Lab course

Hematology 1

1. Blood container, anticoagulant.

ANTICOAGULANTS:

A-Ethylene Diamine Tetra-acetic Acid (EDTA):

 This is the recommended anticoagulant for routine

hematological investigations
 Disodium and dipotassium salts of EDTA are in
common use
 It is used in a concentration of 1.5 mg/ml of blood.
 . Its mechanism of action is chelation of calcium.
 EDTA in excess of 2 mg/ml causes shrinkage of and
degenerative changes in red and white blood cells,
decrease in hematocrit, and increase in mean
corpuscular hemoglobin concentration. Excess EDTA
also causes swelling and fragmentation of platelets,
which leads to erroneously high platelet counts.
 EDTA is used for estimation of hemoglobin,
hematocrit, cell counts, making blood films, sickling
test, reticulocyte count, and hemoglobin electrophoresis
Prolonged storage of blood in EDTA anticoagulant
 B – Heparin

Heparin prevents coagulation by enhancing the activity of

antithrombin III (AT III).

 AT III inhibits thrombin and some other coagulation

factors.
 used in the proportion of 15-20 IU/ ml of blood.
 Sodium, lithium, or ammonium salt of heparin is used.
 Heparin should not be used for total leukocyte count
(since it causes leukocyte clumping) and for making of
blood films (since it imparts a blue background).
 It is used for osmotic fragility test (since it does not
alter the size of cells) and for immunophenotyping.
C -Trisodium Citrate (3.2%)
 This is the anticoagulant of choice for coagulation
studies .
 Use 1:9 (anticoagulant: blood) proportion for
coagulation studies.
D-Plain tubes
 (i.e. without any anticoagulant) are used for chemistry
studies after separation of serum:
 -liver function tests (total proteins, albumin, aspartate
aminotransferase, alanine aminotransferase, bilirubin),
---renal function tests (blood urea nitrogen, creatinine),
calcium, lipid profile, electrolytes, hormones, and
serum osmolality

Erythrocyte Sedimentation Rate

 The erythrocyte sedimentation rate (ESR)

measures the rate of settling (sedimentation) of

erythrocytes in anticoagulated whole blood.

Anticoagulated blood is allowed to stand in a glass

tube for 1 hour and the length of column of

plasma above the red cells is measured in

millimeters; this corresponds to ESR.

ESR test tube

Purpose Of The Test (Indications)
1. It can be done in the occult diseases.
2. For the diagnosis of acute and chronic infections.
3. For collagen vascular diseases.
4. In advanced malignancies.
5. In tissue necrosis and infarction.
6. ESR can be used to monitor disease therapy,
especially for autoimmune diseases. It will
correlate with the severity of the disease or with
the improvement in the disease course.
7. It is useful for the diagnosis and monitoring of
temporal arteritis, and polymyalgia rheumatica.
8. It is also used for monitoring of the Hodgkin’s
lymphoma.
Causes Of Raised ESR:
1. Bacterial infections in the abdomen, pelvic
inflammatory disease, syphilis, and pneumonia.
2. Chronic renal diseases.
3. Malignant diseases like Multiple myelomas,
Hodgkin’s disease, and advanced carcinomas.
4. Inflammatory diseases like temporal arteritis,
rheumatoid arthritis, rheumatic fever, and
systemic lupus erythematosus.
5. Necrotic diseases like Acute myocardial
infarction, gangrene, and necrotic tumors.
6. Tuberculosis.
7. Severe anemia like iron deficiency, and B12
deficiency.

Falsely Lowered ESR Level:
1. Sickle cell anemia.
2. Spherocytosis.
3. Hypofibrinogenemia.
4. Polycythemia.
Factors Affecting ESR:
1. RBCs factors:
1. In case of absence of rouleaux formation, it
will lead to a low ESR level conditions are
like:
1. Sickle cell anemia.
2. Spherocytosis.
3. Acanthocytosis.
2. Plasma factors:
1. In the case of increased protein will lead to
increased rouleaux formation and increased
ESR like:
1. Fibrinogen.
2. Immunoglobulins.
3. Mechanical factors:
1. It depends upon the caliber of the test tubes,
like Wintrobe tubes range from 0 to 100 mm
and has different values as a comparison to
the Westergreen method.
4. Anticoagulants factors:
1. Sodium citrate and EDTA have no effect on
the ESR.
2. Sodium or potassium oxalate shrinks the
RBCs.
3. Heparin also causes shrinkage of the RBCs
and gives rise to increased false ESR value.
4. So EDTA is the choice of anticoagulant.
Principle of ESR : 

This is directly proportional to RBCs mass, and

inversely to plasma viscosity.

1. In abnormal conditions, RBCs form more

rouleaux formation, so that is increased
RBCs mass, which increases the ESR due to
faster settling of RBCs.
2. There are two methods:
1. Wintrobe method.
2. Westergren method.
2. This is a nonspecific but not diagnostic test.
3. ESR is a measurement of the rate at which
the RBC settle in saline solution or plasma over a
specific time, usually one hour.
4. The ESR correlates with the plasma fibrinogen
level and depends on the rouleaux formation
of RBC.
 Manual WBC count.:
Total leukocyte count (TLC) refers to the number of white
blood cells in 1 μl of blood
There are two methods for estimation of TLC:
• Manual or microscopic method
• Automated method

MANUAL METHOD

Principle

A sample of whole blood is mixed with a diluent, which lyses

red cells and stains nuclei of white blood cells.
White blood cells are counted in a hemocytometer counting
chamber under the microscope and the result is expressed as
total number of leukocytes per μl of blood or per liter of
blood
Equipment
1. Hemocytometer or counting chamber with
coverglass:
The recommended hemocytometer is one with
improved Neubauer rulings and metallized surface.
There are two ruled areas on the surface of the
chamber .
Each ruled area is 3 mm × 3 mm in size and consists
of 9 large squares with each large square measuring
1 mm × 1 mm.
When the special thick coverglass is placed over the
ruled area, the volume occupied by the diluted blood
in each large square is 0.1 ml.

In the improved Neubauer chamber, the central large

square is divided into 25 squares, each of which is further
subdivided into 16 small squares.
A group of 16 small squares is separated by closely ruled
triple lines .
Metallized surface makes background rulings and cells
easily visible.
The 4 large corner squares are used for counting leukocytes,
while the central large square is used for counting platelets
and red blood cells.

Only special coverglass, which is intended for use with

hemocytometer, should be used. It should be thick and
optically flat.
 2. Pipette calibrated to deliver 20 μ l (0.02ml, ,

20cmm):

3. Graduated pipette, 1 ml.

4. Pasteur pipette

5. Test tube (75 × 12 mm)

Reagent
WBC diluting fluid (Turk’s fluid) consists of a weak acid

solution (which hemolyzes red cells) and gentian violet

(which stains leucocyte nuclei deep violet).

Its composition is as follows:

• Acetic acid, glacial 2 ml

• Gentian violet, 1% aqueous 1 ml

• Distilled water to make 100 ml

Specimen :

EDTA anticoagulated venous blood

Procedure for Manual White Blood

Cell Counts
Objectives: During this laboratory
exercise, the student will :
1. Perform and interpret the results of
three manual white blood
cell counts within + 15% of the value
obtained from an
automated hematology analyzer.
2. Follow all laboratory safety rules as
outlined in the MLS
student handbook.
Principle: Whole blood is diluted with
a weak acid solution that lyses red
blood
cells and dilutes the blood. The
remaining cells, WBCs and
nucleated RBC precursors and platelets
will not be lysed. From the
number of white blood cells counted,
the total number of white blood
cells per cubic millimeter can be
calculated.
Specimen: Well mixed whole blood
collected in an EDTA tube.
Reagents and Supplies:
Dilution vials containing 1.98 mls of
3% Acetic Acid
Micro-pipette to measure 20ul
Hemacytometer
Petri dish
Alcohol preps
Lens paper
Microscope
Procedure:
1. Add 20 ul of well-mixed anti-
coagulated blood into the vials
containing 1.98 mls of
3 % acetic acid.
2. Allow the blood-diluent mixture sit
together for 5 minutes. This allows for
adequate
lysing of the red blood cells.
3. Clean the hemacytometer and
coverslip with an alcohol prep and dry
them using
lint-free lens paper. Make sure there are
no water marks or lint on the chamber
or
coverslip.
4. Before charging the hemacytometer,
mix the blood-diluent mixture and then
draw
up a sample with a hematocrit tube.
Touch the filled hematocrit tube to the
edge
of the chamber and gently discharge
the diluted blood into the chamber. The
chamber will fill using capillary action.
DO NOT OVERFILL!
Procedure for Manual White Blood
Cell Counts
Objectives: During this laboratory
exercise, the student will :
1. Perform and interpret the results of
three manual white blood
cell counts within + 15% of the value
obtained from an
automated hematology analyzer.
2. Follow all laboratory safety rules as
outlined in the MLS
student handbook.
Principle: Whole blood is diluted with
a weak acid solution that lyses red
blood
cells and dilutes the blood. The
remaining cells, WBCs and
nucleated RBC precursors and platelets
will not be lysed. From the
number of white blood cells counted,
the total number of white blood
cells per cubic millimeter can be
calculated.
Specimen: Well mixed whole blood
collected in an EDTA tube.
Reagents and Supplies:
Dilution vials containing 1.98 mls of
3% Acetic Acid
Micro-pipette to measure 20ul
Hemacytometer
Petri dish
Alcohol preps
Lens paper
Microscope
Procedure:
1. Add 20 ul of well-mixed anti-
coagulated blood into the vials
containing 1.98 mls of
3 % acetic acid.
2. Allow the blood-diluent mixture sit
together for 5 minutes. This allows for
adequate
lysing of the red blood cells.
3. Clean the hemacytometer and
coverslip with an alcohol prep and dry
them using
lint-free lens paper. Make sure there are
no water marks or lint on the chamber
or
coverslip.
4. Before charging the hemacytometer,
mix the blood-diluent mixture and then
draw
up a sample with a hematocrit tube.
Touch the filled hematocrit tube to the
edge
of the chamber and gently discharge
the diluted blood into the chamber. The
chamber will fill using capillary action.
DO NOT OVERFILL!
Using the Neubauer chamber
1. Sample Preparation

 Depending on the type of sample, a preparation

of a dilution with a suitable concentration should
be prepared for cell counting. Typically, the
concentration range for a cell count with
Neubauer chamber is between 250,000 cells / ml
and 2.5 million cells / ml.

 An appropriate dilution of the mixture with regard

to the number of cells to be counted should be
used. If the sample is not diluted enough, the
cells will be too crowded and difficult to count.

 If it is too dilute, the sample size will not be

enough to make strong inferences about the
concentration in the original mixture.

2. Preparing Neubauer Chamber

 Clean the Neubauer chamber and the cover slip

with 70% EtOH. Put the glass cover on the
Neubauer chamber central area. Use a flat
surface to place the chamber, like a table or a
workbench.
3. Introducing the sample into the
Neubauer chamber

 With a pipette, carefully draw up around 20 ml of the

cell mixture (dilution). Place the pipette tip against the
edge of the coverglass and slowly expel the liquid
until the counting chamber is full.
 Capillary action will help to ensure that the counting
chamber is full, but care should be taken not to
overfill the chamber. A volume of 10 ml is sufficient to
fill one counting chamber
4. Microscope focusing and Cell Counting
Place the Neubauer chamber on the microscope stage. Using
the 10X objective, focus both onto the grid pattern and the
cell particles.
As 10X is appropriate for WBC counting, count the total
number of cells found in 4 large corner squares.
To count the RBCs and Platelets, the microscope must be
switched to 40X objective. Count the cells in the respective
areas as stated early.
Write down the amount of cells counted
If cells are touching the 4 perimeter sides of a corner
square, only count cells on 2 sides, either the 2 outer
sides or 2 inner sides.

Procedure for Manual White Blood

Cell Counts
Objectives: During this laboratory
exercise, the student will :
1. Perform and interpret the results of
three manual white blood
cell counts within + 15% of the value
obtained from an
automated hematology analyzer.
2. Follow all laboratory safety rules as
outlined in the MLS
student handbook.
Principle: Whole blood is diluted with
a weak acid solution that lyses red
blood
cells and dilutes the blood. The
remaining cells, WBCs and
nucleated RBC precursors and platelets
will not be lysed. From the
number of white blood cells counted,
the total number of white blood
cells per cubic millimeter can be
calculated.
Specimen: Well mixed whole blood
collected in an EDTA tube.
Reagents and Supplies:
Dilution vials containing 1.98 mls of
3% Acetic Acid
Micro-pipette to measure 20ul
Hemacytometer
Petri dish
Alcohol preps
Lens paper
Microscope
Procedure:
1. Add 20 ul of well-mixed anti-
coagulated blood into the vials
containing 1.98 mls of
3 % acetic acid.
2. Allow the blood-diluent mixture sit
together for 5 minutes. This allows for
adequate
lysing of the red blood cells.
3. Clean the hemacytometer and
coverslip with an alcohol prep and dry
them using
lint-free lens paper. Make sure there are
no water marks or lint on the chamber
or
coverslip.
4. Before charging the hemacytometer,
mix the blood-diluent mixture and then
draw
up a sample with a hematocrit tube.
Touch the filled hematocrit tube to the
edge
of the chamber and gently discharge
the diluted blood into the chamber. The
chamber will fill using capillary action.

Calculating the cell counts :

The total number of cells per microliter of sample can be
calculated from the number of cell counted and area
counted. This is because the ruled areas of the chamber
contain an exact volume of diluted sample.
Since only a small volume of diluted sample is counted, a
general formula must be used to convert the count into the
number of cells/microliter.

The dilution factor used in the formula is determined by the

blood dilution used in the cell count. The depth used in the
formula is always 0.1. The area counted will vary for each
type of cell count and is calculated using the dimensions of
the ruled area.

Estimation Hemoglobin
INDICATIONS FOR HEMOGLOBIN
ESTIMATION:

1. To determine presence and severity of

anemia.
 2. Screening for polycythemia:
3. To assess response to specific therapy in
anemia.
4. Estimation of red cell indices
5. Selection of blood donors.

METHODS FOR ESTIMATION OF

HEMOGLOBIN
1-Colorimetric methods:
In these methods, color comparison is made
between the standard and the test sample,
either visually or by colorimetric methods.:
• Visual methods: Tallqvist chart, Sahli’s
acid hematin method, and WHO hemoglobin
color scale.
• Photoelectric methods:
Cyanmethemoglobin (hemiglobincyanide)
method, oxyhemoglobin method, and
alkaline hematin method.

2-Gasometric method:
Oxygen-carrying capacity of blood is measured in a
Van Slyke apparatus. The amount of hemoglobin is
then derived from the formula that 1 gram of
hemoglobin carries 1.34 ml of oxygen. However, this
method measures only physiologically active
hemoglobin, which can carry oxygen.
 It does not measure carboxyhemoglobin,
sulfhemoglobin, and methemoglobin. Also, this
method is time-consuming and expensive. The
result is about 2% less than other methods

3-Chemical method:
Iron-content of hemoglobin is first estimated. Value
of hemoglobin is then derived indirectly from the
formula that 100 grams of hemoglobin contain 374
mg of iron. This method is tedious and time-
consuming

4-Specific gravity method:

A rough estimate of hemoglobin is obtained from the
specific gravity of blood as determined from copper
sulphate technique. This method is useful in mass
screening like selection of blood donors.

Tallqvist Hemoglobin Chart

Tallqvist hemoglobin chart consists of a series of

lithographed colors said to correspond to hemoglobin

values ranging from 10 to 100%. A drop of blood

obtained by finger puncture is placed on a piece of

absorbent paper. Color produced is matched against

the color on the chart and corresponding reading is

taken. Although this method is cheap and simple,

margin of error is 2050%.

Sahli’s Acid Hematin Method Principle:

Blood is mixed with an acid solution so that

hemoglobin is converted to brown-colored acid

hematin. This is then diluted with water till the brown

color matches that of the brown glass standard. The

hemoglobin value is read directly from the scale.

Equipment

1. Sahli’s hemoglobinometer: This consists of Sahli’s

graduated hemoglobin tube (marked in grams and

percent) and a comparator with a brown glass

standard.

2. Sahli’s pipette or hemoglobin pipette (marked at 20

μl or 0.02 ml).

3. Small glass rod (stirrer).

4. Dropping pipette.

Reagents

1. N/10 hydrochloric acid

2. Distilled water Specimen: EDTA-anticoagulated

venous blood
Method

1. Place N/10 hydrochloric acid into Sahli’s

graduated hemoglobin tube up to the mark

of 2 grams.

2- Take blood sample in Sahli’s pipette exactly

up to 20 μl mark. Blood adhering to the

exterior of the pipette is wiped away using

absorbent paper or gauze.

3. Add blood sample to the acid solution, mix

with a glass stirrer, and allow to stand for 10

minutes.

4. Add distilled water drop by drop till the

color of the solution matches that of the brown

glass standard.

5. Take the reading of the lower meniscus from

the graduated tube in grams.

WHO Hemoglobin Color Scale

This method is rapid, simple, inexpensive, and reliable

within 1 gram/dl for diagnosis of anemia.

The hemoglobin color scale consists of a printed set of

colors corresponding to hemoglobin values ranging

from 4-14 grams/dl .

A drop of blood is placed on a strip of

chromatography paper and the color developed is

matched visually against the printed color scale.

Hemoglobin color scale

Cyanmethemoglobin

(Hemiglobincyanide) Method

This is the method of choice for estimation of

hemoglobin and is recommended by International

Committee for Standardization in hematology.

 This is because

(i) all forms of hemoglobin are converted to

cyanmethemoglobin (except

sulfhemoglobin), and

(ii) (ii) a stable and reliable standard is

available.

Principle

Blood is mixed with a solution of potassium ferricyanide,

potassium cyanide and a non-ionic detergent (Drabkin’s

solution). Erythrocytes are lysed producing an evenly

distributed hemoglobin solution. Potassium ferricyanide

converts hemoglobin to methemoglobin, and methemoglobin

combines with potassium cyanide to form

cyanmethemoglobin (hemiglobincyanide). All forms of

hemoglobin present in blood are completely converted to a

single compound, cyanmethemoglobin. When the reaction is

completed, absorbance of the solution is measured in a

spectrophotometer at 540 nm. At this wavelength,

cyanmethemoglobin has a broad absorbance peak. To obtain

the amount of hemoglobin in the unknown sample, its

absorbance is compared with that of the standard

cyanmethemoglobin solution (the hemoglobin concentration

of which is known) by using a formula or a previously

prepared graph/table. The reaction is linear up to 20 gm/dl.

Equipment

1. Photoelectric colorimeter or spectrophotometer

2. Sahli’s pipette marked at 20 μl (20 cmm).

3. Pipette 5 ml.
Reagents

1. Drabkin’s solution (pH 7.0-7.4):

Potassium ferricyanide 200 mg

Potassium cyanide 50 mg

Potassium dihydrogen phosphate 140 mg

Non-ionic detergent 1 ml

Distilled water to 1000 ml.

2. Cyanmethemoglobin standard solution with known

hemoglobin value.

Specimen:

EDTA-anticoagulated venous blood .

Method
1. In a test tube, take 5 ml of Drabkin’s solution and to it

add 20 μl of blood (1:251 dilution). Stopper the tube, mix by

inverting several times, and allow to stand for at least 5

minutes. This time is adequate for conversion of hemoglobin

to cyanmethemoglobin.

2. Transfer the test sample to a cuvette. Read the absorbance

in a spectrophotometer at 540 nm or in a photoelectric

colorimeter using a yellow-green filter. Also take the

absorbance of the standard solution. Absorbance should be

read against reagent blank (Drabkin’s solution).

3. Hemoglobin value is derived from the formula given

below or from the previously prepared graph or table.

Preparation of graph and table:

If a graph or a table is prepared which correlates

absorbance with hemoglobin concentration, result can be

obtained quickly. This is particularly suitable when a large

number of samples are regularly processed on the same

instrument. Diluted cyanmethemoglobin standards are

available commercially for preparation of a calibration

graph. Alternatively, standard cyanmethemoglobin solution

is diluted serially with Drabkin’s solution. On a linear graph

paper, hemoglobin concentration (horizontal axis) in each

dilution is plotted against the absorbance (vertical axis). A

straight line joining the points and passing through the

origin is obtained. From this graph, a table can be prepared

relating absorbance to hemoglobin concentration .

REFERENCE RANGES (WORLD HEALTH

ORGANIZATION OF HEMOGLOBIN:

• Adult males: 13.0 - 17.0 gm/dl.

• Adult females (non-pregnant): 12.0 – 15.0 gm/dl.

• Adult females (pregnant): 11.0 - 14.0 gm/dl.

• Children, 6-12 years: 11.5 - 15.5 gm/dl.

• Children, 6 months to 6 years: 11.0 – 14.0 gm/dl. •

Children, 2 – 6 months: 9.5 – 14.0 gm/dl.

• At birth (full term): 13.6 – 19.6 gm/dl.

Packed Cell Volume :

is the volume occupied by the red cells when a sample of

anticoagulated blood is centrifuged.

 It indicates relative proportion of red cells to plasma.

 PCV is also called as hematocrit or erythrocyte volume

fraction.

 It is expressed either as a percentage of original volume

of blood or as a decimal fraction

USES OF PCV

• Detection of presence or absence of anemia or

polycythemia

• Estimation of red cell indices (mean cell volume and mean

corpuscular hemoglobin concentration)

• Checking accuracy of hemoglobin value (Hemoglobin in

grams/dl × 3 = PCV).
There are two methods for estimation of PCV: macro

method (Wintrobe method) and micro method

(microhematocrit method).

Micro method is preferred because it is rapid, convenient,

requires only a small amount of blood, capillary blood from

skin puncture can be used, and a large number of samples

can be tested at one time.

MICRO METHOD

Principle

Anticoagulated whole blood is centrifuged in a capillary tube

of uniform bore to pack the red cells. Centrifugation is done

in a special microhematocrit centrifuge till packing of red

cells is as complete as possible. The reading (length of

packed red cells and total length of the column) is taken

using a microhematocrit reader, a ruler, or arithmetic graph

paper
Equipment

1. Microhematocrit centrifuge: It should provide relative

centrifugal force of 12000 g for 5 minutes.

2. Capillary hematocrit tubes: These are disposable glass

tubes 75 mm in length and 1 mm in internal diameter They

are of two types: plain (containing no anticoagulant) and

heparinised (coated with a dried film of 2 units of heparin).

For plain tubes, anticoagulated venous blood is needed.

Heparinised tubes are used for blood obtained from skin

puncture.

3. Tube sealant like plastic sealant or modeling clay; if not

available, a spirit lamp for heat sealing.

4. Microhematocrit reader; if not available, a ruler or

arithmetic graph paper

Specimen

Venous blood collected in EDTA (dipotassium salt) for plain

tubes or blood from skin puncture collected directly in

heparinised tubes. Venous blood should be collected with

minimal stasis to avoid hemoconcentration and false rise in

PCV

Method

1. Fill the capillary tube by applying its tip to the blood

(either from skin puncture or anticoagulated venous

blood, depending on the type of tube used). About

2/3rds to 3/4ths length of the capillary tube should be

filled with blood

2. Seal the other end of the capillary tube (which was not

in contact with blood) with a plastic sealant. If it is not

available, heat-seal the tube using a spirit lamp.

3. The filled tubes are placed in the radial grooves of the

centrifuge with the sealed ends toward the outer rim

gasket. Counterbalance by placing the tubes in the

grooves opposite to each other.

4. Centrifuge at relative centrifugal force 12000 g for 5

minutes to completely pack the red cells.

5. Immediately remove the tubes from the centrifuge and

stand them upright. The tube will show three layers

from top to bottom: column of plasma, thin layer of

buffy coat, and column of red cells

 6-With the microhematocrit reader, hematocrit is

directly read from the scale. If hematocrit reader is not

available, the tube is held against a ruler and the

hematocrit is obtained by the following formula :

To obtain PCV,

the above result is multiplied by 100.

REFERENCE RANGES

• Adult males: 40-50%

• Adult females (nonpregnant): 38-45%

• Adult females (pregnant): 36-42%

• Children 6 to 12 years: 37-46%

• Children 6 months to 6 years: 36-42%

• Infants 2 to 6 months: 32-42%

• Newborns: 44-60%