Prothrombin Time Test- Principle and Interpretation

Prothrombin Time tests the extrinsic coagulation pathway and is useful for detecting coagulation deficiencies, liver disease and disseminated intravascular coagulation (DIC). The prothrombin time (PT) is also the main monitor for coumarin therapy (e.g. warfarin), expressed as a ratio—the international normalized ratio (INR). The test measures the clotting time of plasma in the presence of a tissue extract, e.g. brain (thromboplastin). The test measures prothrombin but also factors V, VII, and X. 

Sample: Citrate

Increase Prothrombin time

• Oral anticoagulation therapy (vitamin K antagonists)
• Fibrinogen deficiency (factor I)
• Prothrombin deficiency (factor II)
• Deficiency of factors V, VII or X (in V or X deficiency the activated partial thromboplastin time APTT will be increase)
• Liver disease especially obstructive
• Vitamin K deficiency
• DIC

Ham’s Acid Lysis Test: Principle and Interpretation

Ham’s Acid Lysis Test is a test for the rare acquired red cell membrane disorder called paroxysmal nocturnal haemoglobinuria (PNH). Its pathophysiology is complex and involves an abnormality of the red cell membrane in PNH making it prone to complement-mediated lysis and episodes of marked intravascular haemolysis leading to free Hb in the urine (haemoglobinuria).

Principle

• Abnormal sensitivity of RBCs from patients with PNH to the haemolytic action of complement.
• Complement is activated by acidification of the patient’s serum to pH of 6.2, which induces lysis of PNH red cells, but not normal controls.

Sample: EDTA, heparin, citrate, oxalate.

Result: Positive result indicates PNH.

Specificity: High—similar reaction is produced only in the rare syndrome HEMPAS (a form of congenital dyserythropoietic anaemia type II), which should be easily distinguished morphologically. 

Sensitivity: Low—as the reaction is crucially dependent on the concentration of magnesium in the serum.

Bleeding Time- Ivy Method (Template Bleeding Time): Principle, Procedure and Reference Value

Bleeding Time- Ivy Method (Template Bleeding Time)

Principle

Bleeding time measures the primary phase of hemostasis: the interaction of the platelet with the blood vessel wall and the formation of a hemostatic plug. Bleeding time is the best single screening test for platelet function disorders and is one of the primary screening tests for coagulation disorders.

This test is of value in detecting vascular abnormalities and platelet abnormalities or deficiencies. It is not recommended for routine presurgical workup.

A small stab wound is made in either the earlobe or the forearm; the bleeding time (the amount of time it takes to form a clot) is recorded. The duration of bleeding from a punctured capillary depends on the quantity and quality of platelets and the ability of the blood vessel wall to constrict.

The principal use of this test today is in the diagnosis of von Willebrand's disease, an inherited defective molecule of factor VIII and a type of pseudohemophilia. It has been established that aspirin may cause abnormal bleeding in some normal persons, but the bleeding time test has not proved consistently valuable in identifying such persons.

Procedure

1. Cleanse the area three fingerwidths below the antecubital space with alcohol and allow to dry.
2. Place a blood pressure cuff on the arm above the elbow and inflate to 40 mm Hg.
3. Select a cleansed area of the forearm without superficial veins. Stretch the skin laterally and tautly between the thumb and forefinger.
4. Start a stopwatch. Use the edge of a 4? × 4? filter paper to blot the blood through capillary action by gently touching the drop every 30 seconds. Do not disturb the wound itself. Remove the blood pressure gauge when bleeding stops and a clot has formed. Apply a sterile dressing when the test is completed.
5. Remember that the end point (by the Ivy or the earlobe method) is reached when blood is no longer blotted from the forearm puncture. Report in minutes and half minutes (eg, 5 minutes, 30 seconds).

Reference Values

Normal 3–10 minutes in most laboratories Duke method (earlobe): 5 minutes (not recommended—not very reproducible with a wide range of normal values) Ivy method (forearm with template): 25–90 minutes Mielke's method (Surgicut): Adults: 1–7 minutes Teens: 3.0–8 minutes Children: 2.5–13 minutes

Clinical Implications

1. Bleeding time is prolonged when the level of platelets is decreased or when platelets qualitatively abnormal:

• Thrombocytopenia (platelet count <80 × 10 3/mm 3)
• Platelet dysfunction syndromes
• Decrease or abnormality in plasma factors (eg, von Willebrand's factor, fibrinogen)
• Abnormalities in the walls of the small blood vessels, vascular disease
• Advanced renal failure
• Severe liver disease
• Leukemia, other myeloproliferative diseases
• Scurvy
• DIC disease (owing to the presence of FDPs)

2. In von Willebrand's disease, bleeding time can be variable; it will definitely be prolonged if aspirin is taken before testing (aspirin tolerance test).

3. A single prolonged bleeding time does not prove the existence of hemorrhagic disease. Because a larger vessel can be punctured, the puncture should be repeated on an alternate body site, and the two values obtained should be averaged.

4. Bleeding time is normal in the presence of coagulation disorders other than platelet dysfunction, vascular disease, or von Willebrand's disease.

5. Aspirin therapy (antiplatelet function therapy): when thrombus formation is thought to be mediated by platelet activation, the patient frequently is given agents to interrupt normal platelet function, which may be monitored by bleeding times or platelet aggregation studies. Aspirin is the most commonly used inhibitor; it inhibits platelet

Interfering Factors

• Normal values for bleeding time vary when the puncture site is not of uniform depth and width.
• Touching the puncture site during this test will break off fibrin particles and prolong the bleeding time.
• Excessive alcohol consumption (as in alcoholic patients) may cause increased bleeding time.
• Prolonged bleeding time can reflect ingestion of 10 g of aspirin as long as 5 days before the test.
• Other drugs that may cause increased bleeding times include dextran, streptokinase-streptodornase (fibrinolytic agents), mithramycin, pantothenyl alcohol.
• Extreme hot or cold conditions can alter the results.
• Edema of patient's hands or cyanotic hands will invalidate the test.

WBC Normal Values and List of Leukocyte Abnormalities and Diseases

White Blood Cell (WBC) Normal Values:

WBC Count= 5,000 – 10,000/cu mm or 5 – 10 x 10^9/L

Differential Count:

Neutrophil = 50 – 70 % 

Segmenter = 50 – 65 % 

Stab = 0 – 5 %

Eosinophil = 0 – 3 %

Basophil = 0 – 1 %

Lymphocytes = 20 – 40 %

Monocytes = 2 – 6 %

Complete Blood Count (CBC) Procedure

Complete Blood Count(CBC) is a basic screening test and is one of the most frequently ordered laboratory procedures. The findings in the CBC give valuable diagnostic information about the hematologic and other body systems, prognosis, response to treatment, and recovery. Most laboratories now are using automated methods which gives more accurate results and faster turn around time.The CBC consists of a series of tests that determine number, variety, percentage, concentrations, and quality of blood cells:

Capillary Puncture (Skin Puncture or Prick Method) Procedure

This method is preferably used in peripheral blood smear preparation but applicable also in other hematologic examinations.

Thick and Thin Blood Films Procedure

Purpose:

Thick and thin blood films stained with Giemsa hematological stains permit the detection of blood parasites including malarial parasites, trypanosomes, and microfilariae.

Principle:

The thick blood film permits the examination of a large amount of blood for the presence of parasites. Since the smear is not fixed with methanol, the red blood cells (RBCs) lyse, permitting better visualization of the organisms. The thin film allows for the observation of RBC morphology, inclusions, and intracellular and extracellular parasites. A larger area of the slide must be examined in the thin smear, although there is less distortion when compared with the thick smear.