PLATELET COUNT MATERIALS PROCEDURE RESULTS

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Neal Lawrence
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1 Platelet count 5 PLATELET COUT hemocytometer, diluting pipette for white blood cells, diluting fluid (Dameshek's solution; cocaine chlorhydrate 3 g, sodium chloride 0.2 g, distilled water ad 00 ml), light microscope, for blood sample collection: cotton balls, alcohol, sterile needles, rubber gloves. Cleanse the hemocytometer and cover glass with a piece of cotton saturated with alcohol and let air dry. Prepare the hemocytometer by placing the cover glass on its mounting supports. cleansed finger (hold firmly between the thumb and forefinger) deep enough so that blood flows freely from the wound. Wipe off the first drop of blood with a piece of cotton; when a second drop has accumulated, proceed with the filling of the pipette. Do not fill the pipettes with blood until sufficient blood has welled up on the fingertip since these pipettes have a very small bore and blood clots extremely easily in them. The finger must not be squeezed. Place the pipette tip just within the drop of blood. Suck up a continuous column of blood in the tube to the 0.5 mark on the pipette by using the mouthpiece. Wipe the excess blood from the tip of the pipette. Immerse the pipette tip in the platelet diluting fluid (Dameshek's solution); and while holding the pipette vertically, suck the diluting fluid exactly to the mark. Dilution should be done very quickly and precisely to prevent clotting of the blood and to insure accuracy. Thus a 20-fold dilution is obtained (the volume of the mixing chamber is μl μl=0 μl, containing 0.5 μl of blood). Close the ends of the pipette with your thumb and middle finger and shake the pipette for 5 minutes. Throw away the first two drops from the pipette (they come from the capillary part of the pipette and contain only diluting fluid) and with the third drop fill the hemocytometer: let the drop fall near the cover glass and the chamber will fill due to capillarity. The excess fluid will flow in the moats. Wait 20 minutes to complete the lysis of the other blood cells. Place the hemocytometer in a light microscope and examine the sample using low-power objective (0 x) and weak light. Count the platelets in 00 rectangles considering the cells inside the squares and from two sides. Report the number of platelets as cells/mm 3. First the average number of platelets in a rectangle must be calculated: (total number of found platelets)/00 (number of rectangles).

2 6 Physiology laboratory exercises This must be reported to the volume corresponding to a rectangle: area x height of the chamber. Last the results must be corrected with the dilution of the blood. Pl Rct Volume Dilution Rct Area Height Dilution Pl DATA ITERPRETATIO ormal range: /mm 3 Higher values thrombocytosis: bone marrow diseases Lower values thrombocytopenia autoimmune diseases, medication, idiopathic

3 Investigation of hemostasis 7 IVESTIGATIO OF HEMOSTASIS Hemostasis is a complex process that causes the bleeding process to stop. Steps: vasoconstriction, mechanical blockage of the injured vessel by a platelet plug, coagulation: formation of the clot (a fibrin protein fiber mesh), the clot retracts and is slowly dissolved. BLEEDIG TIME filter paper, chronometer, to obtain the blood sample: cotton balls, alcohol, sterile needles, rubber gloves. cleansed finger (hold firmly between the thumb and forefinger) deep enough so that blood flows freely from the wound. Start the chronometer in the moment of puncture. Gently blot the blood (touch the finger at the site of puncture) with filter paper every 20 seconds to see if the bleeding has stopped. Stop the chronometer and note the moment when the filter paper is not stained with blood, representing the bleeding time. DATA ITERPRETATIO ormal range: 2-4 min Higher values: impaired platelet function (thrombocytopathy) impaired blood vessel contractility

4 8 Physiology laboratory exercises GLOBAL COAGULATIO TIME The coagulation cascade has two pathways, the contact activation pathway (intrinsic pathway), and the tissue factor pathway (extrinsic pathway), which lead to fibrin formation. The coagulation cascade can be investigated measuring the global and partial coagulation times. The measured value for the coagulation times strongly depend on environmental factors (e.g. room temperature, humidity, etc.). The global coagulation time measures the time between the activation of the coagulation cascade to the formation of insoluble fibrin filaments. clock glass (convex glass) covered with paraffin, bended glass stick, chronometer, for blood sample collection: cotton balls, alcohol, sterile needles, rubber gloves. cleansed finger (hold firmly between the thumb and forefinger), deep enough so that blood flows freely from the wound. Start the chronometer in the moment of puncture. Let one drop of blood fall on the clock glass. Draw the tip of the glass stick through the blood drop every 30 seconds until a thin filament is formed. Stop the chronometer and note the moment when the thin filament is formed (a fibrin filament to which blood cells adhere), representing the clotting time. DATA ITERPRETATIO ormal range: 4-8 min a normal clotting time does not exclude an abnormality of the hemostasis! Higher values: coagulation factor deficiencies.

5 Investigation of hemostasis 9 PARTIAL COAGULATIO TIMES The partial coagulation times investigate different phases of the coagulation cascade. The blood sample is collected through venipucture in a tube containing anticoagulant substance (calcium chelators), the blood cells are removed through centrifugation. Then calcium is added to the plasma and the tests are performed at 37 C. PROTHROMBI TIME The prothrombin time (PT) measures the clotting time from the activation of factor VII, through the formation of fibrin clot. This test measures the integrity of the extrinsic and common pathways of coagulation. It is used to monitor oral anticoagulation. A reagent called thromboplastin (phospholipid with tissue factor and calcium) is added to the plasma, which activates the extrinsic pathway of coagulation. The time until clot formation is measured in seconds. ormal range: varies depending on the reagent-instrument combination and individual laboratory policies. The prothrombin ratio (PR) represents the patients prothrombin time reported to a control prothrombin time, and is reported in percents. ormal range: 85-00% The international normalized ratio (IR) was devised to standardize the results. Each manufacturer assigns an ISI value (International Sensitivity Index) for any tissue factor they manufacture. The ISI value indicates how a particular batch of tissue factor compares to an internationally standardized sample. The IR is the ratio of a patient's prothrombin time to a normal (control) sample, raised to the power of the ISI value for the analytical system used. PT IR PT test control ISI ormal value:, in case of anticoagulation: 2-4. ACTIVATED PARTIAL THROMBOPLASTI TIME The activated partial thromboplastin time (aptt) measures the clotting time from the activation of factor XII, through the formation of fibrin clot. This measures the integrity of the intrinsic and common pathways of coagulation. It is also used to monitor therapeutic heparin anticoagulation. Phospholipid with an intrinsic pathway activator are added to patient plasma, and the time until clot formation is measured in seconds. ormal range: sec.

Bleeding disorders or haemorrhagic diatheses are a group of disorders characterised by defective haemostasis with abnormal bleeding.

Bleeding disorders or haemorrhagic diatheses are a group of disorders characterised by defective haemostasis with abnormal bleeding. Bleeding may be spontaneous in the form of small haemorrhages into the