Holy Grail in the automated counting of the WBC differential has been the enumeration/ quantification of immature granulocytes. This debate continues with clinical colleagues who insist they must

 

¾ These plasma factors cause increased formation of

rouleaux which due to more weight sediment more

rapidly than do single cells

¾ Albumin retards sedimentation

¾ Extreme increase in plasma viscosity slows down ESR

¾ Cholesterol accelerates and lecithin retards the ESR.

2. Red Cell Factors

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

to the surface area

¾ Poikilocytosis retards ESR because abnormal shape

hampers rouleaux formation.

3. Anticoagulants

¾ Sodium citrate and EDTA do not effect ESR but oxalates

and heparin may.

Stages in ESR

1. First 10 minutes—is the period of aggregation. Rouleaux

formation occurs at this stage and sedimentation is

slow.

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.

Interfering Factors

1. The blood sample should not be allowed to stand for

more than 2 hours before the test is started because

rate will increase.

2. In refrigerated blood, the sedimentation rate is greatly

increased. Refrigerated blood should be allowed

to return to room temperature before the test is

performed.

3. Factors leading to reduced rates:

• High blood sugar

• High albumin level

• High phospholipids

• Decreased fibrinogen level of the blood in

newborns

• Certain drugs (see below).

4. Drugs

a. That increase ESR levels:

• Dextran

• Methyldopa

• Methysergide

• Oral contraceptives

• Penicillamine

• Theophylline

• Trifluperidol

• Vitamin A.

b. Those that decrease levels:

• Ethambutol

• Quinine

• Salicylates

• Drugs that cause a high blood glucose level

(cortisone and ACTH).

BLOOD FILM EXAMINATION

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

edged spreader.

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.

FIG. 9.10: Making a spreader

Clinical Hematology 223

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).

Making Thick Smears

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

through it.

Fixing of Blood Films

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.

Staining of Blood Films

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

climate that exists in India.

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

cells a very pale color.

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

Solution No. I

NaOH (sodium hydroxide) 8 g.

Distilled water 1000 cc.

Solution No. II

KH2PO4 (Potassium dihydrogen phosphate) 27.2 g.

Distilled water 1000 cc.

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

water. This has a pH of 6.8.

Stain Preparation and Staining

Wright‘s Stain

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

0.25 or 0.3% solution.

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.

Leishman’s Stain

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.

Method

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’s Stain

Giemsa powder 0.3 g Glycerin 25.0 mL Acetone-free

methyl alcohol 25.0 mL.

This makes stock solution and before use, it has to be

diluted by adding 1 mL (stain) to 9 mL of buffered distilled

water.

Method

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

and dry.

Staining of Thick Films

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

stain and Simeon’s stain.

Field’s Stain

Field’s stain A

Methylene blue 0.8 g

Azure I 0.5 g

Disodium hydrogen

 phosphate (anhydrous) 5.0 g

Potassium dihydrogen

 phosphate anhydrous 6.25 g

Distilled water 500 mL

Field’s stain B

Eosin (yellow eosin, water soluble) 1.0 g

Disodium hydrogen phosphate (anhydrous) 5.0 g

Potassium dihydrogen phosphate (anhydrous) 6.25 g

Distilled water 500 mL.

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.

Method

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

Method

This stain can be used instead of Leishman’s or Wright’s

stain when methyl alcohol is not available to prepare them.

Solution I

Eosin pure 1 g

Distilled water 1000 mL.

Solution II

a. Medicinal methylene blue 1 g dissolves, distilled water

75 mL completely.

b. Potassium permanganate 1.5 g dissolves, distilled

water 75 mL completely.

1. Mix (a) and (b) in a flask. A massive precipitate is

formed.

2. The flask is kept in a water bath at boiling

point for half an hour during which time the

precipitate redissolves.

3. Filter. The stain is now ready for use, it needs no

further dilution.

Clinical Hematology 225

Method for Staining Thin Films

1. Fix the smear by immersion into rectified spirit—1

minute.

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

necessary.

2. Immerse in Sterenel’s blue (solution II)—6 seconds.

3. Wash in tap water.

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

purposes.

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.

RAPID DIAGNOSTICS

Automation in Hematology

Coulter Principle

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 Coulter principle.

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

Hematology cell counters continue to provide an everbroader scope of capabilities. Technologies that were

leading edge a few years ago, such as reticulocyte

enumeration, are now routine. Methods that heretofore

required much manual manipulation—such as CD4

counts—can now be incorporated as part of the randomaccess CBC specimen stream on instruments such as the

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.

226 Concise Book of Medical Laboratory Technology: Methods and Interpretations For 25 years, the Holy Grail in the automated counting

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

clinical use of the “immature granulocyte” channel. This

advance has great potential for the precise and accurate