9.4 Bleeding Time, Clotting Time, Platelet Function, and Special Hematology Tests

Bleeding Time, Clotting Time, Platelet Function, and Special Hematology Tests

The Hematology blueprint lists coagulation tests as including bleeding time, clotting time, prothrombin time, and red cell fragility, and it reserves 5% for special tests. Several of these older tests are rarely ordered now, but they remain on the board because they teach the physiology behind modern assays and are still used where analyzers are limited. This section covers bleeding and clotting times, clot retraction, the tourniquet test (important in dengue), platelet aggregation and the PFA-100, osmotic fragility, PNH testing, G6PD screening, sickling tests, and the LE cell preparation.

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1. Bleeding Time

The bleeding time measures how quickly a standardized skin puncture stops bleeding, which depends on platelet number and function and on vessel wall integrity (primary hemostasis).

• Prolonged in: Thrombocytopenia, qualitative platelet disorders (Glanzmann thrombasthenia, Bernard-Soulier syndrome), von Willebrand disease, aspirin and other antiplatelet drugs, uremia, and vascular disorders.
• Normal in: Coagulation factor deficiencies such as hemophilia A and B (the platelet plug forms normally).
• Limitations: Poor reproducibility and no ability to predict surgical bleeding. Aspirin should be stopped about 7 to 10 days before testing when the test is used to assess an inherited disorder.

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2. Whole Blood Clotting Time and Clot Retraction

• Lee-White (tube) clotting time: Venous blood is placed in three glass tubes at 37 °C; the tubes are tilted at intervals until the blood no longer flows. The normal range is commonly quoted as about 5 to 15 minutes. It is prolonged in severe factor deficiencies, heparin therapy, and afibrinogenemia, but it is insensitive to mild deficiencies and has been replaced by the aPTT.
• Capillary tube (slide) clotting time: A capillary tube filled from a fingerstick is broken at intervals until a fibrin thread appears; the normal range is about 2 to 4 minutes (some texts give up to 5 to 6 minutes). It is a crude bedside test.
• Clot retraction: Blood clotted in a tube at 37 °C normally retracts and expresses serum. Retraction is visible within about 1 hour and largely complete within 24 hours, when the clot has shrunk to roughly half of the original blood volume. Retraction depends on platelet number and on functional glycoprotein IIb/IIIa and the platelet contractile proteins. It is poor in thrombocytopenia and Glanzmann thrombasthenia. Severe anemia increases apparent retraction, and polycythemia decreases it.

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3. Tourniquet Test (Rumpel-Leede Capillary Fragility Test)

• Procedure: Inflate a blood pressure cuff on the upper arm to a pressure midway between systolic and diastolic pressure for 5 minutes. Release the cuff, wait about 1 to 2 minutes, and count the petechiae in a 2.5 cm × 2.5 cm (1-inch) square on the forearm or antecubital area.
• Interpretation: The WHO dengue guidelines for South-East Asia (2011) treat 10 or more petechiae per square inch as positive; older WHO criteria used 20 or more.
• Significance: A positive test reflects capillary fragility and thrombocytopenia. In the Philippines, where dengue is endemic, it is part of the bedside assessment of probable dengue and is interpreted together with the platelet count, hematocrit (hemoconcentration), and NS1 antigen or serology. It is also positive in other thrombocytopenias and vasculitides.

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4. Platelet Aggregation Studies and the PFA-100

Light Transmission Aggregometry

Platelet-rich plasma is stirred at 37 °C in an aggregometer. When an agonist is added and platelets clump, the plasma clears and light transmission increases. Standard agonists are ADP, epinephrine, collagen, arachidonic acid, and ristocetin.

• Specimens are drawn in 3.2% citrate with minimal trauma, kept at room temperature (cold activates platelets), and tested within about 4 hours.
• Ristocetin causes von Willebrand factor to bind platelet GP Ib, so it tests the vWF-GP Ib interaction. Bernard-Soulier syndrome also features giant platelets and thrombocytopenia.

PFA-100 (Platelet Function Analyzer)

Citrated whole blood is drawn under high shear through an aperture in a membrane coated with collagen/epinephrine or collagen/ADP. The closure time is the time taken for a platelet plug to occlude the aperture.

• Prolonged with both cartridges: von Willebrand disease, Glanzmann thrombasthenia, Bernard-Soulier syndrome, thrombocytopenia, and anemia (low hematocrit).
• Aspirin typically prolongs the collagen/epinephrine closure time while the collagen/ADP closure time stays normal.

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5. Red Cell Fragility and Hemolysis Tests

Osmotic Fragility Test

• Red cells are added to a series of hypotonic sodium chloride solutions (commonly from 0.85% down to about 0.10%). Water enters the cells, and they lyse when their volume exceeds their capacity to swell. The percentage of hemolysis in each tube is read photometrically.
• Normal: Hemolysis begins at about 0.45% to 0.50% NaCl and is complete at about 0.30% to 0.35%.
• Increased fragility (lysis at higher salt concentrations): Hereditary spherocytosis and other spherocytic states (warm autoimmune hemolytic anemia), because spherocytes have a low surface-area-to-volume ratio. Incubating the blood at 37 °C for 24 hours before testing makes mild spherocytosis easier to detect.
• Decreased fragility (resistance to lysis): Thalassemia, sickle cell disease, iron deficiency, and other conditions with target cells or flattened cells that have extra membrane.
• The flow cytometric eosin-5'-maleimide (EMA) binding test is now a preferred screening test for hereditary spherocytosis.

Paroxysmal Nocturnal Hemoglobinuria (PNH)

• PNH cells lack GPI-anchored complement-regulatory proteins (CD55 and CD59) and are lysed by complement.
• Historical tests: The sucrose hemolysis test (screen; low ionic strength promotes complement binding) and Ham's acidified serum test (confirmation; acidified serum activates the alternative complement pathway).
• Current standard: Flow cytometry showing loss of CD55 and CD59 on red cells and loss of GPI-anchored proteins on granulocytes and monocytes, often with FLAER (fluorescent aerolysin), which binds the GPI anchor itself.

G6PD Deficiency Screening

• G6PD deficiency is the most common red cell enzyme defect worldwide and is included in the Philippine newborn screening panel. Oxidant stress (fava beans, sulfonamides, primaquine, infections) precipitates hemoglobin as Heinz bodies and causes hemolysis, and bite cells appear on the smear.
• Fluorescent spot test (Beutler): Blood is mixed with glucose-6-phosphate and NADP+. Normal G6PD reduces NADP+ to NADPH, which fluoresces under long-wave UV light. Deficient samples do not fluoresce.
• During or just after a hemolytic episode, the oldest and most deficient cells have already been destroyed, and the reticulocytes have higher G6PD activity, so a falsely normal result is possible. Repeat the test after recovery.
• Heinz bodies are shown with a supravital stain such as crystal violet or brilliant cresyl blue, not with Wright stain.

Sickling and Solubility Tests

• Sodium metabisulfite slide test: A reducing agent deoxygenates hemoglobin under a sealed coverslip, and red cells containing HbS form sickles.
• Solubility (dithionite) test: Deoxygenated HbS is insoluble in a high-phosphate buffer and makes the solution turbid, while other hemoglobins stay in solution and the lines behind the tube remain visible. False negatives occur in infants under about 6 months (high HbF) and with severe anemia. Both tests detect HbS but cannot separate sickle trait from sickle cell disease; hemoglobin electrophoresis or HPLC is required.

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6. The LE Cell Preparation (Historical)

• In the LE cell test, blood from a patient with systemic lupus erythematosus is traumatized and incubated so that antinuclear antibodies coat exposed nuclei. A neutrophil that has engulfed the homogeneous, glassy, purple nuclear material is an LE cell, and the nucleus is pushed to the side.
• A tart cell is a monocyte that has ingested a nucleus that still shows chromatin structure. It is not an LE cell.
• The LE cell test was insensitive and has been replaced by antinuclear antibody (ANA) testing and anti-dsDNA antibodies (see the autoimmune serology section).