glass tubes. They should be scrupulously clean and dry. • The containers should be ideally made out of plastic and not from glass as scratched glass surfaces can activate in vitro the coagulation mechanism within the sample due to contact with silica. While plastic tubes overcome this problem they should be free from leavening chemicals used by the plastic

 

3. Prolonged coagulation time will be noted in afibrinogenemia and marked hyperheparinemia.

Interfering Factors

1. Quality of venipuncture: The venipuncture must be

carefully done because either tissue thromboplastin

obtained as a contaminant when the venipuncture is

done, or hemolyzed red blood cells suctioned when the

blood is drawn, can cause a marked shortening of the

coagulation time. The time required for a severe hemophiliac’s blood to clot can be shortened from 1 hour to

a normal value when a poor venipuncture is done.

Clinical Hematology: Bleeding Disorders 277

2. Type of test tube: The coagulation time will be

lengthened to 20 to 40 minutes if plastic or silicone

coated test tubes are used.

3. Drugs: Increased coagulation time may be seen with:

• Mithramycin

• Tetracyclines

• Anticoagulants

• Azathioprine

• Carbenicillin.

 Decreased coagulation time may be seen with:

• Corticosteroids

• Epinephrine.

Clot Retraction

Principle

When whole blood is allowed to clot spontaneously, the

initial coagulum is composed of all elements of the blood.

With time the coagulum reduces in mass and fluid serum is

expressed from the clot. This is due to an action of platelets

on the fibrin network.

Requirements

¾ Equipment for collecting blood

¾ Clean, dry plain glass graduated centrifuge tube

¾ Timer

¾ Water bath 37°C.

Method

1. 5 mL blood is obtained with a standard two-syringe

technique and transferred to the centrifuge tube.

2. Incubate it at 37°C in vertical position.

3. Record degree of retraction after 1, 2, and 4 hours. It

may be necessary to loosen the clot gently from the

wall of the test tube if contraction is not apparent at

the end of 1 hour. The degree and rate of retraction

should be noted. Note also any digestion of clot or

discoloration of serum.

Clot retraction is directly related to platelet count,

hence, it is impaired in thrombocytopenia, but is normal in

hemophilia. In the method just described, one can remove

the clot by using a hooked long needle and the volume of

serum left behind can be measured. The percentage of clot

can be calculated from the initial 5 mL of blood taken. In

normal individuals, the clot percentage is about 50% at the

end of one hour of the original blood volume taken.

Interpretation

1. Patients with qualitative or quantitative platelet disorders have samples with scant serum and a soft,

plump, poorly demarcated clot.

2. The clot is small and serum voluminous if the patient

has a low hematocrit.

3. Patients with polycythemia have poor clot retraction

because the large numbers of captured red cells

separate fibrin stands and interfere with platelet

contraction.

4. If fibrinogen levels are low, the initial clot is so fragile

that the delicate strands rupture and red cells spill out

into the serum when retraction begins.

5. Serum contamination by red cells is especially striking

if fibrinolysis is abnormally brisk, as often happens

with reduced fibrinogen levels. Sometimes in these

cases, the incubated tube contains only cells and

plasma with no fibrin clot at all.

Errors

1. When fibrinogen is reduced in amount, the clot may

be very small and retraction may be interpreted as

normal even though it is inadequate.

2. In the presence of active fibrinolytic activity, the clot

may dissolve.

3. In normal blood the exuded serum will be clear and

free of RBC’s. The presence of significant number of

RBC’s in the serum suggests fibrinolytic activity.

4. With a low hematocrit value, the mass of the clot will

be proportionately small and may give enormously

high values.

Heparin Therapy

Protocols and Blood Coagulation Tests

1. Heparin combines in the blood with an alpha globulin

(heparin cofactor) for a potent antithrombin.

2. The intravenous injection of heparin will give an

immediate anticoagulant effect, so it is used when

rapid effects are desired.

3. Because of heparin not remaining in the blood very

long, the clotting time is measured before each

injection.

4. The coagulation time is ordinarily maintained at two

to two and one half times the normal limit.

5. To evaluate the effect of heparin, the blood is tested

for coagulation time:

• Before therapy is started for baseline

• One hour before the next dose is administered

• Dependent upon the status of patient during heparin

therapy (signs of bleeding).

6. Protamine sulfate is the antidote for heparin overdose

and hemorrhage.

278 Concise Book of Medical Laboratory Technology: Methods and Interpretations QUALITY ASSURANCE FOR ROUTINE

HEMOSTASIS LABORATORY

Introduction

Coagulation tests for the routine assay parameters in

laboratories are fairly simple to perform and master.

The performance of basic tests require simple apparatus

such as water bath, test tubes, pipettes and stop watch

and/or automatic clot timer. Moreover, It is precisely for

this reason that these techniques appear deceptively easy.

There are a number of pretest variables that affect the

accuracy and precision of coagulation results. These may

relate to collection techniques, processing of samples,

selection and preparation of reagents. In order to achieve

optimum and reproducible results the impact of variables

needs to be understood and controlled so as to reduce variability and errors and improve accuracy and reproducibility.

Various variables that affect the results are discussed, along

with the basis that leads to such recommendation.

Preparation of Patients

Although no special preparation of patients is required

prior to approve techniques, it is preferable that patients

are not heavily exercised before blood collection. Fasting

patients or patients on a light non-fatty meal are preferable.

• Patients who are on fasting or on a light non-fatty

meal prior to blood collection provide samples with

desirable lower opacity this improves the sensitivity

of clot detection especially when photo-optical

instruments are being used

• Turbid, icteric, lipemic or grossly hemolyzed samples

generate erroneous results due to varying opacity.

Sample Collection Techniques (Phlebotomy)

1. Blood should be withdrawn without undue venous

stasis and without frothing into a plastic syringe with

a short needle of 19 to 20 SWG.

2. The venipuncture must be a ‘clean’ one and incase of

difficulty with a new syringe and needle another vein

should be tried. The tourniquet should not be placed

too tightly or for extended lengths of time. Patting the

venipuncture site should also be avoided.

3. Distribute blood into test tubes (preferably plastic)

after detaching the needle from the syringe. Do not

delay mixing blood with anticoagulant by gentle

inversion of the tube.

• ‘Clean’ venipuncture is essential to avoid formation

of microclots at the site of venipuncture and

consumption of factors, which will lead to artificially

prolonged results.

• Usage of short bigger bore needle allows free flow of

blood within the syringe and reducing blood contact

with metal surface. With smaller bore longer needles,

blood will remain in contact with metal surface for

longer time. This will lead to initiation of clotting or

partial consumption of factors being assayed leading

to erroneous results during test procedures.

• Frothing when distributing the blood into anticoagulant tube should be avoided because frothing

induces microclot formation.

Sample Preparation

1. The anticoagulant of choice for most coagulation

procedures is sodium citrate or preferably buffered

sodium citrate.

2. Sodium citrate is an ideal anticoagulant since Factor V

and Factor VII are more stable in citrate. These factors

are more labile in sodium oxalate. Heparin neutralizes

action of thrombin on fibrinogen.

3. The recommended molarity of sodium citrate for

coagulation studies is 0.109M, which equates to 3.2%

of tri-sodium citrate.

4. Use of buffered sodium citrate is preferred over plain

sodium citrate solution.

• After collection of blood in citrate during centrifugation for preparation of platelet poor plasma

(PPP) or platelet free plasma (PFP), the pH of

the solution shifts releasing of carbon dioxide

(CO2). This shift in pH affects the labile factor V

leading to erroneous results during test. Use of

appropriately formulated buffered citrate overcomes

this phenomenon.

• When samples are collected in 3.8% citrate (129 mm)

the prothrombin time of samples especially with

patients receiving oral anticoagulants give prolonged

results. Also the ISI of thromboplastins is lowered.

It is for this reason 3.2% citrate is recommended

universally instead of 3.8% for increasing accuracy

of test results.

5. The optimum ratio of citrate to blood is 1 part of

anticoagulant to 9 parts of blood.

• When the molarity of citrate is accurate the anticoagulant supplied in this amount and ratio is

sufficient to bind all the available calcium in the

collected sample so as to prevent clotting. A shift in

this ratio leads to erroneous results as follows:

• More blood less citrate: The chelating activity of

citrate will not be sufficient to bind the calcium

present in the sample. This will lead to formation

Clinical Hematology: Bleeding Disorders 279

of clots, consumption of factors and subsequent

prolongation of results during test

• More citrate less blood: Excess citrate remaining in

the blood sample would consume the calcium from

the reagents thereby giving prolonged results.

6. The optimum concentration of calcium chloride to be

used for APTT test should be 0.02 M:

• The concentration of 0.02 M CaCl2 replaces the

calcium necessary to activate the intrinsic coagulation cascade. This ultimately generates thrombin

from prothrombin via the coagulation cascade.’

• Appropriate volumes of CaCl2 should be aspirated

for the day's work. Prewarmed CaCl2 should always

be discarded at the end of the working day.

7. The standard ratio of blood to anticoagulant of 9:1 is

for normal hematocrit or PCV:

• For occasional patients with PCV less than 20% (e.g.

microcytic hypochromic anemia) and greater than

50% (e.g. polycythemia vera) the anticoagulant to

blood ratio must be readjusted using the following

formula,

 C = 1.85 L × 10-3 (100-H) V

 Where,

C = Volume of sodium citrate in mL

V = Volume of whole blood–sodium citrate in mL

H = Hematocrit in percentage

• When the PCV is higher than 55% the patient blood

contains so little plasma that excess unutilized

anticoagulant remains and is available to bind

reagent calcium to prolongation of test results.

• On the other hand, if the PCV is less than 20 percent

the patient blood contains excess of plasma but less

of anticoagulant and the chelating activity of citrate

will not be sufficient to bind the calcium present in

sample. This will lead to formation of clots in vitro,

consumption of factors and prolongation of results.

Sample Processing and Storage

¾ Containers for collection and processing of plasma

should be ideally made out of plastic or siliconized

glass tubes. They should be scrupulously clean and dry.

• The containers should be ideally made out of plastic

and not from glass as scratched glass surfaces can

activate in vitro the coagulation mechanism within

the sample due to contact with silica. While plastic

tubes overcome this problem they should be free

from leavening chemicals used by the plastic

industry during molding. These chemicals usually

have an inhibitory effect. Scrupulous washing and

drying overcomes this problem.

• All the containers used for collection, storage and test

suggests that the patient’s blood is hypercoagulable. 3. Vigorous agitation of the tubes will significantly shorten the coagulation time. So tipping should really be very gentle just to see if the blood has clotted.

 

¾ Filter paper

¾ Glass slide

¾ Alcohol sponges.

Method

1. Clean the lobe of the ear or tip of a finger with alcohol

and let dry.

2. For ear—glass slide is placed behind the ear lobe

and held firmly in place. This provides a firm site for

incision.

3. Pierce the lobe of the ear by a firm stroke against the

glass slide (or pierce the finger-tip). Discard the glass

slide if ear lobe has been incised. Start the stop watch

when the stab was made.

4. Bleeding of the wound should be allowed to proceed

without pressure and the blood is allowed to drop on

the filter paper. The paper should be moved so that

each drop will fall on a fresh area. When bleeding

slows, the wound is touched gently with a fresh area

of the filter paper at 30 second intervals. When blood

no longer stains the filter paper, the watch is stopped

and the time recorded.

Normal Values

The normal range is up to 6 minutes. Between 6 and 10

minutes, the results are borderline. Over 10 minutes is

definitely abnormal.

Precautions

1. In children, heel should be used.

2. In suspected cases of a bleeding disorder, the bleeding

may not be controlled easily from the ear lobe hence,

fingertip puncture wounds are better.

3. The area to be punctured should not be congested.

4. The size and depth of the wound may vary if one does

not have a standardized technique.

5. If bleeding persists for more than 15 minutes it should

be stopped by placing a dry gauge sponges over the site

and applying finger pressure (the filter paper used to

collect the drops of blood can be dried and saved as a

record of the procedure).

Ivys’s Method

(Preferred because of greater ease of standardization).

Method

1. Cleanse the inner aspect of the forearm with spirit and

let dry.

2. Place a blood pressure cuff on the upper arm, inflate

at 40 mm Hg, and maintain the same throughout the

test.

3. Select an area on the forearm—Volar aspect which is

devoid of superficial veins. Stretch the skin laterally

between the thumb and forefinger and hold in a taut

position.

4. Take a cork, through which a no. 11 surgical blade has

been inserted with the tip extending 3 mm beyond the

cork surface (both cork and blade should have been

sterilized before), the blade should be withdrawn from

the cork and autoclaved before being used again.

5. Hold the cork with the thumb and forefinger of the

free hand, and with the heel of the hand resting on

the patient’s arm, quickly make two skin punctures

(actually they are small incisions) in the selected area.

It is important that the surface of the cork meet the skin

to ensure a 3 mm deep incision. Holding the skin taut

prevents the test area from being depressed when the

blade enters the skin.

6. Timing is begun as soon as the incisions are made and

bleeding starts.

7. Using the edge of a piece of a filter paper to collect the

blood, gently touch paper to the drop of blood, which

Clinical Hematology: Bleeding Disorders 275

forms over the wound every 30 seconds. Do not rub or

remove the clot. Do not touch the skin. Any disruption

of formed fibrin or clot will prolong the bleeding time.

8. The bleeding time is reported when no blood stain

is seen on the filter paper after a gentle touch. It is

reported in intervals of 30 seconds. One can measure

both wounds and average them, or take the reading of

the last one to stop bleeding.

Normal Values

Normal values are 1 to 6 minutes. More than 6 minutes

should be taken as abnormal.

Interpretation

1. Results of duplicate tests performed on the same

individual should agree within 2 to 3 minutes at most.

2. Bleeding time is prolonged:

• When platelet count < 100,000/mm3

• In patients on aspirin therapy.

• In acquired fibrinogen disorders.

(If the platelets are young even in a thrombocytopenia

patient, the bleeding time may not be raised as young

platelets have enhanced hemostatic capabilities).

When platelet counts are low, one can calculate the

expected bleeding time with the following formula:

Bleeding time =

Platelet count/cu mm

30 5

3850

. ×

A bleeding time longer than that calculated from platelet

numbers alone, suggests defective platelet function in

addition to reduced number. It is also possible to detect

above-normal hemostatic capacity in cases in which

active young platelets comprise the entire population of

circulating platelets.

Clinical Implications

1. Bleeding time is prolonged when the level of platelets

is decreased or when the platelets are qualitatively

abnormal, as in

a. Thrombocytopenia

b. Platelet dysfunction syndromes

c. Decrease or abnormality in plasma factors such as

von Willebrand’s factor and fibrinogen

d. Abnormalities in walls of the small blood vessels—

vascular defects

e. Severe liver disease

f. Leukemia

g. Aplastic anemia

h. DIC disease.

2. Bleeding time can be either normal or prolonged

in von Willebrand’s disease. It will definitely be

prolonged if aspirin is administered prior to testing.

3. A single prolonged bleeding time does not prove the

existence of hemorrhagic disease because a larger

vessel may have been punctured. The puncture should

be done twice (on the contralateral side) and the

average of the bleeding times can be taken.

Interfering Factors

1. The normal range may vary when the puncture is not

of standard depth and width.

2. Touching the incision during the test will break off any

fibrin particles and prolong the bleeding time.

3. Heavy alcohol consumption (as in alcoholics) may

cause bleeding time to be increased.

4. Prolonged bleeding time will result from the ingestion

of 10 g of aspirin up to 5 days before the test.

5. Other drugs that may cause the bleeding time to be

increased include:

• Dextran

• Streptokinase—streptodornase

• Mithramycin

• Pantothenyl alcohol.

Patient Preparation

1. Explain the purpose and procedure of the test to

patient.

2. Warn patient not to consume aspirin for 5 days prior

to test.

3. Advise patient not to consume alcohol in any form.

Coagulation Time

Capillary Tube Method of Wright

Blood is collected in about a dozen capillary tubes from

a finger prick made after aseptic precautions. The tubes

are sealed with plasticine and immersed in water bath at

37o

C. After 4 minutes, remove the first tube from the bath

and expel the blood in it with one end immersed in a dish

containing water. Repeat this every 30 seconds with the

other tubes till the blood is expelled in a worm clot and note

the time.

An alternative way of determining the end point is to

break the capillary tubes every 30 seconds until a clot is

seen between the two broken ends. By these methods, the

normal clotting time is 5 to 10 minutes at 37oC and longer

if performed at room temperature. This test should be

avoided as tissue thromboplastin contaminates the oozing

blood and hence, false reports may be obtained.

Lee and White’s Method

Principle: Whole blood, when removed from the vascular

system and exposed to a foreign surface, will form a solid

276 Concise Book of Medical Laboratory Technology: Methods and Interpretations clot. Within limits, the time required for the formation of

the solid clot is a measure of the coagulation system.

Requirements

1. Stop watch

2. Equipment for collection of blood

3. Clean, dry glass test tubes (10 × 75 mm)

4. Water bath (37°C).

Method

1. Make a clean venipuncture with as little trauma to

(or time spent passing through) the connective tissue

between skin and vein as possible. One may routinely

or in selected cases use the two-syringe technique,

whereby one rinses the needle of all interstitial tissue

fluid by drawing back 1 cc. of blood after entering the

vein. Then remove the first syringe from the needle

and quickly place on a second clean and dry syringe

and draw back blood for the test.

2. Timing is begun when the blood first enters the syringe.

The second syringe in the ‘Two-syringe’ technique.

3. Draw 3–5 mL of blood and withdraw the syringe and

needle. Disconnect the needle. Place approximately

1 mL of blood in each of three (10 × 75 mm) test tubes.

4. Place the tubes in a stand so that they remain upright

and undisturbed, at room temperature for 10 minutes.

If a 37oC water bath is available one may do the entire

test at 37oC, and shorter clotting times will be found

(if the test has been done at 37oC, do not wait for more

than 5 minutes).

5. After 10 minutes (or 5 minutes) take the first of the

tubes and gently tip it every 30 seconds to test for

clotting. Do not tip it further than necessary to get the

information.

6. When the first tube is clotted (can be inverted without

blood running down the edge of the tube), record the

time and start the tipping of the second tube every 30

seconds until it is also found to be clotted. Then do

the same with the third tube (tipping is intended to

allow one to ascertain when blood is clotted—not as

a means of hastening clotting or of assuring mixing of

the blood).

7. The time recorded for the clotting of the third tube is

taken as the clotting time (the purpose of the first two

tubes is to tell one when to start looking in the third

tube, since the agitation of tipping does hasten the

clotting).

 Some choose to tip the tubes in rotation (at 37oC) every

15 seconds, or tip all tubes at once, and average the

results of the three tubes.

Normal Values

Normal times depend on method used. Normal range

at 37oC is usually 5 to 10 minutes. Normal times at room

temperature will vary with the degree of temperature

present and the method used. If one uses the method

which waits 10 minutes before starting to tip, then normal

values may go as high as 22 to 25 minutes, especially in

the cool season. Values shorter than 10 minutes should

be suspected and the test repeated using the two-syringe

technique to rule out contamination by tissue fluid (in the

heat of April, May or June warm tropical climate blood will

clot before 10 minutes without having been contaminated

by tissue fluids). If one uses the method which waits 5

minutes before tipping begins, normal results are between

8 to 18 minutes. Longer than 20 minutes is abnormal. If

clotting occurs in less than 7 minutes, the test should be

repeated using two-syringe technique.

Precautions and Errors

1. The venipuncture must be without trauma to avoid

contamination with tissue thromboplastin.

2. If all three tubes are clotted at 10 minutes (or 5

minutes) when one starts to tip the first tube, the test

is unsatisfactory and should be repeated. If blood was

drawn by single syringe technique, the most likely

explanation is contamination, of the blood by tissue

thromboplastin. If a two-syringe technique is used, it

suggests that the patient’s blood is hypercoagulable.

3. Vigorous agitation of the tubes will significantly

shorten the coagulation time. So tipping should really

be very gentle just to see if the blood has clotted.

Clinical Implications

1. Severe deficiencies of any of the coagulation factors

must be present before the coagulation time will

be prolonged. Fibrinogen for example, needs to

be decreased to 50 mg/100 mL or less before the

coagulation time is affected, the normal range of

fibrinogen is 200 to 400 mg/100 mL.

2. When prothrombin is diminished to a level of 30% of

normal, there will be a small change in coagulation

time.

2. Hyperglobulinemia: Interferes with platelet adherence, release and aggregation. 3. Myeloproliferative disorders: Intrinsic abnormalities of platelet function may occur in patients with essential thrombocythemia and other myeloproliferative disorders.

 

Cumulative Sum (Cusum) Charting was introduced in

1960s. Deviation from the largest is plotted in a cumulative

manner so that each point represents the sum of all the

deviations to date from the mean or target value. This

method of plotting exaggerates trends in data and makes

shifts of the mean much more obvious than by other plots.

The rules for using the Cusum system for quality control

are less well defined than for the L-J system.

Duplicate Tests

A well-known method for checking precision in clinical

analysis is duplicate testing. In this process, a few of the

specimens that were measured in an earlier batch, are

rechecked with the next batch control.

Inbuilt Quality Control

This includes:

¾ These of cumulative reports of a single patient

¾ Clinical correlation: If a physician can not interpret a

report on clinical grounds, a repeat test with a fresh

specimen is indicated

¾ Red cell indices: If reports are giving erroneous rise

or fall in the red cells indices, this usually points to an

error in analysis

¾ Blood film examination ultimately helps in double

checking the analysis done by the instrument.

External Quality Assessment

The college of American Pathologist first introduced

“proficiency testing” survey program in 1960. In the late

60s, the British Committee for Standards in Hematology,

finallydevelopedtheNationalExternalQualityAssessment

Clinical Hematology 271

Scheme (NEQAS) for Hematology. Such methods are used

by various laboratories all over the world to keep up with

international standards.

Standardization

Modern diagnostic systems depends on a calibration

procedure for accurate performance. Calibrators or testing

standards are commercially prepared products, made by a

direct comparison with a primary international standard.

They are used for accuracy and interlaboratory harmonization

of test results. The calibrator has an assigned value as close to

the true value as can be established.

The WHO (World Health Organization) provides a wide

range of biologically important international reference

standard material. Some examples of these which are

available for use in hematology are:

a. Hemoglobin preparation

b. Hemoglobin A2 and F

c. Thromboplastin

d. Blood type sera

e. Various coagulation factors.

Proficiency Surveillance

This is concerned with the pre-analytical parts of the

process that require control, if tests are to be reliable and

effective. This involves following a standard guideline at

various steps of a laboratory analysis.

The steps are:

1. Standard of blood collection tube

2. Phlebotomy technique

3. Identification of sample with special reference to

hazardous specimens

4. Maintenance of transportation standards

5. Data processing of results

6. Establishing normal reference values, assessment of

the significance of results and taking decisions for

further tests.

Technical Proficiency has always been the corner stone

of the laboratory, but in recent years with the advent

of sophisticated instruments and automation, quality

control has assumed an even more important role in good

laboratory practice. It is the duty of the laboratory staff to

ensure that the tests, which are carried out, are appropriate

and to provide reliable analytical results.

10

Clinical Hematology:

Bleeding Disorders

C H A P T E R

PLATELETS, COAGULATION AND BLEEDING

DISORDERS: LABORATORY INVESTIGATIONS

Platelet Count—Dealt in Depth Elsewhere

Capillary Fragility Test of Hess

(Rumpel-Leede Sign, Tourniquet Test)

1. Inflate sphygmomanometer cuff around arm at 80 mm

of Hg pressure for 5 minutes.

2. Look for petechiae in an area 5 cm in diameter just

below the elbow.

3. Under normal circumstances the number of petechiae

should be less than 5, more than 5 indicate a positive

test.

A positive test may be found in reduced capillary

resistance (or increased capillary fragility) as in nonthrombocytopenic purpura and scurvy. It may also be

positive in thrombocytopenia when the platelet count is

below approximately 70,000 mm3

 of blood.

Clinical Implications

1. Increased petechiae formation occurs most commonly

in thrombocytopenia and less commonly in:

(i) thrombasthenia, (ii) vascular purpura, (iii) senile

purpura, and (iv) scurvy.

2. The number and size of petechiae are roughly

proportional to the bleeding tendency and possibly to

the degree of thrombocytopenia. However, the test can

be positive because of capillary fragility in the presence

of normal platelet count.

3. Results will be normal in coagulation disorders and

vascular disorders.

Laboratory Diagnosis of Vascular Bleeding Disorders

Hess’s test is positive in these

Causes and classification

1. Hereditary

• Hereditary hemorrhagic telangiectasia.

2. Acquired

• Simple easy bruising

• Senile purpura

• Purpura of infections

• Henoch-Schonlein syndrome

• Scurvy

• Steroid purpura.

Interfering Factors

1. Menstruation: Capillary fragility is normally increased

before menstruation.

2. Infectious disease: Capillary fragility is increased in

measles and influenza.

3. Age: Women over 40 years with decreasing estrogen

levels may have a positive test that is not indicative of

a coagulation disorder.

4. Readministration: Repetition of test on same arm

within 1 week of the first test may lead to error.

5. Variation: Results may vary because of dif­ferences in

texture, thickness, and temperature of the skin.

LABORATORY DIAGNOSIS OF PLATELET

DISORDERS

Idiopathic Thrombocytopenic Purpura (ITP)

1. Platelet count is usually 10-50 × 109

/L.

Clinical Hematology: Bleeding Disorders 273

2. The blood film shows reduced numbers of platelets,

those present are often large.

3. The bone marrow usually shows increased number of

megakaryocytes.

4. Sensitive tests can demonstrate antiplatelet IgG, either

alone or with complement, on the platelet surface or

in the serum in most patients.

5. Autologous platelet survival studies with 51Cr or DF32Plabeled platelets may be used to show reduced platelet

survival. In severe cases, the mean platelet survival

may be reduced to one hour.

6. Hess’s test may be positive in some cases.

Drug Induced Immune Thrombocytopenia

1. Thrombocytopenia. Platelet count is often <14 × 109

/L.

2. Bone marrow may show normal or increased numbers

of megakaryocytes.

3. Drug dependent antibodies against platelets may be

demonstrated in sera of some patients.

 Drugs usually incriminated are:

• Quinine

• Quinidine

• Sulfonamides

• PAS

• Rifampicin

• Stibophen

• Digitoxin, etc.

Disseminated Intravascular Coagulation (DIC)

1. In acute cases blood may not clot due to gross

fibrinogen deficiency.

2. Platelet count is low.

3. Fibrinogen screening tests, titers or assays indicate

deficiency.

4. Thrombin time is prolonged.

5. High levels of serum fibrin/fibrinogen degradation

products are found in serum and urine.

6. Prothrombin time and partial thromboplastin time are

prolonged.

7. Factor V and factor VIII activity is diminished.

8. Due to microthrombi causing mechanical hemolytic

anemia, RBCs may show crenation and poikilocytosis.

Causes of DIC

1. DIC may be caused by entry of procoagulant material

into circulation, for example,

• Amniotic fluid embolism

• Premature placental separation

• Widespread mucin secreting adenocarcinoma

• Severe falciparum malaria

• Hemolytic transfusion reaction

• Promyelocytic leukemia

• Some snake bites.

2. DIC may also be initiated by extensive endothelial

damage and collagen exposure, for example

• Endotoxemia

• Gram-negative and meningococcal septicemia

• Septic abortion

• Certain viral infections (purpura fulminans)

• Severe burns

• Hypothermia.

3. Massive intravascular platelet aggregation can also

precipitate DIC as occurs in some:

• Bacterial and viral infections

• Immune complexes may have a direct effect on

platelets.

Functional Platelet Disorders

Platelet reactions in the hemostatic process.

Laboratory Diagnosis

1. Platelet count normal.

2. Prolonged bleeding time.

3. Abnormal platelet aggregation studies with ADP,

adrenaline, collagen and ristocetin.

4. Abnormal adhesion studies and nucleotide pool

measurement.

5. Factor VIII clotting assay (for von-Willebrand’s disease).

Abnormal platelet function should be suspected in cases

where bleeding is prolonged despite a normal platelet

count. Various causes included in this are as follows:

Hereditary Disorders

1. Platelet storage pool disease: There is defective release

274 Concise Book of Medical Laboratory Technology: Methods and Interpretations of ADP and 5HT due to an intrinsic deficiency in the

number of dense granules.

2. Thrombasthenia (Glanzmann’s disease): There is

failure of primary platelet aggregation.

3. Bernard-Soulier syndrome: Platelets are larger than

normal, lack surface glycoprotein and fail to make

phospholipid available or to adhere to vessel walls.

4. von Willebrand’s disease: There is defective platelet

adhesion as well as coagulation factor VIII deficiency.

Acquired Disorders

1. Aspirin therapy: It may lead to abnormal bleeding time

although purpura is rare. Aspirin leads to impaired

thromboxane-A2 synthesis. So, there is failure of the

release action aggregation with ADP and adrenaline.

2. Hyperglobulinemia: Interferes with platelet adherence,

release and aggregation.

3. Myeloproliferative disorders: Intrinsic abnormalities of

platelet function may occur in patients with essential

thrombocythemia and other myeloproliferative

disorders.

Bleeding Time

The duration of bleeding from a standard puncture wound

of the skin is a measure of the function of platelets as well

as the integrity of the vessel wall.

Duke’s Method

Requirements

¾ Stop watch

¾ Lancet