Practical Physiology for 1st prof

(Hematology lab technology)

Dr Vivek Nalgirkar
Professor (Physiology)

 Section 1: Collection of blood sample

 Section 2: Use of a microscope
 Section 3: Hemoglobin estimation
 Section 4: Neubauer’s chamber; dilution pipettes
 Section 5: Total leucocyte count (TLC)
 Section 6: Total RBC count
 Section 7: Preparation of blood smear; identification of cellular elements of blood
 Section 8: Differential leucocyte count (DLC)
 Section 9: Arneth count; Absolute white cell counts
 Section 10: Bleeding time
 Section 11: Clotting time
 Section 12: Blood group identification
 Section 13: Blood bank visit; Blood transfusion
 Section 14: Platelet count
 Section 15: Reticulocyte count
 Section 16: Packed cell volume (PCV)
 Section 17: Erythrocyte sedimentation rate (ESR)
 Section 18: Specific gravity of blood
 Section 19: Osmotic fragility test

2
 Section 1
COLLECTION OF BLOOD SAMPLE

 Introduction:
 Obtaining a blood sample is the first important step in hematologic
investigations.
 Blood samples are collected from veins or capillaries; for some
investigations, such as arterial blood gas (ABG), samples have to be collected
from an artery.
 Finding a suitable vein, inserting a needle into it, collection & handling of the
sample require a certain degree of skill.
 Aims & Objectives:
 To learn the various methods of collection of blood samples.
 Materials & Methods:
Lancets, disposable syringe and needle (no. 22G)*, gloves, spirit and cotton (cotton
swabs soaked in 70% isopropyl alcohol), sample containers  a glass bulb or
vacutainer filled with anticoagulant, adhesive dressing tape

* Needles with different gauges (G) have different diameters. As per the standards
established by International Organization for Standardization  19 G = 1.1 mm, 21 G = 0.8
mm, 23 G = 0.6 mm.
- 18G  collection of blood for storage & transfusion
- 20G  collection of smaller samples
For parenteral administration of medication: 22G/23G - for intramuscular injection, 24G -
subcutaneous injection, 26G - for intradermal injection

Blood sample collection:

The samples can be collected from: (a) capillary blood, or (b) venous blood.

Comparison between venous blood and capillary blood:

Capillary blood is obtained by skin puncture. In true sense, it does not remain a capillary
blood sample. Blood from arterioles and venules gets mixed in it. In addition, it may also
contain some interstitial fluid, particularly if the site is squeezed or pressurized while
obtaining the sample.
(a) In capillary blood the PCV is higher. Reason: PCV is the ratio of cells to plasma. As a
matter of fluid dynamics, some amount of plasma fluid leaks out of the capillaries.
Thus, the ratio of cells to plasma would be slightly greater in the capillaries.
(b) In venous blood, Mean corpuscular volume (MCV) of the red cells is slightly
greater. Reason: When blood leaves capillaries and enters venous compartment, a
phenomenon called “chloride shift” has already occurred. That is, chloride from
plasma enters the red cells. Chloride may pull some water into red cells by
osmosis. Consequently, the cell would swell slightly.
(c) Platelet count is lower in capillary blood. Reason: With skin puncture, platelets
adhere to the site of injury, giving a relatively low count in capillary blood sample.

3
Collection of a sample:

(A) Capillary blood 

- Ring finger is the preferred site for skin puncture, for collection of the capillary blood
sample. Ear lobe, sole of the foot (heel) are some of the other sites from where a
sample can be obtained, particularly in infants.
 Ring finger is preferred as it is the least used among the fingers; chances of
infection, after the skin puncture, are less.
 Lateral aspect of the pulp of the finger, being less sensitive, is where skin is
punctured.
Procedure:
- The site should be sterilized with cotton swab soaked in spirit (70% alcohol).
- Allow the site to dry and then puncture the skin with a sterile needle or lancet. The
injury should be 2-3 mm deep. There should be a spontaneous flow of blood. If
necessary, squeeze the finger very gently. (Note: If the finger is milked or squeezed
very firmly, the tissue fluid will get mixed with the capillary blood and the test results
will be unreliable.)
- First drop of blood should be wiped away.
- The next few drops of blood may be collected as per the desired amount for the
specific test. For instance, to prepare blood film, 1-2 drops are sufficient. For Hb
estimation by Sahli’s method, relatively more quantity of blood is needed. The
collected blood may be transferred to a container, filled with an anticoagulant, for
tests to be performed at a later time. Or, test(s) may be performed immediately.
(B) Venous blood 
- Phlebotomy is the procedure of drawing blood from a vein. It has also been termed
venepuncture.
- The most common site for venepuncture is the antecubital vein or any other visible
vein in the forearm. Next preferred site is the veins in the dorsum of hand.
- Blood sample can be collected in a plastic disposable syringe (5 mL), by a sterile
needle attached to it directly or a winged (“butterfly”) needle with a plastic tubing
that can be attached to the nozzle of the syringe. Alternately, blood may be collected
in evacuated tube (vacutainer) filled with an anticoagulant. The tube has needle at
its open end. The end is sealed with a needle holder that secures the needle to the
tube.
Procedure:
- A tourniquet is tightly applied just above the venepuncture site. Since the tourniquet
pressurizes and obstructs the blood flow, the veins below it become engorged and
prominent; finding and piercing the vein becomes easier. [Note: The tourniquet
should be removed immediately once the blood starts flowing out into the
syringe/vacutainer. Longer application of tourniquet results in plasma fluid to shift
out of the engorged vein. Consequently, a possible hemoconcentration may lead to
erroneous results.]

4
 - The part overlying the vein may be tapped gently for a few times. This makes the
area little warm; local blood flow increases slightly and the vein becomes more
visible.
- The area of skin should be sterilized with spirit (70% alcohol). Allow the site to dry
up. Insert the needle gently and obliquely into the vein. The pointed tip of the
needle should be facing up while piercing the skin. This makes the pricking instance
less painful.
- Let the blood fill into the syringe; do not attempt to draw it forcefully.
- Without any delay, the blood should be transferred to the bulb filled with
anticoagulant. To mix the blood with anticoagulant, shake the bulb very lightly.
- Once the sample is collected, remove the needle and keep a cotton swab firmly
pressed against the puncture site. [The swab should be sterile and dry. A spirit-
soaked swab may cause local vasodilation, resulting in excess oozing of blood after
the sample has been collected.] Apply a small adhesive tape to the site of
venepuncture.
 Arterial blood samples are obtained for certain investigations, for instance, the arterial
blood gas (ABG) analysis. Femoral artery is commonly used for the purpose.

Reasons for erroneous results:

(A) Before sample collection:
- food intake (for instance, it will influence the blood glucose estimation)
- Excess water intake (up to an hour prior to the collection of sample) - a
hypotonic plasma may give wrong results for some tests, such as serum
electrolytes, osmolality of plasma
- Physical activity (half an hour prior to collection) - shift of water out of the
capillaries; sweating resulting in loss of ECF water
- Drugs: e.g. opioids, barbiturates influence the TSH levels
(B) During sample collection:
- Excessive/prolonged tourniquet pressure  plasma fluid leaks out of vessels,
leading to hemoconcentration.
- Forcefully drawing out the blood into syringe  excess negative pressure in the
syringe  plasma may get drawn in excess over cells, giving a picture of
hemodilution
- Diurnal variations  e.g. steroid levels are high in morning and low in evening;
tests related to steroid levels in the body will show erroneous results if samples
collected during different times of the day
(C) After the collection of sample:
- Excess anticoagulant
- Inadequate anticoagulant
- Improper storage conditions (e.g. temperature)

5
Storage of blood:
- The collected blood samples are stored in refrigerator, at 40C.
- There is no major effect of storage on the cell counts of the blood samples.
- Platelets rapidly lose their survivability in stored blood. It is therefore advisable to
carry out platelet count as soon as possible.
- Leucocyte count (and especially the absolute lymphocyte count) shows a gradual
decline beyond 24 hours of storage.
- RBCs begin to swell; PCV and MCV will increase in the stored blood. Osmotic fragility
also increases; ESR decreases.

 Anticoagulant: The substance which, when added to blood sample, prevents clotting
of the blood.
NOTE: When tests are to be performed on whole blood, anticoagulant is added to
the sample. However, certain tests or estimations are to be done on serum. For such
tests, anticoagulant is not added to the sample; blood is allowed to clot. Then, the
supernatant serum is separated from the clot, and the tests are performed.

Ca++ (ionic calcium) has a role in almost every step of clotting. One of the mechanisms by
which clotting can be prevented is to make the Ca++ unavailable.

6
  Anticoagulants used commonly:
1. Citrates: (trisodium citrate)
 Mechanism of action: Converts Ca++ in the blood sample into non-ionized form
(Ca++ is required for most steps in clotting)
 Used as anticoagulant in: coagulation studies, ESR estimation using Westergren
tube, and for storage of blood for transfusion purpose.
(NOTE: While doing coagulation studies, the ratio of blood to citrate volume should be 9:1. That is, 9
volumes of blood should be added to 1 volume of sodium citrate. The ratio is absolutely very important
because – (a) citrates may exert osmotic effects, and (b) by chelation or precipitation of Ca ++, they may
alter free Ca++ concentration significantly. Both these effects may influence results in the coagulation
studies.
For ESR estimation, the ratio of blood sample to sodium citrate should be 4:1 volumes.)
2. Oxalates: (double oxalate)
 Double oxalate is a mixture of ammonium oxalate (3 parts) + potassium oxalate
(2 parts).
 Mechanism of action: precipitation of Ca++ as insoluble oxalate crystals
 Used in: PCV & ESR by Wintrobe’s tube. (Wintrobe’s tube is smaller as compared
to Westergren tube; the amount of blood sample taken is lesser for Wintrobe
method. Even a small change in red cell volume, possibly caused by an
anticoagulant, may affect the test results. Double oxalate is a mixture of
ammonium & potassium salts; swelling of cells by one salt is balanced out by
shrinkage of cells by the other. The net effect is – no change in cell size.)
3. Ethylene diamine tetra acetic acid (EDTA):
 Sodium and potassium salts of EDTA are strong anticoagulants
 Mechanism of action: chelation of Ca++ in blood sample.
 Used in: Routine hematology lab tests; anticoagulant of choice for cell counts.
 Pitfalls: Excess EDTA causes shrinkage of RBCs and WBCs. On the other hand,
excess EDTA may cause swelling of platelets; chipping off and fragmentation of
platelets may give an erroneously high platelet count (since the fragments are
large, even they get counted as platelets).
4. Heparin:
 Mechanism of action: Binds to antithrombin; inhibits interactions among several
clotting factors
 Anticoagulant of choice for osmotic fragility testing for red cells. Reason: Heparin
does not alter cell size; chances of lysis (due to anticoagulant) will be minimum.
 Causes clumping of WBCs and platelets (not suitable for cell counts); gives a faint
blue color to the background if blood films are stained by Romanowsky’s stains
(not suitable for making peripheral smears).

7
 Section 2
Use of a microscope

 Introduction:
 Many important lab investigations involve use of a microscope. In fact, a
microscope is the most critical component of many of the lab investigations. Not
only for counting of cells, but correct identification of cells requires a proficient
handling of microscope.
 Identification of WBCs, red cell inclusion bodies, platelets & reticulocytes
counting need a skilled use of the microscope.

 Aims & Objectives:

(1) To get familiar with the components of a microscope,
(2) To learn how to use the microscope in the most effective manner for various
types of objects/specimens
 Materials & Methods:
 There are different varieties of microscopes; each type has its advantages.
E.g. light microscope, phase contrast microscope, fluorescence microscope,
electron microscope, and so on.
 The routine haematology laboratory procedures involve use of the
conventional light microscope; it is a monocular compound microscope.
A. The lens system of a compound microscope: It has two sets of lenses  (a) the eye
piece or ocular lens, and (b) the objectives.
o The objective lens forms a real image of the object.
o This image is then magnified by the eyepiece. What is finally observed is a
virtual inverted & enlarged image.
(a) The eye piece: It has a magnifying power of 10 X.
(b) The objective: The 3 objective lenses have magnifying powers of 10 X, 45 X, and
100 X. They are also referred to as low power, high power, and oil immersion,
respectively.
 Oil immersion lens:
 Its magnification power is 100 X.
 The ‘Cedar wood’ oil is first placed on the slide/specimen. The
objective is then lowered slowly so that oil immersion lens just
touches the the drop of oil on the slide.
When light rays reflected from the mirror have passed through the glass slide, they
enter the gap (air) between the slide and the objective lens. This causes an
additional refraction of the rays before they enter the objective. Cedar wood oil has
the same refractive index as that of the glass. As the objective lens touches the drop
of the oil, the glass slide, the oil, and the lens are in contact so as to make one single
medium. Thus, there is no additional refraction of the light rays; this helps to form a
clear and better image. It is the same principle that is applicable to the use of contact
lenses for vision.

8
  Magnification: The extent of magnification of the specimen being observed is the
product of the eye piece and the objective in use. For instance, when objective of 45
X is being used, total magnification would be 45 X 10 = 450 X.
 Note: A revolving nose piece holds the objective lenses. It is designed to be
‘parfocal’. That is, when the nose piece is rotated to change objective lens from one
power to another, the specimen remains in focus.
 The focus knob: To bring the specimen into sharp focus, two ‘adjustment’ screws are
provided – one called “coarse adjustment” and the other called “fine adjustment”.
One complete rotation (through 3600) of the fine adjustment knob moves the tube
by 0.1 mm. 1 complete rotation of the coarse adjustment moves the tube by 10 mm.
B. ILLUMINATION & CONTRAST: Mirror, condenser & iris diaphragm:


Only a small area on the slide should be illuminated at a time. This is achieved by
the mirror, condenser & iris diaphragm at the bottom of the microscope.
I. Mirror: Concave & plane.
As a general principle, the concave mirror should be used with low
power; the plane mirror can be used with any objective.
II. Condenser: As the parallel rays of light get reflected from the
mirror, condenser converts them into a solid cone of light.
III. Iris diaphragm: It determines the amount of light falling on the
slide, thereby providing various degrees of contrast.
Note: For some of the hematology experiments use of mirror, condenser position, and
contrast are important issues. Particularly, in the practical of TLC the leucocytes appear as
small dots and will be visible when the illumination & contrast is proper.

 Common errors:
 After using course & fine adjustment, if the components on the slide are not clearly
visible, students tend to give up. Use of condenser & iris diaphragm is either
ignored or forgotten. Keep in mind : If after all the efforts to focus a slide, the cells
are still not clearly visible then think of the condenser and iris diaphragm.
 Special precautions:
 Do not change the powers while looking into the microscope.
 When using oil immersion, if the objective needs to be lowered, the movement
should be gentle, with an eye on the distance between the slide and the lens.

9
 Section 3
Hemoglobin estimation

 Introduction:
 This investigation is ordered when a clinician suspects a patient to be
suffering from anemia. A patient with anemia presents with pallor, easy
fatiguability, shortness of breath, etc.
 Anemia is very common in developing countries.
 Oxygen transport to the tissues is the job of hemoglobin contained within the
RBCs. Reduced RBC count and/or reduced Hb levels would result in anemia,
defined as a “decrease in the oxygen-carrying capacity of blood”. Obviously,
the most important investigations in the diagnosis of anemia would be RBC
count and Hb estimation.
 Hb estimation is of critical importance on two counts: (1) Anemia diagnosis
and grading of its severity, (2) the treatment modality and follow up.
Normal Hb levels: 14-18 gms% (males); 12-16 gms% (females)
Hb < 10 gms% is considered as anemia.
 All the methods of Hb estimation, whether basic or advanced, have one thing in
common – all of them are based on matching of the colour. The colour of Hb or
that of compounds formed from Hb is matched with the standards.

 Aims & Objectives:

 To detect the Hemoglobin content (in gms% and percentage) of the given blood
sample.
 Use of Hb in percentage for calculation of color index
 Learn the importance of Hb estimation in the context of anemia.
 Materials and method:
 The material for collection of blood sample – needle or lancet, cotton swab
soaked in spirit
 N/10 HCl, distilled water
 Hb pipette with a mark at 20 mm3
 Sahli’s hemometer tube with markings of gms% on one side and of percentage
on the other side
 A permanent coloured glass standard rack
 A stirrer

10
[Fig: The Hb pipette.]

[Fig: Sahli’s hemometer.]

o Principle:
Hemoglobin is converted into a stable compound (acid hematin), and its color is
matched with the standard.

11
 NOTE: Since the color of acid hematin (and not of hemoglobin) is being matched
with standards, this method is considered to be an ‘indirect’ method.

Procedure:

o Fill N/10 HCl into the hemometer tube up to 20% mark. Place the tube between the
glass standards.
o Take the blood sample in the Hb pipette up to 20 cmm mark. Transfer this blood
sample to the hemometer tube. The blood sample and HCl are mixed well by using a
stirrer.
o Wait for about 8-10 minutes. All the Hb present in the blood sample will get
converted to acid hematin during this time.
o Distilled water is then added drop by drop to the mixture. Stir the mixture regularly
and gently. After the addition of each drop, the stirrer should be lifted out of the
solution to match the color of the mixture with that of the glass standards. The rack
should be held against a good natural light to compare the colors.
o Continue the addition of drops till the colors match.

 Common errors; Special precautions:

While transferring the blood sample from Hb pipette to the
hemometer tube: Make sure that the pipette tip reaches as far
down, into the HCl, as possible. Students tend to blow the
blood sample out into the tube from its top. Even if a single
drop of the blood sample sticks to the walls of the hemometer
tube, it may not get included in the formation of acid hematin.
The eventual result would be an erroneous lower Hb level.
 While matching the colors: The stirrer should be removed
completely out of the mixture before taking the reading. Also,
ensure that the stirrer is completely drained of drops that may
possibly be sticking to it.

 RESULT:
 When the colors are apparently matching, note the reading. Add one more drop.
After the addition of this drop - (a) If the color of the mixture becomes lighter,
take the previous reading as the result, (b) if the color appears to match, take the
reading now as the final result.
 Take the lowest point of the meniscus for reading.
 Result: Hemoglobin in gms% (on one side of the Hb tube) and percent of Hb (on
the other side of the tube).

12
 Note: 14.5 gms% corresponds with 100%. It means, a person with Hb 14.5 gms% is
considered to have 100% Hb. Hemoglobin in gms% is the basis for diagnosis and
grading of anemia; hemoglobin in percentage is of use in calculation of color index,
from which the anemia can be classified morphologically. (Discussed later)
 Discussion:
(A) Technical aspect:
1. About N/10 HCl:
 Taken up to 20% mark. This quantity is needed to convert all the Hb
present in the blood sample into acid hematin.
 What if the quantity is less? Ans:- All the Hb present in the blood
sample will not be converted to acid hematin. The eventual result will
be erroneously low.
 What if the quantity is more than 20%? Ans:- It won’t affect the result.
Once all the Hb is converted to acid hematin, the excess HCl will
simply act as the diluent. In the place of distilled water, as diluent, HCl
can be used. Only in the case of severe anemia, HCl used as diluent will
influence the results.
 Why can’t we use HNO3 or H2SO4 instead of HCl? Ans:- These are
strong acids having corrosive/oxidizing properties. They will not form
a colored acid hematin compound. (Even HCl used is of decinormal
strength.)
2. Inherent errors in Sahli’s method:
a. There are various forms of hemoglobin in RBCs. Some forms of
Hb (such as, sulphemoglobin and methemoglobin) do not get
converted into acid hematin. Thus, Sahli’s method gives a
lower estimate of Hb. The result could be 2-5% less than the
actual Hb present in blood.
b. The compound acid hematin itself is not very stable. Within
the 10 minutes that we wait for its formation, its color reaches
peak and may have begun fading. By the time we note the
reading, the fading color may yield a lower result.
3. Cyanmethemoglobin method: The best method for Hb estimation; it
overcomes both handicaps of Sahli’s method.
 Blood sample is made to react with Drabkin’s reagent that contains a
mixture of potassium cyanide and ferricyanide.
(i) All forms Hb (except sulphemoglobin) in the blood sample get
converted to cyanmethemoglobin or hemoglobincyanide.
(ii) This compound has a very stable color.
4. Other methods for Hb estimation:
 Alkali hematin method – addition of an alkali converts Hb to alkali
hematin.

13
  Talliquist method – a drop of is taken on a blotting paper and is
compared with the color chart depicting colors of different levels of
Hb.
 Oxyhemoglobin method – Blood sample is washed with ammonia; the
solution of HbO2 is formed. Its absorbance is read in a spectrometer at
540 nm or photometer using a yellow-green filter.
 Methods based on estimation of iron content – These are considered
to be most accurate. Iron content of red cells is measured, and then
Hb level is derived from it as follows: 0.347 gm of iron = 100 gm of
Hb.
5. Inherent shortcoming of the methods based on matching of the colours:
 Some substance in plasma may influence the colour of the compound
being formed in the method, and thus give erroneous results.
E.g. High levels of plasma bilirubin will impart yellow colour to the
compound; plasma proteins & lipids can cause turbidity in the
compound.
 Some methods, instead of a freshly prepared standard compound,
employ the permanent coloured glass standards. E.g. Sahli’s method.
The colours of the standards may fade over a period of time.

(B) Physiology point-of-view:

1) What are the normal Hb levels?

 14-18 gm% (men); 12-16 gm% (women).
 In neonates ~ 16-20 gm%. Neonates suffer from a relative hypoxia
(no more placenta for gas exchange, and lungs are yet to be fully
functional); hence the higher red cell count and higher Hb level, in
response to the hypoxic condition.
 Women have a lower RBC count as compared to men (discussed
later); consequently, women have a lower Hb level.
2) Oxygen carrying capacity of blood:
 1 gm Hb (when fully saturated with O2) carries 1.34 mL of oxygen. If,
for instance, a person’s Hb is 15 gms%, the O2 carrying capacity would
be – (15 X 1.34 =) 20.1 mL/100 mL of blood.
3) Anemia: Classification; Significance of Hb estimation:
 Anemia can be classified in various ways –
a) Clinical classification: (discussed above)
b) Morphological classification:
I. Based on RBC size – Microcytic anemia (iron deficiency),
Normocytic (blood loss), macrocytic (vit. B12 and/or folic
acid deficiency)
II. Based on Hb/iron content (color of a red cell is due to its
Hb content) – Hypochromic anemia (iron deficiency);
Normochromic anemia (hemolysis, blood loss)

14
 c) Etiological classification:
i. Anemia due to excess blood loss – acute, chronic
ii. Anemia due to excessive RBC destruction – Congenital
defects (e.g. Spherocytosis); Acquired (e.g. Malaria)
iii. Anemia due to impaired RBC production – protein
deficiency, iron deficiency, bone marrow depression
caused by drugs or radiation
(C) Clinical perspective:

1. What is the clinical importance of Hb estimation?

 As discussed previously, Hb estimation has 2 important applications –
(a) In the diagnosis of anemia, (b) to decide the treatment strategy in
the case of anemia.
 Clinical classification of anemia is based on the Hb levels. A patient
with Hb < 10 gm% is diagnosed as having anemia.

Hb level (gms%) Grade of anemia Treatment advised

10-8 Mild Oral iron therapy
8-6 Moderate Injectable iron
Below 6 Severe Blood transfusion

Utility of Hb estimation in percentage:

RBC count of 5 million/mm3 is considered 100%; Hb level of 15
gms% is taken as 100%.
Patient’s RBC count and Hb level is measured, and the values
are converted to percentage.
 Color index = Hb(%)
RBC(%)
Normal range: 0.8-1

A value less than 0.8 indicates hypochromic anemia;

A value between 0.8 and 1 means normochromic anemia.

15
 Advances in lab technology:
Some methods that make use of technology, and are quicker and yield more accurate
results –
1. Direct spectrometry:
 The Hb of a diluted blood sample is determined by a spectrophotometer.
 The blood sample is diluted with cyanide-ferricyanide reagent, and the
absorbance is measured at 540 nm.
 This method does not need a standard for comparison. However, the
spectrometer should be calibrated correctly.
2. Portable hemoglobinometers:
 These hemoglobinometers have a precalibrated scale for direct reading of
Hb.
 They have built-in filters and battery-operated spectrometers.

16
 Section 4
Neubauer’s chamber; Dilution pipettes

 Introduction:
 Blood cell counts range from a few thousands (e.g., white cells) to a
few millions (e.g., red cells) per mm3 of blood. To count these cells
manually, specially designed counting chambers are required.
 It is practically impossible to manually count the cells that are so
crowded in a cubic mm of blood. Blood samples are collected in the
pipettes that are calibrated so that a certain volume of blood can be
diluted to 20 X or 200 X. Dilution of blood implies dispersion of cells so
that they can be counted manually under microscope. If, for instance,
cells are dispersed by 20 X dilution, the cell count will be 20 times less.
Hence, after the cell count per mm3 is done it has to be multiplied by
20 to arrive at the final cell count.
 Aims & Objectives:
1. Learn to focus the counting chamber with Neubauer’s rulings (simply
called Neubauer’s chamber)
2. Learn the skill of charging of the chamber
3. Calculations associated with counting chamber and dilution pipettes.

 Materials & methods:

 Microscope
 Neubauer’s chamber
 Dilution pipettes

Principle:
More the crowding of the cells per unit area, more is the dilution needed to disperse them.
Thus, for WBC count, blood sample is diluted 20 times whereas for RBC count the dilution is
200 times. Also, the cells which are more crowded are counted in a relatively smaller unit
volume and then the results are extrapolated. For instance, In the case of WBCs, the count is
done in a volume of 4/10 mm3 and then the results are extrapolated for WBCs per mm3 of
blood. RBCs are counted in 1/50 mm3 space and then the per mm3 result is derived.
Let’s take a simple example. If you need to count the number of plants in a garden, count
them in a small unit area and then extrapolate the entire count. However, if you have to
count grass blades in the garden, you will have to make an even smaller unit area for
counting as they are much more crowded.

 The Neubauer chamber:

17
[Fig: Improved Neubauer rulings.]
Naked eye appearance:
 There are grooves that make an ‘H’. This ‘H’ shaped region is slightly elevated from
the rest of the slide. The region contained within ‘H’ is a metallised depressed region
or the counting chamber. On either side of the transverse groove lie the Neubauer’s
rulings.
Depth of the chamber: (The ‘H’ shaped region is an elevated platform so that it can be
easily identifiable as having the counting chambers. This platform itself is slightly
depressed so that when a coverslip is placed onto the chamber, a depth is created
between the coverslip and the platform.)
When an optically plane coverslip is placed on the counting chamber, a depth of 0.1 mm
or 1/10 mm is created.
Look horizontally along the surface of the cover slip. If the chamber and the cover slip
are clean and properly aligned, concentric colored rings will be visible. These are the
so-called “Newtonian rings”.
 Charging of the Neubauer’s chamber: The pipette with diluted blood sample is
brought near the edge of the counting chamber. With the pipette being held very
close to the edge at angle of about 45 degrees, its tip is gently touched to the narrow
slit between the Neubauer’s chamber and the coverslip. A drop of the sample flows
between the coverslip and the chamber by capillary action. [NOTE: Do not try to
blow the sample out of the pipette.]
Procedure:
 Focus the Neubauer’s chamber under low & high powers.
a. When viewed under low power: The entire ruled area is visible; it has dimensions of
3 mm X 3 mm (total area = 9 mm2).
The entire area is divided into 9 equal squares. Thus, each square has dimensions of
1 mm X 1 mm (area = 1 mm2).

18
 Of these 9 squares, the 4 squares on the four corners are WBC squares; the total
WBC count is done at these squares. (NOTE: Each of these squares are further
subdivided into 16 equal small squares.)
The central square is the RBC square.

[Fig: Neubauer’s chamber under low power.]

b. View under high power – RBC square: The central square meant for RBC count, with
an area of 1 mm2, is divided into 25 equal squares. Each of these small squares have
dimensions of 1/5 mm X 1/5 mm (area = 1/25 mm 2). (NOTE: Each of these small
squares are further subdivided into 16 equal small squares. The small subdivisions, in
RBC & WBC counting fields, are not taken into account during calculations. They only
provide for a disciplined counting.)
Also note – The 25 equal squares are demarcated and separated from each other by
closely ruled triple lines.

19
[Fig: Neubauer’s chamber under high power; RBC squares.]

“Improved” Neubauer’s chamber:

 In the original Neubauer’s chamber, the central square (1 mm X 1 mm) for RBC count
was divided into 16 equal squares. In the “improved” Neubauer’s chamber, the
central square is divided into 25 equal squares.
 In the “improved” Neubauer’s chamber, the 25 squares are separated by triple
rulings that are very closely lined.

20
[fig: Original Neubauer rulings (left) and improved Neubauer rulings (right).]
[The original and the “improved” Neubauer chamber: In the original Neubauer chamber,
the central ruled area for RBC counting was of 1 mm X 1 mm. It was further divided into 16
squares. These squares were separated from one another by triple rulings. In the
“improved” chamber, the central RBC square (1 mm X 1 mm) is divided into 25 squares. The
triple rulings that separate these 25 squares from each other are closely ruled, as compared
to those in the original rulings. The 16 squares in the original and 25 squares in the
improved chamber are further subdivided each into 16 small squares.]
 The dilution pipettes: Identifying features; principles of dilution

[Fig: Dilution pipettes ~ for RBC count (above) and WBC count (below).]

21
 o The markings: The RBC pipette has the markings 0.5, 1, and 101. The WBC
pipette has the markings 0.5, 1, and 11. These markings have no units. The
RBC pipette dilutes a sample 200 times; the WBC pipette dilutes a sample 20
times.
o The bead: Red bead inside the bulb of RBC pipette; white bead in the bulb of
WBC pipette. NOTE: A common error – What is the function of the bead?
Most students answer that the bead color helps identify the pipettes.
However, in addition to the beads, the tubes attached to the pipettes also
have identifying colors. The bead helps mixing of the blood sample with the
diluting fluid inside the bulb.
o The bulb: Mixing of the blood sample and the diluting fluid occurs in the bulb.
Relatively, a larger bulb for RBC pipette, having the capacity of 100 volumes;
smaller bulb for WBC pipette, with the capacity of 10 volumes.
 Procedure:
o Let us consider the RBC pipette. The blood sample is taken up to 0.5 mark.
Then, take the diluting fluid up to 101 mark. It means, the bulb is completely
filled with the mixture.
o Capacity of the bulb is 100 volumes out of which 0.5 volume is blood. Thus,
0.5 volume is diluted 100 times. That is, 1 volume is diluted 200 times.
o In the case of the WBC pipette, the capacity of the bulb is 10 volumes. 0.5
volume of blood sample gets diluted 10 times; 1 volume diluted 20 times.
 Note: As one starts taking the diluting fluid into the pipette, the blood sample moves
into the bulb and so does the diluting fluid. As the bulb is filled completely, the stem
portion of the pipette contains only the diluting fluid. Hence, before charging the
Neubauer’s chamber, first 2-3 drops (coming from the stem) should be discarded.
 Common error:
Let’s consider RBC pipette. Since diluting fluid is taken up to 101 mark, many
students tend to believe the dilution factor as follows – 0.5 volumes diluted to 101,
hence 1 volume diluted to 202, and since 2-3 drops are discarded the dilution
becomes 1:200.
It should be borne in mind the capacity of the bulb is 100, in which 0.5 volume is
blood.

 Discussion:
1) Can the dilution pipettes be used for any purpose other than WBC or RBC
counts?
Ans: Yes. Any fluid sample can be diluted 20 times or 200 times by using these
pipettes.
2) Can the RBC pipette be used for WBC count?

22
 Ans: Yes. Leukemia is a condition characterized by uncontrolled or cancerous
proliferation of WBC precursors; the WBC count may be in lakhs per mm 3. The
blood sample, in such a condition, would have to be diluted to a greater extent
(200 times) instead of the usual 20 times, for its WBC count.
3) Can the central RBC square be used for WBC count?
Ans: Yes. Again, for the same reason. Lakhs of WBCs per mm3 in leukemia will
have to be counted in a smaller field (RBC square).
4) How to avoid duplication in cell counting?
The inverted ‘L’ rule: While counting cells under high power (for instance, the red
cells), it is particularly important to avoid cells on the lines getting counted
repeatedly in different squares. The cells lying over the limbs of an inverted ‘L’
(“Γ”) should be counted within the corresponding square.
Also note: In the red cell count, the cells falling on inner two of the triple lines be
counted.

[Fig: Diagrammatic representation of inverted “L” rule. Two cells are shown, falling on the
margins of squares, and the arrows indicate where those cells be counted.]

23
 Section 5
Total leucocyte count (TLC)
 Introduction:
 Infectious diseases, newer generations of antibiotics, antibiotic
resistance, etc are the fields of medicine which have seen a great
research in the recent times.
 A patient suffering from an infection usually presents with fever, one
of the commonest symptoms.
 The white blood cells (WBCs, or leucocytes) form the defence
establishment or immune system of the body against invading
pathogens. Most infections, acute or chronic, are accompanied by a
rise in the total Leucocyte count (TLC). Some infections show a
decrease in the TLC. Hence, when a patient presents with fever, TLC is
the first investigation a clinician thinks of to confirm the presence of
an infection.
 Normal range for TLC: 4000-11000/mm3
 Aims & objectives:
 To determine the total WBC count in a given sample.
 To understand the significance of total WBC count in the context of
infections.
 Materials and methods:
 Materials for collection of blood sample (lancet/needle, cotton swab
soaked in spirit)
 Microscope
 Neubauer’s chamber, coverslip
 WBC pipette
 WBC diluting fluid (also called ‘Turk’s fluid’)
Principle:
 The blood sample will be diluted 20 times; RBCs in the sample will be lysed. The
white cells will be counted in the Neubauer’s chamber under low power. The count
will be converted to per mm3 of blood. Then, this count will be multiplied by 20 to
obtain the final result.
Procedure:
o With a spirit-soaked cotton, sterilize the ring finger.
o Prick the finger by a lancet/needle.
o Collect the blood in the WBC pipette up to 0.5 mark.
o Take the WBC diluting fluid in the pipette up to 11 mark.
o Hold the pipette horizontally between the palms; gently rotate the
bulb portion so as to cause mixing of the blood sample and the
diluting fluid.
o Discard 2-3 drops from the pipette (stem portion; only the diluting
fluid).

24
 o Charge the Neubauer’s chamber (as discussed in the previous
chapter).
o Focus the the WBC fields and count the cells under low power. In the
lab manual, draw the diagram of the WBC counting areas. Enter the
counted number of cells in the respective smallest squares.

 Result:
Calculation:

 Each WBC field has dimensions of 1 mm X 1 mm. Thus, the area of a

WBC field = 1 mm2. Depth of the Neubauer’s chamber is 1/10 mm.
Thus, volume of a single WBC field would be – 1 mm X 1 mm X 1/10
mm = 1/10 mm3.
 WBCs are counted in 4 WBC fields. Hence, total volume in which the
WBCs are counted = 4 X 1/10 = 4/10 mm3.
 Let’s say, the total WBCs counted = “n”. If 4/10 mm3 contains “n”
number of cells, 1 mm3 contains – (10/4 X “n”) cells.
 This is the sample that was diluted 20 times. Hence, finally, the
number of cells per mm3 will be – (10/4 X “n” X 20). Or, (“n” X 50) per
mm3.
 Total leucocyte count (TLC): /mm3.

25
  Common errors; special precautions:
 While filling the pipette: (avoid air bubble formation)
I. Ensure the drop of blood is big enough and the pipette
tip is completely dipped in it.
II. Let the pipette be as vertical as possible.
III. Most imp: Suck the blood very slowly, as you follow the
above two points.
All these precautions will make sure that the blood
sample is taken exactly up to 0.5 mark and is devoid of
air bubble(s).
 While charging the Neubauer’s chamber:
I. There may be ‘overcharging’, or the mixture overflows
into the grooves of the counting chamber. The cells will
have gone into the grooves; no cells would be found in
the counting area. Wipe the chamber & the coverslip
clean; attempt charging again.
 Focusing of the WBCs: (proper contrast is the key factor)
- Very often, the WBCs aren’t visible in spite of following all
the steps meticulously. If this happens, synchronize the fine
adjustment with the mirror & iris diaphragm adjustment.
WBCs WILL BE VISIBLE WHEN THE CONTRAST IS PROPER.
- Artifacts, dirt particles, etc shouldn’t get counted as WBCs.
To confirm, one may simply rotate to high power; nucleus
of a WBC should be identifiable.

 Discussion:
(A) Technical aspect:
1. About the diluting fluid:
(i) What are its constituents?
o The Turk’s fluid contains –
I. Glacial acetic acid (2%) – It causes RBC lysis.
II. Gentian violet – stains the nuclei of the WBCs
III. Distilled water – solvent; to add up to the volume.
Note the importance of glacial acetic acid: Since RBC count is in millions/mm3, their
presence, in a field under microscope, will interfere with the counting of WBCs. RBCs should
be lysed. Distilled water could cause osmotic lysis of the red cells; however, it would take
longer to do so. Glacial acetic acid causes a quick lysis of red cells as also the WBC
membranes.
2. What are the other uses of the WBC pipette?
 RBC count, in severe anemia.

26
  Cell count in CSF
(B) Physiology point-of-view:
1. What is the normal TLC?
 Normal range for TLC: 4000 – 11,000 per mm3
 The count is high in newborn, infancy, and childhood.
What are the physiological variations in TLC?
The TLC increases in following physiological conditions ~ exercise, diet,
pregnancy & parturition, emotional stress

2. What are the functions of WBCs?

 WBCs form the defence system of the body. Different types of WBCs
perform the the defence function in their different ways.
 Neutrophils and monocytes respond in acute illnesses; lymphocytes
form the basis of long-term immunity.

B. Clinical perspective:
1) What are the pathological variations in TLC?
 Pathological variations:
(a) Leucopenia: WBC count < 4,000/mm3; seen in -
a. Typhoid fever (although it is an infection
associated with high grade fever), early
stages of some viral fevers
b. Bone marrow depression; aplastic anemia
(b) Leucocytosis: WBC count > 11,000/mm3; found in –
o Most types of bacterial infections, acute (such as
pneumonia) or chronic (such as tuberculosis);
(c) Leukemia: ‘blood cancer’; it is an uncontrolled proliferation of the
WBC precursors. Immature WBCs will be found in peripheral
blood; the WBC count will be in lakhs/mm3.

27
 Section 6
Total RBC count

 Introduction:
 When does a clinician ask for this investigation? Ans: When a patient
is suspected of having anemia.
 “Pallor” is the sign most definitely indicative of anemia. There is
decreased redness of skin & mucous membranes. It can be noticed on
the face, lips and the oral mucosa, conjunctiva, nail beds, palms.
 Iron deficiency is the most common cause.
 RBC count, by itself, can indicate only the presence of anemia.
However, clubbed with other related investigations (such as Hb &
packed cell volume) it helps establish the type of anemia and its
cause.
 Aims & Objectives:
 To determine the RBC count in a given sample
 Use of the RBC count in diagnosis and categorization of anemia.

 Materials and methods:

 Materials for collection of blood sample (lancet/needle, cotton swab soaked
in spirit)
 Microscope
 Neubauer’s chamber, coverslip
 RBC pipette
 RBC diluting fluid (also called ‘Hayem’s fluid’)

 Principle:
The blood sample will be diluted 200 times. RBCs will be counted in the Neubauer’s
chamber under high power. The count will be converted to per mm3. Then, this
count will be multiplied by 200 to obtain the final result.
Procedure:
 With a spirit-soaked cotton, sterilize the ring finger.
 Prick the finger by a lancet/needle.
 Collect the blood in the RBC pipette up to 0.5 mark.
 Suck the RBC diluting fluid in the pipette up to 101 mark.
 Hold the pipette horizontally between the palms; gently
rotate the bulb portion so as to cause mixing of the blood
sample and the diluting fluid.
 Discard 2-3 drops from the pipette (stem portion; only the
diluting fluid).
 Charge the Neubauer’s chamber.

28
  Focus the the RBC squares, first under low power and then
under high power. In the lab manual, draw the 5 RBC fields
– top left, top right, bottom left, bottom right, and the
central square. Count the cells under high power. Enter
the counted number of cells in the respective smallest
squares of each of the 5 fields.

 Common errors; Special precautions:

 While charging the chamber:
As discussed previously, the fluid should not be forced out of the
pipette. It will flow between the coverslip and the chamber by
capillary action.
Have a look at the charged chamber before placing it under
microscope. If a drop is found to be overlying the coverslip even
after proper charging, gently remove the drop by filter paper.

 Result:
Calculation –
 Of the 25 RBC squares, cells have been counted in 5.
 Each square has the dimensions of 1/5 mm X 1/5 mm. The area of the square
= 1/25 mm2. Depth of the Neubauer’s chamber is 1/10 mm. Thus, volume of
one RBC square = 1/5 X 1/5 X 1/10 = 1/250 mm3.
 The RBCs are counted in 5 such squares. Hence, the volume of 5 RBC squares
= 5 X 1/250 = 1/50 mm3.
 Let’s say, the RBCs counted in this volume = “n”. If 1/50 mm3 contains “n”
RBCs, 1 mm3 contains: (50 X “n”)
 The blood sample was diluted 200 times. Hence, finally, the number of cells
per mm3 will be – (50 X “n” X 200). Or, (“n” X 10,000) per mm3.
 Total RBC count: /mm3.

 Discussion:
(A) Technical aspect :
1. About the diluting fluid:
 What are the constituents of the RBC diluting fluid?
Ans: The Hayem’s fluid contains –

29
 o Sodium chloride [NaCl] 0.9% (0.5 gm) – to make the fluid
isotonic, so that the RBCs do not shrink or swell
o Sodium sulphate [Na2SO4] (2.5 gm) – prevents rouleaux
formation; also preserves the shape of the red cells
o Mercuric chloride [HgCl2] (0.25 gm) – acts as a preservative
o Distilled water – to add up to the volume.
 Some authors mention the role of sodium sulphate as an anticoagulant. However,
when diluted 20 times or more, the blood clotting process is decelerated greatly.
Hence, in these experiments, blood sample does not need addition of an
anticoagulant.
 The Hayem’s fluid is prepared and stored in labs. With storage for a few days, there
are chances of bacterial or fungal overgrowth in the fluid. This is prevented by the
mercuric chloride.
 In some disease conditions, RBC clumping is accelerated. Gelatin (0.01 gm in 100 mL
fluid) prevents clumping of the red cells and hence sometimes added to the fluid.
(B) Physiology point-of-view:
1. What is the normal RBC count?
 Normal range: 5 to 6 million per mm3 (males); 4.5 to 5.5 million per
mm3 (females)
 Neonates have a relatively higher red cell count – 5.5-6.5 million/mm3
(at birth). Neonates suffer from a relative hypoxia; consequently they
have a higher red cell count.
 Why is RBC count less in females as compared to males?
 In males, the hormone testosterone is known to stimulate the process
of erythropoiesis in 3 ways – (i) it causes kidneys to secrete more
erythropoietin, (ii) it upregulates the erythropoietin (Ep) receptor in
the bone marrow, and (iii) in some unknown way, it directly
stimulates erythropoiesis.
 In females, the hormone estrogen has been shown to suppress the
erythropoiesis to an extent.
[Menstrual blood loss in females is not the primary reason for a relatively lower count in
them as compared to their male counterparts. By the logic of “negative feedback”
mechanism that regulates most of the body functions, including the erythropoiesis, the 30-
40 mL blood loss per month should actually stimulate erythropoiesis. However, if the loss is
excessive (polymenorrhea/menorrhagea), it may lead to anemia.]

 Physiological variations in RBC count:

(a) A higher RBC count is seen in the following: (The conditions of relative or
absolute hypoxia; hypoxia is a key stimulus for erythropoietin secretion and
resultant accelerated erythropoiesis.)
 In newborn
 At high altitudes

30
 Increased RBC count is generally accompanied by elevated numbers of WBCs
and platelets.
(b) A lower RBC count is seen in:
ii. Old age
iii. Pregnancy

2. Morphology of the red cells:

 Red cells are non-nucleated biconcave-shaped cells, 8  in

diameter and have a volume of 78-94 3.
 They are saturated up to 32-38% by Hb, and they have a central
pallor.
 Life span of a red cell – 120 days.

(c) Clinical perspective:

1. What are pathological variations in RBC count?

 Decrease in red cell count is seen in anemia; increased count beyond

physiological limit is termed polycythemia.
(i) Polycythemia:
 It may be a true polycythemia in which there is an absolute
increase in the number of RBCs, or a relative polycythemia that
results from a relative decrease in plasma volume as compared to
the volume of the RBCs.
 Excessive numbers of RBCs resulting from a stimulation primarily
of the hematopoietic tissue in the bone marrow – polycythemia
vera.
 Secondary polycythemia (erythrocytosis) – Excess red cell
production due to the altered erythropoietin secretion; secondary
to tissue hypoxia.
 Relative polycythemia – Occurs in conditions, such as dehydration,
in which there is decrease in plasma volume rather than an
absolute increase in red cell count.
(ii) Anemia: When RBC count is below the lower limit of the normal range, it is
labelled as having anemia.
Relevance of the red cell count in anemia –
3 steps in the investigation of anemia:
1. Is anemia present?
 Decreased RBC count will indicate the presence of anemia.
2. What is the severity and type of anemia present?

31
  RBC count, seen in isolation, is of little use in grading the severity
of anemia. It can be inferred though, that lesser the count more
severe is the anemia. (As discussed previously, Hb estimation is of
greater significance in judging severity.)
 Whether the anemia is micro-, normo-, or macrocytic?
This is determined by the mean corpuscular volume (MCV). The
average red blood corpuscle volume: 78-94 3.
To calculate the red cell volume, one needs to have packed cell
volume (PCV) and the RBC count.
 Whether the anemia is hypo- or normochromic?
This is determined by the color index (discussed previously). To
calculate the color index, patient’s Hb level and RBC count are
determined and the values are converted to percentages
respectively.
3. What is the cause of anemia?
 Anemia from blood loss or from hemolysis will have a moderately
decreased RBC count.
 Anemia caused by impaired red cell production, particularly due to
bone marrow depression, will exhibit a greatly decreased RBC
count.
 RBC count helps indirectly in identifying some types. For instance,
megaloblastic anemia (vit. B12 and/or folate deficiency) has a
higher MCV; calculation of MCV involves the red cell count. (Blood
indices; discussed later).

32
 Section 7
Preparation of a blood smear
Identification of the cellular elements of blood

 Introduction:
 Preparing a blood smear is a skill. A properly made and stained smear holds
the key in the accurate counting of the various WBC types.
 Blood smears are also useful in detection of parasites, such as malaria and
filaria.
 Blood smear should ideally be made immediately after the sample has been
taken. If the sample is collected by a technician and is to be sent to the
laboratory where smear will be made, a time gap of up to 3 hours is
acceptable. Beyond this, the time lapse would result in altered morphology
of the cells.

 Aims & Objectives:

 Learn to make an ideal blood smear
 Study the various stains
 Identify the different blood cells from their morphology
 Materials & methods:
 Materials for collection of blood sample
 Glass slides
 Leishman’s stain
 Microscope
o Principle:
A smear is prepared from a drop of blood. The smear is then stained and
observed under the microscope.

o Procedure:
 With a spirit-soaked cotton, sterilize the ring finger.
 Prick the finger by a lancet/needle. Allow a drop of blood to form.
 Touch the drop of blood on a glass slide; the drop should be
placed at one end of the slide in the center.

33
  The slide is placed on a smooth surface (table) in such a way that
the drop on the slide comes to your right side.
 Take another slide, called the “spreader slide”. Hold this slide
along its longer sides, and bring it near the drop of blood. The
spreader should be held such as to make an angle of about 450
with the first slide.
 Hold the ‘spreader’ just in front of the drop and move it backward
slowly till it touches the drop. By ‘capillary action’ the drop will
spread along the shorter side of the spreader. Now quickly, with a
single action and uniform motion, push the spreader leftward on
the slide. The blood will follow the spreader, and a film or smear
will be formed on the slide.
 Wave the slide gently in the air so that smear dries in no time.
Staining of the blood smear:
 Add about 10 drops of Leishman’s stain in such a way that it
covers the entire slide. Keep the stain for about 1 minute.
 The slide should now be flooded with double the volume (about
20 drops) of distilled water. Blow gently on the slide; wait for 5-7
minutes.
 Wash the slide in a stream of running water; the smear now
acquires a bluish pink tinge.
 Wipe the back of the slide. Set the slide upright; allow it to dry.
Note: It is prudent to make 3 such stained smear slides from a single sample. The best
prepared one then may be selected for the cell count.

 Observation/result:
 Observe the stained film first with naked eyes. Note its features.
 Observe the slide under microscope, under high power and then under oil
immersion.
 Note the morphologic features of the blood cells.

[Fig: A blood smear.]

34
  Common errors; Special precautions:
 The glass slide should be clean: The slide should be washed and wiped clean
just before making the smear. Dirt particles or grease, if present, obstruct the
spreading of the drop. In the event, the smear will form with visible gaps; the
cell distribution will be grossly irregular and patchy.
 The “spreader”: In common practice, any glass slide is used as a “spreader”
slide. Ideally, however, the spreader should have a smooth edge. One corner
of the slide should be broken off with a glass cutter, so as to leave about 2 cm
of the edge as the spreader.
 The spreading action: Inaccurate results have one common reason – the
failure in making a good smear. The following points should be kept in mind:
 The drop of blood should be at the center, at one end, about 1 cm
from the (short) edge of the slide.
 Spread the drop with a steady motion, with a light but even pressure
throughout the act of spreading. The smear should end at least 1 cm
before the other end of the slide.
 Do not lift the spreader until the drop has been spread to the other end.
There should be no back & forth movement of the spreader.
 The spreading action shouldn’t be too slow. Slower movement would
cause the smear to be thin; however, it will be uneven.
 The staining procedure:
 Avoid excessive staining of the slide; do not wait for longer than 2
minutes after the addition of stain.

 Discussion:
(A) Technical aspect:
1. How can the thickness of the smear be regulated?
(Thickness of the film determines the separation of the cells. When the smear
is thin, the cells will have spread out with less aggregation or overlap.)

35
 Ans: By varying the –
 Pressure of the spreading hand (more the pressure, thinner
the smear)
 Speed of the spreading (slower motion, thinner the smear)
 Angle of the spreader with the slide
 Importance of the “angle” of the spreader with the slide:
A desired thickness of the smear can be achieved by changing the angle
between the spreader and the slide.
(i) When the cell count is suspected to be lower (anemia) – apply a
wider angle of the spreader slide. The smear will be thick; more cells
will be visible in a microscopic field.
(ii) if the count is suspected to be too high (polycythemia) – use a
narrower angle; the smear will be thin and the cells will have spread
out.
However, this approach is fine only when the goal is to learn the making of smears and
observation of cells.
 Laboratory perspective: In practice, a technician makes the smear and, later on, the
pathologist observes/counts the cells. The technician should stick to 450 angle. An
unintended or deliberate change of angle will modulate the spread of the cells and
may prejudice the pathologist. [Narrower angle -> thin smear -> fewer cells per field
-> observer wrongly predicts anemia/leucopenia.]

 Use of peripheral blood smear for detection of parasites: (utility of thick

smears)
- Malaria is routinely diagnosed by finding its parasites in a blood smear.
- If malarial parasites are fewer in number, use of thick smear first is
advocated. Once the presence of parasite is confirmed, the standard thin
smears are used for determining the species (P. ovale, P. vivax, etc).
- Thick smears are also employed for the detection of microfilaria.
- Other parasites that can be detected on blood smears: leishmania,
trypanosomes

2. What are the criteria for an ideal smear?

Upon naked eye observation:
(i) The smear should neither be too thick nor too thin.
(ii) It should not have patches or gaps; it shouldn’t be streaky or serrated

36
 (iii) The smear should be tongue shaped, with a smooth tail. It should
occupy the middle 2/3rd of the slide.
(iv) An ideal smear has 3 parts – head, body, and tail (Head – where drop
was taken and smear began; tail – Where smear ended.)
(v) The stained smear should look pink in colour.
When observed under microscope:
(i) There is some overlap of the RBCs; the overlap decreases and RBCs
seen separated near the tail.
(ii) On microscopic examination – The RBCs should appear pink, the
platelets should be pink to purplish blue, and the various WBCs should
take appropriate respective colours (for instance, reddish pink
granules of eosinophils).
3. How to notify a cell’s position in a particular high power field?
 Ans: The field is circular. Imagine it to be the dial of clock. The position
of a cell can be notified as “O’clock position”.

4. What are the stains commonly used in hematology labs ?

Paul Ehrlich first used the idea of using a mixture acidic and basic dyes to
stain the basic & acidic components of cells. The idea was further
propounded by Russian physician Dmitri Romanowsky.
 Romanowsky stains are used universally for routine staining of blood
films. The property that makes them greatly useful is – they stain
granules in various cells differentially. This property is imparted by
two components of the stain – azure B (or, modified methylene blue)
and eosin Y.
 The Romanowsky-Giemsa effect: The mixing of the two components
produces an altogether new shade of staining that is not attributable
to either of the component (dye).
 The two components of the Romanowsky’s stain:
(a) A cationic dye: azure B; it binds to anionic molecules of cell
components, and
(b) An anionic dye: eosin Y; it binds to cationic sites on cell’s proteins.
 Various Romanowsky stains that are currently in use:
1. Leishman’s stain
2. Giemsa’s stain
3. Jenner’s stain
4. May-Grünwald stain

37
(B) Clinical perspective:
 Advanced lab technology:
- There are automated machines or methods now available for making and staining of
smears. The instrument spreads the drop, fixes the smear and stains it.
- Different types of machines apply stains in different ways:
(i) “Flat-bed staining” – Slide placed horizontally, as in manual staining
(ii) “Dip-and-dunk technique” – Slide is dipped vertically in a bath of staining solution
(like a ‘piece of bread dunked into a coffee mug’)
(iii) Spraying technique – Slide placed in a cytocentrifuge; stain is sprayed onto it.

1. What observations can be made from the smear? How will they be useful in
the context of diagnosis?
 All the blood cells can be observed for their morphology.
(i) RBCs will appear pink. Their size can be compared to the size
of the nearby WBCs, so as to judge if they are normocytic,
micro- or macrocytic. The central pale area may be observed;
it can give a clue about the Hb content (or, whether it is
hypochromia or normochromia),
(ii) WBCs should be identified; morphology of each type should be
noted. (E.g. Granulocytes with different colours of their
granules, observe the number of nuclear lobes of neutrophils,
distinguish between a large lymphocyte and a monocyte, etc.)
 A rough guesstimate can be made about the numbers of each cell type. (I)
Are there too many or too few RBCs in a single high power field? (II) How
many WBCs are visible per high power field, and in which part of the smear?
(III) How many platelets are visible in the thin part of the smear; what is their
proportion to RBCs? [In a healthy adult, the proportion of platelets to red
cells, on the smear, is about 1:10 to 1:20.]
 NOTE: When the blood films are to be made from a stored blood sample, the time
lapse and possible changes in cell morphology should be borne in mind. No changes
will be visible up to a time lag of 3 hours in the case of WBCs. The changes that
appear after a longer storage – (1) Nucleus – Lobes get separated (neutrophils);
budding off, forming 2-3 lobes (agranulocytes), (2) Cytoplasm – Vacuoles begin to
appear.

38
 Section 8
Differential Leucocyte Count (DLC)

 Introduction:
This practical is about counting of different types of WBCS. Leucocytes are classified
as follows:- (1) Granulocytes : (based on staining properties of the granules in their
cytoplasm) - Neutrophils, Eosinophils, Basophils. (2) Agranulocytes: Monocytes,
Lymphocytes.

 Infections are of 4 types – Bacterial, viral, fungal, and protozoal.

 Not every infection manifests as fever. Bacterial & viral infections
usually cause fever. As discussed previously, with fever as the
presenting symptom, TLC is the first choice of investigation.
 However, various types of microbes can, apart from fever, exhibit
a wide range of manifestations. Bacterial infections may cause
abscess, cough; many viral infections manifest as sore throat,
common cold. Fungal infections result in mucocutaneous
manifestations, skin rashes; protozoal infections show a variety of
symptoms ranging from abdominal pain (worms) to fever with
chills & rigors (malaria).
 Different types of infections are tackled by different types of
WBCs. Increase in a particular type of WBC would thus point
toward a certain type of infection.
 When a clinician suspects a particular infection, some questions
need to be answered – (1) Is the infection acute or chronic? (2)
Which micro-organism is responsible? (3) What is the severity of
the infection?
 To get answers to these questions, the clinician orders the
differential leucocyte count (DLC), in addition to TLC.
 Aims & Objectives:
 identify different WBCs under microscope
 Count the various types of WBCs; notify their percentages
 Calculate the absolute counts for each type of WBC.
 Materials & methods:
 Materials for collection of blood sample
 Glass slides
 Leishman’s stain
 Microscope
o Principle:
A stained smear will be observed under high power and oil immersion. Different
types of white cells will be identified and counted. 100 such WBCs are counted
and the percentage of each type of WBC is noted.
o Procedure:

39
  Wash and wipe clean 3 glass slides; 3 stained smears are to be prepared.
Make sure that the slides are grease-free.
 With usual aseptic precautions, prick the ring finger and take a drop of
blood on the slide. Make the smear with all the specifications mentioned
in the previous chapter.
 Put 10 drops (approx.) of Leishman’s stain on the slide so as to cover the
entire slide with the stain.
 Wait for 1 minute; allow the smear to be fixed.
 Add double the quantity (about 20 drops) of distilled water on the slide;
blow gently on the slide.
 Wait for 5-7 minutes.
 Wash off the excess stain by holding the slide under a stream of running
water.
 Wipe the underside of the slide. Keep the slide vertically inclined; allow it
to dry.
 Counting of the WBCs: (“zig-zag” fashion)
 There are no squares or lines for demarcation. Starting from one end
of the smear, scan each high power field for the WBCs. Observe the
cells in adjacent fields; moving field-to- field when you reach the
other end of the slide, move down and start moving field-to-field in
the opposite direction now. Continue the procedure till you encounter
100 WBCs.
 Observations/Result:
o Draw 100 squares (10 rows, 10 columns) in the lab manual. Note down the
observed WBC types in the successive squares, using alphabets that denote the
respective cells – e.g., N (Neutrophil), E (Eosinophil), and so on.
o Two varieties of lymphocytes will be seen – small and large. They should be
noted as SL and LL, respectively.
o DLC - Note down the count as percentage for each type of WBCs.
o Using the TLC and the DLC, calculate the “absolute” counts for each of the types
of WBCs. (Discussed in the next chapter.)

40
  Common errors; Special precautions:
 Observe all the precautions during making and staining of the smear, as
mentioned in the previous chapter.
 If the drop of blood is large, the smear will be too thick. A part of the drop
may be transferred to another slide using a glass rod.
 You have prepared 3 films; use the one with a lighter hue of bluish pink.
 While you wait for 5-7 minutes after the addition of distilled water, do
not allow the diluted stain to dry. The slide should be rocked gently,
and/or blow on it through a pipette.

 Size of the cell is an identifying feature. For this purpose, the leucocytes should
be compared with the surrounding RBCs.
Diameters of the cells – (1) Red cells = 7-8 , (2) Most granulocytes = 10-14 
(about 1 and ½ times the size of red cells), (3) Monocytes = 18  (largest cell on
the smear; 2 and ½ times the size of red cells)

 Discussion:
(A) Technical aspect:
(1) About Leishman’s stain:
i. What are the contents of the stain?
(Note: “phil” = to love or attract. If a cell or its component is
said to be ‘basophilic’, it means that the contents are acidic
and they attract the basic dye. Conversely, the ‘acidophil’ or
‘eosinophil’ is the cell or its component that has basic content
which attracts acidic dye.)
 Methylene blue: (basic dye) – It stains the acidic
components of a cell – (i) Nuclei of WBCs, due the
presence of nucleic acids and proteins, (ii) Granules of
basophil: contain heparin which attracts the basic dye.
 Eosin: (acidic dye) – It stains the basic components of a
cell – (i) Granules of eosinophil: they contain a spermin
derivative which has a basic grouping.
 Acetone-free methyl alcohol (methanol): It acts as a
“fixative”; it fixes the smear to the slide. Mechanism: It
precipitates the proteins, which then adhere firmly to
the slide.
NOTE: The nucleated RBC precursors are basophilic as they have acidic contents. Once the
Hb appears in them, they become acidophilic as Hb molecule has basic groupings.

41
The term “fixative” has another connotation in pathology lab techniques. When tissue slices
are taken for investigations, there are no more bodily conditions (such as O 2 and nutrient
supply) for cells. The cellular functions will hence alter dramatically and so will their
morphology. The job of a “fixative” is to maintain the cells ‘as it is’, or ‘fix’ the cells at a
certain situation so that there are no further changes in cellular morphology.
(2) About the smear and distribution of the cells:
(Also refer to “criteria for an ideal smear” mentioned in the previous chapter.)

i. Are blood cells uniformly distributed throughout the smear?

Ans: Even in a properly made ideal smear, the cells are not found to
be uniformly distributed. Preponderance of various cells, in various
parts of the smear, is seen as follows:
o Near the tail and at margins – Neutrophils, monocytes
o Body of the smear – lymphocytes
The reason for this kind of uneven distribution: The various cell types differ from
each other in their size, stickiness, and specific gravity.
 Generally speaking, the larger cells (monocytes, neutrophil) tend to be
pushed toward the periphery – i.e., edges and tail end of the smear,
whereas the smaller ones (such as, lymphocytes) tend to gather more
toward centre – the body of the smear.
 It is prudent to always start observing/counting WBCs from the tail end of the smear.
Major search for various cell types should be near the tail region.

(3) Identification of the white cell types:

Granulocytes Size Nucleus Cytoplasm/granules

1. Neutrophil 10-14  Dark staining, Violet pink, fine granules,
deep purplish scattered throughout
blue; cytoplasm
segmented,
(majority will
have lobes,
joined by thin
strands)
2. Eosinophil 12-15  Bi-lobed or Coarse, reddish pink
“spectacle- granules; pale blue
shaped” (two cytoplasm (obscured by
lobes the overlying granules)
connected by a
thin strand of
chromatin
3. Basophil 10-14  Bilobed or S- Coarse, bluish purple
shaped; the granules that obscure the
lobes folding nucleus

42
 on each other;
barely visible
Agranulocytes
4. Monocyte (the 15-18  Horse shoe- Large quantity of pale
largest shaped blue cytoplasm
circulating
WBC)
5. Lymphocyte
1. Small 7-10  Round nucleus; Hardly visible
almost
occupies entire
cell

10-14  Bean-shaped or
2. Large kidney-shaped; A thin rim of cytoplasm
occupies 90% visible
of cell volume

(4) Further discussion on cell morphology:

I. How to differentiate between a large lymphocyte and a monocyte?

Large lymphocyte Monocyte

Size 1 and ½ times the 2 and ½ times the
surrounding red surrounding red
cells cells
Nucleus Bean-shaped, with Horse shoe-
shallow shaped, with deep
indentation; indentation;
occupies 90% of occupies 70% of
the cell the cell
Cytoplasm A thin rim of Greater amount of
cytoplasm; 10% of cytoplasm (about
the cell volume 30% of the cell
volume)

II. Why is it that some lymphocytes are small and others are large? (Why no
other cell shows such morphologic dichotomy)
Ans: These are developmental stages for a lymphocyte. The precursor cell
enlarges (lymphoblast) and then divides into two smaller lymphocytes.
Thus, about 90% of the circulating lymphocytes are small, and 10% of
them are large.
(A common error by students: There are two “functional” types – ‘T’
lymphocyte & ‘B’ lymphocyte. Students tend to think that the small &

43
 large might be related to their functional types. However, there is no such
correlation.)
 Another moot question is – ‘Why such “small & large” variation is found only
with lymphocytes?
1. Among the white cells, lymphocytes have the longest life-
span – 300 days. The other WBC types have short life-spans.
2. Except for lymphocytes, all other white cell types are formed
in the bone marrow. Lymphocyte precursors leave bone
marrow at an early stage; they undergo subsequent
modification in other organs (such as, thymus). Via
bloodstream, the lymphocytes then migrate to secondary
lymphoid structures where they are stationed. Then, they re-
enter circulation whenever needed for immune mechanisms.
Thus, lymphocytes exhibit a complex traffic between vascular
and extravascular compartments. Compare this with, say,
neutrophil. They are formed in bone marrow, enter the
circulation where they remain for about 4-8 hours and then
enter the tissues. Their life-span is 4-5 days and they do not re-
enter the circulation.
Thus, a longer life-span, along with a series of developmental changes and an extensive
travel between bloodstream and tissues, results in the lymphocytes exhibiting different
forms (small & large) in circulation. The lymphoblasts (precursors) enlarge and then divide
into smaller lymphocytes. Thus, small lymphocytes are the more matured ones.
Note: Monocytes too have longer life-spans and can re-enter circulation. However, after a
short stay in circulation, they enter the tissues and remain there for prolonged periods as
macrophages.
(B) Physiology point-of-view:
 Functions of the various WBC types:
Granulocytes
1. Neutrophil: (50-70%)
 “First line defence”
 Phagocytosis: The neutrophils move out of the
circulation, ingest the bacteria and kill them.
 Granules: Contain enzymes involved in the killing of the
ingested pathogens and degradation of the
phagocytosed material. Two types of granules – (1)
Primary granules (basophilic): contain acid hydrolytic
enzymes and peroxidase; (2) Secondary granules
(acidophilic): contain alkaline phosphatase and other
enzymes.
2. Eosinophil: (1-4%)
 Weakly phagocytic;

44
  The granules contain “major basic protein” (MBP), a factor
that is believed to immobilize and kill the parasites (e.g.
Hookworm, roundworm)
 Involved in allergic reactions; discharge the granule
contents on combining with IgE antibodies
3. Basophil: (0-1%)
 Granules contain histamine, heparin, and serotonin
 Involved in allergic reactions
Agranulocytes
4. Monocyte: (2-8%)
 Considered as “second line defence” (the second cell to
attack bacteria)
 A stronger phagocyte compared to a neutrophil (i.e., kills a
much greater number of microbes)
 A larger phagocyte; bigger microbes can be tackled better.
E.g., malarial parasite
5. Lymphocyte: (20-30%)
 “Third line defence”; non-phagocytic
 Body’s long-term immunity is mechanised by lymphocytes
 Cell-mediated and humoral immune responses directed
toward foreign antigens

(C) Clinical perspective:

 When a clinician suspects an infection, the patient is advised TLC and DLC. In
addition, absolute white cell counts and Arneth count are derived (discussed
in the following chapter). For some infections, such as dengue, additional
tests like platelet count are advised.
1. Inference drawn from the DLC:
As a general principle, increased neutrophils indicate acute infections whereas
increased lymphocytes point toward chronic infections.
Decreased count of most white cell types is indicative of bone marrow suppression
(as occurs in radiotherapy, certain drugs, etc.)
(i) Neutrophils: (Normal range ~ 50-70%)
 Neutrophilia – increased neutrophil count; Neutrophilic leucocytosis occurs in
~
 acute inflammatory states
 acute bacterial infections caused by Staphylococci, Streptococci,
etc.
 Acute febrile illnesses
 Pyogenic infections (infections that cause pus formation); abcesses
 Neutropenia – decreased neutrophil count; often seen in ~
 Viral infections

45
  Typhoid fever
 Rickettsial infections
(ii) Eosinophils: (Normal range ~ 1-4%)
 Eosinophilia (increased eosinophils) occurs in ~
 Parasitic infestations (e.g., helminths)
 Allergic conditions
 Tropical eosinophilia (a pulmonary parasitic infestation
syndrome characterized by cough & breathlessness)
 Skin diseases
 Eosinopenia (decreased eosinophils) ~ Steroid administration causes
disappearance of eosinophils from peripheral blood, causing eosinopenia.
(iii) Basophils: (Normal range ~ 0-1%)
 Basophilia ~ Increased basophil numbers; occurs in conditions associated
with uncontrolled proliferation of precursors in bone marrow –
myeloproliferative disorders, viz. chronic granulocytic leukemia.
 [Punctate basophilia: It is a condition that is mistakenly thought to be of basophils. It
is a condition of RBCs. RBC precursors are basophilic. When red cell maturation is
arrested in the basophilic stages, it shows as “basophilic stippling” of RBCs; seen in
lead poisoning.]
(iv) Monocytes: (Normal range ~ 2-8%)
 Monocytosis (increased number) ~ occurs in –
 Malaria
 Some chronic infections, such as tuberculosis
 Chronic inflammatory conditions, like Crohn’s disease
 Leukemias with a monocytic component
(v) Lymphocytes: (Normal range ~ 20-30%)
 Lymphocytosis (increased number) ~
 Chronic bacterial infections
 Infectious mononucleosis
 Autoimmune diseases
 Lymphatic leukemia
[NOTE: (a) “Relative” lymphocytosis – When a decrease in neutrophils gives a picture of a
‘relative’ increase in lymphocytes, (b) “Reactive” lymphocytosis – Viral infections present a
blood picture of ‘reactive lymphocytes’; larger nuclei with open chromatin and abundant
cytoplasm; such reactive lymphocytes are seen in infectious mononucleosis.]


Lymphopenia ~ a decrease in the absolute lymphocyte count; seen in or associated
with:
 Pancytopenia (decreased numbers of all cell types)
due to any cause
 Advanced Hodgkin’s disease
 AIDS
—————————————————————————————————————————

46
 Section 9

Arneth count; Absolute white cell counts

 Introduction:
 TLC & DLC provide useful information that almost clinches the diagnosis, by
providing clues about – presence of infection, organism involved, severity &
prognosis of the disease. Yet, a further analysis of the information is
sometimes necessary.
 On DLC, the first 100 cells are counted. This count of different white cells
often gives a “relative” picture.
 Consider this – If there were 80 neutrophils counted, then obvious conclusion
would be of neutrophilia. But, we scanned only a region of the smear. What if
the smear was not made properly? What if the area that we observed had
more neutrophils incidentally? If the neutrophils have increased, the total
WBC count may also have increased; is that really the case?
Also, if 80 out of 100 cells were neutrophils, it means the other white cell types
will get counted in the remaining 20 cells. It may give a picture of ‘lymphopenia’.
Is the lymphopenia really present, or it’s only a ‘relatively’ low lymphocyte
count? To get clear answers to these questions, the ‘absolute’ counts are
calculated from TLC & DLC. For instance, if your TLC is 10,000/mm 3, and the
neutrophil count is 50%, then the “absolute” neutrophil count would be – (50% of
10,000 =) 5000/mm3.
Arneth count: (or, Arneth index; named after Josef Arneth)
 Neutrophils are ‘polymorphonuclear’ cells. Their nuclear segmentation provides for
a criterion to classify the neutrophils. Arneth count is the classification of neutrophils
based on the number of lobes of their nuclei.
 Aims & Objectives:
 To learn to identify neutrophils and categorize them on the basis of their
nuclear lobes
 To learn to calculate absolute counts for each of the white cell types

 Materials & methods:

ii. The materials used for TLC & DLC.
o Principle:
1. Arneth count: On a smear, 100 neutrophils will be counted and classified as per
their nuclear lobes.
2. Absolute counts: TLC and DLC are performed. The percentage of each white cell
is converted into its ‘absolute’ count (percent of the TLC).
o Procedure:
(1) Arneth count:

47
  With all the precautions mentioned previously, prepare a stained
blood smear (as in DLC).
 Observe the smear under high power first and then under oil
immersion (100 X). Identify the neutrophils; note down the number of
lobes in each of the neutrophil observed.
 Count 100 neutrophils in this manner; observe the same procedure
and rules as were followed for DLC.
 Result:
iii. Draw 100 squares (10 rows X 10 columns).
iv. Note down the neutrophils, with their nuclear lobes, in the squares,
as follows: N1 – single lobe, N2 – two lobes, N3 – three lobes, N4 – four
lobes, N5/6 – five or more lobes.
v. Express the result in terms of the percentage of each of the stages.
(2) Absolute counts:
 Perform the TLC & DLC on the blood sample, with the procedure and
precautions mentioned previously. Obtain the results.
 Result:
 For each of the white cell type, express its absolute number by converting
its percent into percentage of the total leucocyte count (TLC).

 Discussion:
(1) Arneth count:
(A) Physiology point-of-view:
(i) What is the significance of segmentation of the nucleus in a
neutrophil?
1. When a neutrophil is formed, its nucleus is single-lobed, round
or oval. The granulocyte precursors – myeloblast,
promyelocyte, and myelocyte – having this nucleus, undergo
active proliferation. Subsequent to myelocyte stage, the
maturation process continues in non-dividing cells, wherein
nuclear segmentation begins.
2. Segmentation starts with the nucleus first getting indented – a
bean-shaped nucleus in the metamyelocyte. When the degree
of indentation becomes more than 50% of the nuclear
diameter, the cell is said to have reached the band stage (or
stab-form stage).
3. With further maturation, there is increase in the segmentation
(number of lobes). In other words, neutrophils having nuclei
with one or two lobes are young neutrophils. The older or
senile neutrophils have nuclei with 5, 6, or even 7 lobes.
(ii) What is the normal Arneth count?
At any given point of time, the relative percentages of neutrophils, in
peripheral blood, are as follows:

48
 Stage 1 (N1) 5%
Stage 2 (N2) 30%
Stage 3 (N3) 45%
Stage 4 (N4) 18%
Stage 5 (N5+6+7) 2%

[fig: The graphic depiction of Arneth count; shift-to-left and shift-to-right.]

(B) Clinical perspective:
(a) Shift-to-left: (also called ‘regenerative shift’)
 If neutrophils in stages 1+2+3 are found to be more than 80%;
increase in the number of band forms

49
  An overwhelming proportion of younger neutrophils in peripheral
blood; indicates a hyperactive bone marrow resulting in increased
production of neutrophils
 Occurs in - infection, myeloproliferative disorders, myocardial
infarction.

(b) Shift-to-right: (also called ‘degenerative shift’)

 Greater proportion of older neutrophils (stages 4+5 > 20%) in
peripheral blood; indicates a hypoactive bone marrow.
 Hypersegmentation is seen in – megaloblastic anemia, uremia,
chronic liver diseases, bone marrow aplasia.
Thus, Arneth count can help assess the state of bone marrow activity with a simple
procedure like peripheral blood smear.
 What is Schilling’s index or Schilling’s classification?
(Named after Victor Schilling) The Schilling’s index classifies neutrophils into 4
categories:
 Myelocyte - 0
 Metamyelocyte – 0
 Band cell – 2-6%
 Segmented neutrophil – 55-75%

(2) Absolute counts:

(i) What are the absolute counts for various WBC types?
(number of cells per mm3 of blood)
 Neutrophils – 2000-7500
 Lymphocytes – 1500-4000
 Monocytes – 200-800
 Eosinophils – 40-400
 Basophils – 1-100
[The International Council for Standardization in Hematology (ICSH) has recommended that
the DLC should always be expressed as the absolute count of each cell type per unit volume
of blood.]

(ii) What is the significance of the absolute counts?

 Absolute counts are done to avoid misinterpretation of the results
of differential count.
The differential count (in %) should be contextualised with the
total leucocyte count of the patient.
For instance, in a patient, the neutrophil count is 73% and TLC is
well within range (say, 9000 per mm3), it should not be

50
 interpreted as neutrophilia. If the neutrophil count increases,
there should be some increase in the total leucocyte count.
Conversely, a differential neutrophil count of, say, 70% may look
normal. However, if the patient’s TLC is 20,000 per mm3, the
absolute neutrophil count would be (70% of 20,000 =) 14,000 per
mm3, which is way beyond the upper limit of the range for the
absolute neutrophil count. It is neutrophilia, although the
differential neutrophil count was normal.
Another confusing picture would be – an increase in a particular
cell (%) but decrease in the total WBC count. For instance,
increased lymphocyte (%) when total WBC count is actually
decreased. E.g. In typhoid fever. In reality, the WBC count is low
because the neutrophil count is low. And since neutrophils (%) was
low on DLC, there was a ‘relative’ increase in lymphocyte count.
This is called ‘leucopenia with relative lymphocytosis’.
Note:
 If there is change in leucocyte count, it should be denoted as to which cell is
responsible. E.g. Neutrophilic leucocytosis, lymphocytic leucocytosis, and so on.
 If there is a change in the differential count (% of a cell), it should be ascertained
from its absolute number. For instance, neutropenia is confirmed if the absolute
neutrophil count is less than 1000/mm3. (And, severe neutropenia – if the absolute
neutrophil count < 500/mm3.)

—————————————————————————————————————————

51
 Section 10

Bleeding time (BT)

 Introduction:
o There is a battery of tests performed on a patient undergoing surgery. (1)
Blood grouping and cross matching is done, and accordingly the blood bottles
are kept ready in case there is excessive bleeding during the surgery. (2)
Bleeding time and clotting time (BT, CT) are the tests to assess whether the
patient’s blood vessels take normal or longer time to stop oozing after they
have been ruptured. The other tests performed before surgery – Hb & RBC
count (for O2-carrying capacity), TLC & DLC (to rule out any concurrent
infection), and the overall clinical condition is assessed by recording BP, ECG,
and by examination of the cardiorespiratory status.
o A clinician needs to know the bleeding & clotting time in a variety of other
clinical settings, such as – (1) when there is appearance of purple coloured
spots on the skin, or petechiae (small hemorrhagic dots) indicative of
bleeding with faint injuries, or excessive bleeding into the tissues indicating
defective clot formation, (2) when platelet plugs or clots tend to form in
coronary or cerebral vessels, leading to myocardial infarction or stroke, and
when such patients are given platelet aggregation inhibitors or anticoagulant
drugs.
 Aims & Objectives:
 To estimate the bleeding time of the obtained blood sample.
 To study the clinical significance of the bleeding time (BT)
estimation.

 Materials & Methods:

 Lancet or needle
 Spirit-soaked cotton swab
 Filter paper
 Stopwatch
o Principle:
Bleeding is induced and then time lapse is noted for the stoppage of bleeding.
o Procedure:
 With the usual aseptic precautions, prick the ring finger. Immediately
start the stopwatch.
 Touch the filter paper to the drop of blood formed on the finger.
 Thereafter, touch the filter paper on the oozing area every 30 seconds.
This should be repeated till no stain of blood can be seen on the filter
paper, upon touching the injured area. It means, bleeding has stopped.
 Stopwatch is stopped at this instance and time noted.
 Result:

52
 Time (in minutes) should be noted between onset of bleeding and stoppage of
bleeding.
If the stopwatch is not available, wrist watch or a clock with seconds arm can be
used. First drop was taken at zero time, and every drop thereafter was with 30
seconds’ interval. Count the number of drops; divide the number by 2. That will give
bleeding time (minutes).
Note: Add 15-30 seconds to the result obtained from the number of drops. Reason – The
last drop indicates the bleeding was still on at that instance. The definition of bleeding time
is the ‘time interval between onset and stoppage of bleeding’.

 Common errors; Special precautions:

 Noting of the time: If the sample is being collected by a self-inflicted injury,
the observer tends to forget about noting down the time of the onset of
bleeding.
 When puncturing the skin: The injury should be deep enough (not less than
2-3 mm).
Of particular importance is to note that the injured finger shouldn’t be
milked or squeezed, to let out blood, once the puncture has been made.
Squeezing of the finger will interfere with the platelet plug formation that
is expected to occur spontaneously and naturally in the ruptured vessel(s).
 The drop of blood should not be wiped off. The filter paper should just be
touched gently to the tip of the drop.

 Discussion:
(A) Technical aspect:
(i) What are the other methods used to record bleeding time?
 Duke method: A BP cuff is applied to the arm; pressure in the cuff
raised to 40 mm Hg. Using a lancet or a needle, the ring finger is
pricked. Rest of the procedure is similar to the one described
above.
 Ivy method: BP cuff is applied in this method too. This method is
more invasive. Using a scalpel blade, an incision (10 mm long; 1
mm deep) is made on the forearm. Bleeding time is noted using
filter paper or a paper towel.
 Template method: Similar to Ivy’s method; the only variation is in
the instrument used to make the incision.

53
  Tourniquet test: (Hess’s test for capillary resistance)
BP cuff is applied to the arm and pressure raised up to 100 mm Hg for a
period of 5-7 minutes. 2-3 minutes after the cuff is deflated, an area below
the cubital fossa is observed for petechiae (hemorrhagic dots). In the area
with 3 cm diameter – (1) In normal subjects: the number of petechiae is up
to 10. (2) More than 20 petechiae is considered abnormal.

The abnormally high number (positive tourniquet test) is diagnostic of

thrombocytopenia, with or without an increased capillary fragility.
This test is thus a useful indicator for platelet count. Thrombocytopenia is a
decrease in platelet count below the lower limit of their normal range (i.e., less
than 1,50,000/mm3).

(B) Physiology point-of-view:

(i) Hemostasis: 3 events occur after a vessel is ruptured – (1)
Vasoconstriction (within a few seconds): To minimize the amount
of bleeding, (2) Platelet plug formation: In about 1-3 minutes’ time
after the injury, the platelets reaching the area are activated and
get adhered to each other and to the injured vessel wall. The plug
seals the gap temporarily and the bleeding stops. (3) Clot
formation: In about 4-9 minutes after the injury, a clot is formed
on the platelet plug.
(ii) If there is a bleeding disorder, manifested as spontaneous
bleeding or excessive/prolonged bleeding after an injury, it may
be because of abnormalities of one or more of the components of
hemostasis, such as -
(a) Increased vascular fragility: E.g., in severe vitamin C deficiency
and deficient collagen of vessels, systemic amyloidosis, etc.
(b) Deficient platelet plug formation: It may be because of – (i)
decrease in the platelet numbers (thrombocytopenia), or (ii)
defects in platelet function – E.g., as occur in uremia, or in
patients taking aspirin.
(c) Coagulation abnormalities: Clotting disorders, such as
hemophilia. (Discussed later)
 Among the categories mentioned above, bleeding time is abnormal in vascular
abnormalities and platelet defects. In clotting disorders, bleeding time is normal.
(iii) What are the other tests for platelet function?
 Platelet function is indicated by clot retraction time. A clot
is formed on the surface of the platelet plug. The
contractile filaments (actin, myosin, thrombosthenin)

54
 within the platelets then cause contraction of the plug and
reduction in the clot size.
 The normal platelet count: 1,50,000-4,00,000/mm3.

(C) Clinical perspective:

(i) What is the normal bleeding time?
 Normal bleeding time: 1-3 minutes. [Note: The reference
range for bleeding time depends on the method employed;
the range varies from 2 to 9 minutes.]
 It is prolonged in the conditions with low platelet count.
(ii) What is ‘critical’ platelet count?
 A platelet count of 40,000/mm3 or less is considered critical. With
such a low count, bleeding time is prolonged. In other words,
bleeding will continue for much longer periods.
(iii) What are the factors that may influence the bleeding time?
 Bleeding time is mainly the function of platelets, and hence
platelet count is the most obvious factor. Some other factors may
influence the bleeding time, viz. –
a. Size of the injury
b. Condition of endothelium of the vessel and its
adhesion properties
c. Contractile property of smooth muscle in the vessel
wall
d. Blood pressure
(iv) What is purpura?
 Appearance of purple coloured spots on skin, resulting from
bleeding underneath the skin.
 The petechial hemorrhages appear as smaller spots (< 3 mm in
diameter); the spots of purpura are larger (3-10 mm). Ecchymoses
are even larger (> 10 mm).
 A common cause of hemorrhagic spots is a disorder called
idiopathic thrombocytopenic purpura (ITP). It results from an
underlying immune mechanism; antibody formation resulting in
thrombocytopenia.

55
 Anti-platelet agents:
 Patients having suffered from myocardial infarction or stroke, or any
patient shown to have tendency for thrombus formation, are given the
anti-platelet drugs or platelet aggregation inhibitors. The most common
drug is aspirin.
 If a patient is on aspirin, obviously the bleeding time would be prolonged.
In such a scenario, the drug should be stopped for 7 days prior to the
bleeding time test.

56
 Section 11

Clotting time (CT)

 Introduction:
 Blood coagulation is the function of clotting factors present in plasma.
 Coagulation disorders result from deficiencies of one or more clotting factors.
The deficiency may be – (a) congenital: e.g., Hemophilia (hereditary
hemorrhagic disorder due to factor VIII deficiency), or (b) acquired: e.g., due
to liver disease (most clotting factors are synthesized in the liver).
 A coagulation disorder manifests as the bleeding tendency. There may be
bleeding into the tissues after a faintest injury, or there may even be
spontaneous bleeding. Patients often present with hematomas that form due
to bleeding into the tissues.
 When abnormal bleeding is the presenting symptom, the clinician needs to
probe it further along the following lines: (1) Is it a bleeding disorder? If yes,
then – (2) Which component of the hemostasis is affected? The blood
vessels, the platelets, or the clotting system? The bleeding time & clotting
time (BT, CT) estimations will provide answers to these questions. (3) If it is
the coagulation defect, what is the cause? Which factor(s) is/are deficient?
 Clotting time (CT) is thus an important tool in the investigation of
hemorrhagic disorders.
 Aims & Objectives:
 To estimate the clotting time of the obtained blood sample.
 To understand the importance of clotting time (CT) estimation in various
clinical settings.
 Materials & Methods:
 Cotton swab soaked in spirit
 Lancet or needle
 Capillary tube: 1.5-2 mm in diameter and 6 inches long
 Stopwatch
o Principle:
Bleeding is induced and time taken for clot formation is noted.
o Procedure:
 With usual aseptic precautions prick the ring finger. Start the stopwatch.
 Fill the capillary tube with the blood. At least the 2/3rd of the length of the
tube should be filled.
 Every 30 seconds break a small piece of the blood-filled portion of the
tube. Repeat this until a fibrin thread can be seen between the broken
ends of tube. Stop the stopwatch and note down the time.
 Result: Clotting time should be noted in minutes. It is the time interval between the
onset of bleeding and the formation of clot (fibrin thread).
 Normal clotting time: 4-9 minutes.

57
  Common errors; Special precautions:
 The depth of injury: Ideally, the injury should be about 5 mm deep. In
practical terms, however, it should at least be deeper as compared to
the one applied for bleeding time estimation. The reason is two-fold: (i)
For the capillary tube to be filled, a larger drop of blood is needed. (ii)
Coagulation cascade needs to be initiated. (Discussed later.)
 Filling of the capillary tube: One end of the tube should just be touching
the drop of blood. The other end should be open, not blocked by the
fingertip.
 Breaking a piece of the tube: Break a small piece gently, and hold it just
near the remaining portion of the tube. The delicate fibrin thread can be
seen to be bridging the two pieces of the tube.

 Discussion:
(A) Technical aspect:
 Why should the injury be deep in this procedure?
 Hemostasis involves the processes of vasospasm, platelet plug
formation and clot formation.
 In the case of small vessels, hemostasis is mainly achieved by
vasoconstriction and by the platelet plug, rather than a clot. (A man
suffers micro-injuries almost on a routine basis; such injuries are filled
mainly by platelet plugs.) The injury made should be deeper, involving
larger vessels, in which the clotting is the major process that limits the
blood loss.
 What are the other methods for determination of clotting time?
 Lee & White method: Clotting time of venous blood is determined in a
glass test tube of standard bore.
(B) Physiology point-of-view:
[Bleeding time = 1-3 minutes; Clotting time = 4-9 minutes.]
 Why is clot formation necessary, when bleeding has already been stopped by
the platelets? (Ans: To allow for healing of the vessel.)
 Platelets are the cellular elements of blood. The platelet plug is an
important early tool for a quick limitation of blood loss. However, this
hemostatic plug may interfere with fluidity of blood and patency of

58
 the vessels. Hence, the cellular elements (platelets) in the plug are
lysed and replaced by fibrin.
 The platelet plug is a quicker but temporary response. The platelet
aggregation and adhesion properties have a limited time-span.
However, the injury in the vessel wall takes longer to heal. Thus, even
as the platelets are removed from the hemostatic plug, the hole in the
vessel is kept sealed by the fibrin mass which too is gradually digested
and the vessel is healed.
 Why does blood not clot spontaneously within the vessels, despite the
presence of clotting factors in plasma?
 Almost 50 substances or mechanisms related to blood clotting are
present in the body; some are pro-coagulant and others
anticoagulant. Under normal circumstances, the anticoagulant
mechanisms predominate. The pro-coagulant substances or
clotting factors are in inactive forms.
 There are two ways in which the clotting process is initiated: (1)
Extrinsic mechanism: Tissue injury causes release of tissue
thromboplastin, which initiates coagulation. (2) Intrinsic
mechanism: Trauma to blood, or blood (factor XII) getting exposed
to subendothelial collagen initiates coagulation. (When blood is
collected in a test tube, it clots purely by intrinsic mechanism.
Factor XII is activated when it comes in contact with a water-
wettable surface, like glass.)
 Both these mechanisms, by different pathways, form prothrombin
activator (factor Xa or activated factor X). It converts prothrombin
to thrombin; thrombin, in turn, converts fibrinogen to fibrin.

(C) Clinical perspective:

 Clinical importance of the clotting time test: (Clotting disorders)
 Hemophilia is an inherited coagulation disorder. Hemophilia A results
from deficiency of factor VIII. Hemophilia B (Christmas disease) results
from deficiency of factor IX.
 In hemophilias, clotting time is prolonged, whereas bleeding time is
normal.
 Acquired clotting disorders:
e. Clotting factors II, VII, IX, and X are vitamin K-
dependent factors; Vitamin K deficiency results in a
severe clotting defect.
f. Diseases of the liver may also lead to coagulation
disorders since many clotting factors are synthesized in
the liver.

59
 The other screening tests and their clinical significance:
1. Prothrombin time [PT](Quick’s one-stage test):
 Tissue thromboplastin (usually a powdered dried brain extract)
and ionized calcium are added to patient’s plasma. The time
required for activation of prothrombin (clot formation) is noted
as prothrombin time.
 The normal prothrombin time – 11-16 sec.
 The test measures concentrations of prothrombin and of the
clotting factors V, VII, and X. These factors are involved in
extrinsic pathway of coagulation.
 PT is normal in hemophilia and Christmas disease since the
extrinsic pathway does not involve factors VIII & IX for clot
formation.
 PT is sensitive to prothrombin levels, and is prolonged in
deficiencies of factors V, VII, and X, as may occur in vitamin K
deficiency.
 PT is employed to titrate the anticoagulant therapy with vitamin
K antagonists, such as warfarin.
 NOTE: The normal range for prothrombin time varies with different tissue
factors used for the test. Hence, while using a particular tissue extract for a
patient’s PT, the same tissue factor is used on a plasma from a normal
subject (the ‘control’ prothrombin time). The patient’s PT is then denoted
as the ratio with the ‘control’. The patient’s result is regarded abnormal if
the PT is more than 2 seconds longer than the control PT, or if the ratio of
patient’s PT to control PT is more than 1.2.
2. The (activated) partial thromboplastin time [PTT]:
 It measures the production of thromboplastin (prothrombin
activator) by the intrinsic pathway.
 The test involves two steps – (i) Patient’s plasma is incubated
with kaolin or some other surface active agent. This will activate
the contact-activated clotting factors (factors XII & XI) and will
trigger the intrinsic pathway for clotting; (factor XII will be
activated to XIIa, which then activates XI to XIa. Clotting will not
proceed beyond this point as Ca++ is essential for the remaining
steps.) (ii) Platelet substitute and Ca++ are added and the time
taken for clot formation is noted. (Platelet substitute is in the
place of platelet phospholipids which are involved in intrinsic
pathway in the body.)
 Normal range: 35-45 sec.
 Abnormal result is indicative of deficiencies of factor VIII
(hemophilia) or factor IX (Christmas disease).

60
 Section 12
Blood group identification
(Blood typing)

 Introduction:
 Before the start of 20th century, blood transfusions were attempted
randomly, with the belief that all humans have essentially the same type of
blood. Many of the transfusions resulted in severe reactions (sometimes
even leading to death).
 With the discovery of blood group antigens in 1900 by Carl Landsteiner, it
was known that the presence (or absence) of certain antigens is what
separates one person’s blood from another.
 Till date, more than 20 blood group systems have been discovered, each
having its own significance. ABO & Rh systems are clinically the most
important blood group systems. Before any blood transfusion, the blood
groups in these two systems have to be identified in the donor and the
recipient.
 Knowing one’s own blood group is almost obligatory today. It has become a
part of one’s identification (most ID cards have the person’s blood group, in
case he/she needs an emergency transfusion). Before marriage, seeking the
Rh-group of the prospective husband & wife is advisable (possibility of
‘hemolytic disease of newborn’ – discussed later).
 Aims & Objectives:
 To detect the ABO & Rh blood group by detecting the antigens on the red
cells.
 To learn the importance of blood grouping and cross-matching in the context
of blood transfusion.
 Materials & Method:
 Materials for the collection of blood sample
 Anti-sera (anti-A, anti-B, and anti-D)
 1% sodium citrate
 0.9% saline
 Kahn tube
 Dropper
 Glass slides or porcelain tile
 Microscope

61
o Principle:
Blood group antigens to be identified are present on the red cell membrane. The
antisera contain antibodies against those antigens. A drop of the red cell
suspension is mixed with each type of antibody (anti-A, anti-B, and anti-D). The
blood group antigen can be identified from its reaction (agglutination) with the
particular antibody.
o Procedure:
 Take 1-2 mL of sodium citrate (1%) in Kahn tube.
 With usual aseptic precautions prick the finger and obtain 2 drops of
blood; transfer this blood to the Kahn tube.
 Mix the sodium citrate and the blood by gently rocking the Kahn tube. A
red cell suspension has been formed.
 Take two drops each of: (i) anti-A serum, (ii) anti-B serum, (iii) anti-D
serum, and (iv) normal saline (‘control’), in separate depressions, in the
porcelain tile. Use separate droppers for each of the anti-sera. The
depressions should already be marked respectively as ‘A’, ‘B’, ‘D’, and
‘control’. 4 glass slides may be used instead of the tile.

62
  Add 1-2 drops of red cell suspension in each of the depressions (or glass
slides) containing the anti-sera. Blow over the depressions gently, or
shake the slides, so that the red cell suspension mixes with the anti-sera.
 Look for ‘agglutination’; observe all the depressions (or slides) 5-6
minutes after the mixing step. Note if agglutination is also found to have
occurred in ‘control’. If yes, entire procedure is invalidated and should be
repeated.
 Observe the tile or slides under microscope, to confirm the agglutination.
 Observation/Result:
o If agglutination is found in ‘A’:- A antigen present on the red cells
o If agglutination is found in ‘B’:- B antigen present on the red cells.
(i) ABO group can thus be determined as follows:
 Agglutination found only in ‘A’: The blood group is A
 Agglutination found only in ‘B’: The blood group is B
 Agglutination found in both ‘A’ & ‘B’: The blood group is AB.
 Agglutination found in neither ‘A’ nor ‘B’: The blood group is
O.
(ii) ‘Rh’ group should be determined: If agglutination has occurred in
‘D’, the group is Rh +ve; if agglutination wasn’t found in ‘D’, the
group is Rh –ve.
Result: The blood group should be mentioned for both ABO & Rh systems.
For instance, “AB +ve” (when agglutination was found in A, B, and D).

63
 Common errors; Special precautions:
 Mixing of the red cell suspension with anti-sera:
 It is an important step. Tilt or rock the slides. Small glass rods may also be used
for mixing.
 Observing the agglutination:
 Wait for 5 minutes; let the agglutination reaction to occur. Do not wait for too
long as the mixture gets dried up.
 Although visible and identifiable on naked eye examination, agglutination
should also be verified and confirmed under microscope.
 “Pseudoagglutination” should be distinguished from true agglutination:
 RBC clumping, called ‘rouleaux formation’ may resemble agglutination. This
‘pseudoagglutination’ should not be mistaken for agglutination.
(I) When observed under microscope, a rouleaux formation can be identified as
piling up of RBCs in a line (agglutination will be seen as almost circular
aggregates). (II) Addition of saline to pseudoagglutination will cause the
rouleaux to disperse or get lined up as a typical rouleaux.
 The tile should be washed thoroughly every time, preferably using a detergent. There should
be no left-overs of the antigens or antibodies from any previous blood typing, lest it will give
erroneous results.

 Discussion:
(A) Technical aspect:
 About the anti-sera:
 An anti-serum is actually the serum that contains the specific
antibodies. A particular antigen is injected into an animal (e.g. rabbit).
The animal develops antibodies against the antigen; its blood is
collected and serum is separated from it. The serum is now called the
anti-serum.
 Serum is a clear, pale yellow-coloured liquid. However, for the
universal identification purpose, anti-sera A & B are added with blue
and dark yellow colours, respectively.
 “Washing” of the red cells; making a red cell suspension:
 from a technical standpoint, this is the most important step. Blood
sample is gently mixed with citrate-containing saline; the cells are

64
 allowed to settle down and then the supernatant fluid is pipetted out.
(The mixture may be centrifuged to separate plasma from cells.) Saline
is again added to the cells; the procedure thus repeated 2-3 times.
(a) A diluted red cell suspension is being used: because it allows
clumping to be detected easily.
(b) Plasma is removed: to minimize the chances of either false
positive or false negative agglutination due to the proteins
present in the plasma. For instance, in some disease conditions,
certain globulins in plasma may hasten rouleaux formation
(stacking of red cells), which may be mistaken as agglutination.
This is false positive.
(c) Cells are ‘washed’: Patient’s cells may already have been coated
in vivo with antibodies. (Red cell membranes have blood group
antigens. Patient’s plasma may have had some antibodies, other
than blood group agglutinins, which may have coated the blood
group antigen even before the sample was taken out for
grouping.) These agglutinins need to be washed off from the red
cell membrane.
 Importance of the “control”: (Significance of agglutination in control)
 0.9% NaCl is isotonic to red cells, and hence is used as a control. (a)
There would not be lysis of cells in the isotonic saline. (b) There
should be no agglutination in saline.
Agglutination in control invalidates the test. In such
an event, entire procedure will have to be repeated.
Significance: (i) It means the cells were not ‘washed’
properly. If there was in vivo sensitization, the
antibodies (IgG) thence formed may already have
coated the red cells. This may be the reason for
agglutination in control.
 What is “reverse” grouping?
 Once a blood group is determined, it can be confirmed by reverse
grouping. That is, patient’s serum contains agglutinins; the serum is
mixed with the red cell antigens (A & B) from a known sample. Thus,
agglutinins are detected in patient’s plasma, and the blood group is
confirmed indirectly.
 Since the conventional method of detecting the blood groups by red
cell antigens has the possibility of ‘pseudoagglutination’ (false
positive) and false negative results, reverse grouping may additionally
be employed for confirmation.
(B) Physiology point-of-view:
i. About the antigens & antibodies:
 Blood group antigens or agglutinogens are present on red cell
membrane; the antibodies or agglutinins are present in

65
 plasma. In ABO system, the two antigens are A & B. Their
agglutinins are anti-A (or ) and anti-B (or ).
 In Rh system, there are 6 varieties of Rh antigen – C, c, D, d, E,
e. More than 95% of the Rh +ve persons have the variety D.
Hence, Rh antigen is also called ‘D’ antigen; its agglutinin is
called anti-D.
 Landsteiner’s law: “If a particular antigen is present on the red
cells of a person, then its corresponding agglutinin will not be
present in his/her plasma. And, if an antigen is not present on
red cells, then the agglutinin corresponding to it will be
present in the plasma of the person.”
NOTE: (1) The anti-A and anti-B are naturally occurring antibodies (IgM type). It should be
noted that antigens A & B are not present only on the RBCs; they are also present in the
nature. When a child is born, he/she gets exposed to both antigens A & B from the nature
(through food or through microbes, etc). If he/she belongs to A blood group, A antigen will
be recognized as “self” (no antibody will be formed against A); antibodies will be formed
against B antigen as B will be considered to be “non-self”. Converse will happen for persons
of group B. (2) Anti-D antibodies are “induced” antibodies (IgG type); they do not occur
naturally. D antigen is present ‘only’ on the RBC membranes. Hence, the only situation when
anti-D antibody is formed is – when Rh negative persons are exposed to Rh +ve blood.
(C) Clinical perspective:
 What is “cross matching”?
 Blood grouping and cross matching are routinely done before any
blood transfusion.
(a) Blood grouping: The groups are determined in the donor and the
recipient. Both should belong to the same blood group.
Conventionally, ‘O’ group is considered universal donor since the
red cells do not harbour antigens. ‘AB’ is considered universal
recipient, as it doesn’t have agglutinins in the plasma that would
cause agglutination.
(b) Cross matching: Donor’s and recipient’s blood samples are
collected and cells and plasma are separated.

66
 Donor Recipient
Donor’s cells
are mixed

with
recipient’s Cells Cells
plasma; this
is called
Major
major cross-
matching. Minor
Plasma Plasma
Donor’s
plasma is mixed with recipient’s cells; this is called minor cross-matching. (Why is
this needed?

Ans: There are many different types of antigens on red cells; the antigens that are not
detected on routine blood grouping. Hence, before a decision about transfusion with a
particular donor’s blood is to be taken, it needs to be screened for such possible antigens on
his/her red cells, against which the recipient may have or may form antibodies.)
“Donor cells --> mixed with recipient plasma” is called “major” cross-matching. Why?

Explanation: The antigen is on the cells. Donor’s cells (sample) are mixed with the
recipient’s plasma. If agglutination is observed, it means the recipient’s plasma has
antibodies against the antigen on donor’s cells. If transfusion is given with such a finding,
there would be massive agglutination reaction in the recipient, resulting in hemolysis and
other consequences. Hence, this cross-matching is called major (for its major
consequences).
The agglutinins are in plasma. Donor’s plasma (sample) is mixed with recipient’s cells. Now,
if agglutination is found to have occurred, it means donor’s plasma has antibodies against
the antigens on the recipient’s red cells. If the transfusion is given in such a scenario,
donor’s agglutinins are likely to react with the red cell antigen, in the recipient. However,
the resultant hemolysis or other likely complications will be of minor consequence. Reason:
Let’s say, 500 mL of blood is given during a transfusion. Out of this, roughly about 250-260
mL will be plasma, which contains antibodies. When transfusion is given, the antibodies in

67
this small amount of plasma will be greatly diluted in the 5 L blood volume of the recipient;
even if they were to cause agglutination, it would not be severe.
Hence, with minor cross-matching showing agglutination, the blood transfusion may be
allowed to be given, under emergency situations or when an appropriate matching blood is
not available.
When major cross-matching shows agglutination, a transfusion with that blood isn’t
allowed.
 Feto-maternal blood group incompatibility:
[Hemolytic disease of newborn]
 The condition that results from Rh-incompatibility between mother
and fetus; fetus affected due to agglutination reaction.
 When an Rh –ve mother conceives for the first time, the problem will
start if (and only if) the fetus is Rh +ve (i.e., D antigen inherited from
the father). The placenta does not normally allow the transfer of red
cells from fetus to mother. However, when placenta is separated,
during delivery, or due to some attempted obstetric maneuvre,
placental barrier for the antigen may be lost. Sometimes referred to
as feto-maternal bleed, it may allow fetal red cells (and their D
antigen) to enter the mother’s circulation.
 Due to the antigenic exposure, the mother’s immune system will form
anti-D antibodies. The process starts within 48-72 hours; the antibody
titre will reach a peak level by 2-4 months. It is assumed here that this
is the mother’s first ever exposure to D antigen.
 The fetus wil be delivered unharmed. However, mother has anti-D
antibodies. If the mother conceives again, and the fetus is Rh +ve, the
maternal antibodies (IgG type) will cross over to the fetus. (Note: IgG
antibodies can cross the placental barrier.)
 Agglutination reaction, in fetus, will result in hemolysis. The fetus will
be born with anemia and jaundice. It is called hemolytic disease of the
newborn. Hemolysis in fetus results in rapid proliferation of
erythroblasts (immature red cell precursors) in bone marrow.
Presence of such erythroblasts in peripheral blood is the reason that
the condition is called erythroblastosis fetalis.

68
 Section 13
Blood bank visit; Blood transfusion

 Introduction:
 Storage of whole blood and blood products is done in specially designed
places called the blood banks.
 Collection of blood, its handling and storage need certain skill sets and
facilities. Blood banks are manned by personnel trained in collection and
storage of blood.
 After blood is collected for storage, it is screened for its blood group and
certain infections.
 The appropriate blood is stored and utilized for elective surgeries, emergency
procedures, and various other clinical settings that need blood transfusions.
 Aims & objectives:
 To learn the laboratory protocols for blood sampling and storage of blood.
 To learn the procedural details involved in blood transfusion
 Discussion:
 What is blood transfusion? When is it necessary?
 Collection of whole blood from a person and infusion of the blood
into another person is termed blood transfusion.
 When a person has suffered an injury that resulted in blood loss, or if
a person is having severe anemia, blood transfusion becomes
necessary.
 When blood loss is anticipated (e.g., during a surgery), blood is kept
ready for transfusion.
 Persons having congenital hemolytic diseases (e.g., thalassemia) need
repeated transfusions.
o Donor: a person whose blood is collected for transfusion;
o Recipient: a person who receives the blood
(A) Technical aspect:

 Criteria for a donor; Screening of the donor:

 Age: between 18 and 55 years
 Weight: should not be less than 45 Kgs
 Hemoglobin: should be more than 12 gm%
 The donor should be screened for hepatitis B & C and HIV.
 The donor should not be suffering from any communicable
disease; the donor should not have suffered from a febrile illness
(malaria, viral fever, etc) in the recent past
 History of (H/O) major cardiac and pulmonary illness should be
enquired into; also, rule out H/O jaundice during the preceding 3
years.
 Interval between two successive donations: should not be less than 3 months.
 Females should not be donors during menstruation, pregnancy, or lactation.

69
  Collection and storage of blood:
(a) Steps in the collection of blood:
 Note down all the relevant details of the donor. Make sure
that the tray contains all the required materials.
 The area for venepuncture (forearm, around the antecubital
vein) is sterilized with 70% alcohol.
 A sphygmomanometer cuff is applied to the donor’s arm;
pressure in the cuff is kept around (approximately) diastolic
pressure. The skin over the site of the vein may be gently
tapped a few times. The vein will be prominently visible.
 A 21G needle or a winged (‘butterfly’) needle is introduced
into the vein. (The needle is attached to a plastic tubing,
which, in turn, is attached to the bottle for blood collection.)
 Blood starts flowing into the bottle which already contains an
anticoagulant. The bottle is gently shaken periodically, so that
the blood and the anticoagulant get mixed properly. The ratio
should be 350 mL of blood : 50 mL of anticoagulant. (Or, 480
mL of blood : 120 mL of anticoagulant.)

 Anticoagulants used commonly:

o Citrate is the anticoagulant; used in combination with
dextrose – (1) Acid-Citrate-Dextrose (ACD),
(2) Alsever’s solution (a variant of ACD),
(3) CPD-A (Citrate-Phosphate-Dextrose-Adenine)
o Addition of dextrose allows for a longer survival of the red
cells.

(b) Storage of blood:

 The collected blood should be immediately labelled; the label
should include the date of collection, donor’s relevant data (full
name, date of birth, blood group, etc), sample number (pertaining
to the blood bank records). Bar code labelling is preferred.
 The blood is stored in refrigerator at 40C (+/- 20C).

 Indications for transfusion:

70
  Severe anemia (Hb < 6 gm%)
 Hemoglobinopathies (e.g., thalassemias)
 Blood loss due to trauma/accidents or surgery

 Precautions before transfusion:

 Blood grouping and cross matching of the donor’s and recipient’s
blood samples
 Carefully noting the details on the label of the blood bottle – date,
blood group, etc.
 Blood should be warmed to the room temperature.
 Instructions regarding the transfusion should be written legibly on
the recipient’s case file. For instance, rate of the transfusion drip –
20 drops/min. Diuretics are recommended when whole blood
transfusion is being given in elderly or patients with compromised
status of the heart.
 During transfusion: The recipient should be monitored for general condition (GC),
pulse, blood pressure.
 As per the guidelines recommended by British Committee for Standards in
Hematology (BCSH), all stages of the transfusion process must be documented and
the records preserved for 30 years.
(B) Clinical perspective:
(i) What are the hazards of blood transfusion?
The complications after transfusion can be categorized as: (A) Due to
incompatible transfusion, (B) Due to transfusion process (donor-related;
recipient-related)
o Hazards of mismatched blood transfusion:
Mismatched blood means the blood groups not matching; donor’s red
cells have antigens against which the recipient’s plasma has agglutinins.
Such transfusion results in agglutination reaction which manifests as –
 (early or acute) Fever, chills & rigors
 (delayed) Hemolysis, jaundice
 Acute renal shutdown
o Transfusion complications:
 Thrombophlebitis (if the needle is placed in the vein for longer
periods)
 Heart failure (due to circulatory overload; particularly in elderly,
hypertensives, or patients with heart disease)
 Febrile reaction due to transmission of pyrogens from the donor
 Allergic reactions due to allergens in donor’s blood, to which the
recipient is previously sensitized.
(ii) What are “blood products” and “blood substitutes”?
Often, a patient needs only a specific constituent of blood. Instead of whole
blood, it is sometimes desirable to give transfusion of a specific component

71
of blood, called “blood product” or blood fraction. Examples of blood
products are –
 Fresh frozen plasma (FFP) – contains clotting factors; given in patients
with clotting disorders
 Cryoprecipitate – given in factor VIII deficiency
 Packed red cells – preferred in elderly, with heart status being
compromised; in chronic anemia
 Platelet-rich plasma, platelet concentrate – given in
thrombocytopenia
o To avoid allergic and agglutination reactions that may be caused by blood
transfusion, ‘substitutes’ are given. They are also referred to as plasma
expanders. E.g. Dextran, Hemaccel. They contain saccharides that pull
interstitial fluid water into blood vessels by osmosis, thereby increasing
the circulating blood volume.

72
 Section 14
Platelet Count
(Brecher & Cronkite method)

 Introduction:
 Bleeding time test is an indicator for a patient’s platelet count. However,
platelet count itself is done routinely as a part of Complete Blood Count
(CBC).
 Platelet count is ordered separately in cases where there is spontaneous
bleeding or in which there is easy bruising or if bleeding does not stop within
the normal time range. Platelet count is often ordered in conjunction with
bleeding time, PT, and PTT tests.
 There are some clinical conditions in which platelet count becomes a pointer
to the diagnosis. E.g. Dengue. Fever with a low platelet count should arouse a
suspicion in clinician’s mind about such conditions.
 Platelet count also becomes a prognostic indicator. As the diagnosis is made
and treatment started, an increase in platelet count over due course is
anticipated.

 Aims & Objectives:

 To determine the platelet count of the given sample, by Brecher’s method.
 To understand the significance of platelet counts in a variety of clinical
settings.
 Materials & Methods:
 Spirit, cotton, needle/lancet
 Hemocytometer – WBC pipette, Neubauer’s chamber
 Diluting fluid consisting of 1% ammonium oxalate
 Microscope

o Principle:
Blood sample is diluted 20 times (WBC pipette). The platelets are counted
in a smaller field (RBC squares), as the number is expected to be in lacs.
The counted number (‘n’) is converted to per mm3, and then multiplied
by 20 to obtain the final count.
o Procedure:
 Clean the ring finger with a spirit-soaked cotton swab; prick the
finger with a lancet or a needle.
 Take the blood up to 0.5 mark in the WBC pipette. Take the
diluting fluid up to 11 mark.
 Roll the pipette gently after holding it horizontally between the
palms; the blood and and the diluting fluid should be mixed
properly.

73
  Discard the first 2-3 drops (coming out of the stem, consisting only
of the diluting fluid). Further drops coming out of the pipette will
be from the bulb that contains the diluted blood sample. Charge
the Neubauer’s chamber.
 The Neubauer’s chamber is now kept in a moist Petri dish for
about 15-20 minutes; this will allow for settling of the platelets.
 Focus the RBC squares, first under low power and then under high
power. Platelets appear as small refractile dots or shiny particles;
count them under high power.
 Count the platelets in 5 squares – top left, top right, bottom left,
bottom right, and the central square.
 Observation/Result: (Refer to the chapter on ‘RBC count’ for the calculations)
The platelets counted =
Calculations:
Volume of a single RBC square is 1/250 mm3; volume of 5 squares = 5 X 1/250 = 1/50
mm3.
If 1/50 mm3 contains ‘n’ platelets (the number that you counted), then 1 mm3
contains ‘n’ X 50 platelets.
The blood sample was diluted 20 times. So, 1 mm3 blood actually contains ‘n’ X 50 X
20 = ‘n’ X 1,000 platelets.

74
  Common errors; Special precautions:
 While collecting the sample: Do not squeeze the finger excessively.
 Dirt particles may get counted as platelets: Since platelets are only
2-4  diametered small dot-like particles, even artifacts, debris or
dust may be mistaken for platelets. Hence, all the apparatus, and
particularly the Neubauer’s chamber should be diligently washed
and wiped clean. Also, filter the diluting fluid just before its use.
This is done to remove particles.
 Condenser position: To view small refractile particles (platelets),
the focus & illumination should be proper. Condenser should be at
a lower position; condenser-iris diaphragm and fine adjustment
should be manoeuvred synchronously.
 Prevent clumping of platelets:
(i) Blood sample should be quickly diluted,
(ii) Mixing of the blood and the diluent should be thorough; roll
the pipette for 5-10 minutes.
 Allow the platelets to settle: The charged Neubauer’s chamber is
kept under moist Petri dish. The mixture will not evaporate, and
the platelets will settle down.

 Discussion:
(A) Technical aspect:
 Blood sample and the variation in platelet counts:
 Blood samples obtained by skin prick often give lower platelet
counts, and the counts are not consistent. Reason: Platelets tend
to adhere to the site of injury; they do not get adequately
represented in the obtained sample.
Platelet count should be done on venous blood samples, with
EDTA as the preferred anticoagulant.
 Dilution of the sample:
 The diluting fluid contains ammonium oxalate (1%); it performs
following actions: (a) destroys red cells, (b) preserves platelets,
and (c) acts as anticoagulant.
 The principle for dilution is – Higher the cell count expected, greater should be the
dilution of sample, i.e., greater dispersion of the cells being counted. For WBC count
(in thousands), 1:20 dilution is adequate. For RBC count (in millions), the dilution
should be 1:200.

75
 When WBC count is in lacs, e.g., in leukemia, use of RBC pipette is suggested for
dilution purpose. Platelet count is normally in lacs. Yet, dilution used is 1:20.
Explanation: WBCs are larger cells and they are being counted in larger fields (WBC
squares). Hence, it is prudent to use a greater dilution in leukemia. Platelets are very
small. They are being counted in smaller fields (RBC squares). These two factors will
make it appropriate for 1:20 dilution, even though the number is in lacs.
 What are the other methods for platelet count?
 Rees-Ecker method (direct method; uses hemocytometer)
 On peripheral smears: (indirect method) – Platelet to RBC ratio is
estimated. Normal range is about 1:10 to 1:20.
 Automated counting.
(B) Physiology point-of-view:
 Normal platelet count:
 1,50,000 to 4,00,000/mm3.
 What is “plateletcrit” (PCT)?
 Hematocrit (HCT) is the volume of blood cells relative to the
plasma volume. 38-45% is the body hematocrit; it is mainly the
volume of red cells. The remaining 55% is the volume of plasma, in
a unit quantity of blood.
 “Plateletcrit” is about 0.15-0.3%.
 “Threshold” for platelet transfusion:
 Platelet count < 20,000/mm3 is considered as an indication for
platelet transfusion.
 What are the functions of platelets?
Platelets (thrombocytes) perform following functions:
1. Arrest of bleeding – Formation of the hemostatic plug ensures stoppage
of bleeding about 1-3 minutes after a vessel is injured.
2. Release of platelet factors: The activated platelets release certain factors
that initiate intrinsic pathway for clot formation.
3. Stabilization of fibrin:
4. Clot retraction: The contractile filaments cause the contraction of the
hemostatic plug; this reduces the clot size and brings the broken ends the
vessel closer.

(C) Clinical perspective:

 Variations in platelet count:
(a) Thrombocytopenia: Reduction in platelet count below 1.5 lacs/mm3. It
results from: (i) Decreased production of platelets – leukemias, some
types of anemia (resulting in a compensatory proliferation of erythroid
precursors that encroach the bone marrow), viral fevers, bone marrow
depression as occurs in chemotherapy. (ii) increased destruction of

76
 platelets: idiopathic thrombocytopenic purpura (ITP), thrombotic
thrombocytopenic purpura (TTP), dengue fever, hemolytic uremic
syndrome; hypersplenism – platelets trapped in and destroyed by spleen.
—————————————————————————————————————————

Section 15

Reticulocyte count

 Introduction:
 Reticulocyte is the immediate precursor stage of a matured RBC. It
is at this stage that the developing red cell leaves the bone
marrow. Over the next 24 hours in the circulation the reticulocyte
matures into an erythrocyte.
 There are 5-6 millions of RBCs per mm3 of blood. Of the total red
cell count, about 1% cells are reticulocytes. The reticulocyte count
becomes an indicator of the rate of RBC production. For instance,
in conditions of hypoxia the rate of red cell production and the
rate of their release from the marrow would be accelerated,
resulting in a higher reticulocyte count.
 Aims & objectives:
 To determine the reticulocyte count of blood.
 To calculate the corrected reticulocyte count or reticulocyte production
index.
 Materials & methods:
 EDTA-anticoagulated blood sample
 Reticulocyte stain
 Glass slides
 Microscope
o Principle:
A stained smear is made from the blood sample. The smear is scanned
under microscope; the reticulocytes are counted in successive fields and
their ratio or percent is determined with respect to the matured red cells
on the smear.
o Procedure:

77
  2-4 volumes of the blood sample and 2-3 drops of the stain are
taken in a tube. Mix the sample and the stain properly.
 Incubate the mixture at 370C for 15-20 minutes.
 Prepare a smear on the glass slide; allow the smear to dry.
 Observe the smear under oil immersion. Eyepiece with an
adjustable diaphragm should be used, if available. Observing the
reticulum and identifying a reticulocyte is becomes easier with
such an eyepiece.
 Observation/Result:
(i) Identifying the reticulocytes:
Reticulocytes can be distinguished from the matured RBCs by the presence of
reticulum; the reticulum takes a deep blue stain. (Matured red cells are
stained pale greenish blue with a central pallor; they lack the reticulum.)
(ii) Counting of the reticulocytes:
 Observe the successive 100 fields on the smear; identify and count
the reticulocytes in those fields.
 The matured RBCs should be counted in every 10th of those 100 fields.
Total of 10 fields will be observed for RBCs.
 Number of reticulocytes found in the 100 fields =
 RBCs counted in 10 fields = “n”; Therefore, total number of RBCs in 100 fields
= [“n” X 10]
 Reticulocyte count can thus be calculated as % of the RBCs in 100 fields.
 Absolute reticulocyte count = % reticulocyte count X RBC count.

 Common errors; Special precautions:

 Too thin a smear: Since reticulocytes are relatively fewer in
number, a very thin smear will disperse them even further, making
it difficult to find them.
 Amount of blood to be added to the stain: For optimal staining of
the cells, the volume of blood added to the stain should be
adjusted as per the expected red cell count. In case of anemia, a
larger proportion of the blood sample should be added; in case of
polycythemia, a smaller volume of blood should be added. (If the
cell count is low, more stain per cell will result in overstaining;
converse will happen with high cell count.)

78
 Discussion:
(A) Technical aspect:

1. What is the composition of the reticulocyte stain? How is the stain

prepared?
 The stain contains New methylene blue, which differs chemically
from methylene blue.
 1.0 gm of New methylene blue is dissolved in 100 mL of citrated
saline solution (3.8% sodium citrate + 0.9% NaCl in a ratio of 1:4).
Once the dye is dissolved, the mixture is filtered; now it is ready to
use.
 New methylene blue stains the reticulum deeply and uniformly.
 Azure B can be a substitute for New methylene blue. Brilliant
cresyl blue is not a satisfactory alternative.
2. What is supravital staining?
 Stainining of the living cells, outside the body, is called supravital
staining. That is, the cells are alive when they are stained. Staining
of reticulocytes with New methylene blue, when mixture of the dye
and blood sample is incubated at 370C, is the example of supravital
staining.
 Vital staining is the one in which the living cells extrude the dye
out of the cells, and only the dead cells take up the dye and get
stained. Thus, living cells can be seen by exclusion (or “negative”
staining).
(B) Physiology point-of-view:
1. What is the normal reticulocyte count?
 0.2-2% in peripheral blood. Thus, only about 2% red cells in
peripheral blood are at reticulocyte stage.
 In the conditions of hypoxia, bone marrow will be stimulated for a
faster production of red cells. More red cells will appear in
circulation (at reticulocyte stage), and the reticulocyte count will
be higher. Thus, reticulocyte count indicates bone marrow
activity.
 Since neonates and infants suffer from relative hypoxia after birth,
they have a higher reticulocyte count (2-6%).
(C) Clinical perspective:
1. What is increased reticulocyte count called?
Ans: Reticulocytosis. It occurs in conditions of hypoxia (e.g., high altitude).
The hypoxic stimulus, acting via erythropoietin, causes a greater
production and faster release of red cells into blood in their precursor
forms.
Reticulocytopenia- a decreased reticulocyte count
2. What is “corrected” reticulocyte count, or reticulocyte production index?

79
  Reticulocyte count is not an absolute number of cells; it is a
relative percent of red cells that are in reticulocyte stage. Since it
is only a relative count, it may sometimes lead to erroneous
conclusions, particularly in anemias.
 For instance, in anemia, the RBC count and packed cell volume
(PCV) is expected to be low. As the matured red cell count is
lower, in peripheral blood, the reticulocyte count may be
relatively high. Also, reticulocytes are little larger as compared to
matured red cells. So, when PCV is done on the peripheral blood
sample, the larger reticulocytes may yield a higher PCV result.
 Hence, it is important to know if reticulocyte count is only
relatively high or anemia has caused a proliferation of bone
marrow precursors and resulted in reticulocytosis. For this reason,
a “corrected” reticulocyte count is calculated, as follows:

Reticulocyte production index =

Reticulocyte% X [(observed PCV)/(normal PCV)]

 Erythropoietin doping in sports: Use of reticulocyte production index

as “Biological passport” for athletes -
 Athletes use erythropoietin for a faster, greater red cell
production, so as to enhance oxygen delivery to muscles.
 Reticulocyte production index may be used as a test to detect
the recent stimulus on the bone marrow, and thus to detect
the so-called ‘erythropoietin doping’.

80
 Section 16
Packed Cell Volume (PCV)
[Macrohematocrit method/Wintrobe method]
 Introduction:
 Two frontline investigations in a suspected case of anemia would be Hb
estimation and RBC count. However, equally important (if not more) is
determination of the packed cell volume (PCV) in the patient.
 PCV or hematocrit is the total volume of red cells relative to plasma, in a unit
quantity of blood. Though it is the volume of all cells, most of the volume of
cells is due to the RBCs (as platelets are very small and WBCs are relatively
less). PCV is expressed as the percentage of the total blood volume.
 Increase or decrease in red cell count will be reflected in the PCV.
 Classification of anemia needs calculated blood indices (discussed later);
some of the blood indices are based on the PCV value.
 Since it is a relative expression of ratio of cells to plasma, any increase or
decrease in plasma volume will also affect the PCV value.
 Materials & Methods:
 Materials for collection of blood sample
 Anticoagulant – Double oxalate
 Glass pipette
 Wintrobe tube
 Centrifuge

81
 o Principle:
When blood sample is placed in a vertical tube and a specific centrifugal
force applied to it, the blood cells are separated from plasma and get
packed near the bottom of the tube.
o Procedure:
 With usual aseptic precautions collect the venous blood sample in
oxalate bulb.
 Using a glass pipette, transfer the sample to the Wintrobe tube;
the tube should be filled up to the highest mark, i.e., 10 cm mark
or zero mark.
 Place the tube in the centrifuge; the blood sample should be
centrifuged at 3000 rpm for 30 minutes.
 Remove the tube from centrifuge at the end of 30 minutes and
note down the reading.
 Observation/Result:
 Red cells will have got packed near the bottom half of the Wintrobe tube.
 PCV is noted as the height of the column of the red cells. If the column is up
to 4 cms (out of the total 10 cms of the sample), the PCV is 40% or 0.4.

82
  Common errors/Special precautions:
 Fill the Wintrobe tube carefully with the glass capillary
pipette specifically meant for the purpose.
 While placing the tube in the centrifuge, make sure that
the buckets in the centrifuge are fitted with rubber
adapters. If there is no rubber adapter in the bucket, the
Wintrobe tube may break during centrifugation.
 Balance the opposite bucket in the centrifuge.
 Seal the upper end of the tube with clay.
 The upper end should not be pointing toward the centre of
the centrifuge; it should be placed so as to point away
from the centre.

 Discussion:
(A) Technical aspect:
1. About the centrifugation:
 The centrifugal force should be appropriate in order to achieve a
complete separation of the cells from plasma. 3000 rpm for 30
minutes creates the desired force.
 Microhematocrit method: A capillary tube is filled with blood
sample, and centrifuged at 10,000 rpm for 5 minutes. The method
has certain advantages: (i) Blood is obtained by skin-prick; lesser
amount of sample is needed, (ii) The method is quicker, and (iii)
Results obtained are more reliable.
o “Relative centrifugal force” or “Relative centrifugal field” (RCF) is
expressed as follows:
RCF = 0.00001118 X [r] X [RPM]2
Where,
r is the radius (cms) of the rotation,
RPM is the rotational speed or rotations per minute.
2. About the layers seen after separation of cells from plasma:
 From the bottom end upward, following layers can be seen: (i) The
layer of packed red cells at the bottom half, (ii) “Buffy coat” layer
above the packed red cells: It is whitish in appearance, about 0.5-1
mm in thickness. The layer is formed of packed WBCs (below) and
platelets (above), (iii) The uppermost layer of plasma: It is pale
yellow or straw-coloured. If it appears pink or reddish in colour,

83
 either there was hemolysis during the procedure or cells have not
been completely separated from the plasma; the entire procedure
may have to be repeated.
(iv) There is a thin black line between packed red cells and buffy
coat. It appears due to hemolysis or reduction of Hb in the red
cells.
 “True” PCV: The plasma entrapment – It is expected that the plasma gets completely
separated from the cells. However, even with the appropriate centrifugal force, the
separation is not absolute; some amount of plasma remains entrapped among the
cells, particularly in the lower part of the red cell column. Due to this inherent
shortcoming of the method, the observed PCV value is slightly higher. Although the
red cell dehydration during centrifugation tends to balance out the plasma
entrapment factor, some correction in the observed PCV is needed. The obtained PCV
is about 1-3% higher, and hence the “corrected” PCV should be 97-99% of the
obtained PCV.
Corrected PCV = (0.97) X observed PCV.

(B) Physiology point-of-view:

1. Normal PCV in health:

45% (+/- 5%) in men,
41% (+/- 5%) in women.
2. Variations in PCV: (Physiological changes in the ratio of red cells to
plasma) -
 Body hematocrit is lower than the observed value of PCV; it shows
following variations – (a) In capillary blood, the ratio of red cells to
plasma is higher. Reason: Some amount of plasma moves out of
capillaries, on their arteriolar ends. This increases the ratio of red
cells to plasma in the capillary blood. (b) ‘Chloride shift’ occurs in
capillary blood. When blood enters veins, the already occurred
chloride entry into the red cells will cause some amount of water
to move into the cells by osmosis. Thus, the swollen red cells will
yield a greater volume of packed red cells.
In addition to the ‘trapped plasma’ factor, these influences of
circulation give a higher value of PCV.
Average body hematocrit = (0.89) X observed PCV.
(C) Clinical perspective:
1. Utility of the PCV investigation:
(a) The layer of packed red cells: Increase or decrease in its length is a
reflection of increase or decrease in red cell count.

84
 (b) The buffy coat layer: Increase in its thickness may indicate an
increase in white cell count. Films can be made from the buffy
coat. Buffy coat films are useful in detection of atypical white
cells, microbes within white cells, or abnormally high or low cell
counts.
(c) Plasma layer: A deeply yellow coloured plasma hints at an
increase in plasma bilirubin – jaundice.
2. Clinical applications of the PCV investigation:
(a) Anemia – Lower value of PCV
(b) Polycythemia – Higher value of PCV
(c) Calculation of blood indices for classification of anemia – Mean
corpuscular volume (MCV) can be calculated from PCV & RBC
count. [discussed later]
(d) Hemoconcentration and hemodilation – PCV is the ratio of cells to
plasma, in a unit quantity of blood. Thus, changes in plasma
volume may cause a relative change in PCV. For instance, loss of
plasma volume, as happens in dehydration, will lead to a relative
increase in PCV. Conversely, increase in plasma volume
(‘hemodilution’) will cause a relative decrease in PCV.

 Advanced lab technology:

 Hb is measured on a whole blood sample. Then, some blood from the same
sample is taken in a capillary and centrifuged for 5 minutes. Hb of the
packed red cells is measured. PCV is calculated the ratio of Hb of the whole
blood to Hb of the packed red cells.
 International Council for Standardization in Hematology (ICSH) advocates a
“surrogate reference method” in which the blood samples are taken in
duplicate in glass capillary tubes and are centrifuged. The capillary tubes
are then aligned horizontally against edges of a glass slide; the preparation
is then observed under microscope (low power). With the vernier scale the
lengths of various layers is noted; surrogate reference PCV is calculated.

85
 Section 17

Erythrocyte Sedimentation Rate (ESR)

 Introduction:
 When a clinician starts treatment for a chronic disease, he/she
needs to assess the effectiveness of the treatment and
progression/regression of the disease. Apart from the subjective
improvement in signs & symptoms, there has to be an indicator
that will objectively indicate the progress & success of the
treatment; Erythrocyte sedimentation rate (ESR) is such an
indicator.
 If done on the first visit of the patient, ESR does not provide a
concrete proof for the diagnosis; many diseases exhibit a rise in
ESR. However, it gives a starting point for the future assessment of
the treatment strategy and its outcome. Hence, ESR is said to be
of “prognostic” value, rather than any diagnostic importance.
 When blood is collected and placed in a vertical tube, the red cells
settle down with a certain pace. This settling rate, called
sedimentation rate, increases with certain chronic inflammatory,
granulomatous, and connective tissue disorders. In that sense, ESR
is a non-specific screening test.
 Aims & Objectives:
 To learn the methodologies (Wintrobe and Wetergren) for determination of
ESR
 To understand the prognostic significance of ESR
 Materials & Methods:
o Principle: Two methods – Wintrobe and Westergren – are being
employed; principle for methodology is the same for both. Blood sample
is made to stand in the vertical tube. The red cells settle down in the
tube. The reading is taken at the end of one hour; the length by which the
red cells have settled is noted.
(1) Wintrobe method:
Apparatus & Reagents – (i) Materials for collection of blood sample, (ii) Wintrobe
tube: It is a glass tube having 3 mm internal diameter and 110 mm in length; the
calibrations on the tube are 0 to 100 mm, with 1 mm being the smallest interval,
(iii) Anticoagulant: Double oxalate, (iv) Wintrobe stand.
o Procedure:
 Collect the venous blood sample after having observed the usual
aseptic precautions.
 The collected blood is mixed with the anticoagulant – ammonium
potassium oxalate – in a ratio of 2 mg of anticoagulant/mL of

86
 blood. [NOTE: The blood sample may first be anti-coagulated with
EDTA and then diluted with citrate, or the sample may be directly
mixed with citrate.]
 Using a long nozzle dropper, fill the Wintrobe tube up to zero
mark.
 Place the tube vertically in Wintrobe stand; leave it there for 1
hour.
 Observation/Result:
 At the end of 1 hour – Height of the clear plasma is noted (in mm) above the
upper level of sedimenting cells.
 Result is expressed as ESR (mm) at the end of 1 hour.

(2) Westergren method:

Apparatus & Reagents – (i) Materials for collection of blood sample, (ii)
Westergren tube: It is glass tube, 300 mm long, having 2.55 mm diameter. Lower
200 mm of the tube show calibrations of 1 mm (from 0 to 200). Note: The tube is
open at both ends, (iii) anticoagulant: Sodium citrate (3.8%), (iv) Specially
designed racks: The rack has adjustable screws that hold the Westergren tube
firmly in place and exactly vertical.

87
Procedure:

88
  With usual aseptic precautions collect the venous blood sample.
 The anticoagulant – sodium citrate (3.8%) – is added to the blood sample
with the ratio of 4 volumes of blood : 1 volume of sodium citrate.
 The blood sample is then drawn into the Westergren tube up to 200 mm
mark by using a mechanical device. Taking of blood sample by mouth
suction is not advisable.
 Place the tube in the rack; make sure the tube is exactly vertical. Leave
the tube in the position for 1 hour.
 Observation/Result:
 At the end of 1 hour: Read the height of clear plasma above the uppermost
level of the column of sedimenting red cells.
 Result is expressed as ESR (mm) at the end of 1 hour.

 Common errors/Special precautions:

(1) For Wintrobe method:
 While transferring blood into the Wintrobe tube –
Use a long nozzle dropper that reaches up to the
bottom of the tube. The filling of tube should be
done skillfully; formation of air bubbles or frothing
of blood should not happen.
(2) For Westergren method:
 A greater skill is required. The tube is open at
both ends. Blood is drawn up to just above the
highest (200 mm) mark; some amount of blood
may drop off the tube before the top end is
quickly closed with a finger. (Anticipating this
draining of blood before it is transferred to the
rack, blood should be sucked up to above the
highest mark. As the column of blood is falling
back into the bulb, the fingertip is placed at the
upper end just when the column reaches the 200
mm mark.)
 The tube should be aligned exactly vertical in the
rack. Even a slight tilt will influence the final
reading.

89
  Discussion:
(A) Technical aspect:
 How will the rate of sedimentation be influenced if the tube is not exactly
vertical?
 The red cells will quickly settle vertically toward the nearest wall of the
sloped tube, and then settle toward the bottom of the tube. This will give
a faster rate of erythrocyte sedimentation.
 What are the advantages and disadvantages of Wintrobe and Westergren
methods?
 Wintrobe method has following advantages – (i) It is not an open-ended
tube; filling the tube is easier. Also, blood sample does not leak from the
tube. (Westergren tube is open-ended; a greater degree of skill is
required to fill the tube exactly up to the highest mark. Also, the tube
may leak the sample from the bottom end if it is not properly fitted in the
rack.) (ii) Wintrobe tube is shorter; a smaller amount of blood suffices in
the method.
 However, the shorter length has an inherent shortcoming; the red cells
will sediment quicker to the bottom of the tube. This may yield
erroneously high ESR, particularly in conditions of low red cell counts.
(B) Physiology point-of-view:
1. What is ESR? What are its normal values?
 ESR is the rate of settling down of RBCs when blood is made to
stand in a vertical tube.
 Rate of RBC settling primarily depends on the difference in specific
gravity between red cells and plasma; red cells, being heavier,
tend to settle down toward the bottom of the tube.
 Normal range:
(A) Wintrobe method:
(I) Men: 0-9 mm at the end of one hour
(II) Women: 0-20 mm at the end of one hour
(B) Westergren method:
(I) Men: 0-5 mm at the end of one hour

(II) Women: 0-7 mm at the end of one hour

[Note: (i) ESR is expressed as mm at the end of one hour, and not as mm/hour. It is taken as
a value at the end of first hour only. (ii) With Wintrobe method, the ESR is higher as
compared to Westergren’s. The reason, as discussed previously, is the shorter length of the
Wintrobe tube; erythrocytes settle faster toward the bottom of the shorter tube.]
2. What is rouleaux formation? How does it influence the ESR?

90
  Red cells pile on each other (like a stack of coins) when blood is
drawn out of the body. This piling of red cells is called rouleaux
formation.
Within the body, the circulating RBCs do not come together to
form such stacks or rouleaux; they are repelled from one another
due to the presence of negative electrostatic charges on their
membranes or zeta potential.
When blood is drawn out of the body, certain factors in plasma
cause overcoming of the zeta potential resulting in the coming
together of the RBCs to form clumps or piles – the rouleaux.
 Sedimentation occurs in 3 phases: (i) Red cell clumping or
rouleaux formation in first few minutes, (ii) Settling down of the
clumps at a steady rate, and (iii) Slow-down in the sedimentation
rate as the clumps pack toward the bottom of the tube.
 In certain clinical conditions, the rouleaux formation is hastened.
This will result in an increased ESR.

 Rouleaux formation and rate of RBC settling:

 A red cell has a surface are of about 150 pm2; thus, 10 red cells (when
not formed a rouleaux) will have a surface area of 1500 pm2. However,
when red cells form a rouleaux, those 10 red cells will have a surface
area of only about 600 pm2, whereas the mass of the same red cells
will have increased by 10 times. In other words, with rouleaux
formation there is much more increase in the mass relative to increase
in the surface area. A lower ratio of surface area to volume allows the
piles of red cells to settle even faster as it overcomes the viscous
resistance offered by plasma. Thus, greater the extent of rouleaux
formation, greater will be the ESR.

3. Factors that hasten rouleaux formation; the factors that increase the ESR:
i. Asymmetric macromolecules in plasma: Presence of such
molecules, e.g. Fibrinogen & globulins, hastens rouleaux
formation by overcoming the zeta potential on red cells; the result
is an increase in ESR. Many infections, chronic inflammatory
conditions, states of tissue destruction, and wasting diseases
cause increased fibrinogen and -globulin, and an increase in ESR.
Transport globulins or acute-phase proteins in plasma (such as C-
reactive protein, ceruloplasmin, haptoglobin, etc) promote
rouleaux formation. Albumin tends to retard rouleaux formation.

91
 In conditions with decreased plasma albumin, e.g. nephrotic
syndrome, ESR is increased.
ii. Bacterial toxins, products of inflammation and tissue destruction:
These factors cause alterations in plasma proteins, thereby
increasing the rouleaux formation.
4. Some other factors that influence the ESR:
 Anemia: A decrease in red cell count increases ESR. Lesser number
of RBCs makes it easier for them to aggregate and settle down
faster.
 Polycythemia: Crowding of red cells retards their settling rate.
 Red cell morphology: Larger RBCs (increased MCV/megaloblasts)
will have lower tendency for rouleaux formation; ESR will
decrease. Spherocytes (sphere-shaped red cells) have lower rate
of aggregation; ESR decreases in spherocytosis.
 Viscosity of plasma also has an influence on the sedimentation
rate. Proteins in plasma, particularly fibrinogen, increase the
plasma viscosity which increases ESR.
 Temperature: ESR increases with a rise in temperature.
(C) Clinical perspective:

1. What is the clinical significance of ESR?

 Not much a diagnostic value; significance as a prognostic indicator –
Since there are hundreds of diseases in which ESR increases, a rise in
ESR value, by itself, does not carry any diagnostic importance. It may,
however, indicate the presence of a chronic infection or an
inflammatory condition. When clubbed with other relevant
investigations, ESR may offer a clue to the diagnosis. ESR, by itself, is
useful in diagnosis of some conditions; E.g., temporal arteritis,
polymyalgia rheumatica.
 Early stage of myocardial infarction (MI) shows a rise in ESR.
 ESR decreases in: Consumptive coagulopathy or hypofibrinogenemia
(low fibrinogen in plasma; low ESR), heart failure.
 Once the diagnosis is established, ESR may be used to analyze the
progression or regression of the disease. Thus, ESR is valuable to
judge the prognosis of the disease. It is particularly useful in
conditions associated with a rise in acute-phase proteins in plasma.
 Prognosis of chronic infectious conditions (e.g., tuberculosis), chronic
inflammations (e.g., rheumatoid arthritis), or granulomatous
disorders can be assessed on the basis of periodic successive values of
ESR.

92
 Section 18

Specific Gravity of Blood

(Copper sulphate method)

 Introduction:
 Specific gravity of blood is the ratio of the weight of a unit volume of blood to
the weight of same volume of water at 40C.
 Specific gravity of whole blood is 1.060.
 Specific gravity of plasma and blood cells is 1.030 and 1.090 respectively.
 In conditions resulting in loss of plasma, and increased ratio of red cells to
plasma, specific gravity of blood increases. Conversely, if plasma volume
increases relative to red cells, the specific gravity of whole blood will be lower
than normal.
 Thus, specific gravity is a simple investigation that will reveal the relative
changes in red cell volume vis-à-vis the plasma volume.
 Aims & Objectives:
 To learn to determine the specific gravity of the whole blood.
 To understand the importance of specific gravity as a simple method to
estimate the red cell count or volume and the volume of plasma.
 Materials & Methods:
 Test tubes
 Pipettes (10 mL)
 CuSO4 stock solution of specific gravity 1:1
 Distilled water
o Principle: (Falling drop method)
 Blood is added drop-by-drop in a series of CuSO4 solutions of
increasing (known) specific gravities. If a drop of blood floats in a
solution, specific gravity of blood is lower than that of the
particular solution. If the drop of blood sinks in the solution,
specific gravity of blood is higher than that of the solution.
o Procedure:
 Prepare copper sulphate solutions of known specific gravities in a
series of test tubes; specific gravities of the solutions, starting with
1.050, should be in increasing order of 0.002. [1.050, 1.052, and so
on.]
Procedure for preparation of the copper sulphate solutions is
given below:

93
 Test 1 2 3 4 5 6 7 8 9
tube
number
CuSO4 4.9 5.1 5.3 5.5 5.7 5.9 6.1 6.3 6.5
solution
(mL)
Distilled 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5
water
(mL)
Specific 1.050 1.052 1.054 1.056 1.058 1.060 1.062 1.064 1.066
gravity
of
solution

 The assumption is that the specific gravity of blood is between 1.050 to 1.066.

 Collect the blood sample with usual aseptic precautions; add the
anticoagulant (oxalate) to the blood sample.
 Take the oxalated blood in a dropper. Let a drop of blood fall in
test tube no.1. Blood should be dropped into the test tube from a
height of 1 cm. With the momentum of the fall, the drop of blood
will sink some distance into the solution; it will then either rise
toward surface or sink deep into the solution. Observe the
movement of the drop, whether sinks or floats, within 14 seconds.
 If the drop floats in the first test tube, the specific gravity of the
blood is less than that of the solution. In such a case, prepare the
CuSO4 solutions of specific gravities less than 1.050 and repeat the
procedure from the lowest specific gravity (solutions prepared
newly).
 If the drop of blood sunk to the bottom of the test tube no.1, the
specific gravity of the blood sample is more than 1.050. Move on
to the next test tube and repeat the procedure.
 Observation/Result:
 In one of the test tubes, the drop of blood will neither float nor sink; it will
remain static midway into the solution for about 10-20 seconds. The specific
gravity of the blood sample is equal to the specific gravity of the particular
CuSO4 solution.
[Also note: In all the next test tubes, the drop of blood will float to the surface as the specific
gravity of blood is less than that of the remaining solutions.]

94
  Discussion:
(A) Technical aspect:
1. What are the advantages of the CuSO4 method, or of using the CuSO4
solution?
 (I) Upon addition of drop of blood, the drop does not immediately get
mixed with the CuSO4 solution. Rather, a film of copper proteinate
surrounds and forms a shell around the drop, at least for 15-20
seconds. Behaviour of the drop (rise or fall in the solution) can hence
be observed. (II) The temperature coefficient of expansion for blood
and the CuSO4 is almost the same; correction for temperature is not
required. (III) With CuSO2, solutions that have small differences in
specific gravity can be prepared easily; results obtained with such
solutions will be accurate. (IV) The drop of blood eventually sinks and
settles down at the bottom. Thus, the solution becomes clean and can
be used repeatedly.
(B) Physiology point-of-view:

1. What is the normal range for specific gravity of whole blood ?

 Normal range: 1.052 to 1.063
 Specific gravity of plasma ~ 1.025 to 1.030
 Specific gravity of red cells ~ 1.090
[Note: Specific gravity (SG) of cells is 1.090, and that of plasma is 1.030. The average of the
two SGs is the SG of blood ((1.060). If red cell count decreases, or plasma volume increases,
the SG will decrease. If plasma volume decreases, SG will increase.]
(C) Clinical perspective:
1. What is the clinical significance of this investigation?

95
  It is a simple investigation; cheap and easy to perform. It hints at
the changes in blood composition – red cell count/Hb level and
plasma volume.
 If large numbers of samples are to be screened for anemia in quick
time, for instance, in health check-up camps, estimation of specific
gravity may be a preferred method in the absence of other
specialized equipments and techniques.
 Specific gravity increases in: conditions of loss of plasma volume,
dehydration (vomiting, diarrhea), severe exercise,
hemoconcentration.
 Specific gravity decreases in: conditions of increased plasma
volume (pregnancy*, administration of steroids), hemodilution.
 Specific gravity can be an indicator of anemia. Decrease in red cell
count results in a decrease in specific gravity.
[* - In pregnancy, there is an increase in red cell volume by 20%, but plasma volume
increases by 40%. Hence, it results in a condition of relative hemodilution.]

96
 Section 19
Osmotic Fragility Test

 Introduction:
 The diagnosis of anemia can be established on the basis of combination
of clinical manifestations and lab tests (RBC/Hb/blood indices). A major
cause for anemia is ‘congenital corpuscular defects’. The membrane of
the red blood corpuscle may have some congenital defect, or the globin
chains of Hb may be deviant. Examples – Spherocytosis, thalassemia, etc.
 In such congenital defects, the RBC count and Hb levels will be low,
indicating the presence of anemia. In addition, blood indices may be
needed to pinpoint the cause for anemia (congenital red cell defects). The
places where automated analyzers or other specialized equipments and
materials are not available, blood indices (MCV, MCH) can not be
measured accurately. Osmotic fragility testing provides a simple and
cheap alternative that provides clue to the diagnosis of congenital red cell
defects.
 A normal biconcave-shaped RBC is lysed after a certain amount of water
moves into it by osmosis. If, for instance, the RBCs are sphere-shaped
(spherocytosis), they will be lysed early, with lesser amount of water
having entered them osmotically. This is the basis for osmotic fragility
testing.
 Aims & Objectives:
 To learn to determine the osmotic fragility of red cells in a given blood
sample
 To understand the clinical significance of osmotic fragility testing.
 Materials & Methods:
 Test tubes
 Metal rack
 Droppers
 NaCl – 0.9% & 0.5%
 Distilled water
o Principle:
The solutions of know tonicity are prepared and red cells are placed in
them drop-wise. The tonicity of the solution at which the red cells begin
to be lysed osmotically is noted.
o Procedure:
 NaCl solutions with different tonicities (from 0 to 0.88) should be
prepared. Refer to the table given below for preparation of the
NaCl solutions.
 Number the test tubes from 1 to 12; keep them in the metal rack.
 Preparation of NaCl solutions of different tonicities:

97
 Test 1 2 3 4 5 6 7 8 9 10 11 12
tube
number
No. of 22 16 15 14 13 12 11 10 9 8 7 0
drops
of 1%
NaCl
No. of 3 9 10 11 12 13 14 15 16 17 18 25
drops
of
distilled
water
Tonicity 0.88 0.64 0.60 0.56 0.52 0.48 0.44 0.40 0.36 0.32 0.28 0
of NaCl
(in %)

[The assumption is that the osmotic fragility will lie somewhere between 0.64% and
0.28%. Test tube no. 1 & 12 are just ‘controls’. In spherocytosis and some other hemolytic
anemias, osmotic fragility is markedly increased; fragility may even start in test tube no. 2,
that is, at 0.64% NaCl. Hence, additional hypotonic solutions of 0.7% and 0.8% may also be
prepared.]
 Add one drop of the blood sample each to the test tubes 1-12.
Each tube should be inverted gently after addition of the drop; it
allows for mixing of the contents.
 All the test tubes should now be left undisturbed in the metal
racks for one hour. During this period, the unhemolysed red cells
will settle down at the bottom.
 Obervation/Result:
 All the test tubes should be observed at the end of one hour. Hold the rack at
the eye level and observe the test tubes one-by-one.
 Test tubes with no hemolysis: Red cells will have settled at the
bottom, and the supernatant saline will look clear, unaltered.
 Test tubes with partial hemolysis: The unhemolysed red cells will have
settled at the bottom; the hemolysed red cells will give a pink tinge to
the supernatant saline.
 Test tubes with complete hemolysis: No RBCs will be seen settled at
the bottom; the saline will look uniformly red.
o Result: Hemolysis began at % of saline, and was complete in
% of saline.
[Note: Test tubes no. 1 and 12 are “controls”, for no hemolysis and complete hemolysis,
respectively.]

98
99
  Common errors/Special precautions:
 The blood sample delivered into the test tubes should be exactly of the
same amount for all. Exceptional care should be taken in observing this
rule.
 Take the blood with a marked pipette; same pipette should be
used for all the test tubes.
 There should be no air bubbles when blood is taken in the
pipette.
 The pipette tip should be carefully wiped of any blood that may
be sticking after sample is taken for each of the test tubes.
[If the amount of blood sample for each test tube is different, the amount of plasma,
in the blood sample being added in each test tube will also vary. Plasma has its
tonicity. When it is added to the solutions, the tonicity of the solutions may also get
altered. Hence to allow the maximum uniformity in this factor, the amount of blood
sample added to each test tube should be exactly the same.]

 Discussion:
(A) Technical aspect:

1. Why are the test tubes no.1 and 12 taken as ‘controls?

 Test tube no. 1 has the solution which is almost isotonic to red cells.
In this solution, there would not be any osmotic movement of water
into the cells; no hemolysis will occur here.
 Test tube no. 12 has only distilled water; it has 0% tonicity (no saline).
Since it is very hypotonic to red cells (0.9%), water will move into the
cells and hemolysis will occur rapidly and completely.
 Hence, to compare the complete, partial, and no hemolysis, two
extreme tonicity solutions (0% and 0.88%) are taken as controls.
2. What are the technical factors that may influence the results of this test?
i. Volume of blood added to test tubes and its relative proportion to
the saline solution: (as discussed previously) The blood contains
plasma, and the plasma has a certain tonicity. When this plasma in
blood sample gets added to the saline solutions, it could have
altered their tonicity, but for the fact that the amount added is
very low.

100
 ii. pH of the blood sample; ambient temperature during the test:
Venous blood shows greater osmotic fragility as compared to
arterial blood. When blood leaves capillaries and enters veins, CO2
has entered blood and entered the red cells; it forms H+ ions that
lower the pH of blood. Decrease in pH causes an increase in
osmotic fragility.
Ambient temperature should be held constant during the entire
duration of the test. An increase in temperature leads to a
decrease in osmotic fragility.
3. What is median corpuscular fragility [MCF]?
 It is the tonicity of saline at which there is 50% red cells are
osmotically lysed.
 Normal range for MCF ~ 0.40-0.45%

(B) Physiology point-of-view:

1. What is isotonic saline?


Tonicity of fluid inside red cells is 0.9%. If the fluid surrounding the
cells also has the same tonicity, there would be no movement of
water, by osmosis, either into or out of the cells; the cell size will
remain unaltered. Such a solution is termed ‘isotonic’.
2. Define the terms “osmolarity” and “tonicity”.
 Osmolarity: It is the number of moles of solutes per liter of the
solution. All the solutes are taken into account when we calculate
osmolarity.
 Tonicity: It is the number of moles of non-penetrating solutes per
liter of the solution.
So, what is the difference between the two concepts?
In tonicity, we consider only those solutes to which the cell
membrane is relatively impermeable. Since the membrane is
impermeable, the solutes do not equilibrate across the
membrane; their concentrations remain unequal on either side of
the membrane (that is, in ICF & ECF). The unequal concentrations
cause movement of water, from low solute concentration to high
solute concentration. Movement of water either into or out of the
cell will result in changes in cell volume. Thus, tonicity refers to
those osmoles that may cause changes in cell volume.
3. What are ‘hypotonic’ and ‘hypertonic’ fluids?
 Hypotonic fluid has relatively low solute and high water content,
with respect to the fluid inside the red cells. When a red cell is

101
 placed in a hypotonic solution (< 0.9%), the water will enter the
cell by osmosis.
 Hypertonic fluid has relatively more solute and less water. When a
red cell is placed in a hypertonic fluid, water will move out of the
red cell.
4. What is osmotic fragility?
 It is an index of resistance or susceptibility of red cells to undergo
osmotic hemolysis..
 If a cell is placed in a hypotonic solution, as discussed previously,
the water will move into the cell and the cell will swell. More the
solution is hypotonic, more is the amount of water moving in (to
cause osmotic equilibrium between the intracellular &
extracellular fluids). The low hypotonicity has such a limit that, a
further lower tonicity will cause so much amount of water to
enter that the cell will burst. This is termed as “osmotic lysis”.
5. What is the normal range for osmotic fragility? Why should there be a
range when all cells have intracellular fluid with essentially the same
tonicity?
 Normal range: Hemolysis starts in the hypotonic solutions with
tonicity between 0.45 and 0.39% NaCl. Hemolysis is completed at
0.32 to 0.30% NaCl.
 All RBCs have essentially the same intracellular fluid, with tonicity
of about 0.9% (of NaCl). All the RBCs, therefore, should behave
the same way in the context of osmotic lysis. That is, at a certain
tonicity of the hypotonic solution, all of them are expected to
swell and lyse at one instance. However, the osmotic lysis ‘begins’
at a certain tonicity and there is complete lysis in a further
hypotonic solution. Reason: In circulation, there are red cells
having different ages. Older red cells have lower activity of Na +/K+-
ATPase activity in their membranes. In addition, there are other
factors that make their membranes already fragile. Such cells will
begin to lyse first, in solutions which are less hypotonic. With
further reduction in tonicity, even the younger cells with normal
membranes will begin to undergo osmotic lysis. In other words,
younger cells are more resistant to osmotic lysis and older cells
are more fragile osmotically.
(C) Clinical perspective:

1. What is the importance of osmotic fragility testing, in the context of

anemia?
 Congenital or hereditary defects in red cell membrane or
abnormal hemoglobins due to globin chain alterations constitute
an important category of hemolytic anemias.

102
  A biconcave-shaped normal red cell has a high ratio of surface
area: volume. If the red cell becomes sphere-shaped, this ratio is
decreased. Such a red cell will imbibe less water, by osmosis,
before it is lysed. In other words, its osmotic fragility is increased.
Osmotic fragility test is most useful in the diagnosis of hereditary
spherocytosis.
 Spherocytes are lysed earlier as compared to normal red cells (for
normal red cells the lysis starts at a hypotonic solution of 0.45%).
 The osmotic fragility testing may be useful in the screening for
thalassemia.
Osmotic fragility test is a useful guide in various hemolytic anemias.
i. Osmotic fragility increases in:
 Hereditary spherocytosis
 Hereditary elliptocytosis
 Hereditary stomatocytosis
 Autoimmune hemolytic anemia
ii. Osmotic fragility decreases in:
 Thalassemia
 Iron deficiency anemia
 Hereditary xerocytosis
 Red cell enzyme abnormalities
 Unusually flat red cells (leptocytes) have a higher surface area: volume ratio; such
cells are greatly resistant to osmotic lysis. These cells have lower volume or MCV.
Iron deficiency anemia and thalassemia, in which the red cells are flattened, have a
decreased osmotic fragility.

 Advanced lab technology:

 What are the other methods for osmotic fragility testing?
 The red cell membrane defects can be detected by
membrane protein analysis, and other tests such as
glycerol lysis time, cryohemolysis, etc.
 Dye binding test (or flow cytometric test) can specifically
detect the defects in the red cell membrane proteins,
present in spherocytosis. The dye EMA reacts specifically
with the band 3 protein in the red cell membrane.

===========================================================================

103
104
105
106