¾ These plasma factors cause increased formation of
rouleaux which due to more weight sediment more
¾ Albumin retards sedimentation
¾ Extreme increase in plasma viscosity slows down ESR
¾ Cholesterol accelerates and lecithin retards the ESR.
¾ Anemia is responsible for accelerated ESR. The change
in erythrocyte-plasma ratio favors rouleaux formation
¾ Microcytes sediment more slowly and macrocytes
somewhat more rapidly than normocytes. The
sedimentation rate is directly proportional to the
weight of the cell aggregate and inversely proportional
¾ Poikilocytosis retards ESR because abnormal shape
¾ Sodium citrate and EDTA do not effect ESR but oxalates
1. First 10 minutes—is the period of aggregation. Rouleaux
formation occurs at this stage and sedimentation is
2. Next 40 minutes—is period of fast settling, during this
period rate of fall is constant.
3. Last 10 minutes—is the final period of packing.
1. The blood sample should not be allowed to stand for
more than 2 hours before the test is started because
2. In refrigerated blood, the sedimentation rate is greatly
increased. Refrigerated blood should be allowed
to return to room temperature before the test is
3. Factors leading to reduced rates:
• Decreased fibrinogen level of the blood in
b. Those that decrease levels:
• Drugs that cause a high blood glucose level
Preparation of a Thin Blood Film
A thin blood film is made by spreading a drop of blood
evenly across a clean grease free slide, using a smooth
Making of Spreaders (Fig. 9.10)
¾ Select a slide which has smooth edges
¾ Using a glass cutter and a ruler, mark off 4 equal
divisions, each measuring 19 mm
¾ Break off at each division to give 4 spreaders
¾ Readymade spreaders are available.
For anemic blood, a rapid smearing is needed; whereas
for thick concentrated blood, smearing should be done
slowly. A well-spread smear shows no lines extending
across or downwards through the film and the smear
should be tongue shaped (Figs 9.11A and B).
While the thin smears are used for describing blood cells,
the thick smears are used for detecting malarial parasites
and microfilariae. A large drop of blood is taken on the
center of a slide and with the aid of a needle or slide corner
spread the drop over ½ an inch square area. When dry, the
thickness should be such that printed matter can be seen
Before staining, the blood films need to be fixed with
acetone-free methyl alcohol for ½ to 1 minute in order to
prevent hemolysis when they come in contact with water
while staining them with aqueous (water-based) stains
or when water has to be added subsequently. Alcohol
denatures the proteins and hardens the cell contents.
For Wright’s stain and Leishman’s stain, no prefixation is
required as these contain acetone-free methyl alcohol;
but for Giemsa’s stain, prefixation is a must because the
alcohol content is only 5% in the ready-to-use stain.
Blood cells have structures that are acidophilic and some
basophilic structures, so they vary in their reaction (pH).
The nuclei are basophilic and stain blue. The highly
basophilic (acidic) basophil granules also stain blue.
Hemoglobin (being basic) stains acidophilic or red.
Stains that are made up of combinations of acid and basic
dyes are called Romanowsky stain and various modifications
are available, e.g. Wright’s, Leishman’s, Giemsa’s, and
Jenner’s stains. Most use methylene blue as the basic stain,
though toluidine blue is used in some. Most use eosin as the
acid stain, though Azure I and Azure II are also used.
The dried film can stay for a couple of days in hot dry
weather, but gets bad if they are not fixed in hot and humid
It is best to use neutral distilled water for diluting the
stain. Stale distilled water becomes acidic after absorbing
CO2 from atmosphere. If the distilled water is alkaline
RBCs stain a dirty bluish green color, the parts of WBC
which should stain blue will be slightly purplish, the
granules of eosinophils bluish or greenish instead of pink
and granules of neutrophils overstained. If the water is
acidic RBCs stain bright orange and nuclei of the white
The ideal pH is 6.8 and in order to maintain this buffered
distilled water is used. Buffer water is a solution which
tends to keep its original pH even on addition of small
amount of alkali or acid (Buffer tablets ready for use, to be
dissolved in distilled water).
Buffer Solution used in the Laboratory
KH2PO4 (Potassium dihydrogen phosphate) 27.2 g.
Take 23.7 cc of solution I, add to it 50 cc of solution II,
add 20 cc of the above mixed solution to 1000 cc of distilled
Stain Preparation and Staining
Wright’s stain (powder) 0.2 g.
Acetone free methyl alcohol 100 cc.♥
Let stand this solution for a few days.
If the WBC granules do not stand out clearly, try out a
FIG. 9.11A: Direction of spread
FIG. 9.11B: A thin peripheral blood smear
224 Concise Book of Medical Laboratory Technology: Methods and Interpretations Method
Cover the slide with stain for 1–2 minutes taking care that
it does not dry on the slide. Now dilute this with equal
amount of buffer water (if the stain is ripe, a scum or film
with a metallic sheen will form on the surface of the diluted
stains on the slide). The diluted stain is allowed to act for
3–5 minutes and then flooded off with buffer or tap water.
The stain should never be poured off or a precipitate of
the stain will be deposited on the slide. Should this occur,
it can sometimes be removed by flooding the slide with
undiluted stain for 10–15 seconds and then washing it off
again by flooding the slide once more with buffered water.
Powdered Leishman’s stain 0.15 g.
Acetone-free methyl alcohol 133 mL.
All the stain should be dissolved (better if the stain
crystals are well ground before), keep the stain in a glass
stoppered bottle. Do not filter.
Like that for Wright’s stain but with double dilution of the
buffer water; (i) Pour few drops (about 8) on the slide. Wait
for 2 minutes, (ii) Add double the amount (16 drops) of
buffered water. Mix by rocking and not by blowing and wait
for 7–10 minutes, (iii) The stain is flooded off with distilled
water and this should be complete in 2-3 seconds. Longer
washing will remove stain, and (iv) Stand in a rack to drain
and air dry. A fan will expedite the process.
Giemsa powder 0.3 g Glycerin 25.0 mL Acetone-free
This makes stock solution and before use, it has to be
diluted by adding 1 mL (stain) to 9 mL of buffered distilled
The blood film is fixed with methyl alcohol for 3–5 minutes
and dried. Pour on diluted stain and keep for 15 minutes
or longer. Wash off with tap water or neutral distilled water
Thick films have to be dehemoglobinized before staining
with one of the previously mentioned stains. The slide is
kept in distilled water for 10 minutes, then taken out, dried
and stained with any of the stains already mentioned. They
must not be fixed before staining, or the water will not
hemolyze the cells. The stains commonly used are Field’s
Eosin (yellow eosin, water soluble) 1.0 g
Disodium hydrogen phosphate (anhydrous) 5.0 g
Potassium dihydrogen phosphate (anhydrous) 6.25 g
Grind all solids well and dissolve in the said solvent,
keep the stains for 4 hours for ripening and filter before use.
Keep the stains in covered jars. The depth of the solution
should be about 3 inches, the level should be maintained
by adding more of the stain solution.
1. Dip the film for one second in solution A.
2. Remove from solution A and immediately rinse by
waving very gently in clean water for a few seconds,
until the stain ceases to flow from the film and the glass
of the slide is free from stain.
3. Dip for one second in solution B.
4. Rinse by waving gently for 2–3 seconds in clean water.
5. Place vertically in a rack to drain and dry.
Simeon’s Modification of Boye’s and Sterenal’s
This stain can be used instead of Leishman’s or Wright’s
stain when methyl alcohol is not available to prepare them.
a. Medicinal methylene blue 1 g dissolves, distilled water
b. Potassium permanganate 1.5 g dissolves, distilled
1. Mix (a) and (b) in a flask. A massive precipitate is
2. The flask is kept in a water bath at boiling
point for half an hour during which time the
3. Filter. The stain is now ready for use, it needs no
Method for Staining Thin Films
1. Fix the smear by immersion into rectified spirit—1
2. Rinse with tap water—4 seconds.
3. Immerse into solution I—10 seconds.
4. Rinse with tap water—4 seconds.
5. Immerse into solution II—15 seconds.
6. Rinse with tap water—4 seconds.
7. Immerse again into solution I—5 seconds.
8. Rinse with tap water—4 seconds.
9. Allow to dry in an upright position.
Procedure for Staining Thick Smears
1. Dehemoglobinize by immersion into tap water, if
2. Immerse in Sterenel’s blue (solution II)—6 seconds.
4. Immerse in eosin solution (solution I)—12 seconds.
5. Wash in tap water, allow it to dry in air. Examine
under microscope. The stains are useful for screening
Mounting and Preservation of Films
Unstained films cannot be preserved well. Due to hardening
of plasma, they do not stain well after some time. Stained
films if left unmounted tend to fade away rapidly. Canada
balsam should not be used as it decolorizes the smear.
Gurr’s neutral mounting medium is quite satisfactory. Use
only thin coverslips for mounting.
The Coulter principle states that particles pulled through
an orifice, concurrent with an electrical current, produce a
change in impedance that is proportional to the size of the
particle traversing the orifice. The Coulter principle was
named for its inventor, Wallace H Coulter.
Wallace was an electrical engineer by training with a
passion for radio technology. During the Second World
war, Wallace joined the US Navy. While working on a
technique to detect submarines using sonar, he frequently
detected large echos where no submarines were operating.
In an attempt to determine the source, Wallace lowered a
series of small bottles with remote trap doors to various
depths. The bottles were constructed such that the remote
door could be opened and shut at predetermined depths,
filling the bottle with seawater from that depth. The source
of the false echos turned out to be high concentrations of
plankton. In order to count the number of plankton cells
per milliliter of seawater accurately and reproducibly,
Wallace created a device that would become the basis for
The device consisted of a dual chambered container
whose two sides were separated by a thin membrane. A
small hole in the membrane called an aperture was the only
connection between the two chambers. Electrodes from a
battery were placed in the chambers, positive on one side
and negative on the other. An ohmmeter was connected
to the circuit so as to measure the resistance to the flow
of current (impedance) from one electrode, through the
orifice, and to the other electrode. Both chambers were
filled with seawater from the trap bottles. Then one of the
two chambers was partially drained, forcing seawater to
flow from the opposite chamber, through the orifice to
balance the level of liquid in the two sides. As the seawater
passed through the orifice so did the plankton cells, which
created momentary changes in impedance that were seen
on the ohmmeter. By counting the number of impedance
pulses per unit of seawater, Wallace’s device was able to
count the number of plankton particles.
This technology found commercial success in the
medical industry where it revolutionized the science
of hematology. Red blood cells, white blood cells and
platelets make up the majority of the formed elements in
the blood. The average salinity of human blood is very close
to that of seawater, and mixture of salt (NaCl) and water
with the same salinity as seawater is said to be isotonic
with whole blood. When whole anticoagulated human
blood is diluted with isotonic saline, the Coulter principle
can be applied to count and size the various cells that
make up whole blood. The first commercial application
of the Coulter principle to hematology came in 1954 with
the release of the Coulter Counter Model A (developed by
Wallace and brother Joseph R. Coulter). Within a decade,
literally, every hospital laboratory in the United States had
a Coulter Counter, and today every modern hematology
analyzer depends in some way on the Coulter principle.
The Basics of Hematology Analyzers in a Nutshell
leading edge a few years ago, such as reticulocyte
enumeration, are now routine. Methods that heretofore
required much manual manipulation—such as CD4
Abbott Cell-Dyn series. Food and Drug Administration
approval of quantitative nucleated red blood counts on
several instruments now permits automated handling of
patients with a variety of pathologic states.
of the WBC differential has been the enumeration/
quantification of immature granulocytes. This debate
continues with clinical colleagues who insist they must
have a manual differential because they want to know
if “bands” are numerous. It does not faze them that
study after study demonstrates that the “band count” is
terribly imprecise and non-reproducible. At least one
manufacturer has submitted applications to the FDA for
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