THROMBIN TIME (TT) TEST

Thrombin time (TT) test
The TT test is sensitive to a deficiency of fibrinogen or inhibition of thrombin. It measures the formation of a fibrin clot by the action of thrombin on fibrinogen.

Principle of test
Thrombin is added to citrated plasma at 37°C. The time taken for the mixture to clot is measured and the appearance of the
clot noted.

Reagent
The use of a thrombin time test kit is recommended.
This provides the correct concentration of thrombin to use in the test, i.e. that which gives a clotting time of 12–15 seconds with pooled normal plasma.

Collect venous blood into citrate
anticoagulant and centrifuge to obtain platelet poor plasma as described for the APTT test. Perform the test with as little delay as possible.

See also:

Procedure for TT test

PROTHROMBIN TIME (PT) TEST


The PT is a screening test for the extrinsic clotting system, i.e. factor VII. It will also detect deficiencies of factors, prothrombin, V, X, and fibrinogen. It is mainly used to monitor patients receiving warfarin
anticoagulation.

Principle of the test

Plasma or capillary blood is added to a thromboplastin and calcium chloride reagent at 37°C and the time taken for a clot
to form is measured. The clotting time in seconds is converted to the International Normalized Ratio (INR), usually by reference to a table provided by the manufacturer of the reagent OR by the formula:
INR= (PT PATIENT/PT CONTROL)^ISI
ISI is provided by reagent manufacturer

Reagents

Thromboplastin calcium chloride combined reagent.
Several different thromboplastin calcium combined reagents are available depending on the source of the thromboplastin and whether the test is to be performed using plasma or capillary whole blood. Some manufacturers supply a thromboplastin calcium reagent that can be used with both capillary blood and plasma.

Capillary blood PT testing:

This avoids the need to collect venous blood when monitoring patients being treated with warfarin. It should not be used
however when a patient is anaemic or polycythaemic. Plasma should then be used.

See also

Prothrombin time test procedure

PROTHROMBIN TIME (PT) TEST PROCEDURE

  1. Pipette 0.25 ml of the thromboplastin/calcium reagent into a small glass tube. Place in a 37°C water bath for 1–2 minutes.
  2. Using a calibrated capillary or delivery pipette, add (0.05 ml) of capillary blood or plasma, mix, and start the stop-watch. Hold the tube in the water bath and tilt the mixture back and forth looking for clot formation. When a clot forms, stop the stop-watch and record the time
  3. Convert the clotting time to the INR using the table provided by the manufacturer. Separate INR tables are provided for capillary blood and plasma.

The INR conversion table provided by the manufacturer is specific for The batch of thromboplastin supplied with it.

What is the normal range for Prothrombin time?

11 – 16 seconds

What are Causes of Prolonged PT?

  • Treatment with oral anticoagulant drugs (vitamin K antagonists) such as warfarin.
  • Liver disease
  • DIC
  • Haemolytic disease of the newborn
  • Rarely a deficiency of factor VII, X, V, or prothrombin.

See also

The principle of prothrombin time test

DISORDERS OF BLOOD COAGULATION

Can result in uncontrolled haemorrhaging into joints, muscles and deep tissues with the formation of haematoma. Coagulation disorders may be:

Hereditary

  • haemophilia A due to a deficiency of clotting factor VIII (commonest hereditary coagulation disorder),
  • haemophilia B (Christmas disease) due to a deficiency of factor IX
  • Von Willebrand’s disease caused by a deficiency or abnormality of the von Willebrand factor resulting in a defect in platelet adhesion. Haemophilia is carried by the female and affects males.

Acquired

  • Vitamin K deficiency
  • Severe liver disease, and disseminated intravascular haemolysis (DIC) associated with infections.
  • Obstetric complications (septic abortion, eclampsia, fetal retention, ruptured uterus).
  • Haemorrhagic disease of the newborn.
  • Snake envenomation.
  • Malignancies.
  • Overdose of anticoagulant drugs, e.g. warfarin.

Laboratory investigations

HAEMOGLOBIN ELECTROPHORESIS


Haemoglobin electrophoresis is used to separate and identify the different haemoglobins by their migration within an electric field. Haemoglobin variants separate at different rates due to differences in their surface electrical charge as determined by their amino acid structure.

Alkaline cellulose acetate electrophoresis

Several techniques are available to separate haemoglobin variants by electrophoresis. For routine work, electrophoresis in an alkaline buffer at pH 8.4–8.6 using a cellulose acetate membrane is adequate.
This gives good separation of HbA, HbF, HbS, and HbC. On alkaline electrophoresis HbD and HbS have the same mobility and HbC, HbE and HbO also co-migrate. In specialist laboratories agarose gel
electrophoresis at an acid pH (6.0) can be used to separate these haemoglobins and also to provide a clear separation of HbF from HbS and HbC.

MYCOBIOTIC OR MYCOSEL AGAR

Mycobiotic or Mycosel selective medium principally formulated for the isolation of dermatophytes but also used for the isolation of other pathogenic fungi from specimens contaminated with saprophytic fungi and bacteria.

The medium consists primarily of peptones from a pancreatic digest of soybean meal and dextrose. The selective agents are cycloheximide and chloramphenicol.

Cycloheximide inhibits the faster-growing saprophytic fungi. Chloramphenicol inhibits Gram-negative and Gram-positive organisms. Susceptibility to cycloheximide may be used for identification of fungi.

WATER AND CORNMEAL AGAR

WATER AGAR


Water agar is a nutritionally-deficient media known to enhance production of spores and conidia of sporulation. Media Preparation


• Mix 20 grams of agar in 1 litre of distilled water.


•Bring reagents to a boil.


• Autoclave at 15 lb/in2 for 15 minutes.


• Cool in slanted position or store as butts to be melted down and used as slants or plates as needed.

CORNMEAL TWEEN-80 AGAR


CMT agar is a well-established medium used for cultivation of fungi as well as to study chlamydospores production by Candida species and other yeasts.

CHROMOGENIC BISMUTH GLYCINE GLUCOSE YEAST (BIGGY)

Agar BiGGY agar is a partially selective and differential medium for the cultivation and identification of Candida species from pure cultures or clinical specimens.

Candida species, through a process of substrate reduction, reduces the bismuth salt to bismuth and sulfite to sulfide. Bismuth and sulfide combine to form a brownish to black precipitate that stains colonies and may diffuse into the medium.

Also bismuth and sulfur compounds are inhibitory to many bacteria. Yeast extract and glucose provide essential nutrients for growth. Glycine is an additional nutrient, but also inhibits many bacterial species at the high concentration used in this medium.

POTATO DEXTROSE AGAR (PDA)

Is a general purpose medium for a wide range of yeasts and molds.

The nutritionally rich base (potato infusion) encourages mold sporulation and pigment production in some dermatophytes like Trichophytan rubrum.

Most fungi thrive on Potato Dextrose Agar (PDA), but this can be too rich for many fungi, so that excessive mycelial growth is obtained at the expense of sporulation.

Is composed of dehydrated potato infusion, dextrose and agar. Many standard procedures use a specified amount of sterile tartaric acid (10%) to lower the pH of this medium to 3.5 +/- 0.1, inhibiting bacterial growth.

It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

BRAIN HEART INFUSION (BHI) BROTH AND AGAR

It is a non-selective fungal culture medium that permits the growth of virtually all clinically relevant fungi. It is used for the primary recovery of saprophytic and dimorphic fungi.

The medium contains proteose peptone and infusions from calf brain and beef heart which serve as sources of carbon, nitrogen, essential growth factors, amino acids and vitamins. Dextrose is used as a source of energy. Disodium phosphate helps in maintaining the buffering action of the medium whereas sodium chloride maintains the osmotic equilibrium of the medium.

BHI with 10% defibrinated sheep blood is useful for isolation and cultivation of Histoplasma capsulatum and other fungi.

One can culture food, blood and water samples at 35–37°C for 24–48 hours for the isolation of Candida albicans. Some fungi might require more incubation time.

After the incubation period, examine tubes for turbidity which is an indication of growth. If desired, a loopful of the culture broth can then be sub-cultured onto an appropriate solid medium to observe colony morphology.

It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.