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