The Effect of Thrombocytopenia on the Determination of Platelet Aggregation P E T E R H. L E V I N E ,

M.D.

Blood Coagulation Laboratory, Hematology Service, New England Medical Center Hospital, and Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts

ABSTRACT

T H E MEASUREMENT of platelet aggregation is an increasingly widely used study of platelet function. With control of certain known variables, 4,5 it is a relatively simple and reproducible method of detecting both congenital and acquired thrombopathies. 13 The study is performed on platelet-rich plasma (PRP) obtained by differential centrifugation. 11 Such PRP is generally diluted to a standard count in the range of 200,000-400,000 platelets per cu. mm. of plasma. 9 It has been common knowledge that platelet aggregation cannot be reliably determined in blood from thrombocytopenic subjects, owing to the low number of platelets in the PRP prepared from such blood. 8 This has greatly limited the use of this test of platelet function in many groups of patients known to be at risk for platelet disorders. With the advent of a more sensitive and Received March 3, 1975; received revised manuscript April 15, 1975; accepted for publication April 15, 1975. Address reprint requests to Dr. Levine: Memorial Hospital, 119 Belmont Street, Worcester, Massachusetts 01605.

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self-calibrating aggregation detector, the study of thrombocytopenic PRP was reexamined. The results presented here suggest that measurement of platelet aggregation is indeed feasible in spite of thrombocytopenia. Materials and Methods Venous blood was collected in plastic syringes from healthy adult volunteers who had abstained from all medications for at least ten days. The blood was immediately mixed (9:1) with acid-citrate anticoagulant 2 in plastic tubes, centrifuged at 200 X g for 6 minutes at 22 C , and the resultant PRP removed. The remaining blood was then centrifuged at 850 X g for 20 minutes to yield platelet-poor plasma (PPP). Using autologous PPP as the diluting fluid, each PRP sample was diluted to the following platelet counts: 200,000,150,000, 125,000, 100,000, 75,000, 50,000, and 25,000 per cu. mm. of plasma. Samples were counted in a Coulter model ZBI counter, and were excluded from further

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Levine, Peter H.: The effect of thrombocytopenia on the determination of platelet aggregation. Am J Clin Pathol 65: 7 9 - 8 2 , 1976. Platelet aggregation has been widely assumed to be unmeasurable in thrombocytopenic samples. Using a sensitive differential self-calibrating aggregometer, and standard aggregating agents, aggregation was measured in serially diluted plateletrich plasma. Aggregation induced by ADP or collagen was reproducible at platelet counts as low as 50,000 per cu. mm., and with epinephrine aggregation was reproducible at platelet counts as low as 75,000 per cu. mm. (Key words: Adenosine diphosphate; Blood platelet; Collagen; Epinephrine; Platelet aggregation; Thrombocytopenia.)

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FlG. 1. Percentage maximum platelet aggregation induced by 1 x 10~5M adenosine diphosphate, in serially diluted platelet-rich plasma from six normal donors (N = 82 - 96%).

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FIG. 2. Percentage maximum platelet aggregation induced by 1 X 10~6M adenosine diphosphate, in serially diluted platelet-rich plasma from six normal donors (N = 31 - 9 6 % ) .

study if not within 20% of the predicted prior to testing was exposed to epinephrine as above. platelet count. Aggregation was calculated in terms of Aggregation was performed using standard methods 10 on a Platelet Aggregation maximum percentage change in light Profiler, Model PAP-2A (Bio/Data Corp., transmittance. The light transmittance of a Willow Grove, Pa.). This device simultane- sample of PPP was regarded as 100%, and ously accepts both platelet-rich and plate- that of the autologous PRP as zero, prior let-poor samples, and automatically cali- to the addition of the aggregating agent. brates an integrated recorder for full-scale The normal range of platelet aggregadeflection, based on the differential of the tion given is the mean ± S.D. of plasma initial optical densities of the test speci- samples from 60 healthy unmedicated volunteers. mens. Twenty-four sets of serially diluted plateResults let specimens were studied. Six sets were exposed to each of the following four agUsing ADP, 1 X 10_5M, as the aggregregating agents: (1) adenosine diphos- gating agent, a series of six sets of serially phate (ADP) at 10~5M; (2) ADP at 10~6M; diluted PRP was studied. Results are shown (3) epinephrine at 5 x 10_6M; (4) collagen in Figure 1. No significant change in platesuspension by Hovig's method 7 at .05 mg. let aggregation occurred at or above 50,000 per ml. final concentration in PRP. An ad- platelets per cu. mm. ditional set of serially diluted specimens, At a lower concentration of ADP, 1 prepared from six donors who took 600 x 10~6M, more individual variability of mg. acetylsalicylic acid by mouth 24 hours response was seen. However, for any given

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FlG. 3. Percentage maximum platelet aggregation induced by 5 X I0"6M epinephrine, in serially diluted platelet-rich plasma from six normal donors (N = 69 94%).

individual, serial dilution of PRP to concentrations as low as 50,000 per cu. mm. caused no significant alteration of aggregation response (Fig. 2). With epinephrine as the aggregating agent, aggregation remained normal at or above 75,000 platelets per cu. mm. in PRP (Fig. 3). In one of six samples, aggregation was clearly diminished at 50,000 platelets per cu. mm. With collagen suspension as the aggregating agent, aggregation remained normal at or above 50,000 platelets per cu. mm. (Fig. 4). Using platelets from donors who had taken aspirin, epinephrine-induced aggregation remained consistently abnormal at or above 50,000 platelets per cu. mm. (Fig. 5). For each aggregating agent, the mean maximum percentage of aggregation at 200,000 platelets per cu. mm. was compared with the mean maximum percentage aggregation at 75,000 platelets per cu. mm.

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FIG. 4. Percentage maximum platelet aggregatHon inducedi by collagen suspension, suspension in serially diluited platelet-rich plasma from six normal donors (N = 78 - 92%).

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FlG. 5. Percentage maximum platelet aggregation induced by 5 x 10_6M epinephrine, in serially diluted platelet-rich plasma from six aspirin-treated normal donors.

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Table 1. Aggregation with Four Agents Aggregating Agent ADP lfr 5 M

ADP 10"6M

Epinephrine

Collagen

Mean percentage maximum aggregation at 200,000 per cu. mm.

87.5

71.1

91.0

88.0

Mean percentage maximum aggregation at 75,000 per cu. mm.

94.3

73.7

85.7

87.0

Results are shown in Table 1. None of these differences was significant when analyzed by Student's t test for paired data (p > .05). Discussion

The data presented here indicate that platelet aggregation as induced by ADP or collagen is unchanged by dilution of the PRP to counts as low as 50,000 platelets per cu. mm. Epinephrine-induced aggregation was reliable at 75,000 or more platelets per cu. mm. Platelets exposed to aspirin in vivo showed abnormal platelet aggregation; the abnormality was not significantly altered by serial dilution of the platelets to concentrations as low as 50,000 per cu. mm. It should be noted that a fixed amount of aggregating agent has been used in these studies, which results in a greater amount of aggregating agent per platelet in the

References 1. Bang NU, Heidenreich RD, Trygstad CW: Plasma protein requirements for human platelet aggregation. Ann NY Acad Sci 201: 2 8 0 299, 1972 2. Canoso RT, Rodvien R, Scoon K, et al: Hydrogen peroxide and platelet function. Blood 43: 645-656, 1974 3. Cardamone JM, Edson JR, McArthur JR, et al: Abnormalities of platelet function in the myeloproliferative disorders. JAMA 221:270-273, 1972 4. Goldenfarb PB, Cathey MH, Cooper GR: T h e determination of ADP-induced platelet aggregation in normal men. Atherosclerosis 12: 335-340, 1970 5. Harrison MJG, Emmons PR, Mitchell JRA: T h e variability of human platelet aggregation. J Atheroscler Res 7:197-205, 1967 6. Horowitz HI, Stein IM, Cohen BD, et al: Further studies on the platelet-inhibitory effect of guanidinosuccinic acid and its role in uremic bleeding. Am J Med 49:336-345, 1970 7. Hovig T: Aggregation of rabbit blood platelets produced in vitro by saline extract of tendons. Thromb Diath Haemorrh 9:248-263, 1963 8. Hutton RA: Test of platelet function and their use in the investigation of bleeding disorders. J Med Lab Technol 23:161-185, 1966 9. Levine PH: An acute effect of cigarette smoking on platelet function. Circulation 48:619-623, 1973 10. Levine PH: A qualitative platelet defect in severe vitamin B 12 deficiency. Ann Intern Med 78: 533-539, 1973 11. Mustard JF, Hegardt B, Rowsell HC, et al: Effect of adenosine nucleotides on platelet aggregation and clotting time. J Lab Clin Med 64: 548-559, 1964 12. Tangiin Y: Platelet aggregation and platelet factor 3 activity in myeloproliferative disorders. Thromb Diath Haemorrh 25:241-251, 1971. 13. ten Cate JW: Platelet function tests. Clin Hematol 1:283-294, 1972

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Many disorders that lead to decreased platelet number are further complicated by the presence of qualitative defects of these cells. Typical examples are multiple myeloma, 1 uremia, 6 vitamin B 12 deficiency, 10 a n d myeloproliferative syndromes. 3 With myeloproliferative syndromes such as polycythemia vera, aplastic anemia, or chronic granulocytic leukemia, at least 80% of patients can be expected to have abnormal platelet aggregation. 3,12 Such platelet abnormalities are therefore useful diagnostic clues in mild or early myeloproliferative syndromes. Thrombocytopenia is often present in myeloproliferative syndromes, however. A major limitation has thus been the technical difficulty, lack of sensitivity and variability of platelet aggregation studies on thrombocytopenic samples.

thrombocytopenic samples. This may explain, for example, the tendency to slightly greater ADP-induced aggregation in the thrombocytopenic samples (Table 1). These data indicate that thrombocytopathy may be accurately diagnosed in spite of the presence of significant thrombocytopenia.

The effect of thrombocytopenia on the determination of platelet aggregation.

Platelet aggregation has been widely assumed to be unmeasurable in thrombocytopenic samples. Using a sensitive differential self differential self-cal...
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