has a hematocrit of 50, then:
5 ml 5
N= = = 10 ml
(1- 0.50) 0.50
So, the sample blank is prepared by adding 20 µl of patient’s plasma to 10 ml

Drabkin’s solution.
Hemoglobin concentration is expressed in g/dl (gram per deciliter)
Reference Hemoglobin Concentration Ranges:

Males : 13.5 – 18.0 g/dl
Females: 12.0 – 16.0 g/dl
Newborn: 16.5 – 19.5 g/dl
Children : 11.2 – 16.5 g/dl (varies with age)
27
Spun Microhematocrit
Principle
Hematocrit is the ratio of the total volume of RBC’s to that of whole blood
expressed as percentage(%) (whole blood = total volume of cells + plasma). The
second synonym for hematocrit is PCV (Packed Cell Volume). The procedure is
easy to perform, whole blood is centrifuged in a narrow tube (capillary tube),
cellular elements will be separated from the plasma, after centrifugation blood will
be separated into 3 layers : (1) Bottom layer contains packed RBC’s, (2) Middle
layer contains WBC’s and Platelets (on top of RBC’s), (3) Upper plasma layer.
The hematocrit value is determined by comparing the volume of RBC’s to the total
volume of the whole blood sample, it is usually reported as a %.
Sample:
♦ EDTA anticoagulated whole venous blood (correct volume is highly
required. When EDTA is in excess, cell shrinkage occurs, and as a result a falsely
low hematocrit is obtained, with corresponding increase in MCHC, and decrease in
MCV).
♦ Heparin
♦ Or directly from a finger prick, to a heparin coated capillary tube.
28
Apparatus and Materials:

1- Microhematocrit centrifuge.
2- Modeling clay (seal material).
3- Capillary tubes (7 cm long, 1mm diameter)
4- Hematocrit measuring device reader or a conventional ruler.
Procedure:
1- Fill the capillary tube with blood by capillary attraction. Either from free
flowing finger punctured by a sterile lancet/ or from a well mixed anticoagulated
whole venous blood (this requires only few microliters of blood).
2- Seal with the modeling clay the empty end of the capillary tube.
3- Place and position the capillary tube in the radial grooves of the
microhematocrit centrifuge with the sealed end away from the center (pointed
toward the outside).
4- Centrifuge for 5 minutes at 12000 g, so that additional centrifugation does
not pack the red blood cells more.
5- The height of the RBC column, and the total column should be measured
with the aid of a ruler in cm and mm, then divide the RBC column height over the
total column height (total height = RBC column + buffy coat + plasma column), or
simply with the aid of a special HCT reader device.
6- Express the results in percentage (%).
Buffy coat is the layer where WBC’s and Platelets are collected to, after
centrifuging a whole blood sample, this is the middle whitish-tan colored
layer.
Plasma Layer
Buffy Coat Layer
Red Blood Cell Layer
Buffy coat layer will contain all nucleated cells, including the nucleated red
cells, which are not normally found in the peripheral blood, but are seen in
pathological conditions. Also, all abnormal cells, including leukemic cells
are found in this layer, i.e. the buffy coat layer.
29
Reference intervals:-
Males : 40 - 53%
Females : 37 - 47%
Newborns: 51 - 60%
Children : 34 - 49%
Notes:
1- Higher values than the reference intervals is called polycythemia.
2- Lower values than the reference intervals is called anemia.
3- In cases of very high HCT, additional centrifugation for 5 minutes is
recommended to reduce plasma trapping. In general the higher the hematocrit, the
greater the centrifugal force required.
4- Adequate centrifugation time and speed are important for accurate
hematocrit.
5- Cells should be packed so that additional centrifugation does not alter or
reduce HCT reading.
6- Plasma trapping is slightly more in macrocytic anemia’s, spherocytosis,
hypochromic anemia’s, and in sickle cell anemia.
7- Errors may occur as a result of:
• Inadequate mixing of the blood.
• Improper reading of the column lengths.
• Inclusion of buffy coat height with RBC column height (in leukocytosis or in
thrombocytosis, the buffy coat column height will be increased).
• Plasma trapping is still one of the causes of erroneously increased HCT
results.
• Hemolysis of blood sample (due to improper collection, delay in processing)
will cause erroneously decreased HCT.
8- Increased anticoagulant to red cell ratio (short EDTA sample), will cause red
cell shrinkage and the hematocrit will be erroneously decreased.
Clinical Significance:
HCT is used to detect anemia’s, polycythemias, hemodilution, hemo-
concentration, and also is used in the laboratory to calculate the MCV, and the
MCHC manually.
If you direct the capillary tube towards the microhematocrit centrifuge center,
the sealed material will be removed, and at the end of centrifugation you will
find an empty capillary tube, blood will go out from the tube!!
30
Nowadays, Hct is supplied by the widely used automated hematology

analyzers. But this Hct is calculated rather than measured, these analyzers
are not equipped with centrifuges, Hct is calculated from the MCV, and RBC
count, by using the following formula:
MCV x RBC
Hct=
10
The sealed end of the capillary tube should be directed to the outside.
• The microhematocrit should be read at the top of red cell layer – not at the top of
the buffy coat.
There are two types of capillary tubes, red banded and blue banded capillary
tubes. The red banded are heparin coated, and use it when doing finger prick
hematocrit. Blue banded capillary tubes are plain, and use it with EDTA blood
samples.
Manual hematocrit is slightly more than the calculated automated hematocrit,

because of the trapped plasma which will be included with manual hematocrit,
and excluded with automated hematocrit (because it is calculated not
measured).
31
Red Blood Cell Indices

Red blood indices are calculated parameters which determine red
blood cell size, hemoglobin content of red cells, and hemoglobin
concentration of red cells. These parameters are useful in classifying
anemia’s into microcytic, normocytic, or macrocytic; and hypochromic
or normochromic. These parameters are calculated from total red cell
count, hematocrit and hemoglobin.
1- MCV
Mean Cell (Corpuscular) Volume, is the average volume of red cells.
This parameter is useful in classifying anemia’s into: Microcytic,
normocytic, and macrocytic. MCV is calculated from the hematocrit
(HCT), and the Red Blood Cells Count (RBC count).
HCT
MCV = x 10
RBC
The results of MCV are expressed in femtoliters (fl). 1 fl = 1 x 10-15 L.
In automated hematology analyzers measure (not calculating) MCV from

the area under the RBC histogram, and then calculating the HCT from
MCV and Total RBC count.
MCV Normal Range: 80 – 96 fl

♦ If results are less than 80 fl, the red cells are said to be
Microcytic:
a. Slight Microcytosis 75-79 fl
b. Moderate Microcytosis 70-74 fl
c. Marked Microcytosis <70 fl
♦ If results are within 80-96 fl, the red cells are said to be
Normocytic.
♦ If results are higher than 96 fl, the red cells are said to be
Macrocytic:
a. Slight Macrocytosis 96-105 fl
b. Moderate Macrocytosis 106-110 fl
c. Marked Macrocytosis > 110 fl
32
2- MCH
Mean Cell Hemoglobin, is the hemoglobin content in the average red
blood cell, or in other words, the average weight of hemoglobin per
RBC. It is calculated from the hemoglobin concentration (Hb), and the
total RBC count.
Hb g/dl
MCH = x 10
RBC
Results of MCH are expressed in picograms (pg). 1 pg = 1 µµg = 10-12 g.
MCH Normal Range: 27 – 32 pg

• Macrocytic red cells have higher MCH, because they are larger and
contain more hemoglobin.
• Microcytic red cells have lower MCH, because they are smaller and
contain less hemoglobin.
3- MCHC
Mean Cell Hemoglobin Concentration, is the average hemoglobin
concentration in 100 cc red blood cells. It indicates the average weight
of hemoglobin as compared to the cell size. It correlates with the degree
of hemoglobinization of the red cells on the peripheral blood film. MCHC
is calculated from the hematocrit and hemoglobin.
Hb g/dl
MCHC = x 100
HCT
OR
MCH in picograms
MCHC =
MCV in femtoliters
♦ Results of MCHC are expressed in percentage (%) or gm/dl.
Normal Range: 32 – 36 g/dl (%)
33
♦ If results are within this range, it is said that red cells are
Normochromic.
♦ If results are less than normal, red cells are said to be Hypochromic,
which is seen in microcytic hypochromic anemias e.g. iron deficiency
anemia.
Notes:
The only highly comparable red cell parameter between automated cell
counters and manual hematology tests is the MCHC, because MCHC
needs hemoglobin, and hematocrit in order to calculate it, which are easy
to perform manually with high reproducibility and accuracy.
Red cells can’t accommodate more than 37 g/dl of hemoglobin, which is

seen only in cases associated with spherocytosis. Macrocytic anemias
have normal MCHC. If you have a case with high MCHC, and you checked
the blood film and you didn’t find spherocytes, this may indicate an error in
hemoglobin and/or Hct. Since Hct is a calculated parameter, it is derived
from RBC and MCV, so may also indicate an error in RBC count and / or
MCV. Solutions to resolve this error include: retesting the specimen,
perform a spun microhematocrit, performing a manual hemoglobin
determination, and checking the quality control and other patients results.
Hematology Automated Analyzers nowadays can perform all of the

following:
Count RBC
Measure hemoglobin spectrophotometrically.
Directly measure MCV, from the area under the RBC histogram.
Calculate Hematocrit, which is derived from MCV and RBC count.
Calculate MCH, which is derived from Hb, and RBC count.
Calculate MCHC, which is derived from Hct, and Hb.
34
Preparation of Blood Films
Principle:
Blood film enables us to evaluate WBC, RBC, and PLT morphology,
also, allows us to perform differential WBC count, furthermore estimation
of WBC and platelets counts can be done on blood films. Blood films are
made on glass microscopic slides.
Sample:
v Finger stick blood or EDTA anticoagulated venous whole blood
may be used. Films of peripheral blood must be made immediately.
Films may be made from EDTA anticoagulated blood as long as two to
three hours after collection. All specimens should be free of clots.
Procedure:
1- Use clean standard size glass slides (3 inch x 1 inch = 7.5 cm x
2.5 cm), wiped from dust just immediately before use.
2- Place a small drop of well mixed anticoagulated whole blood, in
the center line of the slide, about 1.5 to 2 cm from one end, with
the aid of a capillary tube.
3- Immediately, without delay, with the aid of a second clean slide
with uniform smooth edges (spreader slide), with a 30 –40
degrees angle, move back so blood drop will spread along the
edge of the spreader slide, when this occurs, spread, or smear
the film by a quick, unhazizating, uniform forward motion of the
spreader.
35
Notes:
q Before preparing the films, you must check that blood samples are
free from clots, and this is done with two wooden applicator sticks. If
clots are present the specimen is unsatisfactory.
q Films can be labeled with patient’s name and /or Lab. No. on the
thick end of the film itself, after being dried, by using a pencil.
q With anemia (low Hct, reduced viscosity), the spreading angle
should be greater, to avoid running off the slide.
q With polycythemia (high Hct, increased viscosity), the spreading
angle should be less, to avoid short, too thick films.
q With large blood drops, increase the spreading angle.
q With small blood drops, decrease the spreading angle.
36
q If the anemia is too severe, let the blood specimen settle, so that
blood is divided into two layers, plasma layer and red cell layer, then
discard part of the plasma layer, then mix the blood specimen, by
doing this you have increased the viscosity of blood, by this you will be
able to prepare a nice blood film.
DO NOT ATTEMPT TO CENTRIFUGE TO DISCARD
PLASMA, THIS MAY DISTORT AND DISINTEGRATE THE

CELLS, WHICH ARE OUR INTEREST!
Staining Blood Films

With Romanovsky Stains
Blood films are stained so that morphology of blood cells become

more easily viewed, identified, and evaluated. In addition, blood films may
be examined for the presence of blood parasites (Malaria, Trypanosoma,
Babesia). Furthermore, stained blood films can provide important
information about a patients health, they may lead to a diagnosis or verify a
diagnosis, or they may rule out a diagnosis. Evaluation of stained blood
films also may lead to the decision of performing other hematology special
blood stain procedures in order to identify specific cell components.
As soon blood films are air dried, it is best to stain them as soon
as possible. Blood films are stained with one of the Romanovsky stains,
which are universally used for staining blood films. There remarkable
property is creating distinctions in shades of staining granules
differentially and this is dependent on two staining components: Azure B
37
(the basic dye) and Eosin Y (the acidic dye). Other factors which affects
the staining results include : 1) Staining time, 2) Ratio of Azure B to
Eosin Y, 3) pH of the staining solution.
• Azure B will stain the acidic cell Components (e.g. nucleus, because
it contains nucleic acids; basophilic granules also take the Azure B
staining because they contain heparin, which is acidic in origin),
• while Eosin Y will stain the alkaline basic components (e.g.
Eosinophilic granules in eosinophils, because these granules contain
spermine derivatives, which are basic in origin). Red cells have
affinity for acidic Eosin Y dye, because it contains hemoglobin which
is basic in origin.
Romanovsky stains include:

× Giemsa Stain
× Wright’s Stain
× Leishman Stain
× May-Grünwald Stain
The widely and popular used Romanovsky stains are:
Ø Leishman Stain
Ø Wright’s Stain
Leishman Stain Procedure:
1- Let the films be air dried.
2- Put the films on a staining trough rack.
3- Flood the slides with the stain.
4- After 2 minutes (or more, if the stain in newly prepared), add
double volume of water, and blow to mix the stain with water,
until a shiny layer is seen.
38
5- After 5-7 minutes, wash with a stream of water.

6- Wipe the back of the slides with gauze.
7- Set the films in upright position on a filter paper to dry.
8- Read the blood films microscopically.
If delay in staining blood films may occur, fix the films in absolute
methanol, for 1-2 minutes, but do not stain the slides until completely
dried.
Romanovsky Stain Blood Cell Characteristics

No. Cell Structure Staining characteristics
1 Red cells Red or pinkish red
2 Nuclei of all cell types Purple/violet
3 Lymphocyte cytoplasm Blue
4 Monocyte cytoplasm Grayish blue
5 Platelets cytoplasm Light blue
6 Neutrophilic granules Violet-pink
7 Eosinophilic granules Orange-red
8 Basophilic granules Purplish black/ Deep blue
9 Platelets granules Purple
Sources Of Errors In Staining

1- Stain Precipitate: May obscure cell details, and may cause confusion
with inclusion bodies. Filter the stain before use.
2- pH of the buffer or water:
♦ Too acidic pH causes too pinkish slides.
♦ Too basic pH causes too bluish slides.
39
3- Improper stain timing may result in faded staining or altered colors:

♦ Too long staining time causes too blue slides
(overstaining).
♦ Too short staining time causes too red slides.
4- Forced drying may alter color intensities and/or distort cell
morphology.
5- Non-stain related errors:
1st- EDTA causes crenation of the cells after blood collection.
2nd- Severely anemic blood samples causes slower drying
(before staining) due to excessive plasma.
3rd- Old blood specimens may cause disintegration in WBC’s
and decrease in their numbers.
4th- Collection of blood in heparin causes blue staining of
RBC’s with bluish background, which makes heparin
unsatisfactory for routine hematology testing, also heparin
induces platelet aggregation and clumping, with subsequent
erroneous platelet count with automated counters.
Always filter the stain before each use, to eliminate stain

precipitates.
Automatic stainers are available in the market, in which slides are

moved automatically and they are stained as they move.
40
WBC (Leukocyte) Manual Counting

Principle:
Blood sample is mixed and diluted with weak concentration of
hydrochloric acid (HCl), or acetic acid (in specified known volumes). Weak
acids will lyse red blood cells, and will darken WBC’s to facilitate counting
by the hemacytometer.
Manual WBC counting is used in cases of very low WBC count
(leukopenia) with automated hematology cell counters, and when
automated cell counters are not available.
Sample:
q EDTA anticoagulated whole venous blood.
Reagent and Supplies To Prepare Diluting Fluid:

1- Volumetric Flask 100 cc.
2- Serological pipettes.
3- Concentrated HCL
4- Glacial Acetic Acid
Preparation of Diluting Fluid:

Diluting fluid is either:
v 1% hydrochloric acid in distilled water (1 ml Conc. HCL + 99 ml
Dist. water).
v 2% Acetic Acid in distilled water (Turk’s solution) (2 ml glacial
acetic acid + 98 ml distilled water).
Glassware, Apparatus, Equipment :
1- Neubauer improved hemacytometer.
41
2- Clean cover slip slide (especially made for the hemacytometer).

3- Automatic micropipette (20 µl, 380 µl are the required volumes).
4- Gauze 10 x 10 cm
5- Glass/Plastic tubes- (12x75 mm).
6- Handy tally counter.
7- Conventional light microscope.
Procedure:
1- Mix the blood sample gently but thoroughly by inversion, manually or
by mechanical rocking mixer.
2- Pipette 0.38 ml (380 µl) of diluting fluid into a 12x75 mm tube.
3- Pipette 0.02 ml (20 µl) of well mixed blood to be counted and wipe
the tip with gauze into the tube containing diluting fluid and mix the
tube.
4- Let the tube stand for 2-3 minutes to ensure complete RBC lyses,
then mix well.
5- Prepare the clean hemacytometer and cover it with the designed
coverslip.
6- Load one side of the hemacytometer with the aid of a capillary tube or
micropipette, do not attempt to overload or underload the
hemacytometer.
7- Allow the hemacytometer to sit for several minutes to allow the
WBC’s to settle in the counting chamber, to avoid drying effect, place
the loaded hemacytometer in a covered Petri dish with a moist gauze,
until counting.
8- Place the hemacytometer in the microscope stage.
42
9- Focus with x10 objective lens (low power), with lowering the
condenser.
10- The WBC’s are counted in the 9 corner large squares, with the aid of
hand tally counter.
11- Follow the counting pattern shown in the figure below. During
counting, do not count cells that touch the right or bottom boundaries
to ensure unduplicated counting.
Begin
12- The total counted WBC’s in the 9 squares are added together.
Fig. WBC’s are

counted in the 9
hemacytometer
squares
q If the number of cells

in a square varies from
any other square by more
than 9 cells, the count
must be repeated,
because this represents an
uneven distribution of
43
cells, which is may be caused by improper mixing of the dilution or

improperly filled hemacytometer.
Calculations:
No. of WBC’s counted/

64 big square 1 1
3
WBC’s Count/mm Blood = -------------------------- X ----------------- X --------------
No. of big square Volume of Dilution
(64) big square
• No. of WBC’s counted 64 small square = ??? /

• No. of big square = 64
• Volume = Length X Width X Height = 1/4 X 1/4 X 1/10 = 1/160
• Dilution = 1:20 = 1/20
i.e. Factor = 50
WBC’s Count/mm3 Blood = ??? X 50
Reference Range
♦ Adults : 4.5 – 11.0 x 109 /L
♦ Six years: 4.5 – 12.0 x 109 /L
♦ One year: 6.0 – 14.0 x 109 /L
♦ Newborn: 9.0 – 30.0 x 109 /L
q WBC count varies according to age but not to sex.
44
45
Sources of Error:
1- Contaminated diluting fluid.
2- Incorrect dilution.
3- Uncalibrated Micropipettes.
4- Uneven distribution of WBC’s.
5- Presence of clumped WBC’s.
6- Unclean hemacytometer or cover slips.
7- Presence of air bubbles.
8- Incompletely filled hemacytometer.
9- Over flow.
10- Presence of debris.
11- Drying of the dilution in the hemacytometer.
1 ml of gentian violet can be added to the diluent to color the white

blood cells, thus counting will be easier.
In leukopenia ( decreased WBC count), with a total
WBC count below 2500/cumm, the blood is diluted 1:10,

whereas in leukocytosis (increased count), the dilution
may be 1:100 or even 1:200.
46
WBC Differential Count
White blood cells are evaluated by a differential count, which reports

percentages of the types of WBCs present. These are neutrophils which fight
infection (also known as polys and bands, polymorphonuclear leukocytes, PMN’s,
grans, segs and nonsegs), lymphocytes which produce antibodies and other
immune system activities (lymphs, ly), monocytes which also fight infection
(mono’s), eosinophils (eos) and basophils (basos) which are involved with
allergies. The red cells are also evaluated for size, shape, color and the presence
of any abnormalities
Manual differentials are performed by taking a drop of blood, spreading it

on a slide, staining it, and evaluating 100 cells individually for quality and
changes in morphology. For patients with elevated white cell counts, differentials
of 200 cells or greater might be done. Automated differentials are performed by
either testing for specific compounds within the cells or comparing their size,
shape, and content. Most instruments will count thousands of cells. For both types
of differentials, the numbers are reported in percentages.
In some patients percentages might be misleading so absolute values of the

types of WBC, i.e., the number of white blood cells multiplied by the percentage
seen are valuable in diagnosing illness or following therapy. Persons receiving
chemotherapy often have decreased WBC. If a patient’s absolute granulocyte
count (ANC or AGC) goes below 2,000 cells, then physicians become concerned
about the possibility of infection. A number below 1,000 is cause for greater
concern and less than 500 usually lands the patient in the hospital.
Principle:
Testing a Romanovsky stained blood films in order to determine and assess
the percentage of various classes of WBC’s present, and to assess red blood cells
and platelets morphology.
Increased or decreased normal WBC’s class/subpopulation counts or the presence
of immature precursors of WBC’s or RBC’s in the peripheral blood film are of
diagnostic importance in various inflammatory and disease states.
47
Morphological red blood cells abnormality are important in various

anemia’s (spherocytes, sickle cells, acanthocytes, burr cells, microcytes,
macrocytes, target cells, ….. etc).
Platelet morphology, distribution, and size abnormality are suggesting a platelet
disorder.
Sample:
v EDTA anticoagulated whole venous blood film, bone marrow film, and
body fluid sediments (e.g. CSF).
Reagents, Supplies, and Apparatus:

1- Differential Tally Counter.
2- Conventional Binocular Microscope.
3- Oil immersion.
4- Well Stained Blood Film/s.
Procedure:
1- Focus the film under x10 lens, and scan the film to check cell
distribution.
2- Add a drop of oil, and move to the x100 oil immersion lens.
3- Choose a suitable area, where cells are evenly distributed without
appreciable overlapping- the monolayer cell zone.
4- Count the WBC’s using tracking pattern.
5- Each cell identified should be immediately tallied as:
• Neutrophil- segmented.
• Neutrophil – band
• Lymphocyte
• Monocyte
• Eosinophil
• Basophil
• Immature cells: blast, promyelocyte, myelocyte, metamyelocyte,
promonocyte.
• Variant atypical lymphocyte.
48
6- Morphological abnormalities of WBC’s, RBC’s and platelets should be

noted.
7- Nucleated red blood cell precursors (nucleated red blood cells-NRBC) are
not included in the differential count, but are counted per 100 WBC’s, and
if they are more than 10 NRBC/100 WBC’s, a corrected WBC count
should be made (because as discussed before that NRBC’s are counted as
WBC’s, and will be included in the total WBC count erroneously) by
applying the following formula:
8-
Uncorrected WBC X 100

Corrected WBC count =
NRBC + 100
• NRBC is the number of NRBC seen per 100 WBC during differential
process.
9- Express the results as percentage for each cell class/ subpopulation.
! Count in the monolayer
zone
Method of Differential Counting Pattern- Tracking Pattern
If total WBC count is high (more than 20 x 109/L), a 200 or 300 cell
differential is advisable.
v Blood film made for WBC differential counting should be evaluated for red
cells. Red cells are evaluated for;
1- Variation in red cell volume/size,
2- Variation in shape,
3- Variation in staining properties,
4- Alteration in distribution,
5- Presence of intracellular inclusions and
6- The presence of extracellular or intracellular parasites.
49
v Blood film for WBC differential counting should be evaluated for;

1- Variation in platelet size (large, giant),
2- Presence of megakaryocyte fragments,
3- Dwarf megakaryocytes, or megakaryocytes.
4- Also, platelet distribution (satellitism, aggregation, clumping) which
produce erroneous platelets count results with impedance technology
electronic counters, should be noted (causing thrombocytopenia, and an
increase in WBC count and/or RBC count, and affects RBC and WBC
histograms).
v Blood film is evaluated for abnormal WBC’s inclusions,

1- Toxic granulation,
2- Alder-Reilly granules,
3- Chediak Higashi granules,
4- Döhle bodies, and 6- toxic vacuolization.
All of the above may indicate specific disease process of acquired or inherited
origin.
Most of abnormal / immature cells tend to be accumulated at the blood

film edges, do not forget to scan these areas. All nucleated red cells,
especially megaloblasts also tend to be accumulated at these edges.
Scan the blood film edges !!!
Normal Values for Differential WBC count in Adults:

Cell Type/ Population %Normal Differential Normal Absolute Count * x 109
Neutrophils- Segmented 50-70% 2.0-7.0 x 109/L
Neutrophil- Band 0-10% 0.0-1.0 x 109/L
Lymphocytes 15-45% 0.6-4.0 x 109/L
Monocytes 0-10% 0.0-1.0 x 109/L
Eosinophils 0-6% 0.2-0.7 x 109/L
Basophils 0-1% 0.0-0.2 x 109/L
* Absolute count is obtained by multiplying the % differential of the cell type

in concern by the total WBC count, obtained either by manual or automated
50
methods. automated CBC counters supply us with this absolute count, for the main
three cell types (i.e. the neutrophils, the lymphocytes, and the monocytes). So,
differential count can be expressed as percentage and also as absolute count.
Automated Differential Counting

Nowadays, hematology laboratory is equipped with automated hematology
analyzers capable of automated differential counting, which are more quicker,
precise, and accurate than the manual time consuming differential count, but this is
true when the sample contains normal cell populations. When the sample contains
abnormal or immature cell populations, your eyes are not substituted. Although
current laser cell counters identify abnormal and immature cell populations in a
sample, but this does not substitute looking to a nice looking blood film to identify
these abnormal cell populations or subpopulation.
Clinical Significance Of Increased Normal WBC Counts:

Neutrophilia: Bacterial Infections, Inflammation , Stress, Chronic
Myelocytic Leukemia.
Eosinophilia: Invasive parasite, Active Allergy, Myeloproliferative
diseases/disorders..
Basophilia: Ulcerative Colitis , Myeloproliferative Diseases,
Hyperlipidemia.
Lymphocytosis : Viral Infections, Whooping cough, Chronic
Lymphocytic Leukemia.
Monocytosis: Tuberculosis, Rheumatoid Arthritis, Pyrexia of
unknown origin.
Blood Film WBC and Platelet Quantitative Estimation

• Total WBC count can be quantitatively estimated from blood films (under x
40 objective lens), also this estimation may be used for quality control
purposes, and as delta check for manual and automated WBC counts, follow the
table below for estimating WBC count in blood films:
Number of WBC cells seen per x40 field Estimated total WBC Count
2–4 4,000 – 6,000 /cumm
4–6 6,000 – 10,000 /cumm
51
6 – 10 10,000 – 13,000 /cumm

10 –13 13,000 – 20,000 /cumm
v Also WBC count can be estimated quantitatively from blood film, by the
following formula: Average number of nucleated cells per field at x100
magnification = nucleated cell count x 109/L.
v Platelets can be estimated quantitatively from blood films, each platelet seen
under oil immersion lens approximately equals to 20,000 PLT/cumm.
Normally, blood film from healthy individuals usually shows 7-22 platelets per oil
immersion field. When platelet aggregates or clumps are present in the blood film,
then platelet estimation would be absolutely unreliable.
Differential Tally Counter
52
Interpretation
Certain disease states are defined by an absolute increase or decrease in the number of a
particular type of cell in the bloodstream. For example:
Type Of Cell Increase Decrease

Red Blood Cells Erythrocytosis or Anemia or
(RBC) Polycythemia Erythroblastopenia
White Blood Cells
Leukocytosis Leukopenia
(WBC):
-- Lymphocytes -- Lymphocytosis -- Lymphocytopenia
-- Granulocytopenia or
-- Granulocytes: -- Granulocytosis
Agranulocytosis
-- --Neutrophils -- --Neutrophilia -- --Neutropenia
-- --Eosinophils -- --Eosinophilia -- --Eosinopenia
-- --Basophils -- --Basophilia -- --Basopenia
Platelets Thrombocytosis Thrombocytopenia
All Cell Lines - Pancytopenia
Granulocytes
Cells that contain large visible granules are sometimes called granulocytes. They
can be separated into 3 distinct cell lines, based on the reaction of the granules to
the most commonly used stain in Hematology, the Wright stain. The stain is a pH
based stain. Structures that favor the basic stain stain dark blue or basophilic;
while those that favor the acid stain, eosin, stain bright red-orange. Some
structures seem indifferent to the stain and are called neutral.
Neutrophils cells are usually between 50 - 70% of all of the

cells seen in a normal differential performed on an adult.
53
Cells whose granules stain bright red orange are called

eosinophils and are part of the allergic response. Those granules
contain histamine among other proteins. They should constitute
between
0 - 4% of a normal differential for an adult.
Cells whose granules stain dark blue are called basophils and
are also involved in allergic reactions.
Agranulocytes
Monocytes are a type of cross over cell. They are primarily

responsible for removing dead or damaged cells and constitute
less than 10% of the adult differential.
Lymphocytes are cells that do not contain large numbers of any

granules. They are responsible for producing antibodies against
foreign material such as antigens found in viruses and some
bacteria. They also are active against malignancy. In the adult,
they range between 20 and 45% of the cells seen in the
differential.
When a lymphocyte is actively defending against an antigen,

there will be changes seen in the cell and the cell is called a
"reactive" lymphocyte.
54
Red blood cells in the differential
Normal red cells should be monotonous. They should all be

about the same size, biconcave in shape, well filled with
hemoglobin, and be about the same color.
Changes in these descriptions are clues to many disease

processes. Changes are usually graded as slight/moderate/
marked or 1+, 2+, 3+, and 4+ with 4+ being the most unusual.
Terms that are used to describe these changes include: anisocytosis (changes in
size), poikilocytosis (changes in shape), hypochromia (less than adequate
hemoglobin) and polychromasia/polychromatophilia (changes in color).
Platelets in the differential

Platelets cannot be counted with any accuracy when
viewing the blood smear. They are only be estimated and no
comments about they ability to act in clotting can be made.
So you are left with comments such as adequate or appears
decreased/increased. Comments can be made about their
size but since platelets swell when they come into contact
with the anticoagulants used in the collection of blood for a
CBC, this may sometimes not be very important,
Reticulocyte count (Retic count)
Reticulocytes can correlate with the ability of the marrow to

produce red cells. Elevated retic’s may mean that he marrow is
capable of producing increased amounts of red cells. Decreased
retic counts may mean that there is some damage to the marrow’s
red cell making apparatus. This test aids in the diagnosis of
anemia and is an indicator of response to therapy for anemia.
Either blood from a lavender topped tube or fingerstick can be
used. Reticulocyte counts may be done manually at the microscope or by
automated methods. The normal range is approximately 0.5 - 2.0 %, but varies
with age and population.
55
Red blood cell distribution width
The red blood cell distribution width, or RDW, is a measure of the

variation of red blood cell (RBC) width that is reported as part of a standard
complete blood count. Usually red blood cells are a standard size of about 6-8μm.
Certain disorders, however, cause a significant variation in cell size. Higher RDW
values indicate greater variation in size. Normal reference range in human red
blood cells is 11 - 14%. If anemia is observed, RDW test results are often used
together with mean corpuscular volume (MCV) results to figure out what the cause
of the anemia might be. It is mainly used to differentiate an anemia of mixed
causes from an anemia of a single cause. Vitamin B12 deficiency produces a
macrocytic (large cell) anemia with a normal RDW. However, iron deficiency
anemia initially presents with a varied size distribution of red blood cells, and as
such shows an increased RDW. And in the case of a mixed iron and B12
deficiency we will have a mix of both large cells and small cells hence the RDW
will usually be elevated. An elevated RDW, i.e. red blood cells of unequal sizes, is
known as anisocytosis.
The "width" in RDW is actually misleading, since it in fact is a measure of

deviation of the volume of RBCs, and not directly the diameter.
Mathematically the RDW is calculated with the following formula:
RDW = (Standard deviation of MCV ÷ mean MCV) × 100
there is no known cause of decrease red cell distribution width.
56
Platelets (PLT)
Platelets are essential for the clotting of blood. One could think of them as
tying together the clotting mechanism and the damaged area. When platelets are
low, it may take longer for the blood to clot. When platelet counts are too high,
unnecessary blood clots may occur. Strangely enough, bleeding can also happen if
the platelets interfere with each other! Platelet function is affected by many drugs;
one of the most well known is aspirin. Easy bruising may be a sign of a decreased
platelet count. Bruising like Goldilocks’ companions comes in three "sizes".
The largest is called hematoma; it’s multicolored, raised, has very well defined
edges, and painful. The middle one is flat, usually red/blue and not well defined
edges. Many normal women get these on their thighs from bumping into furniture,
etc. The smallest is called petechiae. These looks something like freckles. They
occur in multiples, may be itchy, and are caused by very small blood vessel
damage. Petechiae can be seen in nosebleeds and gum bleeding.
57
Erythrocyte sedimentation rate
The erythrocyte sedimentation rate is a non-specific indicator of

inflammation. It is measured by the degree of settling of red blood cells in a
specific time period, usually one hour. There are several methods for determination
of the ESR; the most common are the Wintrobe and the Westergren, named for
the developers of the procedure. Blood for these procedures is drawn into either a
lavender or blue top tube, depending on the procedure. There is also an automated
method for determining the sedimentation rate. There is no special patient
preparation.
ESR are best used when comparing changes over time. A single test does not
give much usable information. Some things that can cause an elevated ESR include
exercise, arthritis, rheumatic fever, myocardial infarct (MI), infections, some
malignancies, menstruation, and normal pregnancy after the third month. The
normal range varies by the method used.
The erythrocyte sedimentation rate (ESR), also called a sedimentation rate or

Biernacki Reaction, is the rate at which red blood cells precipitate in a period of 1
hour. It is a common hematology test which is a non-specific measure of
inflammation.
To perform the test, anticoagulated blood is placed in an upright tube, known as a

Westergren tube, and the rate at which the red blood cells fall is measured and
reported in mm/h. There is also a wintrobe method.
Since the introduction of automated analyzers into the clinical laboratory, the ESR
test has been automatically performed.
The ESR is governed by the balance between pro-sedimentation factors,

mainly fibrinogen, and those factors resisting sedimentation, namely the negative
charge of the erythrocytes (zeta potential). When an inflammatory process is
present, the high proportion of fibrinogen in the blood causes red blood cells to
stick to each other. The red cells form stacks called 'rouleaux' which settle faster.
58
Rouleaux formation can also occur in association with some

lymphoproliferative disorders in which one or more immunoglobulins are secreted
in high amounts. Rouleaux formation can, however, be a normal physiological
finding in horses, cats and pigs.
The ESR is increased by any cause or focus of inflammation. The ESR is

increased in sickle cell anemia, and decreased in polycythemia, and congestive
heart failure. The basal ESR is slightly higher in females.
This test was invented in 1897 by the Polish doctor Edmund Biernacki. In
some parts of the world the test continues to be referred to as the Biernacki Test or
Westergren test, which uses sodium citrate-anticoagulated specimens.
Normal Values
Note: mm/hr. = millimeters per hour.
Westergren's original normal values (men 3mm and women 7mm) made no
allowance for a person's age and in 1967 it was confirmed that ESR values tend to
rise with age and to be generally higher in women. Values are increased in states of
anemia, and in black populations.
Adults
The widely used rule for calculating normal maximum ESR values in adults (98%
confidence limit) is given by a formula devised in 1983:
ESR reference ranges from a large 1996 study with weaker confidence limits:
Age (years)
(ESR 95% limits)
20 55 90
Men 10 14 19
Women 15 21 23
59
Blood Typing
Blood typing involves the two

types of molecules called antigens and
antibodies. When an antigen (foreign
substance) is present in the body, an
antibody reacts with it.
ABO System
The standard test to determine
ABO and Rh blood type consists of
putting a drop of anti-A antibodies,
anti-B antibodies, and anti-Rh
antibodies separately on a slide. To
each of these three antibody solutions, a drop of the person’s blood is added. a. If
agglutination occurs, as seen in the top right photo, the person has this antigen on
red blood cells. b. Several possible results. The ABO blood type is indicated by
using letters, and the Rh blood type is indicated by using the symbols (+) and (–).
60
Worked example
1. Hemoglobin (Hb)
2. Hematocrit (Hct)
3. Red Blood Cell Count (RBC)
4. RBC indices (Mean Cell Volume/MCV, Mean Cell Hemoglobin/ MCH,
Mean Cell Hemoglobin Concentration/MCHC)
MCV = Hct / RBC
MCH = Hb / RBC
MCHC = Hb / HCT
5. Relative or Red cell Distributive Width (RDW)

6. White Blood Cell Count (WBC)
7. Platelets (PLT)
8. Sedimentation Rate (ESR, Sed rate)
Ø Principle:
Ø Sample:
Ø Reagent and Supplies To Prepare Diluting Fluid:
Ø Diluting Fluid:
Ø Glassware, Apparatus, Equipment :
Ø Procedure:
Ø Calculations:
Ø Refere Range
Ø Sources of Error:
61
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Practical Hematology Dr.

Dr. Alaa-Eldin Salah-Eldin

Associated Professor of Molecular Biology and

Anticoagulants Used In The Hematology Laboratory

1] Ethylene Di-Amine Tetra-Acetic Acid (EDTA):

EDTA is the most commonly used anticoagulant in the hematology laboratory,

hematocrit (HCT), and subsequent increase in MCHC and decrease in MCV

• Heparin is used therapeutically as an in vivo anticoagulant.

No. Anticoagulant Hematology Laboratory Use Universal Color Code

Improved Neubauer Hemacytometer

specifications, it is very thick and non-flexible. There are many types of

Figure1: Top view of the hemacytometer

Figure 2: Cover glass position on the hemacytometer

Hemacytometer Counting Areas

Figure 3 - Red Blood Cells Counting Area

Using The Hemacytometer

The Counting Pattern

Figure 4 Counting Pattern

Calculating The Cell Counts

1. The average number of cells counted.

Hematology, also spelled haematology, is the branch of internal medicine,

Hematology includes the study of etiology, diagnosis, treatment, prognosis, and

In a clinical laboratory the hematology department performs numerous

derived values: Plasma osmolality · Serum osmolal gap

clotting factors: Prothrombin time · Partial

Hematology/ fibrinolysis: Euglobulin lysis time · D-dimer

ratios: Mean corpuscular hemoglobin · Mean corpuscular

viral infection: HIV (HIV test, BDNA test) · Epstein-Barr

protozoan infection: toxoplasmosis (Sabin-Feldman dye

Manual blood count

A complete blood count will normally include:

RED BLOOD CELL (RBC’s) COUNT

4. Continuing to hold the pipette as horizontal as possible, draw Ringer's solution

6. Empty ~1/2 of pipette into waste container

add a small amount of the diluted blood to one chamber of the

7. Let the preparation sit for a minute (for cells to settle).

Calculate the RBCs/cmm by adding the cells in the 5 groups and

Ringer's Solution, per 100 mL:

No. of RBC’s counted/

• No. of RBC’s counted 80 small square = ??? /

i.e. Factor = 10000

RBC’s Count/mm3 Blood = ??? X 10000

Hemacytometer Counting Areas

The Counting Pattern

Decrease in hemoglobin concentration beyond established normal ranges for

Drabkin’s solution contains the following:-

Hemoglobin + Potassium Ferricyanide Methemoglobin (Hi)

Methemoglobin + Potassium Cyanide Cyanomethemoglobin

Hemoglobin Concentration Determination

Whole blood is diluted in a solution containing Potassium Ferricyanide and

v Sample: EDTA anticoagulated venous whole blood.

v Working Drabkin’s Solution Preparation:

v Hemoglobin Standard Curve Preparation:

Hemoglobin Standard Curve

Preparation Of Sample Blank

Where N = ml of Drabkin’s to be added to 20 µl of patient plasma.

So, the sample blank is prepared by adding 20 µl of patient’s plasma to 10 ml

Hemoglobin concentration is expressed in g/dl (gram per deciliter)

Reference Hemoglobin Concentration Ranges:

Apparatus and Materials:

Buffy Coat Layer

Red Blood Cell Layer

Nowadays, Hct is supplied by the widely used automated hematology