(brilliant cresyl blue or new methylene blue). In 1–2 days, it loses its basophilia and becomes a mature erythrocyte. Control of erythropoiesis: Erythropoietin (formed in kidneys) is released in response to lowered tissue oxygen tension. Erythropoietin is a glycoprotein and stimulates primitive cell differentiation to pronormoblasts. It affects the rate of multiplication and maturation. It acts up to early normoblast stage and also affects the rate of hemoglobinization.

 

quantitation of immature granulocyte forms (the collective

total of promyelocytes, myelocytes, and metamyelocytes).

Ironically, the clinical significance of automated immature

granulocyte counts is difficult to measure at present, since

the existing literature is heavily weighted toward only band

counts and not extended immature granulocyte counts.

We do hope to see these immature granulocyte counts take

hold and, finally, eliminate the use of the manual band

count.

Bayer’s reticulocyte hemoglobin measurement is useful

in the early diagnosis of iron deficiency and in monitoring

response to treatment.

Another interesting new channel is hematopoietic

progenitor cells, or HPCs, available on the Sysmex XE2100. In some settings, this will permit stem cells to be

quantitated (for example, in an apheresis product) without

requiring a direct CD34 study on a flow cytometer. This

study is based on differential membrane lipid content.

HPCs have lower membrane lipid content than mature

leukocytes and are preserved after treatment with a lysing

agent.

With increasing routine automation of assays that

previously required the use of flow cytometers, we may see

flow cytometers redirected to more in-depth analyses of cell

structure and function—the emerging field of cytomics.

The rate-limiting step on the introduction of new

diagnostic modalities is no longer a matter of how quickly

the technology can be developed, licensed, and deployed.

Far more important is how quickly medical practitioners

embrace the new technologies and incorporate them into

their routines.

Those selecting hematology instruments can no

longer base their decisions solely on the lowest-price

instrument. Medical considerations should and may

dominate. Perhaps the patient mix requires a parameter

that is available only on certain instruments, for example,

Operational considerations may be paramount—reliable,

high-throughput, easy-to-use instrumentation may be

more crucial than having all the newest parameters on

a more difficult-to-use instrument. The fiscal effect of

eliminating flow cytometry for high-volume studies, such

as CD4 or CD34, may outweigh a higher cost-per-test on

CBCs.

¾ Three-dimensional VCS technology provides the

highest sensitivity, specificity and efficiency in

abnormality detection

¾ Compact, bench top analyzer saves valuable laboratory

space

¾ 75 samples-per-hour throughout maximizes productivity

¾ Detailed reports and histograms for operator review

¾ Automatic calibration and Zero-routine-maintenance

maximizes uptime

¾ Data management system stores up to 5,000 patient

records

¾ Closed vial sampling, automatic cap piercing and probe

wipe minimize biohazards

¾ Walkaway automation frees up valuable operator time

¾ Positive patient ID makes sample tracking easy

¾ No routine maintenance

¾ Built-in Quality Assurance ensures accuracy.

Coulter MAXM and MAXM AL Hematology

Flow Cytometry Systems (Fig. 9.12)

The MAXM is the easiest hematology system to learn and

operate. It features walkaway operation, positive patient

identification, automatic calibration, auto-probe wipe,

single-operator interface and continuous computer

monitoring of system performance. Best of all, the MAXM

requires no routine daily maintenance. Your staff is free

to handle more complex tasks. The optional Autoloader

allows walkaway operation. Load 25 bar-coded samples

and then just walk away. The MAXM automatically

analyzes both patient samples and controls-providing

automatic printouts of the finished reports at a throughput

of up to 75 samples per hour unsupervised.

Coulter MAXM AL together with Coulter STKS and

the Coulter GEN-STM System offer the only fail-safe

FIG. 9.12: Coulter MAXM

Clinical Hematology 227

sample management system with positive patient ID and

monitoring of sample integrity both pre and postsampling.

This means peace of mind for you, no reports incorrectly

distributed because of short samples and no mix-up in the

identity of the patient.

Instrument Specifications

Parameters

¾ White blood cell count

¾ Lymphocyte % and #

¾ Monocyte % and #

¾ Neutrophil % and #

¾ Eosinophil % and #

¾ Basophil % and #

¾ Red blood cell count

¾ Hemoglobin concentration

¾ Hematocrit

¾ Mean corpuscular volume

¾ Mean corpuscular hemoglobin

¾ Mean corpuscular hemoglobin concentration

¾ Red cell distribution width

¾ Platelet count

¾ Mean platelet volume.

Throughput

¾ 75 samples per hour

¾ 30 samples per hour for Retics.

Sample Requirements

¾ 185 μL primary mode

¾ 125 μL secondary sample mode

¾ 50 μL predilute mode.

Patient Result Storage

¾ 1,000 sets plus sample analysis screen displays

¾ 5,000 sets plus all sample analysis screen displays for

Retic units.

Barcode Symbology

¾ Code 39

¾ Codabar

¾ Interleaved 2 of 5

¾ Code 128.

0–24 Hours Sample Stability

¾ Near-native state analysis of WBC using four reagents

that are safe to use and discard

¾ Printouts via standard graphics printer with optional

color kit, or single ticket printer.

High Efficiency through Comprehensive Flagging

Instrument-defined suspect abnormalities (User defined

abnormalities).

¾ Definitive flags

¾ High and low laboratory action limits

¾ RBC morphology Gradient.

DEVELOPMENT OF BLOOD CELLS AND SITES OF

BLOOD FORMATION

Normal Sites

Fetus: Less than 2 months—yolk sac. From 2–7 months:

Liver, with minimal hemopoiesis in spleen.

After 3 months: Hemopoiesis starts in bone marrow.

Full-term infant: Bone marrow is the only site for

production of granulocytes and monocytes. Occurs mainly

in the spleen, lymph nodes and other lymphoid tissues,

though liver and bone marrow produce these in much less

numbers.

After birth: Same as above except that the monocytes

are provided by the bone marrow, spleen and lymphoid

tissues contribute minimally.

Abnormal Sites

Extramedullary hemopoiesis (myeloid metaplasia): In

certain disorders the fetal, organs revert to their old

function supported by the reticulum cells, which retain

their potential hemopoietic activity. This occurs when

bone marrow cannot any further fulfil the requirements or

demand imposed upon it, e.g. in:

¾ Growing children with hemolysis

¾ Myelosclerosis

¾ Secondary carcinoma of the bone.

Development of Blood Cells (Flow chart 9.1)

Blood formation has to undergo three stages:

1. Multiplication of precursor cells (1% of all marrow cells

are in dividing phase).

2. Gradual maturation (both structural and functional).

3. Release into the peripheral circulation. The exact

release mechanism is ill understood, granulocytes

achieve this by their motility and RBCs by diapedesis.

Erythropoiesis (Fig. 9.13)

Erythroblast is a nucleated red cell.

Normoblast implies normal (reaction) erythropoiesis.

Normoblastic maturation involves:

• Reduction in cell size

• Ripening of cytoplasm, i.e. hemoglobinization.

Maturation time from pronormoblast to RBC is 7 days.

Mitotic division occurs till the intermediate normoblast

stage.

228 Concise Book of Medical Laboratory Technology: Methods and Interpretations Pronormoblast: 12–20 μm, large nucleus surrounded by a

rim of deep basophilic cytoplasm and has a perinuclear

halo. Nucleus is round and has several nucleoli.

Early normoblast: 10–16 μm, nucleus still large, chromatin

coarser and deeply staining nucleoli disappear.

Intermediate normoblast: 8–14 μm, nucleus smaller,

hemoglobinization commences, cytoplasm takes an

acidophilic tint, chromatin becomes coarser and very

deeply staining.

Late normoblast: 8–10 μm, cytoplasm is acidophilic,

nucleus becomes much smaller, later it becomes pyknotic

and is eccentrically placed, ultimately it is lost by extrusion.

Reticulocyte: Flat, non-nucleated, disc shaped, slightly larger

than mature RBC. It shows diffuse pale basophilia, which

appears in the form of a reticulum with supravital stains

(brilliant cresyl blue or new methylene blue). In 1–2 days, it

loses its basophilia and becomes a mature erythrocyte.

Control of erythropoiesis: Erythropoietin (formed in kidneys)

is released in response to lowered tissue oxygen tension.

Erythropoietin is a glycoprotein and stimulates

primitive cell differentiation to pronormoblasts. It

affects the rate of multiplication and maturation. It acts

up to early normoblast stage and also affects the rate of

hemoglobinization.

FLOW CHART 9.1: Development of blood cells

Erythropoietin levels are reduced in:

¾ Acute starvation

¾ Hypophysectomy

¾ Transfusion-induced polycythemia.

Erythropoietin levels are increased in:

¾ All anemias except those of renal origin

¾ Aplastic anemia

¾ Polycythemia.

Leukopoiesis (Fig. 9.14)

The Myeloid Series

Specific granules are developed at the myelocyte stage,

which determine the nature of the mature cell.

Development of a Mature Neutrophil

Maturation involves:

1. Development of specific granules

2. Loss of basophilia of the cytoplasm

3. Nuclear ripening till the segmented stage

4. Ability to be motile and to phagocytose (Mitotic

division occurs till the myelocyte stage only).

Myeloblast: 15–20 μm has a large round or oval nucleus,

evenly stained chromatin in strands or granules with

reticular appearance, 1–6 nucleoli. The cell is peroxidase

negative.

Clinical Hematology 229

Promyelocyte: It is like myeloblast except that it contains

azurophilic granules, which are peroxidase positive.

Nuclear chromatin becomes condenser and nucleoli are

less well defined.

Myelocyte: Specific neutrophilic granules appear, nucleus

shows no nucleoli. N:C ratio reduces, cytoplasm is pale

pink, chromatin thicker and deeply stained.

Metamyelocyte: Nucleus is smaller and indented,

cytoplasm is pink with neutrophilic granules (purplish).

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

13. Salpingitis, appendicitis (often normal), due to absorption of purulent necrotic material. FIG. 9.9: Wintrobe’s ESR tube with stand 222 Concise Book of Medical Laboratory Technology: Methods and Interpretations 14. Infected, necrotic or malignant tumors. 15. Liver disease (depends upon blood proteins). 16. Menstruation (slight acceleration). Slow ESR is Usually Seen in

 

Normal Values

Males—0 to 9 mm at the end of 1st hour. Females—0 to

20 mm at the end of 1st hour.

Microsedimentation (Landau) Method

Capillary blood can be taken.

Materials Required

1. 5.0 g/dL sodium citrate solution.

2. Landau pipette: This pipette resembles RBC pipette.

It is graduated from 0 to 50 mm.

FIG. 9.8: Westergren’s ESR pipette with stand

Contd...

Normal range, unit SI units

16 years 2800/µL or mm3 2800 × 106

/L

18 years 2700/µL or mm3 2700 × 106

/L

20 years 2500/µL or mm3 2500 × 106

/L

Platelets

Adults 150,000–400,000/ 150–400 × 109

/L

µL or mm3

Panic/Levels <30,000/µL or mm3 <30 × 109

/L

>l,000,000/µL or mm3 >1000 × 109

/L

Children

Newborn 100,000–300,000/

µL or mm3

100–300 × 109

/L

3 months 260,000/µL or mm3 260 × 109

/L

1–10 years 250,000/µL or mm3 250 × 109

/L

Panic/Levels <20,000/µL or mm3 <20 × 109

/L

> 1,000,000/µL or mm3 >1000 × 109

/L

Clinical Hematology 221

3. Landau pipette stand.

4. Suction device for drawing blood into the pipette.

5. Capillaries for blood collection.

Procedure

1. Attach Landau pipette to the suction device.

2. Draw 5.0 g/dL citrate up to first line on the stem.

3. Now draw blood by suction device up to second mark

on the stem (avoid air bubbles).

4. Wipe excess blood on the external side of the pipette.

5. Draw citrate solution and blood into the bulb of the

pipette. Mix the contents thoroughly.

6. Force back the mixture into the stem of the pipette.

7. Set the upper level of the mixture of the zero mm mark

at the top.

8. Detach the suction device.

9. Place the pipette in vertical position on the stand, set

time to one hour.

10. Note the reading (the distance red cells have fallen or

the extent of plasma column) after one hour.

Normal Values

Male: 0–5 mm after Ist hour. Female: 0–8 mm after Ist hour.

Zeta Sedimentation Rate (ZSR)

The zeta sedimentation rate (ZSR) is performed using a

special small-bore capillary tube that is filled with blood

and span for 3–4 minutes in a centrifuge called Zetafuge

(Beckman Coulter). This centrifuge alternately compacts

and disposes the RBCs under standardized centrifugal

force. The tube is then read on a special reader to obtain a

value called the Zetacrit, which represents the percentage

of sedimented erythrocytes. The Zetacrit value is divided

into the hematocrit value (also a percentage) and the

result in the ZSR expressed as percentage.

The ZSR’s advantages are that it is rapid, correct for

anemia, and requires only a small blood sample which

is desirable for pediatric patients. However, a special

centrifuge and reader are needed to perform the test.

Many other automated systems/devices are also

available.

Sources of Error for any ESR Method

1. Improper anticoagulant.

2. Tube not vertical, an inclination of 3° raises ESR by

almost 30%.

3. Dirty tube.

4. Bubbles caused by too vigorous mixing.

5. Hemolysis may modify ESR.

6. Prolonged storage of blood after withdrawing it, the

test should be performed within 3 hours.

7. Pipette/tube kept on a vibrating surface (vibration

prevents rouleaux formation).

Interpretation of ESR

The value of ESR is that it indicates the possible presence

of organic disease, or to follow the course of disease. Its

main use is as a prognostic tool. It is used as a diagnostic

criterion (minor) in rheumatic fever only.

Rapid ESR is Found in

1. In any chronic infection, e.g. tuberculosis (maximum

in miliary tuberculosis), has prognostic value.

2. Any extensive inflammation, cell destruction or

toxemia.

3. Pregnancy, after the second month.

4. Puerperium, returns to normal within 2 months.

5. Active myocardial infarction (rapid rise).

6. Acute myocardial infarction (rapid rise).

7. Active rheumatoid arthritis (not much elevated in

osteoarthritis).

8. Nephrosis (low blood albumin, anemia).

9. All types of shock.

10. Active syphilis (moderate acceleration).

11. Postoperative states (for variable periods).

12. Any active infectious disease, acute or chronic.

13. Salpingitis, appendicitis (often normal), due to

absorption of purulent necrotic material.

FIG. 9.9: Wintrobe’s ESR tube with stand

222 Concise Book of Medical Laboratory Technology: Methods and Interpretations 14. Infected, necrotic or malignant tumors.

15. Liver disease (depends upon blood proteins).

16. Menstruation (slight acceleration).

Slow ESR is Usually Seen in

1. Newborn infants.

2. Polycythemia.

3. Congestive heart failure.

4. Allergic states.

5. Sickle cell anemia (poikilocytosis).

Factors that Play a Role in ESR

1. Plasma Factors

¾ An accelerated ESR is favored by elevated levels of

fibrinogen, and to a lesser extent, of globulins (a and b

globulins are more effective than g globulin)