A nnals of C linical Laboratory Science, Vol. 2, N o . 1 Copyright © 1972, Institute for Clinical Science Laboratory Tests of Platelet Function H. JAMES DAY, M.D. and HOLM HOLMSEN, Ph .D. Hematology Laboratories, Temple University School of Medicine Philadelphia, PA 19140 Introduction portant adenine nucleotide released is ADP, There has been growing interest in the a powerful platelet aggregating substance. role of platelets in the hemostatic process. Platelets also contribute a major source of Following injury to the blood vessel wall, phospholipids required for platelet coagula­ the platelets are exposed to some substance tion and this has been identified as platelet in the basement membrane, which may or factor 3 or platelet thromboplastic factor. may not be collagen, which leads to the This phospholipid is not normally available rapid formation of a platelet plug. The on the platelet surface and only becomes steps involved here are those of adhesion so when the platelets are altered or acti­ to the site of injury and exposed basement vated. Hardisty and Hutton1 have shown membrane, then aggregation of the plate­ that Kaolin makes this thromboplastic activ­ lets during which time the platelets un­ ity available and Spaet and Cintron24 have dergo a release reaction. Formation of a shown that connective tissue suspensions permeable platelet plug then occurs and are equally active. Platelets are also re­ finally, the subsequent development into a quired for clot retraction, the energy for hemostatic plug with the incorporation of which is derived from the breakdown of fibrin strands. It is important to note that platelet ATP to ADP. During this time, the time needed for the formation of a plate­ fibrin strands form which tend to radiate let plug correlates well with the bleeding between the clumps of platelets which act time measured from the beginning of the as the nidus of clot retraction. To date, clot infliction of the wound to the time the retraction has only been shown to be abnor­ bleeding ceases, i.e., approximately 6 min­ mal in those patients who have a defect utes. Thus, in performing a bleeding time, known as thrombasthenia and in those who the steps are measured which are necessary lack a normal number of platelets, i.e., for platelet plug formation. In vitro the thrombocytopenia. It is uncertain which function of platelets is tested by measuring role clot retraction plays in the mainte­ platelet adhesion to glass, aggregation with nance of normal hemostasis. various agents, the release reaction, clot re­ 5-Hydroxytryptamine (5-HT), a power­ traction, platelet-phagocytosis and platelet ful vasoconstrictor, is also carried by the uptake of numerous substances. platelets and released during the platelet Platelets contain large amounts of ade­ release reaction. The role of 5-HT in nor­ nine nucleotides which are released during mal hemostasis is not known, but recent the platelet release reaction, an important evidence has shown that a low platelet step in hemostasis. Possibly the most im­ serotonin level is often associated with low 63 64 DAY AND HOLMSEN releasable nucleotide levels. Thus, plate­ disease, etc. It is not in the scope of this lets are unable to release aggregating mate­ paper to discuss these in detail. More rials and hemorrhagic manifestations result. detailed general review papers on this Such a defective release reaction is typical subject should be consulted.12- ls- 16- 20 of thrombopathy or thrombocytopathy, a clinical condition characterized by hemor­ Methods rhagic manifestations. This paper will describe those tests of By definition, patients having defects in platelet function which are simple to per­ platelet function (also called qualitative form and yield the desired information. All platelet disorders) are those who have a are now available for use in clinical normal platelet count, but a prolonged laboratories. bleeding time owing to abnormal function­ ing platelets. It is generally accepted that Collection of Blood Sample bleeding phenomena do not occur unless the platelet count is below 2 0 ,0 0 0 per mm3. It is important to collect a sample of However, some patients, specifically those blood from a normal subject at the same with thrombopathy, may have platelet time as that of the patient if detailed tests counts that are in the range of 75 to 150,000 of platelet function are to be performed. per mm3 and have bleeding phenomena The volume of blood to be drawn for study even with this relatively minor thrombocy­ of platelet function may vary anywhere topenia. Under these circumstances, the from 9 to 38 ml. As performed in our platelets can often be shown to have a laboratory, 18 or 38 ml of blood are drawn functional defect. by gravity into plastic conical tubes by free The major diseases of platelet function flow into 2 ml or 4 ml of 0.11 M sodium that have been identified to date are classi­ citrate (31.3 g per 1 trisodium citrate 'H^O) fied in the following manner: with a teflon coated needle attached to a 1. Thrombasthenia — a disease which plastic tubing. The tube is then capped seems to be characterized by a defec­ with parafilm and inverted several times tive platelet membrane resulting in a for adequate mixing. Two blood smears failure of platelet aggregation. are also made at this time for staining with 2. Thrombopathy — in which there is a Wright’s stain and examined with particular failure of the release mechanism in reference to platelet number, platelet mor­ platelets and a failure to make plate­ phology and the presence or absence of let factor 3 (PF3) available. platelet aggregates. A sample of the whole 3. Von Willebrands disease or pseudo­ blood is then submitted for platelet count­ hemophilia—which is characterized by ing by standard electronic counting tech­ niques. Plastic equipment or siliconized a prolonged bleeding time and a low glassware is used for all platelet transfers. factor VIII. Strictly, this is not a platelet functional abnormality in as Preparation of Platelet-Rich and much as the platelets function per­ fectly well, but is included mainly Platelet-Poor Plasma because of a prolonged bleeding time. Citrated whole blood is centrifuged for These patients lack a plasma factor ten minutes at 2 0 0 x g at room temperature. called von Willebrand’s factor. It is important that the platelets not be Many platelet functional abnormalities subjected to extreme cold. The platelet-rich are seen as secondary to other disease plasma (PRP) is then transferred to a sec­ states. These include renal diseases, para- ond plastic tube, care being taken to avoid proteinemias, thrombocythemia, iatrogenic transferring any red cells. A platelet count LABORATORY TESTS OF PLATELET FUNCTION 65 is performed on this PRP which is kept Bleeding Time at room temperature until tested. Approxi­ Introduction mately 5 ml of PRP is re-centrifuged at 4°C for 30 minutes at 2,000 xg to remove The bleeding time is perhaps the best platelets and obtain platelet poor plasma screening test for defects of platelet func­ (PPP), which is collected and kept at 22 tion. As mentioned previously it measures to 25 °C until tested. the entire sequence of events in the forma­ tion of the hemostatic platelet plug. When Capillary Resistance the bleeding time is abnormal, other tests of platelet function are performed which Principle yield specific information as to the specific The test for capillary resistance, called type of hemostatic defect. the tourniquet test, is performed every time a venous blood specimen is obtained with Principle venostasis. It is best quantitated by putting A standard incision is made through the a blood pressure cuff on the arm and inflat­ skin and the time necessary for stopping of ing it to a point mid-way between systole bleeding is carefully measured. As useful and diastole (about 100 mm Hg). The as is this test, it must be rigidly controlled. cuff is left in place for 3 to 4 minutes with The method of Ivy et al. 13 is preferred in an area approximately the size of a half- as much as it is better controlled than any dollar, circumscribed on the volar aspect others. Recent modifications1’ 4- 17 have in­ of the forearm. The number of petechiae troduced even further methods for quanti­ in this area are then counted following tating the bleeding time. Strict attention deflation of the cuff. Some petechiae also must be paid to detail and each laboratory occur on the dorsum of the hand, this area must establish its normal range. should be observed closely. At the same time petechiae may develop in the area to Procedure1, 4 which the cuff is applied. A sphygnomanometer is placed around Discussion the patient’s arm and inflated to 40 mm of mercury. Following adequate cleansing of The test is not very useful in assessing the volar surface of the forearm, three platelet function because the results may parallel cuts are made on the forearm with be abnormal in many disease states, only a #11 Bard-Parker blade. These cuts some of which are related to abnormal should be 5 to 6 mm long, 1 mm deep and hemostatic processes . It is generally abnor­ 1.5 cm apart. Blood is carefully absorbed mal in thrombasthenia and von Wille- with the edge of a filter paper every 30 s brand’s disease, but more commonly it is until bleeding ceases, the wound itself not pathological in patients with severe throm­ being touched. Wounds which bleed pro­ bocytopenia or increased vascular fragility. fusely from the start probably represent Patients who develop petechiae under the perforated veins. Those which do not cuff or on the dorsum of the hand usually bleed for at least 2 minutes are assumed to have increased vascular fragility.