MANUAL OF BASIC HEMATOLOGY

TECHNIQUES FOR RESIDENTS

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 Manual Of Basic Hematological Techniques For Residents

CONTENTS
Subject: Page

Cell counters:Principle, interpretation & pitfalls……………………… 4

Stains in common use in

10
hematology…………………………………...

Routine stains of blood & bone marrow………………………….. 10

Staining for red cell inclusions…………………………………… 10

Leucocyte cytochemistry…………………………………………. 11

Tests for hemolytic

12
anemia……………………………………………...

Fragility tests……………………………………………………... 12

RBC enzyme assay……………………………………………….. 16

Hemoglobin stability test…………………………………………. 19

Hemoglobin electrophoresis……………………………………… 22

Tests for PNH…………………………………………………….. 24

Tests for immune hemolytic anemia……………………………... 28

Other
33
tests………………………………………………………………..

Test for cryoglobulins…………………………………………….. 34

Plasma viscosity………………………………………………….. 35

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 Manual Of Basic Hematological Techniques For Residents

CELL COUNTERS:PRINCIPLE, INTERPRETATION & PITFALLS:

Several types of electronic counters are currently avialable, many are capable of
maesuring hematologic variables other than cell counts. Many of these instruements are
supplemented by histograms& scattergram garphics.
Principle
Automated cell counting and sizing of blood cells is generally based on one of 2
main principles; namely the aperture impedance & the light scattering technologies.

Aperture Impedance Technology: First described by Coulter, 1952

It depends on the fact that in any saline-based diluent ,cells are relatively bad
conductors in relation to the diluent. If the cells pass in the sensing zone they can be
detected by increase in the electrical resistance. This transient elevation in resistance
creates an electric impulse. These electrical pulses can then be counted. The
magnitude of each impulse is proportional to the cell size.
Types of instruments: Coulter, Sysmex& Cell-Dyn.
In practice this type of counters is formed simply of a tube with a small orifice
(e.g. 100 μm in diameter and 70 μm in length) immersed in a dilution of blood cells.
Cells are aspirated through the orifice. Impedance is measured between a negative
electrode inside the orifice tube and a positive electrode outside in the dilution of
blood cells. An external vacuum initiates movement of a mercury siphon that causes a
measured volume of the sample to flow through the aperture tube.

Although the sheath flow was originally described in the light-scatter systems,
it can also be applied to aperture impedance system to enhance their ability to measure
cell size.

Light scattering technology:

It is based on the passage of cells through a narrow beam of light. The sensing
zone is restricted partly by narrowness of the light beam and partly by delivering the
cells in a very narrow stream. As the cells pass through the sensing zone they scatter
light. The scattered light can then be collected by a suitable optical system and
measured by a photometer. The transient increases in scattered light create impulses
from the photometer, which can then be counted electronically. The cell counters can
measure the cell size, as the amplitude of the impulse is proportional to the size of the
cell.

In this technology it is mandatory to use the sheath fluid to obtain a narrow

stream of cells. Cells enter the sensing zone are surrounded by the sheath flow which
is adjusted to create hydrodynamic focusing effect which forces the cell suspension to
remain in a narrow stream as it passes through the center of the sensing zone.

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 Manual Of Basic Hematological Techniques For Residents

Type: Technicon.

Difficulties and solutions

Some difficulties are met with, in automated blood cell counting, for which
special designs are introduced:-

 The first difficulty is to count cells once and only once i.e. not to lose
a cell and not to count a cell more than one time. The loss of one cell can occur in
what is called coincidence in which two cells can pass through the sensing zone in the
same time and so they are counted as one cell. The pulse can alternatively be
generated at the dead time of the circuit or the cells may agglutinate due to the
presence of cold agglutinins. These later will give a large impulse and considered as
one large cell. Warming of blood samples and application of suitable mathematical
algorithm to edit out any abnormally large impulse or measuring different dilutions
from the same sample can correct this phenomenon. Counting of a cell more than once
can be due to a phenomenon called recirculation. This can occur mainly in aperture
impedance counters. Counting of bubbles or extraneous particles as cells is another
cause of false increase in the counts of blood cells. These can be corrected by the use
of sheath flow, application of a sweep flow (forcing fluid across the rear of the orifice
to wash its back) or by editing out.

イ The second difficulty is to discriminate the cell of interest from other cells or
electronic noise and debris. The solution of the problem is the process called thresholding. In
red cell count we set only a lower threshold to discriminate between small red cells and large
platelets. No need for an upper threshold to discriminate red cells from leucocytes due their
relative low count. As regards to platelets, we set an upper threshold to eliminate small red
cells and a lower threshold to exclude electronic noise and debris. White cells can be also
discriminated from red cells by applying a lower threshold but after adding a hemolysing
agent to destroy erythrocytes. This is applied mainly when there is sheath flow. When sheath
flow is not used in aperture impedance a theoretical distribution curve is made to platelet
volume histogram and platelet count is extrapolated from the area under the theoretical curve
to avoid the too much overlap between platelet and small erythrocytes and electronic noise
and debris. In light scatter it is necessary to add compounds that stop the debris causing
optical interference.

ロ The third is the difficulty to count blood cells per unit volume. In stead most
counter count cells per unit time and then convert them to cells per unit volume by
multiplication by specific factor. This factor is calculated by calibration.

Use of cell counters in counting different blood cells

1. The red blood cells and platelets are counted by the same channel and can be
differentiated from each other by their size.

2. As regards to red cell indices, the main difficulty in measuring the mean corpuscular
volume (MCV) is that the impulse produced by any cell is only approximately
proportional to the volume of the cell. Aberrant impulses, which do not truly represent
the cell size, can occur but often these impulses have unusual characteristics, which
enable them to be identified by suitable electronic ciruits and edited out so that they do
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 Manual Of Basic Hematological Techniques For Residents

not affect average impulse magnitude that is used as the measure of MCV. This is
more important in aperture impedance without sheath flow.

Recirculating cells give larger impulses than cells passing in the center of the
orifice. The impulses can be edited out easy as they are longer than they should be due
to longer transit at the edge of the orifice. Two cells passing through together give
oversize pulse and so can be edited out. When manual MCHC is reduced there is a
tendency to underestimate the MCV.

In aperture impedance, MCHC that determines the internal viscosity of the

erythrocytes also determines its shape as they pass through the orifice. They take a
cigar shape. When the internal viscosity of this cigar diminishes, it becomes thinner
and it creates a smaller impulse than it should be (shape factor).

In light scatter, both the shape of the cell and internal refractive index affect
the red cell volume. In these systems, they sphere and fix the red cells prior to their
entry into the sensing zone. This minimizes these effects. In addition they apply light
from high angle (5-15°), which is more affected by the internal refractive index and a
low angle (2-3°), which is more affected by cell volume on cell-by-cell basis.

3. All counters can make histograms that are useful for measuring anisocytosis and
characterizing situation in which two populations are present. In dual angle light
scatter it is possible to measure the individual hemoglobin concentration of a red cell
leading to the possibility constructing histogram of the distribution of cell hemoglobin
concentration.

4. Platelet volume distribution histograms can be used to determine platelet count, mean
platelet volume and platelet anisocytosis. Red cell and platelet anisocytosis and mean
platelet volume have little clinical value due to great differences between different
instruments but they are important in the laboratory as they determine a blood film
should be examined or not.

5. Hemoglobinometry is done in many instruments by a modification of the

cyanmethemoglobin method using Drabkin’s type reagent. This method has two
disadvantages:
a. Measurement of absorbance is made after a set time interval after mixing of blood and
reagent but before reaction is completed so a detergent material is included to ensure
rapid cell lysis and reduce the turbidity due to cell membrane and plasma lipids. We
must calibrate the instrument by using samples with of known hemoglobin
concentration.
b. Biohazardous method due to disposal of cyanide containing wastes. In some
instruments this method is replaced by the use of a non-toxic chemical” sodium
laurylsulphate”.

6. In automated reticulocyte counting, these cells are stained by various dyes and are
counted by reticulocyte counters, conventional flowcytometer or as an option in recent
blood cell counters in a separate channel.

7. White blood cells are counted by another channel after lysing erythrocytes. Total and
differential leucocytic counts are carried out at the same channel in most cell counters.
Some instruments use more than one channel for counting leucocytes.
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 Manual Of Basic Hematological Techniques For Residents

Light scatter alone can lead to misleading results in differential leucocyte count
due to the fact that the cell volume is not the only factor that determines the apparent
cell volume. Another equally important factor is the granules, which in turn
determines the internal refractive index of the leucocyte. For this reason neutrophils
appear larger than eosinophils despite that they have the same size or the eosinophils
are even larger. They have the same volume if they are measured by aperture
impedance. Another technology is used now to correct this defect in combination with
light scatter, which is the light absorbance. Leucocytes are stained by peroxidase and
the two technologies are applied together at the same time on cell-by-cell basis. 5
classes of leucocytes can be discriminated i.e. neutrophils, lymphocytes, monocytes,
eosinophils and large unstained cells (LUCs). This occurs in the same channel and so
it is called simultaneous multiparameter measurement.

The single peroxidase channel was proved to be inadequate for basophil

measurement. Thus, many cell counters were provided with additional channel for
basophils. These instruments are now in wide use and are called multichannel
counters.

Other approaches using a single channel are used in other instruments. In

aperture impedance systems the use of high frequency electromagnetic probe at the
same time as measuring volume provides more information about the internal
constituents of white cells and this improves the differential counts in aperture
impedance counters.

In light scatter systems, examination of scatter at different angles at the same

time on cell-by-cell basis enhances the differential count in these counters. It is now
possible to combine both light scatter and aperture impedance technologies in the
same instrument in a single channel.

Calibration, control and internal quality assessment

Calibration:

In direct measuring instruments i.e. instruments that measure counts per unit
volume the operator cannot recalibrate the instrument. He can only check that the
instrument is working correctly and call the service for any problem. As the majority
of instruments are indirect i.e. measure counts per unit time and need to be calibrated
to correct it to count per unit volume. The operator can calibrate them by testing blood
samples whose cell counts per unit volume has been determined independently. The
calibration factor is then determined using the following formula:

count/unit volume on
Calibration known blood
factor = count/unit time on known
blood

When this is stored in the memory of the cell counter it can then calculate
counts per unit volume as such:

Counts/ unit volume on unknown=counts/ unit time on unknown × calibration factor

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 Manual Of Basic Hematological Techniques For Residents

Although the impulses produced by the passage of a cell through the sensing
zone is approximately proportional to the cell volume, the MCV and mean platelet
volume cannot be measured directly but calculated. The calibration factor is calculated
as follows:

Calibration factor= volume of unknown cell / size of impulse produced by unknown

cell
So all instruments must be calibrated in respect to cell volume measurements
and other measurements based on it i.e. PCV and MCHC
PCV = MCV × red cell counts
MCHC = hemoglobin / PCV
In hemoglobinometry instruments have to be calibrated for hemoglobin
measurement because none of them produce an absolute reading i.e.
spectrophotometric absorbance of hemoglobin that is fully converted to
cyanmethemoglobin. So MCH must be calibrated due its link to hemoglobin, whether
or not the erythrocytes are counted directly.

Fresh blood and preserved blood can be used as calibrants. It is

more preferred to use fresh blood calibrants. Assigning values for fresh
blood calibrators are done by reference methods or by suitable selected
methods as shown in table:
Measurement Method
Red cell count Semi automated aperture- impedance counter aspirating a
known volume through orifice using calibrated manometer
and validated coincidence correction.

PCV Carefully performed microhaematocrit

Hemoglobin Cyanmethemoglobin determination

Red cell indices Calculate from above measurements

White cell count The same as used in red cell but after their lysis

Platelet count Use a counter with a sheath flow to estimate the relative
numbers of red cells and platelets. Red cells are calculated
as above and then platelets are calculated.
The methods recommended by ICSH for assigning values to fresh blood calibrants

Fresh calibrants are stable only for four hours. It will be both tedious and cost
ineffective to prepare fresh calibrants daily. Thus, operator must use preserved blood
calibrants, which are stable for 30 days or more. They are expensive and instrument
specific.

Controls:

They are stable preserved blood samples, easy to manufacture in house and
cheaper to purchase because they do not need to have pre-assigned values. They can
be used as follows:

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1. Calibrate the instrument either with fresh blood or with preserved blood calibrants.
2. Make 20 measurements on the control and calculate the mean and standard deviation
SD.
3. Construct a shewhart control chart.
4. Test the control after every 20 patients and calculate how many SD the results differ
from the mean.Plot the results on the shewhart control chart and look for drifts.
5. Make calibration if the results of quality control show excessive drifts as stated above.
Patient means:

They are adjuncts to using preserved blood controls. They are considered
sometimes as alternatives.

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External quality assessments:

They are periodical governmental programs for assessment of the reliability of

results of different laboratories according to specific schemes.

Sources of errors:
Disorders and Conditions that may Adversely Affect the Accuracy of
Blood Cell Counting
Co Disorder/Conditio Effect on Cell Count Rationale
mp n
one
nt
Microctosis or May underestimate Lower threshold of RBC
schistocytes RBC counting window is
greater than microcyte
size
Re
d Howell-Jolly May spuriously elevate Howell-Jolly bodies
cell bodies platelet count (in whole similar in size to platelets
s blood platelet counters
only)

polycythemia May underestimate Increased coincidence

RBC counting
Leukocytosis underestimate WBC Increased coincidence
counting

Acute leukemia May spuriously lower Increased fragiljity of

and choronic WBC leukocytes, including
Wh
lymphocytic immature forms
ite
leukemia
cell
s
Viral infections

Chemotherapy of May artifactually Leukemic cell nuclear or

acute leukemia increase “platelet” count cytoplasmic fragments
identified as “platelets”
Platelet May underestimate Platelet clumping.
Plat agglutinins platelet count, Aggregates may be
elet sometimes with identified as leukocyte
s spurious increase in
WBC
Cold agglutinins May underestimate Red cell doublets, triplets,
Pla RBC with spurious etc.have increased
sm microcytosis volume
a Cryoglobins, Variation in platelet Protein precipitates may
cryofibrinogens count be identified as “platelets”
Some of these examples only affect counts when certain instruments are used. The sffects depend on dilution, solutions
employed. or specimen temperatures.

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 Manual Of Basic Hematological Techniques For Residents

STAINS IN COMMON USE IN HEMATOLOGY

Routine stains of blood & bone marrow:
Giemsa Stain:

Stock solution: 1 gm Giemsa added to 66 ml Glycerol in a conical flask, heat at 56°Cfor 2

hours, then add 66 ml methanol. Mix well & leave for 7 days at RT, filter & use.

Staining solution: dilute 1/10 with DW.

Leishman Satin:

Add 0.2 gm leishman powder to 100 ml methanol, warm to 50°Cfor 15 min. & filter.

Brilliant Cresyl Blue :

add 1 gm BCB to 100 ml 0.9% saline, containing 0.4 gm trisodium citrate.

For reticulocyte staining : incubate 2 volumes of EDTA or heparin anticoagulate fresh

blood sample & 1 vloume of the dye at 37°Cfor 20 minute & then spread films. If the patient
is anemic increase the proportion of the blood to the stain.
Iron Stain:
Reagents:
1. Fixative: metahnol (analar).
2. 1 gm Kferrocyanide dissolved in 100 ml 0.1 N HCl immediately before use.
3. Aquaous safranin 0.1%.

Method:
1. Fix smears in methanol at RT for 15 min. Blot dry.
2. Immerse in K ferrocyanide HCl mixture for 30 min. at RT.
3. Wash in running tap water for 20 min.
4. Rinse in DW & counter stain in 0.1% safranin for 3 min.

Staining for red cell inclusions:

Hemoglobin H inclusions:

Procedure:
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 Manual Of Basic Hematological Techniques For Residents

Mix 2 volumes of freshly blood with 1 vloume of brilliant cresyl blue staining solution
(used for reticulocyte satining), incubate at 37°Cfor 2 hours. Mix & spraed films & examine
for Hb H inclusions which appear as multiple greenish-blue dots like the pitted pattern of golf
ball.

Interpretation & comments:

In alpha tahl. trait only very ocacsional H bodies are seen. In Hb H disease inclusions
are found in more than 30% of red cells.Absence of demonstarble inclusions doesn’t preclude
a diagnosis of alpha thal. trait.

Heinz body preparation:

Reagent: Methyl violet: dissolve 0.5 g methyl violet in 100 ml of 9 g/l NaCl & filter.

Staining: add 1 volume of blood on any anticoagulant to 4 volumes of the staining solution,
allow to stand at RT for 10 min., then prepare films. Leave to dry in air.

Interpretation: Heinz bodies stain an intense purpule.If negative repeat the staing after 24
hours incubation of the blood at 37 ˚C.

Leucocyte Cytochemistry:
Always use directly spread un-anticoagulated blood or BM smears & always run a
control with the test sample.

Peroxidase staining:

Reagents:

Bezidine solution: dissolve 0.3 gm benzidine in 99 ml ethyl alcohol , then add 1 ml of

30% saturated K or Na nitroprusside.

H2O2 : add 3 drops H2O2 raegent to 12.5 ml DW in a test tube immediately before use.

Method:
1. In a staining rack add 10 drops benzidine reagent to cover the surface of the silde.
Leave for 1.5 min.
2. Add ½ the volume i.e 5 drops H2O2 reagent, mix by gently blowing into the mixture
taking care the smear remains generally covered all the time. Leave for 4.5 min. &
tehn wash with tap water.
3. Air dry, then counter satin with Giemsa for 10 minutes.

Other Leucocyte cytochemical stains:

Commercially available kits are usually used . Exactly follow the instuctions of the kit
used.

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TESTS FOR HEMOLYTIC ANEMIA:

Fragility Tests:-
Osmotic fragility test
Sample: -
 2-3 ml blood are withdrawn & added to 2 separate tubes with heparin ; one for the
fresh test & one kept at 37 oC in a sterile tube for the incubated test.
 The fresh test should be carried out within 2hs of collection of blood; stored at room
temperature or within 6hs ; if the blood has been kept at 4 oC.
 The sterile tube is kept for 24 hs at 37 oC for the incubated test.

Diagnostic significance:-

The osmotic fragility test gives an indication of the surface area/ volume ratio
of erythrocytes. Its greatest usefulness is in the diagnosis of hereditary spherocytosis. The test
may also be used in screening for thalassemia. Red cells that are spherocytic for whatever
cause, take up less water in a hypotonic solution before rupturing than normal red cells .

Principle :-

Small volumes of blood are added to large excess of serial hypotonic buffered
saline solutions. The fraction of red cells lysed at each saline concentration is determined
colorimetrically.

Reagents :-
 1% buffered Na Cl solution;
 Prepared from 1mL of stock solution of buffered Nacl 10% on 9 mL distilled water.
 Phosphate buffered Saline 10% stock sloution for OF:
 90gm NaCl +13.65gm Na2HPO4 + 2.43gm NaH2PO4.2H2O in DW to a final vloume
of 1 L.
N.B. Always dissolve the precipitated crystals in the stock solution before making the
dilution by incubating for some time at 37 oC.

Technique :-
1. Make serial dilutions of saline as follows:

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 Manual Of Basic Hematological Techniques For Residents

D.W. 0.4 0.5 0.6 0.7 0.8 0.9

NaCl 1% 1.6 1.5 1.4 1.3 1.2 1.1 2 ml D.W.
13 12 11 10 9 8 + ve
No.
1 2 3 4 5 6 7 -ve
NaCl 1% 0.4 0.5 0.6 0.7 0.8 0.9 1 ml 2 ml saline
D.W. 1.6 1.5 1.4 1.3 1.2 1.1 1 ml
+ Ve control tube 2 mL D.W.
- Ve control tube 2 mL normal saline (0.9%)
2. Put I drop of the patient blood in each tube by Pasteur pipette or 20 μl and mix
immediately by inverting the tubes.
3. Incubate & leave the suspensions for 30 mins at room temperature , mix again, and
then centrifuge for 5 min. ( at 1200g ) & compare with - ve & +ve tubes for
haemolysis.
4. Expressing the results of O.F. :-
a) Hold - ve control tube & start comparing the supernatent from tube number 13
& record the first tube in which discolouration of the sueprnantent starts. Then
continue looking in sequence at the rest of tubes for residual RBCs sediment at
the bottom & record the first tube showing no residual RBCs as the
concentartion of complete hemolysis.
Normal values:Haemolysis starts at 0.45 % NaCl conc. & is complete
at 0.35% NaCl conc.
b) Estimate the amount of lysis in each tube spectrophotometerically at a wave
length of 540 nm. Use as a blank the supernatant from – ve control tube &
assign a value of 100 % lysis to the reading with the supernatant of + ve
control tube then plot the results against the Nacl concentration.
c) From the O.F. curve; we can deduce the median corpuscular fragility (conc. At
which 50% hemolysis occurs) as well as any deviation in the shape of the
curve.

Interpretation:
The osmotic fragility of fresh red cells reflects their ability to take up a certain amount
of water before lysing. This is determined by their volume to surface area ratio. The ability of
the normal red cell to withstand hypotonicity results from its biconcave shape which allows
the cell to increase its volume by about 70 % before the surface membrane is stretched; once
this limit is reached lysis occurs. Spherocytes have an increased volume to surface area; their
ability to take in water before stretching the surface membrane is this more limited than
normal and they are therefore particularly susceptible to osmotic lysis. The increase in
osmotic fragility is a property of the spheroidal shape of the cell and is independent of the
cause of the spheroytosis.

Decreased O.F. indicates the presence of unusually flattened red cells (leptocytes) in
which the volume to surface area ratio is decreased. Such a change occurs in iron deficiency
anemia and thalassemia in which the red cells with a low MCH & MCV are unusually
resistant to osmotic lysis.

Reticulocytes and red cells from splenctomized patients also tend to have a greater
amount of membrane compared with normal cells and are osmotically resistant. In liver
disease, target cells may be produced by passive accumulation of lipid & these cells, too, are
resistant to osmotic lysis.

Pitfalls :-
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 Manual Of Basic Hematological Techniques For Residents

1. The blood must be delivered into the 13 tubes with both +ve & -ve control tubes with
great care. The critical point is not the amount be exactly 20 ul but rather that amount added
to each tube must be equal .Two methods are recommended:-
a) Using an automatic pipette. After aspirating the blood gently, the outside
should be wiped with tissue paper taking care not to suck out any blood from
the tip by capillary action.

b) The blood is then delivered into the saline solution and the pipette rinsed in and out
several times until no blood is visible inside its tip. The tip has to be changed before
moving on to the next tube. This procedure takes time and may result in an increased
exposure for the first few tubes. It is therefore advisable to start the timing on the
addition of the sample to the first tube.
c) Using a Pasteur pipette with a perfectly flat end 1mm in diameter. About I mL of
blood should be sucked up, avoiding any bubbles, and the outside of the pipette wiped.
With the pipette held vertically above tube1, a single drop ( about 20 ul ) is delivered
without the blood touching the wall of the tube. Further single drops are then delivered
into the remaining tubes.

Method (b) appears to be primitive, but with practice it is perfectly satisfactory. It is also more
economic and much faster than method (a).

2. Even when a normal range has been established, it is essential always to run a normal
control sample along with that of the patients to be tested inorder to check, for example, the
saline solutions.

The sigmoid shape of the normal osmotic fragility curve indicates that normal red
cells vary in their resistance to hypotonic solutions; indeed, this resistance varies gradually as
a function of red cell age, with the youngest cells being the most fragile. The reason for this is
that old cells have a higher sodium content and a decreased capacity to pump out sodium.

Incubated Osmotic fragility

Osmotic fragility after incubating the blood at 37˚C for 24 hs. exposes the RBCs to a
metabolic stress. The increased osmotic fragility of normal red cells which occurs after
incubation is mainly caused by swelling of the cells associated with an accumulation of
sodium which exceeds loss of potassium. Such cation exchange is determined by the
membrane properties of the red cell which control the passive flux of ions and the metabolic
competence of the cell which determines the active pumping of cations against a
concentration gradient during incubation for 24h.,the metabolism of the red cell becomes
stressed and the pumping mechanisms tend to fail; one factor being a relative lack of glucose.
The fragility of red cells which have an abnormal membrane, such as those of hereditary
sperocytosis & hereditary elliptocyosis, increases abnormally after incubation .The results
with red cells with glycolytic pathway deficiency such as those of pyruvate kinase deficiency,
are variable. In severe deficiencies; O.F. may increase substantially but in other cases the
fragility may decrease due to a greater loss of potassium than gain of sodium. In thalassaemia,
O.F. is frequently markedly reduced after incubation due to loss of potassuim. A similar,
though usually less marked change is seen in iron deficiency.

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 Manual Of Basic Hematological Techniques For Residents

Acidified Glycerol lysis time test (A G L T)

AGLT is a one tube test designed to measure the time taken for 50% haemolysis of a
blood sample in a buffered hypotonic saline–glycerol mixture to occur. It is useful as a
screening test for hereditary spherocytosis.
Sample: Blood sample on EDTA or heparin.
Principle:-

Glycerol present in a hypotonic buffered saline solution slows the rate of entery of
water molecules into the red cells that the time taken for lysis may be more conviently
measured. Like the osmotic fragility test, differentiation can be made between spherocytes
and normal red cells.

Reagents :-
1. Phosphate buffered saline ( PBS): Add 9 volumes of 9 g /L ( 154 mmol /L) Nacl to l
vol of 100 mmol /L phosphate buffer & Adjust the PH to 6.85 +/- 0.05 at room temp.
2. Glycerol reagent (300 mmol/l ):Add 23 ml of glycerol (27.65 g AR grade ) to 300 ml
of PBS & bring the final Vol. to 1L. by DW.
Technique :

1. Add 20 ul of whole blood to 5.0ml of PBS PH 6.85 & mix the suspension carefully.
2. Transfer 1ml of the previous suspension to a standard cuvette of a spectrophotometer.
3. Adjust the wave length at 623 nm then add 2ml of glycerol reagent rapidly to the
cuvette with a 2ml syringe. Immediately start a stop watch & simaltaneously record
the initial absorbance.
4. Record the absorbance again at short intervals until you reach ½ of the initial
absorbance & immediately record the time. The end point is 30 minutes if ½ the
absorbance is not reached.
5. The rate of haemolysis is measured by the rate of fall of turbidity of the reaction
mixture. The results are expressed as the time required for the optical density to fall to
half the initial valve (AGLT50).

Interpretation

Normal blood takes more than 1800sec ( 30 min ) to reach ( AGLT 50). In patients
with hereditary spherocytois the AGLT50 is <180 sec. For the fresh test. A short AGLT 50
may also be found is chonic renal failure, chronic leukaemia autoimmune haemolytic anemia
and in some pregnant women.

Significance

The same principles apply as with the osmotic fragility test. Cells with a high volume
to surface area ratio resist swelling for a shorter time than normal cells; this applies to all
spherocytes whether the spherocytosis is caused by hereditary spherocytois or other
mechanims.

Incubated AGLT50

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 Manual Of Basic Hematological Techniques For Residents

This have the same significance as the incubated OF test. The blood should be kept
sterile. A diagnostic result for HS is <120 sec.

Red Cell Enzyme Assay:-

G-6-P-D Deficiency

Qualitative Screening Test

Sample :

Blood samples may be anticoagulated with heparin, EDTA or ACD. In any of these
anticoagulants the enzyme is stable for 6 days at 4 ºC & for 24 h at 25 ºC.

Principle:
 G6PD catalyses the oxidation of glucose 6 phosphate (G6P) into 6-phosphogluconate
with the simaltaneous reduction of NADP into NADPH
G6P + NADP PG + NADPH
 NADPH is an important reducing substrate for the conversion of oxidized glutathione
(GSSG) into GSH & under the conditions of stress the reduction of Hbi to Hb.
 Sceening tests for G.6.PD deficiency depend upon the ability of RBCs to convert
oxidized substrate into a reduced state.
The methemoglobin reduction test:

Sodium nitrite converts Hb into Hbi. When methylene blue is added it acts as an
artificial electron acceptor, an intact GP shunt will reconvert Hbi into the reduced state. If
G6PD is deficient, Hbi will remain in the oxidized state.

Reagents:

Dextrose nitrite: 5 gm D-glucose + 1.25 gm Na nitrite in 1 L DW.

Methylene blue: 0.4 mmol/L. Dissolve 150 mg methylthionine chloride (methylene
blue chloride, Sigma) in 1 L DW.

N.B. Glass tubes are better used because plastic may adsorb some reagents.

Technique:

1. Add 1 ml of blood to the tube containing 0.2 ml of the combined reagent {0.l ml Na
nitrite & 0.1 ml MB}. Close the tube with a stopper & gently mix the contents by
inversion.

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 Manual Of Basic Hematological Techniques For Residents

2. Prepare a negative control tube by adding 1 ml of blood to a tube containing 0.2 ml

saline & a positive control tube by adding 1 ml of blood to a tube containing 0.1 ml
Na nitrite& 0.1 ml saline (omitting the MB).
3. Incubate the 3 tubes at 37 oC for 3 hrs.
4. After the incubation, remix the tubes & add 0.1 ml from each tube to 10 ml distilled
water in a separate test tube.
5. Mix the contents genlty & compare colours.

Interpretation:

1. Normal blood yields a colour similar to the nomal reference tube ;clear red.
2. Blood from G6PD-deficient subjects gives a brown colour similar to that in the
deficient reference tube .
3. The degree of deficiency can be semiquantitatively expressed according to the colour
given by the test.
NB: A markedly anemic blood may give false result without an actual enzyme deficiency.
To overcome this, you can adjust the hematocrit of the blood before testing.

G6PD Enzyme Assay

Sample :

Whole blood is collected with EDTA, heparin or ACD. Red cell G6PD is stable in
whole blood for a week when refrigerated at 2-8°C, but unstable in red cell haemolysate.
Freezing of blood is not recommended.

Priniciple:

The rate of formation of NADPH is proprtional to the G6PD activity& in measured

spectrophotometrically at 340 nm.

Procedure: a variety of commercially avialable kits are used. Follow the instructions
of the used kit exactly.

Normal value :

146 – 376 (U/1012 RBC) or 4.6-13.5 U/g Hb.

Interpretation of result :

1. The gene for G6PD is on the X chromosome & therefore males can be either normal
or deficient hemizygote. By contrast females can be either normal, homozygotes or
heterozygotes with intermediate enzyme activity.
2. Red cells are likely to hemolyze on account of G6PD deficiency only if they have
<20% of normal enzyme activity.
3. G6PD activity falls off markedly as red cells age ,therefore whenever a blood sample
has a high popualtion of young red cells; G6PP activity will be higher than normal
sometimes to the extenet that a genetically deficient sample may yield a value within
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 Manual Of Basic Hematological Techniques For Residents

the normal range. This will be uasally, but not always associated with a high
reticulocytosis.
4. A value is the low normal range in the face of reticulocytosis should raise the
suspicion of G6PD deficiency, because with reticulocytosis G6PD activity should be
higher than normal. In such cases the deficiency can be confirmed by repeating the
assay when the reticulocytosis has subsided & sufficient old population is present
(about 6 weeks) or alternatively by assaying the old RBCs after fractionation by
denisty.
5. In females, heterozygozity can be better defined by cytochemical tests rather than
G6PD assay, where the value may range between 10 to 90%.
Assay Of Pyrovate Kinase Defciency

Sample :

One ml. blood on EDTA, heparin, or ACD.

Separation of RBCs from blood samples :

Leucocytes and platelets have a high PK enzyme activity . It is therefore necessary to

separate the RBCs rapidly by washing sample 3 times with normal saline.

Principle :

PK is an important enzyme for the mebabolism of glucose in RBCs:

PEP +ADP pyruvate +ATP

PK activity is measured as the rate of fall in absorbance at 340 nm.

Reagents & procedure:

The test is often done by commercially available kits. Preparation of hemolysate &
enzyme assay are done exactly according to the instructions of the kit ( attached).

Normal Values:

Normal range at 30 ˚C is 8.3-12.3 eu/g Hb.

Interpretation:

Pk deficiency uasually presents as chronic hemolytic anemia. Once the enzyme

activity is deficient, the diagnosis is established. Difficulty in diagnosis may arise in:
1. Abnormal variants with lower enzyme activity; which can be picked by use of a
low substrate concentartion in the test (see instructions).
2. High reticulocytic count as reticulocytes have a higher enzyme activity. In this
case PK deficiency can be suspected by finding a normal enzyme activity in spite
of high reticulocytosis. A family study may help in this case.

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 Manual Of Basic Hematological Techniques For Residents

Tests For Unstable Hemoglobin:

Several methods are available for the demonstration of hemoglobin instability,

heat instability test, isopropanol stability test, n-butanol stability test.

Sampling:

Samples used should be as fresh as possible and no more than one week old. Choose
samples for controls of the same age as the test sample; a normal cord blood sample can be
used as positive control.

HEAT INSTABILITY TEST

Principle:

When hemoglobin in solution is heated the hydrophobic van der Waals bonds are
weakened and the stability of the molecule is decreased. Under controlled conditions unstable
hemoglobins precipitate while stable hemoglobin remains in solution.

Sample:

Samples taken into any anticoagulant are satisfactory. EDTA is the most convenient.
Cells freed from clotted blood can also be used if necessary.

Reagent:

Tris-HCL buffer, pH 7.4, 50 mmol/l. Tris 6.05 g, water to 1 liter. Adjust the pH to 7.4
with concentrated HCL.

Method:

1. Preparation of lysate: (Lyse 1 volume of washed packed cells in 4 volumes of lysing

reagent prepared as follows:
 g EDTA, tetrasodium salt.
 0.7 g potassium cyanide (KCN).
 Water to 1 liter.
2. Add 0.2 ml of lysate, freshly prepared to 1.8 ml of buffer. Include a positive (Hb F)
and a negative (Hb A) control of the same age as the test sample.

3. Such a lysate will not keep for more than 1-2 days at 4 °C as it tends to gel. If
necessary it can be frozen at –20 °C for up to 1 month. Avoid repeated freezing and
thawing).
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 Manual Of Basic Hematological Techniques For Residents

4. Place the tubes in a water-bath at 50 °C for 120 min. examine the tubes at 60, 90, and
120 min for turbidity and fine flocculation.

Interpretation & comments:

The normal control may give minimal cloudiness at 60 min but a major unstable
hemoglobin will have undergone marked precipitation at 60 min and gross flocculation at 120
min.

ISOPROPANOL STABILITY TEST

Principle:

When hemoglobin is dissolved in a solvent such as isopropanol, which is more non-

polar than water, the hydrophobic van der Waal’s bonds are weakened and the stability of the
molecule is decreased. Under controlled conditions unstable hemoglobins precipitate while
stable hemoglobins remains in solution.

Reagent:

Tris-HCL buffer, pH 7.4, 100 mmol/l. Tris 12.11 g, water to 1 liter. Adjust the pH to
7.4 with concentrated HCL.

Isopropanol, 17%. 17 volumes of isopropanol are made up to 100 volumes with

buffer.The resultant 17% isopropanol solution may be stored in a tightly stoppered glass
bottle for 3 months at 4°C.

Method:

イ Add 0.2 ml of lysate, freshly prepated by the CCl4 method given above, to a tube
containing 2.0 ml of 17% isopropanol. Include a positive (Hb F) and a negative (Hb A)
control of the same age as the test sample. Stopper each tube and mix by inversion.

ロ Place the tubes in a water-bath at 37 °C for 30 min. examine the tubes at 5, 20 and 30
min for turbidity & flocculation.

Interpretation & comments:

The normal control will remain clear at 20 min. At 30 min minimal cloudiness
should be apparent, but significant precipitation will not occur until 40 min. A major unstable
Hb will have undergone marked precipitation at 5 min and gross flocculation at 20 min. A
slightly unstable hemoglobin such as Hb E will show diffuse precipitation at 20 min.

n-BUTANOL STABILITY TEST

Principle:

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 Manual Of Basic Hematological Techniques For Residents

When hemoglobin is dissolved in a solvent such as n-butanol, which is more non-polar

than water, the hydrophobic van der Waals bonds are weakened and the stability of the
molecules is decreased. Under controlled conditions unstable hemoglobins precipitate while
stable hemoglobin remains in solution.

Reagent:

Stock sodium phosphate buffer, 0.1 mol/l. NaPo4 15.6 g, EDTA 3.7 g, water to 1 liter,
adjust the pH to 7.4 with concentrated HCL. The buffer may be stored at room temperature.
 n-Butanol
 Working solution.

The working buffer is temperature-dependant; prepare as follows:

18-20 °C n-butanol 6.5 ml, stock buffer to 1 liter.
21-23 °C n-butanol 6.0 ml, stock buffer to 1 liter.
24-26 °C n-butanol 5.5 ml, stock buffer to 1 liter.

Method:

1. Add 0.2 ml of washed packed cells to a plastic tube containing 2.0ml of working
solution. Include a positive (Hb F) and a negative (Hb A) control of the same age as
the test sample.

2. Stopper each tube and mix by inversion; then remove the stopper. The RBCs should
lyse giving a clear solution.

3. Place the tubes at room temperature and examine at 30, 60, 90 and 120 min for
turbidity and fine flocculation.

Interpretation & comments:

1. The normal control will remain clear at 120 min.

2. A major unstable hemoglobin will have undergone marked precipitation at 90 min and
gross flocculation at 120 min.

3. A slightly unstable hemoglobin such as Hb E will show diffuse precipitation at 120

min. The test should be continued until the positive control shows precipitation.

4. Positive results may be given by samples containing as little as 10% Hb F or by

samples containing increased methemoglobin as a result of prolonged storage.

5. False negative results should be avoided by continuing the incubation overnight.

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 Manual Of Basic Hematological Techniques For Residents

HAEMOGLOBIN ELECTROPHORESIS (CELLULOSE ACETATE,

PH 8.5):
Principle:

At alkaline PH, Hb is negatively charged & in an electric field will migrate towards
the anode (+). Structural variants with surface charge differences will separate from Hb A;
those without a change will not.

Sample preparation:
 0.5-1 ml blood on EDTA or heparin.
 Wash 3 X with normal saline & discard the supernatant.
 Prepare hemolysate by adding one drop of packed RBCs to 6 drops hemolysate
reagent (ready made from HELENA Co). If the patient is markedly anemic, the
amount of hemolysate reagent is reduced to 4 drops or the Hb of the hemolysate is
measured & adjusted to 10 gm/dl.

Electrophoresis technique:

1. With the power supply disconnected, fill the 2 compartements of the chamber with
TEB buffer (ready made powder from Helena Co. to be dissolved in DW).The buffer
should not be used for more than 1 month or if the PH is changed at any time. Don’t
use expired buffer packets.
2. Soak 2 strips of filter paper into the buffer & place one on each bridge of the cahmber.
3. In a separate dish soak the cellulose acetate paper (purchased from Helena Co)slowly
(to avoid bubble formation) in the same buffer sloution & leave for at least 5 minutes (
to ensure even saturation of the membrane).
4. Blot the cellulose acetate membrane gently between 2 filter papers, but do not let to
dry out before sample application.
5. Place 10 μl of each hemolysate into a sample well. Include a control sample with each
run.
6. Dip the applicator into the sample wells & apply to a filter paper.
7. Dip the applicator again into sample wells & apply to cellulose acetate membrane
place on the paper support with the edge at the cathodal application line & allow the
applicator tip to remain in contact with membrane for about 3 sec.
8. Place the membrane upside down across the bridge of the tank touching the filter
paper strips& apply a glass slide on the membrane to tighten the contact.
9. Close the chamber& connect to the power supply & adjust the volt to 250-350 for 20
minutes.
10. Disconnect from the power supply& remove the membrane, placing it in the Panceau
S stain ( ready made from Helena to be dissolved in DW), with the cellulose acetate
surface up & leave for 3-5 min.

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11. Elute the excess stain by placing in 3 changes of desatining solution ( 5% acetic acid),
2 minutes each.
12. Dehydrate in absolute methanol for 5 minutes.
13. Immerse in claering solution for 6-8 min. or until the paper becomes clear.
14. Dry at 56°Cfor 5 minutes then scann the cleared paper.

Interpretation:

 Types of separated HB are identified in comparison to the control.

 Concentration of each type is obtained as a % on scanning.

Reagents:

 Electrophoresis buffer: tris / EDTA/borate (TEB):- PH 8.5 Tris-hydroxymethyl

aminometahne (Tris), 10.2 g, EDTA 0.6 g, boric acid 3.2 g & DW to 1 liter.Buffer
should be stored at 4 ˚C .
 Panceau S: 5 g, trichloracetic acid 7.5 g& DW to 1liter.
 Destaining solution (5% acetic acid): 50 ml glacial acetic acid to 950 ml DW.
 Absolute metahnol.
 Clearing solution:Glacial acetic acid 6 ml, absolute methanol 14 ml, 0.8 ml clear aid
( purchased from Helena lab. Co).
 Control sample:either puchased commercially available control hemolysate or
prepared from hemolysate with kown HB variants (add few drops of 0.3 mol/L KCN ;
20 g/L to stabilize the HB)

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 Manual Of Basic Hematological Techniques For Residents

TESTS FOR PAROXYSMAL NOCTURNAL

HEMOGLOBINURIA(PNH)
Paroxysmal nocturnal hemoglobinuria is an acquired disorder in which the
patient’s red cells are abnormally sensitive to lysis by normal constituents of plasma.
In it’s classical form it is characterized by hemoglobinuria during sleep (nocturnal
hemoglobinuria), jaundice and hemosiderinuria. Not uncommonly, however, PNH
presents as an obscure anemia without obvious evidence of intravascular hemolysis or
develops in a patient suffering from aplastic anemia or more rarely from
myelosclerosis or leukemia.

PNH red cells are unusually susceptible to lysis by complement. This can be
demonstrated in vitro by a variety of test, e.g. the acidified-serum (HAM), sucrose,thrombin,
cold-antibody lysis, inulin and cobra-venom tests.

Acidified-Serum Test (Ham Test)

Principle:

The patient’s red cells are exposed at 37 C to the action of normal or the patients own
serum suitably acidified to the optimum pH for lysis (pH 6.5-7.0).

Sample:

The patient’s red cells can be obtained from defibrinated, heparinized, oxalated,
citrated or EDTA blood, and the test can be satisfactorily carried out even on cells which have
been stored at 4C for up to 2-3 weeks in ACD or Alsever’s solution, if kept sterile. The
patient’s serum is best obtained by defibrination, for if in PNH it is obtained from blood
allowed to clot in the ordinary way at 37C or at room temperature it will almost certainly be
found to be markedly lysed. Normal serum should similarly be obtained by defibrination, but
serum derived from blood allowed to clot spontaneously at room temperature or at 37C can
be used. Normal serum known to be strongly lytic to PNH red cells is to be preferred to
patient’s serum, the lytic potentiality of which is unknown. However, if the test is positive
using normal serum it is important, particularly if the patient appears not to be suffering from
overt intravascular hemolysis, to obtain a positive result using the patient’s serum, in order to
exclude HEMPAS. The activity of a single individual’s serum also varies from time to time
and it is always important to include in any test, as a positive control, a sample of known PNH
cells or artificially created “PNH-like” cells.

The sera should be fresh, i.e. used within a few hours of collection. Their lytic potency
is retained for several months at -70C, but at 4C, and even at -20C, they deteriorates within
a few days.

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Technique:

1. Deliver 0.5 ml samples of fresh normal serum, group AB- or ABO-compatible with
the patient’s blood, into six (three pairs) of glass tubes.

2. Place two tubes at 56C for 10-30 minutes in order to inactivate the complement.

3. Keep the other two pairs of tubes at room temperature.

4. Add to the serum in two of the tubes one-tenth volumes (0.05 ml) of 0.2 mol/l HCL.
5. Add similar volumes of acid subsequently to the inactivated serum samples.
6. Place all tubes in a 37C water-bath.
7. Wash samples of the patient’s red cells and of control normal red cells (compatible
with the normal serum) twice in 9.0 g/l NaCL.
8. Prepare 50% suspensions in the saline.
9. Add one-tenth volumes of each of these cell suspensions (0.05 ml) to single tubes
containing unacidified fresh serum, acidified fresh serum and acidified inactivated
serum, respectively.
10. Mix the contents carefully and leave the tubes at 37C for one hour.
11. Centrifuge.

Test (ml) Controls (ml)

Tubes
1 2 3 4 5 6

Fresh normal serum 0.5 0.5 0 0.5 0.5 0

Heat-inactivated normal serum 0 0 0.5 0 0 0.5

0.2 mol/l HCL 0 0.05 0.05 0 0.05 0.05

50% patient’s red cells 0.05 0.05 0.05 0 0 0

50% normal red cells 0 0 0 0.05 0.05 0.05

Trace +++
Lysis (in a positive test) No Hemolysis
(2%) (30%)

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 Manual Of Basic Hematological Techniques For Residents

In case of quantitative measurement of lysis

1. Add 0.05 ml of each cell suspension to 0.55 ml of water so as to prepare a standard.

2. Retain 0.5 ml of serum to use as a blank.

3. Deliver 0.3 ml volumes of the supernatants of the test and control series of cell-
serum suspensions, and of the blank serum and of the lysed cell suspension equivalent
to 0% and 100% lysis, respectively, into 5 ml of 0.4 ml/L ammonia or Drabkin’s
reagent.
4. Measure the lysis in a photoelectric colorimeter using a yellow-green (e.g. Ilford 625)
filter or in a spectrophotometer at a wavelength of 540 nm.

Interpretations:

If the test cells are from a patient with PNH:

1. They will undergo definite, although, as already mentioned incomplete lysis in the
acidified serum.
2. Very much less lysis, or even no lysis at all, will be visible in the un- acidified serum.
3. No lysis will be brought about by the acidified inactivated serum.
4. The normal control sample of cell should not undergo lysis in any of the three tubes.
In PNH 10-50% lysis is usually obtained, when lysis is measured as liberated
hemoglobin. Exceptionally, there may be as much as 80% lysis or as little as 10%.

The red cells of a patient who has been transfused will undergo less lysis than before
transfusion, because the normal transfused cell, despite circulation in the patient, behave
normally. In PNH, it is characteristic that a young cell (reticulocyte-rich) population, such as
the upper red cell layer obtained by centrifugation, undergoes more lysis than the red cells
derived from mixed whole blood.

Acidified-Serum Test With Additional Magnesium (Modified Ham Test)

Principle:

The sensitivity of the Ham test can be improved by the addition of magnesium to the
test to enhance the activation of complement.

Technique:

The technique is identical to that for the standard Ham test (see above) with the
addition of 10l of 250 mM magnesium chloride the acidified-serum test is positive. The
addition of magnesium chloride increases the sensitivity of the acidified-serum test, and it
remains specific for PNH. (Whenever the acidified-serum test is positive it is recommended
that a direct antiglobulin test should be carried out. If this is positive, it could be due to a lytic
antibody, which has given a false positive acidified-serum test. This can be confirmed by
appropriate serological studies. In addition, in such complex cases a more definitive test for

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 Manual Of Basic Hematological Techniques For Residents

PNH, which is now available, is flow cytometry after reaction of the red cells with ante-
CD59).

The only disorder other than PNH that may appear to give a clear-cut positive test is a
rare congenital dyserythropoietic anemia, CDA Type II or HEMPAS. In contrast to PNH,
however, HEMPAS red cells undergo lysis in only a proportion (about 30%) of normal sera;
moreover, they do not undergo lysis in the patient’s own acidified serum and the sucrose lysis
test is negative. The expression of GPI-linked proteins in HEMPAS is normal. In HEMPAS,
lysis appears to be due to the presence on the red cells of an unusual antigen which reacts with
a complement-fixing IgM antibody (anti-HEMPAS) present in many, but not in all, normal
sera.

Heating at 56C inactivates the lytic system and, if there is lysis in inactivated serum,
the test cannot be considered positive. Markedly spherocytic red cells or effete normal red cell
may lyse in acidified serum, probably due to the lowered pH, and such cells may lyse, too, in
acidified inactivated serum.

It must be stressed that PNH red cells are not unduly sensitive to lysis by a lowered
pH per se. The addition of the acid adjusts the pH of the serum-cell mixture to the optimum
for the activity of the lytic system. It is possible to construct pH-lysis curves, if different
concentrations of acid are used. The optimum pH for lysis is between pH 6.5 and 7.0
(measurements made after the addition of the red cells to the serum).

Sucrose Lysis Test

Principle:

The sucrose lysis test is based on the fact that red cells absorb complement
components from serum at low ionic concentrations. PNH cells because of their great
sensitivity will undergo lysis but normal red cells do not.

Sample:
1. Patient sample on EDTA.
2. Clotted sample (ABO compatible with the patient) to have compatible serum as
control.
Technique:

Isotonic solution of sucrose = 0.924 g in 10 ml distilled water (92.4 g/l)(can be stored

at 4 °C for up to 2-3 weeks).
1. Two test tubes are prepared for the test and the control.
2. Put the following in these tubes.

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 Manual Of Basic Hematological Techniques For Residents

Test Control

Solute / Saline 850 l 850l

Compatible Serum 50l 50l

Patient’s washed RBCs (50% suspension) 100l 100l

1) Incubate 30 minutes in 37 °C.

2) Centrifuge then examine for hemolysis.

3) If lysis is visible in sucrose containing tube, measure this in a spectrophotometer (WL

546), using a tube containing serum diluted in saline as a blank and a tube containing
0.1 ml of red cell suspension in 0.9 ml of 0.4 ml/l ammonia in place of the sucrose-
serum mixture as a standard for 100% lysis.

Interpretation:
1. Red cells from some cases of leukemia or myelosclerosis may undergo a small amount
of lysis, almost always <10%; in such cases the acidified-serum test is usually
negative and PNH should not be diagnosed.
2. In PNH, lysis varies from 10% to 80%, but exceptionally may be as little as 5%.
Sucrose lysis and acidified-serum lysis of PNH red cells are fairly closely correlated.
3. The sucrose lysis test is typically negative in HEMPAS.

Tests For Immune Hemolytic Anemia:-

Direct Coomb’s Test

Sample:

2 ml blood on EDTA

Principle:

Demonstration of incomplete auto–antibodies coated to the patient’s red cells, which

become agglutinated on addition of anti–human globulin (AHG) serum. The type of the
coating globulin can then be determined by use of specific anti globulin reagent for IgG, IgM
and IgA or complement. It is thus considered a test for in vivo sensitization.

Reagent (Anti globulin reagents) :-

a) Poly specific reagents:-

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 These should contain both anti IgG and anti complement, if plasma is used, only anti
IgG is necessary as EDTA prevent complement activation.
 The majority of red cell antibodies are non- – complement – binding IgG, and IgG is
therefore an essential component of any poly specific reagent.
 Anti IgA is not required as, IgG antibodies of the same specificity always occur in the
presence of IgA antibodies.
 Anti IgM is also nost required because clinically significant IgM allo –
antibodies that don’t cause agglutination in saline are much more easily
detected by the complement they bind.

b) Mono specific reagent: -

 These can be prepared against the heavy chains of IgG, IgM and IgA, antibodies
&against C3, C4 complement components.
 The main clinical application of those antibodies is to define the immune chemical
characteristics of antibodies.
Technique:
 The EDTA sample is washed 3 times (add saline then centrifuge then pour the
supernatant fluid till the saline is clear).
 10% suspension is prepared as follows: 1 drop washed RBc5 + 9 Drops saline or 100
u washed RBc5 + 900 u saline then
 2 drops of the suspension are added to 2 drops of AHG & the mixture is incubated for
20 min. at 37 °C .
 Centrifuge & examine for agglutination both by nacked eye & by microscope.

Interpretation:-

A positive DAT doesn’t necessarily mean that the patient has AIHA.A positive test is
seen in:
1. Auto antibody on RBC surface with or without hemolytic anaemia.
2. An allo – antibody on RBC surface as in hemolytic disease of newborn or after
incompatible transfusion.
3. Antibodies provoked by drugs adsorbed to the red cells.
4. Normal globulins adsorbed to the red cell surfaces as the result of damage by certain
drugs.
5. Adsorption of immune complexes to the red cell surface as in hospital patients.
6. Sensitization in vitro, this occurs if blood is allowed to stand in refrigerator at 4% or
even at room temperature and the test is then done ;due to adsorption of incomplete
cold antibodies and complement .
7. + ve DAT may occur in a small % of normal people .
8. 20% of patients on long term treatment of methyldopa develop + ve DAT.

Indirect Coombs & Cold Agglutinin Tests

Sample :

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 Manual Of Basic Hematological Techniques For Residents

3 ml clotted venous blood sample is centrifuged and serum is separated. This is better
carried out at 37 °C rather than at room temperature to prevent adsorption of a cold auto
antibody.

Principle:

Detection of free auto-antibodies in serum ,either warm antibodies (which are able to
combine with their corresponding red cell antigen at 37 °C) or cold antibodies (which
combine with red cell antigen at lower temprature)

Technique:

1. Set up a series of 12 tubes , 10 for the test & 2 for control.

1 2 3 4 5 6 7 8 9 10 + ve - ve

Serum(ul) 200 100 300 200 ---

Saline(ul) - 300 200 200 200 200 200

Washed
200 200 200 200
O+ve(50%)

2. In case of demonstration of cold agglutinin incubate the tubes at 4 °C (in refrigerator)

for overnight (N.B all materials used in preparation as saline, serum, tubes are placed
in 4 °C before work).
3. Wash all tubes 3 time with saline & prepare a 10% suspension of washed RBCs.
4. Mix one drop of the cell suspension & one drop of A HG .
N.B ( AHG is not added for demonstration of cold agglutinin & the suspension is
examined directly for agglutination).
5. Incubate all tubes at 37 oC for 10 minutes then centrifuge & examine for agglutination
in case IDAT.
Interpretation: The last titer showing agglutination is recorded.

positive test is present in :

1- Auto immune hemolytic anaemia including:

a. Warm auto antibodies (of IgG type ;IgA, IgM warm antibodies are much less
common& if present; usually in addition to IgG , they are associated with
complement adsorbtion to the red cells.
b. Cold auto–antibodies are nearly always IgM in type. Hemolysis is due to
destruction of the red cells by complement adsorbed to the red cell surface.

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 Manual Of Basic Hematological Techniques For Residents

c. Cold type auto – antibodies have anti I specificity (i.e they react strongly with
the vast majority of adult red cells and only weakly with cord blood red cells.
2- Drug induced immune hemolytic anemia.
3- RH incompatibility
 False negative anti-globulin test:-
1- Failure to wash the red cells properly – the antisera may then be neutralized by immuno-
globulins or complement in the serum or plasma.
2- The use of impotent – antisera so that weakly sensitized cells are not detected.
3- The use of antisera lacking the antibody corresponding to the subclass of immune
globulin responsible for the red cell sensitization.
4- The antibody being readily dissociated in the washing process.
5- DAT –ve AIHA: In about 2-6% of patients with AIHA, the DAT is –ve due to low
concentration of antibody.

Other Tests:

Test For Cryoglobulins

Sample:

Serum. 3 ml blood should be withdrawn with a warm syringe & kept a 37 °C until
clotted.

principle:

Cryoglobulins are a group of proteins that had the common property of forming a
percipitate or gel in the cold. This phenomeon is reversible by raising the temprature. This
group of proteins are classified according to purification & immunochemical analysis into:-

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Immunochemical
Type of cryogbulin Associated diseases
Composition
* Type I: monoclonal cyoglobulin  Ig M  Myeloma
consist of a single monoclonal Ig  Ig G  Waldenstrom’s
 Ig A macrooglobulin emia
 Bence Jones protein Chronic lymphocyic leukaemia
 Ig M – Ig G
* Type II: mixed cryoglobulin  Ig G – Ig G Myeloma
monoclonal immunoglobulin with 2 antibody  Ig A – Ig G waldenstron macroglobulinemia
activity against a polyclonal immunoglolulin (RF C CLL
activity) R RA
Sjogren’s syndrome ix mixed
essential ercryogldulinemia
Ig M – Ig G  Hepatitis C
Ig M – IgG- IgA 
*Type III: mixed polyclonal cryoglablin are SLE
mixed polyclonal cryoglobulin with RF activity . RA
Sjogren’s syndrome
IMN
CMV
Acute viral hehepatitis
Chronic active hehepatitis
Hepatitis C
primary biliary rrcirrhrosis
po poststreptococcal
glomerulonephritis
gi infective endocarditis
Leprosy
Kala – azar
Tropical sp lenomegaly
syndrome

Technique :

1- After clotting the sample at 37 oC

2- Separate the serum by centrifugation at 37 oC,then store it at 4 oC
3- when cryoglobulin is present , a white preciptate or gel appears in the serum after a
variable period, usually 24-72 hours, but the serum should be observed for I week to be sure
that late cryoprecipitation does not go undetected.
4- The reversibility of cryopercipitation should be tested by rewarming an aliquot of
precipitated serum.
5- The cryoprecipitate can be quantitated by several ways:-
a. Centrifugation of the whole serum in a hematocrit tube at 4 oC allows
determination of the relative amount of the cryoglobulin (Cryocrit).
b. Alternatively the protein concentration in the serum before & after
cryoprecipitation may be compared.
c. The precipitate formed in an aliquot of serum may be isolated, dissolved in an
acidic buffer & the cryoglobulin level estimated by absorbance at 280 nm.
After isolation & washing of the precipitate the components of the cryoglobulin can
be identified by immunoelectrophoresis , immunofixation or immunodiffusion.These analyses
are performed at 37 oC using:-
 antiserum to whole human serum

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 Manual Of Basic Hematological Techniques For Residents

 antisera specific for alpha (α), gamma (γ), mu (μ), Kappa (κ), lambda (λ) chains for
classification
 antiserum to fibrinogen may be used to determine the presence of cryofibrinogen ppt.
Interpretation & significance:

 Type I & II:

Cryoglobulins are usually present in large amount in serum (often more than 5
mg/ml).

In general they are present in patients with monoclonal paraproteinemias e.g. in

lymphoma or multiple myeloma ;sometimes however they are found in patients lacking an
evidence of lymphoid malignancy just are “benign” paraproteins.

 Type III:

Cryoglobulins indicate the presence of circulating immune complexes & are the result
of immune responses to various antigens. They are present in relatively low concentrations
(usually less then 1mg/ml) in rheumatoid disease & chronic infections. All Types of
cryoglobulins may be responsible for specific symptoms that occur as a result of changes in
the cryoglobulin induced by exposure to cold; symptoms include:
 Raynaud’s phenomenon
 Vascular pupura
 Bleeding tendencies
 Cold – induced urticaria
 Distal arterial thrombosis with gangrene

Since type II & type III cryoglobulin are circulating soluble immure complexes, they
may be associated with serum sickness – like syndrome charaterized by :
 polyarthritis.
 vasculitis .
 glomerulonephritis
 neurologic symptoms.
In patients with mixed essential IgM – IgG cryoglobulinemia a rather distictive
syndrome may occur that is associated with arthralgia, purpura, weakness, lymphadenopalhy
or hepatosplenomegaly .This synchome may be a sequela of hepatitis B infection.
Glomerulonephritis is common. In some instances is occurs in rapidly progressive form & is
an ominous prognostic significance.

Cryoglobulins may cause a srious error in a variety of laboratory tests by precipitating

at ambient temperatures & thereby removing certain substances from serum; Complement
fixation& inactivation& entrapment of immunoglobulins in the precipitate are common
examples. Redissolving the cryoprecipitate usually dose not fully restore activity to the serum
especially that of complement.

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 Manual Of Basic Hematological Techniques For Residents

Plasma Viscosity
Measurement of the acute phase response is a helpful indicator of the presence &
extent of inflammation & its response to ttt.
Useful tests include:
 CRP
 ESR
 Plasma viscosity
Plasma viscosity is dependent on the conc. of plasma proteins .Change in viscosity
seems to reflect the clinical severity of the disease more than ESR.

principle:

The time taken for a given volume of plasma to pass through a length of narrow tube
is compared to the time for an equal volume of DW.

Sample :

The test requires 0.3 – 0.5 ml of plasma obtained from EDTA. Anticoagulated blood.

Technique:

Preparation:
Blood after being collected on EDTA, is centrifuged as soon as possible at 3000 rpm
for 5 min. The plasma can be stored at room temp for up to 1 week without change to its
viscosity.

Steps:

a. Fill the descending limb of visicometer with DW. With stopwatch in hand allow the
fluid to run through the descending capillary to the lower reservoir bulb.

b. Start stopwatch as fluid level passes the lower mark (L) and stop it when it reaches the
upper mark (U).

c. Record time taken.

1- The procedure should be done in duplicate for:

 DW & Normal plasma
 DW & test plasma
 Relative viscosity is calculated as the time taken for plasma divided by time
taken for water.

Results

Normal range: 1.4-1.8 Values above 50 may be associated with severe symptoms
requiring plasmapharesis.

Interpretation:

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 Manual Of Basic Hematological Techniques For Residents

Cases with increased viscosity are:

 Increased immunoglobulins as a consequence of prolonged antigenic Stimulation:

 Rh. Arthritis

 Liver diseases

 Neoplastic increase of Igs as in Multiple myelma

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