¾ The correct use of the formula to compute the INR.

¾ Uniform understanding of the INR system by clinicians

as well as laboratorians.

Patient Variables in PT/INR Testing

There are many factors that can influence the results of the

PT/INR tests so that they do not reflect the patient’s usual

coagulation state. Coagulation tests are susceptible to

errors introduced by suboptimal specimen quality because

of a number of factors such as blood collection technique,

labile state of several coagulation proteins, and laboratory

transportation factors. In order to get acceptable accuracy

it is important to understand and control these factors as

much as possible.

Factors that Influence Coagulation Test Results

Age and Gender

Age specific reference ranges are critical for correct

interpretation of coagulation data. Bleeding time declines

with age and many coagulation factors increase with age

as do markers of coagulation activation. Age and gender

can also influence platelet function. Females tend to have

longer bleeding times than males.

Blood Type

Type O individuals have significantly lower von

Willebrand factor and factor VIII activity than subjects

with type A, B, or AB. This causes increased bleeding and

dotting times.

Within Day Variation

Incidences of platelet activation are highest in the

mornings, resulting in increased coagulation activation.

Seasonal Variation

Increased coagulation activity has been described in cold

weather.

Intraindividual Variability

Many coagulation analytes are less precise than other

analytes and thus can give variable results within the same

individual.

Diet, Alcohol and Smoking

Cardiac risk factors can increase coagulation factor level/

activation. Smoking elevates plasma fibrinogen. Von

Willebrand factor, thrombin generation and platelet

activation may all have an effect causing variability.

Moderate ethanol intake inhibits platelet reactivity and

increases fibrinolysis and INR.

Medications

A number of other medications, including hormone

replacement therapy, selective estrogen receptor,

modifiers and oral contraceptives can alter coagulation

and raise the INR. In addition, non-steroidal antiinflammatory drugs, antibiotics and fluoroquinolones

can also alter the INR.

Menstrual Cycle, Pregnancy

Significant hormonally determined changes in coagulation

factors, inhibitors, fibrinolysis and activation markers

must be considered as interpretation of the results.

Diseases

States, which lead to anemia, polycythemia or hemolysis

or uremia, can also interfere with coagulation tests.

Physical and Emotional Stress

These are commonly associated with increased coagulation

and platelet activation.

Clinical Hematology: Bleeding Disorders 293

Posture

Values can change from supine to upright positions

due to the shift of water and subsequent reduction in

plasma volume. Hence, standardization of posture is

recommended.

Venous Occlusion

Traumatic or prolonged phlebotomy accentuates the

hemostatic activation, producing artificially altered

coagulation times.

Vitamin K

Certain fat substitutes in some snackitems contain

unspecified amount of vitamin K. Green, leafy vegetables

and green tea also contain high levels of vitamin K. This

can have an impact on serum vitamin K levels and the INR

can drop as a result. Alternative medicines: According to

the AANA (American Association of Nurse Anesthetists)

some sources, certain herbal drugs can cause interference

in coagulation cycles, falsely elevating the INR.

Anticoagulant Therapy

It is of utmost importance to bear in mind that patients on

heparin will show inaccurate INR results.

While certain pre-analytical factors are not entirely

controllable, every effort must be made to ensure that most

conditions have been stable for a period of time. Patient

preparation and blood collection should be standardized

according to the guidelines.

Prothrombin Determination (Two-stage Method)

Principle

Prothrombin in the presence of optimal procoagulants

and calcium will form thrombin. The amount of thrombin

formed can be calculated by determining the dilution of

plasma that will clot a standard fibrinogen reagent in a

specific period of time. The amount of thrombin formed

is a measure of the amount of prothrombin present in the

starting sample.

The test consists of two stages. In the first stage,

prothrombin is incubated with a standard mixture

containing thromboplastin, calcium, a buffer and a source

of procoagulants. In the second stage, samples of the

incubating mixture are added to a standard fibrinogen

solution and the clotting time is determined.

Results

1. The object of the procedure is to determine the dilution

of plasma from which will evolve one unit of thrombin

under optimal conditions. A unit of thrombin is defined

as that amount which will form a clot of 1 mL of fibrinogen in 15 seconds under standard conditions.

2. If varying amounts of thrombin are added to standard

amounts of fibrinogen the clotting time of the mixture

is an index of the thrombin concentration within a

specific range. When thrombin concentrations are

plotted against clotting times, the results describe

a hyperbolic curve. With thrombin concentrations

between 0.80 and 1.34 units, there is good correlation

between thrombin concentration and clotting time.

With greater amounts of thrombin, there is little

change in the speed of clotting, with relatively large

changes in thrombin concentration. With lesser

amounts of thrombin, small changes in thrombin

concentration result in large changes in the speed of

clotting.

APTT/PTTK CEPHALOPLASTIN REAGENT FOR

PARTIAL THROMBOPLASTIN TIME (APTT)

DETERMINATION USING ELLAGIC ACID AS

ACTIVATOR LIQUICELIN-E®

(Courtesy: Tulip Group of Companies)

Summary

The arrest of bleeding depends upon primary platelet plug

formed along with the formation of a stable fibrin clot.

Formation of this clot involves the sequential interaction of

a series of plasma proteins in a highly ordered and complex

manner and also the interaction of these complexes with

blood platelets and materials released from the tissues.

Activated partial thromboplastin time is prolonged by a

deficiency of coagulation factors of the intrinsic pathway of

the human coagulation mechanism such as factor XII, XI,

IX, VIII, X, V, II and fibrinogen. Determination of APTT helps

in estimating abnormality in most of the clotting factors of

the intrinsic pathway including congenital deficiency of

factor VIII, IX, XI and XII and is also a sensitive procedure

for generating heparin response curves for monitoring

heparin therapy.