Basic haematology technique

Dr. Shubhra Pramanik
1st Year MDS
SEMINAR

HAEMATOLOGY
It is a branch of medicine concerned with the study of the cause, prognosis,
treatment and prevention of diseases related to blood and blood coagulation.
BLOOD AND IT’S COMPONENTS
Blood is a body fluid in humans and other animals that delivers necessary
substances such as nutrients and oxygen to cells and transports metabolic waste
products away from those same cells.
Blood is composed of plasma and blood cells suspended in blood plasma.

The red, oxygen-carrying pigment in the red blood cells of vertebrates is
Haemoglobin. The molecular weight of haemoglobin is 64,450.
HAEMOGLOBIN
146
amino
acids
141 amino
acids
HbA


OXYHAEMOGLOBIN(HbO2):
Oxygen combines loosely with iron (ferrous state) in haemoglobin. The loosely
attached oxygen diffuses into the tissues for oxidative processes. The
haemoglobin then binds carbon dioxide and exists as reduced haemoglobin.
The colour of this compound is bright red.
Haemoglobin Derivatives:

CARBOXYHAEMOGLOBIN(COHb):
Haemoglobin combines with carbon monoxide reversibly to form
carboxyhaemoglobin. Carbon monoxide has 200 times greater affinity for
haemoglobin than O2 does. Haemoglobin, in this combination, is incapable of
oxygen transport. Normal range for general people is 0.16%.

METHAEMOGLOBIN(Methb/Hi):
This compound is formed when the ferrous state of heme is oxidised
to ferric state. This compound is incapable of oxygen transport
because of that ferric stage. It is dark brown in colour. Normal
concentration of methaemoglobin is 1-2%.
SULPHAEMOGLOBIN(SHb):
This compound results from the combination of inorganic sulfides
and haemoglobin creating a green coloured compound. This
compound is incapable of oxygen transport. This is an irreversible
reaction formed by the action of certain drugs and chemicals such as
sulphonamides and presence of sulphur in the air.
CYANOMETHAEMOGLOBIN(CNHb):
This compound results when methaemoglobin combines with the
cyanide radical. This compound is used in haemoglobinometry.


It is a technique of using a haemoglobinometer to estimate the blood
haemoglobin concentration after the haemoglobin has been converted to
cyanomethaemoglobin. There is normally a mixture of haemoglobin derivatives
present in whole blood with different absorption spectra; therefore, it is
necessary to dilute whole blood with an appropriate reagent.
There are four basic ways to measure the haemoglobin concentration are:
(1)Colorimetric measurement
[A]PHOTOELECTRIC METHOD
(a)CYANOMETHAEMOGLOBIN METHOD
is the method of choice and is most widely used
(b)Oxyhaemoglobin Method
[B]DIRECT VISUAL METHOD
(a)Acid Haematin Method
(b)Alkaline Haematin Method
(c )Haldane’s Carboxyhaemoglobin Method
(d)Tall Quist Method
(e )Dare’s Haemoglobinometer
HAEMOGLOBINOMETRY

(2)Measurement of the oxygen combining capacity of blood
[GASOMETRIC]
(3)Measurement of the iron content
[CHEMICAL METHOD]
(4)Specific Gravity Method
[GRAVITATIONAL METHOD/CUSO4 METHOD]


1) COLORIMETRIC MEASUREMENTS

Colorimetry or colourimetry can be defined simply as “the
measurement of colour”. A colorimetric method is a light-sensitive
technique which is used to evaluate an unknown colour in reference to
the known colour. The colorimeter is a device which measures the
transmittance and absorbance of light passing through a liquid sample.
The devices are based on Beer-Lambert’s Law according to which the
absorption of light transmitted through the medium is directly
proportional to the medium concentration.
According to the function there are two types of designs present—
colour densitometers, which measures the density of primary colour
and colour photometers which measure the colour reflection and
transmission.
In colorimetry, there are two methods used—
A)Photoelectric method
B )Direct visual method

Photoelectric colorimeter is a device that is used for the quantitative
analysis of different substances like glucose, blood urea, blood
enzymes etc. This is also known as spectrophotometer or simply
spectrometer. Precisely, spectrometer is an upgraded device than
colorimeter as colorimeter operates only in the visible portion of the
electromagnetic spectrum whereas spectrometer works with infrared
as well as visible light and spectrometer also measures the
transmittance and reflectance as a function of wavelength.
Two methods are in common use—1)Haemoglobinocyanide or
Cyanomethemoglobin (HiCN) method and 2)Oxyhaemoglobin (HbO2)
method.

a) CYANOMETHAEMOGLOBIN METHOD
The haemoglobinocyanide (cyanomethaemoglobin,HiCN) method or Drabkin’s
method is the internationally recommended method for determining the
haemoglobin concentration of blood. The basis of the method is dilution of blood in a
solution containing potassium cyanide and potassium fericyanide. The absorbance of
the solution is 540nm. The optical density is proportional to the concentration of Hb.

DILUENT
Blood is diluted in a solution called Drabkin reagent. The original Drabkin reagent
had a pH of 8.6. The reagent, used for this purpose, is a modified form, having a
pH of 7.0-7.4 [recommended by the International Committee for Standardization in
Haematology (ICSH)].
Substituting dihydrogen potassium phosphate[H2KPO4] in this reagent for sodium
bicarbonate[NaHCO3] in the original Drabkin reagent, shortens the time needed
for complete converstion of Hb to HiCN from 10 minutes to 3 minutes. The
detergent enhances the lysis of erythrocyte.
The diluent keeps well in the dark bottle at the room temperature, and should be
prepared fresh once a month. The pH must be maintained and must be checked for
atleast once in a month.
However alternate of KCN, non hazardous substances like sodium azide and
sodium lauryl sulphate can be used which convert the haemoglobin into
haemoglobinazide and haemoglobinsulphate
COMPONENTS AMOUNTS
Potassium ferricyanide (0.607 mmol/l) 200mg
Potassium cyanide (0.768 mmol/l) 50mg
Potassium dihydrogen phosphate (1.029 mmol/l) 140mg
Non-ionic detergent 1ml
Distilled or deionizing water Upto 1L

HAEMOGLOBINOCYANIDE REFERENCE STANDARD:
The International Committee for Standardization in Haematology (ICSH) has
defined specifications on the basis of a relative molecular mass (molecular weight)
of human haemoglobin of 64,458 (i.e. 16,114 as the monomer) and ¼ millimolar
area absorbance (coefficient extinction) of 11.0 (that is, the absorbance at 540 nm of
a solution containing 55.8 mg of haemoglobin iron per litre). These specifications
have been widely adopted and WHO International Standard has been established.
A new lot of the haemoglobincyanide or haemoglobin standard was released in
2008. This newly released standard replaces the previous lot and was produced
using the same methodology previously specified by ICSH. The current standard
has an assigned concentration value of 574.2 (± 5.1) mg/l or 35.63 (± 0.32) μmol/l.
METHOD:
 Make a 1:201 dilution of blood by adding 20 μl of blood to 5 ml of diluent.
 Adjust the stopper of the tube and invert it several times. Let the test sample stand for at
least 5 min to ensure the complete conversion of haemoglobin to haemoglobincyanide
and then pour it into a cuvette.
 The absorbance is measured at 540 nm. The absorbance of a commercially available
HiCN standard (brought to room temperature if previously stored in a refrigerator)
should also be compared in the same spectrometer or photoelectric colorimeter as the
patient sample.
The test sample must be analyzed within 6 hours of dilution. The standard must
be kept in the dark when not in use and discarded at the end of the day.

CALCULATION OF HAEMOGLOBIN CONCENTRATION:
Hb (gm/L)=[A540 of test sample/A540 of standard] x Concentration of
standard x [Dilution Factor201/1000]
STANDARD CURVE OR CALIBRATION CURVE:
It is a graph showing a straight line produced when cyanomethemoglobin
standard concentration and optical density (O.D.) of haemoglobin standard
solution are plotted against x-axis and y-axis respectively.
REAGENTS and EQUIPTMENTS=
1)Cyanomethemoglobin standard=60 mg/dl, commercially available.
2)Test tubes
3)Micropipette, 20 μl (0.020ml)
4)Micropipette tips
5)Volumetric pipettes: 1ml, 2ml, 3ml, 4ml, 5ml
6)Colorimeter
7)Graph paper
[Concentration of Haemoglobin Standard in gm%= 60 mg/dl x Dilution
Factor
=(60/1000)gm/dl x
(5000 μl /20 μl)
=15 gm%
(since whole blood=20 μl and Drabkin’s solution=5ml or 5000 μl) ]

PREPARATION=
1. Cyanomethemoglobin Standard Solution containing 60 mg/mL of
appropriate hemoglobin, prepared in Drabkin’s Solution is stored tightly
capped and refrigerated (2–8 °C) in the dark.
2. Prepare standard dilutions mentioned in the following table in 6 clean, dry,
labeled test tubes.
TABLE:PREPARATION OF WORKING STANDARDS
TUBE
NO
CYANOMETH. STANDARD DRABKIN’S SOLUTION CONCENTRATION STANDARD
1 0ml 5ml 0
2 1ml 4ml 1/5x15 mg%=3 mg%
3 2ml 3ml 2/5x15 mg%=6mg%
4 3ml 2ml 3/5x15 mg%=9 mg%
5 4ml 1ml 4/5x15 mg%=12 mg%
6 5ml 0ml 5/5x15 mg%=15 mg%

4. Read absorbance of Tubes 2–6 versus Tube 1 as the reference at 540 nm.
5. Record the absorbance values.
6. Plot a calibration curve of absorbance values versus the
cyanomethemoglobin concentration (mg/ml). The curve is linear and
passing through the origin.

As referred to earlier, lauryl sulphate or sodium azide can be used as
nonhazardous substitutes for potassium cyanide. However, no stable standards
are available for these methods so HiCN method needs to be used as a secondary
standard.
NORMAL VALUES:
At birth: 15 to 20 g/dL.
Males: 13 to 18 g/dL.
Females: 12 to 16 g/dL.
The main advantages of the HiCN method for haemoglobin determination are
that it allows direct comparison with the reference standard and that the readings
need not be made immediately after dilution so batching of samples is possible. It
also has the advantage that all forms of haemoglobin, except SHb, are readily
converted to HiCN.
The disadvantage is some abnormal plasma proteins or a high leucocyte count
may result in turbidity when the blood is diluted in the Drabkin-type reagent. The
turbidity can be avoided by centrifuging the diluted sample or by increasing the
concentration of potassium dihydrogen phosphate to 33 mmol/l (4.0 g/l).

b)OXYHAEMOGLOBIN METHOD
The Oxyhaemoglobin method (HbO2) is the simplest and quickest method for
general use with a photometer.
METHOD:
 Fill 20 μl of blood into a tube containing 4 ml of 0.4 ml/l ammonia (specific gravity 0.88)
to give a ×201 dilution. Use a tightly fitting stopper and mix by inverting the tube several
times.
 The solution of HbO2 is then ready for matching against a standard in a spectrometer at
540 nm or a photometer with a yellow-green filter. If the absorbance of the haemoglobin
solution exceeds 0.7, dilute the blood further with an equal volume of water and read
again.
Fresh ammonia solution must be made up each week. Once diluted, the blood
sample is stable at 20°C for about 2 days.
STANDARD:
A standard should be prepared from a specimen of normal anticoagulated whole
blood. Its haemoglobin concentration is first determined by the HiCN method.
The blood is then diluted as 1:201 by pipetting 20 μl of the well-mixed blood into 4
ml of ammonia; sequential dilutions are made in ammonia and absorbance is read
in a spectrometer at 540 nm or photometer using a yellow-green filter.
If the HiCN method is not available, a neutral grey filter of 0.475 density can be
used as a calibration standard.

The method is advantageous as it takes less time but also has
disadvantages that it is not possible to prepare a stable HbO2
standard, so the calibration of these instruments should be checked
regularly using HiCN reference solutions or a secondary standard of
preserved blood. The reliability of the method is not affected by a
moderate increase in plasma bilirubin, but it is not satisfactory in the
presence of HbCO, Hi or SHb.

a)ACID HAEMATIN METHOD
Haemoglobin is converted to acid
haematin by N/10 HCl, producing a
brown coloured element which is
compared with standard brown
coloured reference blocks.
The intensity of brown colour depends
on the amount of acid haematin
produced, which in turn depends on
the amount of haemoglobin present in
the blood sample.
This method is also known as Sahli’s
Method.
EQUIPTMENTS=
1) Sahli’s Haemoglobinometer
2) Sahli’s Pipette, marked at 20 μl or 0.02 ml
3) Stirrer
4) Dropping Pipette
REAGENTS=
1) N/10 Hydrochloric Acid (HCl)
2) Distilled water

PROCEDURE=
 Fill the marked Sahli’s tube upto 20 with N/10 HCl.
 Add 20 μl of blood by immersing the nozzle of the pipette in the bottom of the tube and
gently and carefully blow the blood.
 Using the provided stirrer, the blood is thoroughly mixed.
 Incubate the whole for 5-10 minutes.
 Add distilled water with dropping pipette until the colour of the diluted blood is
matched with the solid glass standard.
 Then the result is read on Sahli’s Scale (100%=14gm).
This technique requires less time and depends on comparison of the
colours with naked eyes though it is not recommended as colours
develops slowly and unstable, begins to fade immediately after it reaches
its peak.

b) ALKALINE HAEMATIN METHOD
In this method, the haemoglobin is converted to alkali haematin by addition of
N/10 NaOH. The alkali haematin gives a brown colour that can be read against
comparator standards or in a colorimeter.
EQUIPTMENTS=
1) Photoelectric meter with green filter
2) 0.05 ml Pipette
REAGENTS=
1) N/10 Sodium Hydroxide (NaOH)
2) STANDARD(GIBSON’S AND HARRISON’S) : This is a mixture of chromium
potassium sulphate, cobalt sulphate, potassium dichromate in aqueous solution. The
solution is equal in colour to 1:100 of blood containing 16.0 gm Hb/dl.
PROCEDURES=
 Add 0.5 ml of blood to 4.95 ml of N/10 NaOH.
 Mix well and boil for 4 minutes, along with 5 ml standard solution.
 Cool quickly in cold water and match the test against standard using colorimeter, using
the green filter. If the test gives too high value then have to add 5 ml of water and read
again.
The advantage of the test is that it gives a true estimate of total Hb even if HbCO,
Hi or SHb is present. The disadvantage is some Hb resist denaturation with
NaOH and participate in crystals.

c)HALDANE’S CARBOXYHAEMOGLOBIN METHOD
The haemoglobin is converted to carboxyhaemoglobin by the
action of coal gas in diluted blood.

EQUIPTMENTS=
1) Haldane’s scaled tube
2) 0.02 ml pipette
REAGENTS=
1) Standard solution
2) 0.4% ammonia within distilled water
3) 20 μl of blood sample
PROCEDURES=
 Fill the graduated tube with ammoniated distilled water mix upto 20
mark.
 Add 20 μl of sample blood and mix well.
 Pass coal gas through the solution for 2-3 minutes by means of a pipette.
The end of the pipette is dipped into caprylic acid before it is used for
gas supply. Caprylic acid prevents frothing.
 Add 0.4% of ammonia drop by drop and stir simultaneously until the
colour of the blood mixture matches with the standard under the
daylight.
 Read and calibrate the result of Hb concentration in percentage.

d)TALLQUIST METHOD
Tallquist method involves direct visual
matching of red colour of a drop of whole
blood on a filter paper with standard
colours on paper.
The technique is totally unsatisfactory
with high degree of error ranging 20%(+/-
) to 50%(+/-).
e)DARE’S
HAEMOGLOBINOMETRY
In this method, blood is spread in thin
films between glass discs for direct
matching. But this method is totally
inaccurate.


2)GASOMETRIC METHOD
 This is an indirect method of measuring oxygen combining
capacity.
 Based on the assumption that 1gm Hb can carry approximately
1.34 ml of O2.


3) CHEMICAL METHOD
 It is an indirect method of measuring the iron content.
 Based on the assumption that 1gm Hb contains approximately
3.47 mg iron.


4)SPECIFIC GRAVITY METHOD
 The other name of this method is Gravitational method or CuSO4
method.
 Specific gravity of copper sulphate is 1.053 which is equivalent to
12.5 gm% of Haemoglobin.
 The procedure is to collect a drop of blood in a solution and within
12 seconds and the behavior of the blood drop has to describe.


PORTABLE HAEMOGLOBINOMETER
The portable hemoglobinometer is a micro processed photometer. It is generally based on
the HbO2 method. A number of instruments are now available that use a light-emitting
diode of appropriate wavelength and are standardized to give the same results as with the
HiCN method.
Examples are The HemoCue system (HemoCue AB, Ängelsholm, Sweden), Chempaq
(Chempaq A/S, Hirsemarken 1B, Farum, Denmark), DiaSpect Haemoglobinometer.
The procedure is: after cleaning the skin, a puncture is done and a 10 μl blood sample is
collected with a micropipette and transferred to the reagent vial. After 30 seconds of
mixing, the vial is inserted in the equipment and a button is pressed. The sample
hemoglobin content is exhibited in the display in g/dl.
The portable hemoglobinometer is very advantageous and shows the same accuracy of the
golden standard methods with more toughness, low cost and very user friendly.


RANGE OF HAEMOGLOBIN CONCENTRATIONS
IN HEALTH
AGE CONCENTRATIONS
MALE 13.5-17.5 gm/dl
FEMALE 12.0-15.5 gm/dl
NEWBORN 14.0-24.0 gm/dl
INFANT 9.5-13.0 gm/dl
PREGNANCY 11.0-12.0 gm/dl
ADULT
CHILDREN

The term hematocrit comes from the ancient greek words ’haima’ (blood) and
‘kritēs’ (judge). Haematocrit (Ht or Hct) literally means “blood separation”. It is
the %volume of RBC per liter of the blood. It was coined in 1891 by Swedish
physiologist Magnus Blix as ”haematokrit”.
There are other names for the hematocrit, such as packed cell
volume (PCV), volume of packed red cells (VPRC), or erythrocyte volume
fraction (EVF).
The PCV can be determined by centrifuging heparinized blood in a capillary
tube (also known as haematocrit tube). The red cells become tightly packed in
the bottom of the tube . Since a tube is used, this can be calculated by measuring
the lengths of the layers. It is impossible to completely pack the red cells
together; therefore, about 3 to 4% of the plasma remains entrapped among the
cells and the true hematocrit is only about 96% of the measured haematocrit.
Another way of measuring hematocrit levels is by optical methods such
as spectrophotometry.
There are two methods present to measure haematocrit :
A) Macrohaematocrit method using Wintrobe tube
B) Microhaematocrit method using Capillary tube
C) Automated Analyzer


A)MACROHAEMATOCRIT METHOD
PRINCIPLE:
Anticoagulated sample blood is centrifuged at
a standard speed (3500 rpm). RBCs which are
heavier than white cells, platelets and plasma,
sediment at the bottom and the volume of
red cell mass denotes the hematocrit.
WINTROBE TUBE:
It is a special thick walled glass tube measuring
11 cm in length and an internal diameter of 2.5
mm, with a capacity of 1 ml. This is calibrated
at 1 mm intervals with bold markings from top
as “100” and “0” at the bottom for PCV, in
descending order for the sake of measurement.
ANTICOAGULANTS USED:
EDTA or dry heparin or double oxalate.

EQUIPTMENTS=
1) Wintrobe tube
2) Long necked pasture pipette or a special type of syringe, used for filling
Wintrobe tube.
3) Centrifuged machine with known speed.
4) Anticoagulant
PROCEDURE=
 Fill the Wintrobe tube with anticoagulated blood (2ml blood and 0.4 ml EDTA)
solution upto upper most mark with the help of pasture pipette or syringe.
 Place the tube in the centrifuge and run it at 3,500 rpm for 30 minutes.
 After switch the centrifuge off and allowing it to stop by itself, the Wintrobe
tube is taken out and PCV is read directly with the help of graduation mark
given on the tube.
RESULT=
 Lower layer : Packed red cells.
 Middle Layer : Thin red-gray layer called buffy coat.
 Upper Layer : Straw coloured layer called plasma consisting of white cells and
platelets.
CALCULATION%=[Vol. of the RBCs / Vol. of the Blood] X 100
NORMAL VALUES=
o MALE—42-50 %
o FEMALE— 36-38%


PRINCIPLE:
The blood mixed with anticoagulant is placed in a long capillary tube
and centrifuge. The level reached by the column of erythrocytes is read
with a scale reader. This method is preferable for that using a macro
scale : it’s quicker and blood from the finger can be used. The other
names of this method are Capillary tube method or Meyerstein method.
EQUIPTMENTS:
1) Capillary tube : 75 mm in length
2) Microhaematocrit Centrifuge : a special type of centrifuge having speed
of 15,000 rpm.
3) Microhaematocrit reader : different types of readers are used for
measurements. Card reader is the simplest.
B)MICROHAEMATOCRIT METHOD

4) Anticoagulant : EDTA or double oxalate
5) Blood sample
PROCEDURE=
 Draw the blood sample into appropriate capillary tube with capillary
action. Use plain tube for anti-coagulated blood. Fill the tube about
3/4th length. Seal the another end of the tube with clay or wax. The
sealing should be about 2 mm deep.
 Place two hct tubes in the grooves of centrifuge exactly opposite to each
other.
 Centrifuge at 13,000 (+/-) 2000 rpm for 5 minutes.
RESULT=
After completion of the procedure, the tube is taken out and placed on the
card reader scale to measure the length of RBC packed cell-layer.


PRINCIPLE:
The Automated Haematology Analyzer uses the principle, where blood is
passed between two electrodes through an aperture so narrow that only
one cell can pass through at a time. The change in impedence is
proportional to cell volume resulting in a cell count and measure of volume.
The Sysmex Haematology is an Automated Analyser containing racks
holding ten 4.5 ml tubes with a notch so that the collected sample blood can
only go in one way. The tubes are thin enough that blood cells pass only
one at a time. The characteristics about the cell are measured using lasers
(fluorescent flow cytometry) or electrical impedence.
Haematology autoanalyzers provide quick and accurate results in most
situations. But, despite the sophistication of present day Autoanalyzers,
there are needs to depend on manual techniques for primary calibration.
Auto analyzers are prone to errors as platelet aggregates or hypolobulated
neutrophil may give rise to false high PCV.
C)AUTOMATED ANALYSER

ABNORMAL HAEMATOCRIT LEVEL
HIGH LEVEL LOW LEVEL
 Polycythemia
 Hypoxia
 Heart Problems
 Lungs Problems (eg. Chronic
Obstructive Pulmonary Disease)
 Dengue fever
 Intake of anabolic steroids
 Diarrhoea
 Dehydration
 Burns
 Smoking
 High altitude
 Anemia
 Haemorrhage
 Haemolysis
 Iron Deficiency
 Nutritional Deficiency
 Chronic Kidney Diseases
 Over-hydration


RED CELL INDICES
Red cell indices are a group of blood tests which provide the
information about the haemoglobin and size of the red blood cells.
Abnormal values of these indices are indicative of blood diseases.
The indices are :
1) MCV (Mean Corpuscular Volume)
2) MCH (Mean Corpuscular Haemoglobin)
3) MCHC (Mean Corpuscular Haemoglobin Concentration)


The Mean Corpuscular Volume or Mean Cell Volume (MCV) is a
measure of average volume of the single red blood cell.
CALCULATION=
MCV is calculated by dividing the haematocrit (Hct) by total number of
erythrocytes (RBC Count).
MCV=[Hct(%) x 10]/[RBC]
(Normal RBC count for male- 4.7-6.1 million/μl; female-4.2-5.4 million/μl)
The unit for MCV is Femtoliter (fL) [= 10−15 L]
NORMAL RANGE=
The normal range of MCV is 80-100fL.
1) MEAN CORPUSCULAR VOLUME

RESULT=
In patients with anemia, it is the MCV measurement that allows three
classifications and they are :
a) Microcytic Anemia— When the measured MCV is less than the normal
range. This type of conditions are seen in the cases of inadequate dietary
intake, thalassemia, iron deficiency anemia, sideroblastic anemia, lead
poisioning. In these cases MCV can be as low as 60-70 fL.
b) Normocytic Anemia— When the measured MCV is within normal range,
the condition is called normocytic. These cases are seen in haemolytic,
haemorrhagic conditions.
c) Macrocytic Anemia— When the MCV is higher than the normal range, is
called Macrocytic. These are seen in pernicious anemia, megaloblastic
anemia (vitamin b12 and folic acid deficiency), aplastic anemia,
myelodysplasia, chronic alcoholism. MCV can be elevated upto 150 fL.
MICROCYTIC ANEMIA MACROCYTIC ANEMIA


The Mean Corpuscular Haemoglobin (MCH) or mean cell haemoglobin is
the average mass of haemoglobin in red blood cell in a sample blood.
CALCULATION=
MCH=[Hb(gm/dl) x 10] / [RBC Count]
The unit of MCH is picogram [1pg=10-12 gm].
NORMAL RANGE=
The normal range for adult is 26-32 pg.
RESULTS=
High MCH level normally correlates with macrocytic anemia and low level
MCH, with microcytic anemia.
b) MEAN CORPUSCULAR HAEMOGLOBIN


Mean Corpuscular Haemoglobin Concentration or MCHC is the average
concentration of haemoglobin in the RBC volume.
CALCULATION=
MCHC=[Hb(gm/dl)x100] / Hct(%)
The unit of MCHC is gm/deciliter
NORMAL RANGE=
Normal range of MCHC for adult is 32-36 gm/dl.
RESULT=
MCHC can be elevated (polychromatic) in hereditary spherocytosis, sickle
cell disease and some megaloblastic anemia. MCHC can be falsely elevated
when there is agglutination of red cells, hyperbilirubinemia,
hypertriglyceridemia, and free hemoglobin in the plasma.
c) MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION


ERYTHROCYTE SEDIMENTATION RATE
When anticoagulated blood sample is allowed to stand in a narrow
vertical glass tube, undisturbed, the RBCs, under the influence of
gravity, settle down from the plasma. The rate at which they settle
is measured as the number of millimeters of clear plasma present
at the top of the column after one hour (mm/hr). The settling rate
is called Erythrocyte Sedimentation Rate (ESR).

This sedimentation involves three stages :
a) Stage of Aggregation : The first 10-15 minutes of one hour, the red
cells are start piling like stack of coins which
is called Rouleaux Formation.
b) Stage of Sedimentation : Acute settling of RBCs are seen in this
stage. It occurs in 30-40 minutes out of
one hour, depending on tube-length.
c) Stage of Packing : In final 10 minutes, the settling of RBCs are
going to settle for overcrowding.

METHODS OF ESR DETERMINATION
There are two main methods to determine ESR :
a) WINTROBE’S METHOD :
This method uses—
 Wintrobe’s tube which is one side opened tube having 11 cm length and 2.5 mm
diameter. The tube is calibrated as 0, at the top to 10, at the bottom; opposite to that of
PCV.
 Wintrobe’s stand
 Pasteur pipette
 Blood sample mixed with anticoagulant (like EDTA, oxalate)
Procedure is—
 Pour the anticoagulated blood inside the tube with the help of pasteur pipette.
 With the help of the stand leave it vertically for atleast one hour.
 At the end of one hour, read the result. The result is read as how much clear plasma
present at the top of the tube as in millimeter in one hour.
RESULT –
o MALE:-- 0-9 mm/hr
o FEMALE:-- 0-20 mm/hr

WINTROBE’S
TUBE FOR ESR
MEASUREMENT
METHOD TO MEASURE THE SEDIMENTATION

100
40
METHOD TO
MEASURE PCV
0

b) WESTERGREN METHOD:
This method uses:-
 Westergren tube which is 2.5 mm diameter and 30 cm long double side-opened tube. The
lower 20 cm is marked with 0 at the top and 200 at the bottom.
 Westergren stand
 Rubber bulb
 Anticoagulated blood (0.4 ml of 3.13% trisodium citrate solution + 1.6 ml blood)
PROCEDURE:-
 Fill the anticoagulated blood upto 0 mark with the help of rubber bulb.
 Set the tube upright in the stand and place it tightly so that there is no leakage.
 Leave it undisturbed for one hour. At the end of one hour, read the result.
RESULT:-
o MALE : 0-10 mm/hr
o FEMALE : 0-15 mm/hr
Wintrobe’s method is more sensitive when ESR is low and when ESR
is high Westergren method is more preferable.

ABNORMAL ESRs
ELEVATED DECREASED
1. Acute infections (eg. fungal,
bacterial, viral, necrotizing )
2. Elevated fibrinogen level
3. Ischemic stroke
4. Malignancy
5. Renal insufficiency
6. Low serum albumin level
7. Anemia
8. Pregnancy
9. Old age
1. Infections (eg. uninary tract,
gastrointestinal tract, lung and
bloodstream)
2. Myocardial infarction
3. Venothromboembolic diseases
4. Rheumatoid arthritis
5. Low serum albumin level
6. Polycythemia

ERYTHROCYTE FRAGILITY TEST
Erythrocyte fragility refers to the tendency of the RBCs to rupture under
stresses which are physical and osmotic and/or mechanical.
According to the type of stress applied, erythrocyte fragility can be of two
types—
a) Erythrocyte Osmotic Fragility This is seen when RBCs are placed in a
hypotonic solution. Osmotic fragility is
affected by membrane composition, integrity
as well as the cells’ sizes or surface-area-to-
volume ratios. Test for osmotic fragility is
done in cases like hereditary spherocytosis,
sickle cell anemia, thalassemia etc.
b) Erythrocyte Mechanical Fragility Mechanical fragility of RBCs are seen when
the cells are subjected to shear stress. Test
for mechanical fragility is done to detect
damages in stored RBC, leading to appli-
cation in blood transfusion and blood
banking.

Osmotic fragility test is an important test to determine the erythrocyte resistance to
haemolysis. This test is performed in the symptoms like fatigue, shortness of breath,
dizziness, palpitation, decreased ability to exercise. The procedure was described by Parpart et
al.
REAGENTS:
1) Buffered saline: 10% NaCl solution, 90gm + Na2HPO4, 13.65gm +
NaH2PO4, 2.43gm + 1L distilled water (pH=7.4)
[0.36%=isotonic]
2) 12 Test tubes (levelled as #1-12)
3) Anticoagulated blood, 20 uL.
METHOD:
-20 uL of anticoagulated (EDTA) whole blood is pipetted into each of 12 test tube
containing 4 ml of buffered saline sol of decreasing concentration from #1 to tube#12
(mentioned in the graph).
-Shake each tube and leave at room temperature for 30 minutes. Then centrifuge each
at 1200 to 1500 rpm for 5 minutes.
-Read the optical density of each test tube on a spectrometer at 540 nm. Set the o.d. as
‘0’ or haemolysis as 0% in test tube #1 and 100% haemolysis in test tube #12.
-The %haemolysis is calculated = [(O.D. of tube #1 /O.D. of tube #12) x 100]
The result can be read by placing the test tubes on written page if words can be read.
RESULT:
The osmotic fragility of a cell depends on a)volume, b)surface area and c)functional
state of the cell membrane.
 The result is expressed as the range=haemolysis at %NaCl begins and haemolysis
at %NaCl ends.

The osmotic fragility is increased in hereditary spherocytosis, hypernatremia where
the surface-to-volume ratio and the fragility resistance are decreased so cell rupture
and reduced in thalassemia, sickle cell anemia, iron deficiency anemia, hyponatremia
where the RBCs are resistant to break and due to increased cell surface/volume ratio,
the cells absorb more and become spherical before they rupture. Either way, the colour
of blood mixed solution changes due to release of haemoglobin from ruptured RBCs.
The test is advantageous as this is cheap and easy to perform.
The result is graphed, plotting the NaCl concentration along the “x-axis” and
%haemolysis along the “y-axis”.
Normal Range : 0.45+/-
0.05% to 0.30+/-
0.05%NaCl

Isotonic
solution; the
solution is
clear.
Hypertonic and
Hypotonic solution;
solutions are hazy thus
the text behind the
solution can’t be read


TOTAL WBC COUNT
Total WBC count is an important blood test which measures the total number of
white blood cells (WBC) in the body.
WBCs, also called the leucocytes, are important part of the immune system.
Their main function is to defend against the invading bacteria, viruses and fungi.
The classification of WBCs is:-
A) Granulocytes B) Agranulocytes
I. Neutrophil
II. Eosinophil
III. Basophil
I. Lymphocyte
II. Monocyte

The white cells are found in the Buffy Coat which is derived after centrifugation.
Leucocytes are found throughout the blood and lymphatic system. The normal
ranges of total white blood count, according to age are:
Abnormal WBC count are seen in :
AGE WBC per µl of blood
Newborn 9,000-30,000
1 month 5,000-20,000
1 year 6,000-11,000
2 years 5,000-12,000
5 years 4,000-12,000
12 years 3,500-9,000
Adult 3,500-10,500
LEUCOCYTOSIS (INCREASED) LEUCOCYTOPENIA (REDUCED)
 Leukemia
 Infections (eg. Tuberculosis)
 Inflammations (eg. Bowel disease)
 Tumors (eg. In Bone marrow)
 Asthma
 Stress
 Exercise
 Pregnancy
 Corticosteroids
 HIV
 Autoimmune Disorders
 Bone marrow disorders
 Lymphoma
 Liver and spleen diseases
 Lupus
 Rheumatoid arthritis
 Radiation therapy
 Certain antibiotics, chemotherapeutic agents


Differential WBC count is done to determine the count of different types of
leucocytes in a blood smear and to detect different inflammatory and infectious
diseases.
DIFFERENTIAL WBC COUNT
WBCs ABSOLUTE VALUES(/µl of blood) PERCENTAGES
Neutrophil 2000-7000 40%-80%( in barcterial inf)
Eosinophil 200-500 1%-6%( in parasitic inf)
Basophil 0-100 0%-2%( in allergies)
Lymphocyte 1000-3000 20%-40%( in viral inf)
Monocyte 200-1000 2%-10%( in viral inf)

To determine the differential count:--
-A drop of blood is thinly spread over a glass slide with the spreader in such a
manner that the smear starts 1-2mm of the slide edge and 3-4mm long, tongue
shaped uniformly thickened. There should not be any striations or air bubble in the
smear.
-Then the smear is air dried placed horizontally.
-Romanowsky stain (Leishman or Wright or May-Grunewald-Giemsa technique
containing Methylene Blue or Toluidine as basic and Eosin or Azure-I or Azure-II as
acidic component) is poured on the slide for fixation and leave that for 2 minutes.
-At the end of 2 minutes, the buffered saline water is added to the slide which should
not be spilled and mix with the stain with mild intermittent air blow. Leave the stain-
water mix on the smear for 10 minutes.
-After 10 minutes, pour off the stain over the smear.
-Wash the slide under tap water for average 2 minutes gently till the smear gets
pinkish tinge
-Wipe the back of the slide and place the slide upright to dry.
-Place a cedar-wood oil drop on the blood film. Focus the microscope’s oil immersion
objective such that it touches the drop. Then raise the objective lens and then adjust
the microscope for fine adjustment.
-Study the different parts of the smear under the microscope.
DIFFERENTIAL WBC COUNT PROCEDURE

HAEMOCYTOMETER
The Haemocytometer is a counting-chamber , designed for counting blood cells.
This was invented by Louis-Charles Malassez. A well known type of
haemocytometer is the Neubauer counting chamber.
COMPONENTS=
1) Haemocytometer with loading chamber, which is a gridded area consisting of nine 1mm x
1mm (1 mm2) squares. These are subdivided in 3 directions; 0.25 x 0.25 mm (0.0625 mm2),
0.25 x 0.20 mm (0.05 mm2) and 0.20 x 0.20 mm (0.04 mm2) and the central square is
subdivided into 0.05 x 0.05 mm (0.0025 mm2) squares.
2) Coverglass, which is hold 0.1mm off the gridded area by the edge of the chamber.
3) Thoma pipette
4) Rubber sucking tube
1
2
3
4

RBC COUNT
PROCEDURE
• The anticoagulated blood sample is filled
upto “0.5” mark and diluting fluid upto
“101” of RBC pipette (has a red spot)
rapidly.
• After 2 minutes, after discarding few drops
of blood solution, the tip of the pipette is
hold under the cover glass in order to fill the
counting chamber with small amount.
• Allow the cells to settle down for 2-3
minutes.
• Place the haemoglobinometer under the
microscope at 40X.
• The four corners and central small box at the
central square of the grid are seen for the
count.
CALCULATION
TOTAL RBC/cubic mm=(No. of RBC counted x Dilution
factor x Depth factor)/No. of chambers counted
WBC COUNT
PROCEDURE
• The anticoagulated blood sample is filled
upto “0.5” mark and dilution fluid (WBC
Dilution Fluid or Truck’s Fluid: 1ml 3% acetic
acid+ 1% gelatin violet+ 1-2 drops methelene acid,
which destroys RBCs) upto “11” mark in
1:20 dilution of WBC pipette having white
spot.
• Then the counting chamber is filled with the
solution
• The haemoglobinometer is placed under
microscope and is seen under low power.
• The outer four corners are seen for the
count.
CALCULATION
TOTAL WBC/cubic mm=(No. of WBC counted x
Dilution factor x Depth factor)/No. of chambers counted
RBC
WBC

PLATELET COUNT
A platelet count is the enumeration of platelets which are the major components
of blood coagulation. Platelets help in formation of primary haemostatic plug.
EQUIPTMENTS :
1. Haemocytometer with improved Neubauer chamber (Hawksley counting chamber).
2. Micropipette.
3. Reagent, ammonium oxalate (10 gm/L) is used to destroy the RBCs in the sample blood.
4. Anticoagulated venous blood sample, 20 μl (blood sample from vein is collected due to
the nature of the test).
PROCEDURE :
• Mix properly the diluent with blood sample in 1:20 ratio.
• With the help of micropipette the solution is filled in the chamber.
• Wait for 15-20 minutes.
• Lastly the chamber is seen under the microscope at 40X.
The counting is done in the RBC counting areas of the grid.
CALCULATION :
TOTAL PLATELET/cubic mm=
(No. of Platelet counted
x Dilution factor x Depth factor)
/ No. of chambers counted
Normal Value= 1,50,000-4,50,000 per microliter of blood

ABNORMAL PLATELET COUNT
THROMBOCYTOSIS
(INCREASED PLATELETS)
THROMBOCYTOPENIA
(DECREASED PLATELET)
 Reactive
-Acute bleeding
-Burns
-Haemolytic anemia
-Iron deficiency
-Cardiac stroke
-Infections (eg. Tuberculosis)
-Inflammations (eg. Rheumatoid
arthritis, celiac disease, inflammatory
bowel syndrome)
-Pancreatitis
 Medication induced
-Heparin sod.
-Epinephrine
 Decrease production
-Dehydration
-Vitamin B12 and folic acid def.
-Liver failure
-Leukemia
-Sepsis (viral or bacterial)
-Hereditary (eg. Fanconi anemia)
 Increased destruction
-Thrombocytopenic purpura
-Post-transfusion purpura
-Dengue fever
-Zika virus
-SLE
-DIC
 Medication induced
-Valproic acid
-Methotrexate
-Interferon
-Isotretinoin
-PPI

PLATELET AGGREGATION CASCADE
PLATELET
FUNCTION
HAEMOSTASIS

CLOTTING FACTORS
Clotting factors are important components of blood plasma responsible for
haemostasis (i.e cessation of blood loss from a damaged vessel followed by repair).
There are thirteen clotting factors (but, except factor VI) present in blood:-
FACTORS NAMES
Factor I Fibrinogen
Factor II Prothrombin
Factor III Tissue Thromboplastin
Factor IV Calcium ions
Factor V Labile factor
Factor VII Stable factor
Factor VIII Anti-Haemophilic factor
Factor IX Christmas factor or PTC
Factor X Stuart power factor
Factor XI Plasma thromboplastin antecedent
Factor XII Hageman factor
Factor XIII Fibrin Stabilizing Factor

THE CLOTTING CASCADE
EXTRINSIC PATHWAYINTRINSIC PATHWAY
(surface factor) (tissue factor)
FVII
FV
FVIII
FXII
FXI
FX
FIX
Prothrombin(FII) Thrombin (FIIa)
Fibrinogen (FI) Fibrin (FIa)
FXIII
PTT : for IP
PT : for EP
The tests which are related to body haemostasis are Bleeding Time (BT),
Clotting Time (CT), Prothrombin Time (PT), International Normalized
Ratio (INR), Activated Partial Thromboplastin Time (aPTT).

BLEEDING TIME
Bleeding time is a test done to assess the platelet function.
PROCEDURE: (DUKE METHOD)
 Sterilize the puncture site (like finger tip) with an antiseptic solution to minimize
the risk of infection.
 At the puncture site, prick should be done deep enough (3-4mm) that it cause
slight bleeding.
 The puncture site should be wiped at every 15 minutes.
 Start the stopwatch at the moment bleeding starts and record the time when
bleeding stops.
 Cover the cut point.
(In IVY’S Method, 1mm deep incision is made instead of cut)
NORMAL RANGE:
2-5 minutes (in Ivy’s method=3-10 minutes)
INTERPRETATION:
Increased BT means the platelet function is affected. In certain vascular disorders
like purpura, von Willebrand disease, haemophilia, thrombocytopenia and certain
drugs like antiplatelet drug (aspirin), anticoagulant drug (warfarin), bleeding time
is increased.

CLOTTING TIME
Clotting time is the time required for a sample of blood to coagulate. The blood
clotting time is a rough measure of all intrinsic clotting factors in the absence of tissue
factors. Its chief application is in monitoring anticoagulant therapy.
METHOD: (Capillary Tube Method)
 Clean the tip of a finger with spirit.
 Puncture it upto 3 mm deep with a disposable needle.
 Start the stopwatch.
 Fill two capillary tube with free flowing blood form the puncture after wiping the
first drip of blood.
 Keep these tubes at body temperature.
 After 2 minutes, start breaking the capillary tube at 1 cm distance to see whether a
thin fibrin stand is formed between the two broken ends.
 Stop the watch and calculate the time from average of the tow capillary tubes.
NORMAL RANGE : 1-5 minutes
Other method is Lee and White Method where the clotting time range is 5-10
minutes.
INTERPRETATION:
Clotting time is prolonged in deficiency of clotting factors (haemophilia), vitamin K
deficiency (F-II, VII, IX, X), administration of drugs such as anticoagulants,
administration of heparin.

PROTHROMIN TIME
Prothrombin Time (PT) is a blood test that measures how long it takes blood to clot.
A PT test looks at one set of these factors (extrinsic) to see how well these factors are
working.
METHOD:
 Anticoagulated blood sample is collected in a test tube.
 The sample is then centrifuged to collect the blood plasma (as PT is most
commonly measured in plasma).
 The plasma from the sample is extracted and collected in a measured test tube.
 Calcium solution is added to reverse the function of anticoagulant.
 Finally, in order to activate the extrinsic pathway of clotting, tissue factor (factor
III) is added and the time the sample takes to clot is measured.
NORMAL RANGE : 12-13 seconds
INTERNATIONAL NORMALIZED RATIO (INR) :
The result (in seconds) for a prothrombin time performed on a normal individual
varies according to the type of analytical system employed. The INR is devised to
standardize the result. INR=(PTtest/PTnormal). Normal range is 0.8-1.2.
INTERPRETATION:
• Monitoring warfarin level in blood.
• Prolonged PT is seen in vitamin K deficiency, warfarin therapy, liver disease (as
except F-III, IV, XII every clotting factor protein is synthesized by liver) , DIC.

ACTIVATED PARTIAL THROMBOPLASTIN
TIME
The partial thromboplastin time (PTT) or activated partial thromboplastin time
(aPTT) is a test that characterizes blood coagulation by intrinsic factors.
METHOD:
 Anticoagulated blood sample is collected in a test tube.
 The sample is then centrifuged to collect the blood plasma (as PT is most
commonly measured in plasma).
 The plasma from the sample is extracted and collected in a measured test tube.
 Calcium solution is added to reverse the function of anticoagulant.
 Finally, in order to activate the intrinsic pathway, an activator (silica, celite, kaolin,
ellagic acid) is added and the time the sample takes to clot is measured optically.
NORMAL VALUES : 30-50 seconds
INTERPRETATION:
• Detection of F-I, II, V, VIII, IX, X, XI and XII
• Prolonged aPTT may indicate use of heparin.
• Presence of antibodies against coagulation factors.

COMPLETE BLOOD COUNT
Complete blood count or CBC is combination of Hct, MCV,
MCH, MCHC, RBC Count, ESR, Total WBC Count, Differential
WBC Count, Platelet count, BT, CT, PT, INR, aPTT. This is
important for evaluation of RBC, WBC and Platelet before
starting of any kind of treatment to understand the health of the
patient.

OTHER TESTS
-GLUCOSE TOLERANCE TEST:
The glucose tolerance test is a medical test in which glucose is given and blood
samples taken afterward to determine how quickly it is cleared from the blood. This
test is used for diabetes, insulin resistance, impaired beta cell function, carbohydrate
metabolism disorder.
-GLUCOSE TEST:
Blood glucose monitoring is a way of testing the concentration of glucose in the
blood. This test is advised for the patients having diabetes mellitus to determine the
insulin dose and assess the effectiveness of prior insulin dose.
-THYROID FUNCTION TEST:
Thyroid function test is a collective test of T3, T4, TSH to check the function of the
thyroid (overactive or underactive). In hyperthyroidism T3, T4 is increased and TSH is
decreased and vice versa for hypothyroidism.
-LIVER FUNCTION TEST:
Liver function tests (LFTs or LTs) are groups of blood tests which give information
about the state of a patients’ liver. These tests include PT, aPTT, albumin, bilirubin.
The liver transaminases aspartate transaminases (AST or SGOT) and alanine
transaminases (ALT or SGPT) are useful biomarkers of liver diseases.

QUICK QUESTIONS
--How is the ESR increased in inflammation?
The ESR is governed by the balance between pro-sedimentation factors, mainly
fibrinogen, and those factors resisting sedimentation, namely the negative charges of
the erythrocytes (zeta potential : the potential difference between the dispersion
medium and the stationary fluid attached to the surface of dispersed particle). When
an inflammatory process is present, the high proportion of fibrinogen in the blood
causes red blood cells to stick to each other. The red cells form stacks called
'rouleaux' which settle faster, due to their increased density.
--Why the intrinsic factors are named as “intrinsic” and extrinsic factors as
“extrinsic”?
The intrinsic and extrinsic, two pathways were named that way because of the way
blood clots in a test tube in the lab. The extrinsic pathway is kicked off by tissue
factor (IIIa) combining with VIIa. Tissue factor is not normally present in the blood
(it’s in little closed-up particles, or it’s in the subendothelium, or it’s in inflammatory
cells). To get blood clot in a test tube via the extrinsic pathway, tissue factor (which is
extrinsic to the blood) has to add to the test tube. To get the intrinsic pathway to run,
everything that it needs is already in the blood to clot, thus intrinsic.

--In Neubauer’s chamber, how many chambers have to count for RBC and WBC
count?
All the 4 chambers are counted for WBCs and the 4 corners and the central division
of the central chamber (total 5 boxes) are counted for RBCs.
Total Surface Area for WBC
count=4 x 1mm =4mm
Total Surface Area for RBC
count=5 x 0.04mm=0.2mm
WBCs are counted in a “snake
like” line in each chamber.
2 2
2 2

--How to count in Neubauer’s Chamber if the blood sample spread from the
counting chamber?
If the blood sample spreads throughout the chamber going outside the counting site,
WBCs touching the upper and right-hand lines are counted whereas the cells touching
bottom and left-hand lines are not to be counted. In case of Platelets and RBCs
upper and left-hand lines are not to be counted.
RBC Count
Platelet Count
Don’t count the RBCs
and Platelets along
the GREEN Line.

Basic haematology technique

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