COAGULATION AND TRANSFUSION MEDICINE Original Article
Platelet Aggregation in Platelet-Rich Plasma and Whole Blood in 120 Patients with Myeloproliferative Disorders CARLO L. BALDUINI, M.D., GIAMPIERA BERTOLINO, M.D., PATRIZIA NORIS, M.D., AND GIAN CARLO PILETTA, M.D.
platelet count because it also occurred in samples with normal platelet content (at variance with 13 subjects with reactive thrombocytosis, in which SPA was observed only in samples with high platelet concentration). Platelets from patients with idiopathic myelofibrosis had the highest tendency to undergo SPA. (Key words: Platelet aggregation; Idiopathic myelofibrosis; Essential thrombocytosis; Polycythemia vera; Chronic myelogenous leukemia; Myeloproliferative disorders) Am J Clin Pathol 1991;95:82-86
Platelets in myeloproliferative disorders are abnormal in many ways. One common abnormality is a reduction of platelet aggregation in platelet-rich plasma (PRP) after stimulation with many physiological stimuli.1 Such evidence has been obtained with the optical method of Born, which assesses in vitro platelet function in experimental conditions quite different from those occurring in vivo. One of the most relevant differences is the absence of red cells, which are known to interfere in many ways with platelet aggregation.2"4 For this reason, we previously studied platelet function in whole blood (WB) in 18 myelofibrotic patients by the impedenzometric method of Cardinal and Flower and compared these results with those obtained in PRP by the optical method of Born.5 While PRP studies revealed that 33% and 66% of patients were hypo-aggregating and normo-aggregating, respectively, WB studies demonstrated spontaneous platelet aggregation (SPA) in 55% of cases. On the basis of these
preliminary data, we extended our study to include a larger number of patients with myeloproliferative disorders. MATERIALS AND METHODS Patients
One hundred twenty consecutive subjects with myeloproliferative disorders observed in our department from January 1987 to December 1989 were investigated. Thirtyone had idiopathic myelofibrosis (IM), 32 had essential thrombocytosis (ET), 23 had polycythemia vera (PV), and 34 had Phi-positive chronic myelogenous leukemia (CML) in chronic phase. Thirteen subjects with reactive thrombocytosis (RT) after withdrawal of cytotoxic drug therapy or splenectomy also were investigated. Table 1 describes some characteristics of these patients. All patients underwent bone marrow trephine biopsy of the posterior iliac crest. Red cell mass was measured in subjects with ET and PV. Any medication interfering with platelet function was withdrawn at least one week before From the Institute of Clinical Medicine 2, Department of Interna! the study of platelet function. Medicine, University of Pavia-IRCCS S. Matteo, Pavia. Italy. Received January 8, 1990; received revised manuscript and accepted for publication April 9, 1990. Supported by grants from MPI and CNR, Italy. Address reprint requests to Dr. Balduini: Clinica Medica II, IRCSS S. Matteo, Piazzale Giolgi, 27100 Pavia, Italy. 82
Platelet
Aggregation
Blood obtained by venipuncture of the antecubital vein was mixed with 3.8 g/dL trisodium citrate (blood/anti-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
In vitro platelet aggregation in platelet-rich plasma (PRP) and in whole blood (WB) was assessed in 31 patients with idiopathic myelofibrosis, 32 with essential thrombocytosis, 23 with polycythemia vera, and 34 with chronic myelogenous leukemia. In PRP most subjects showed normal or reduced platelet aggregation, whereas in WB the majority of patients showed increased platelet function. Spontaneous platelet aggregation (SPA) was observed frequently in WB, whereas it was seldom observed in PRP. SPA in WB was inhibited by in vitro addition of aspirin and apyrase, and SPA was only partially dependent on high
83
BALDUINI ET AL. Platelet Aggregation in PRP and WB TABLE 1. CLINICAL AND LABORATORY DATA OF THE PATIENTS WITH MYELOPROLIFERATIVE DISORDERS AND REACTIVE THROMBOCYTOSIS Sex
IM ET PV CML RT 3
No. of Patients
M
F
Age (yr)
WBC (X 10'IL)
Hb* (gldL)
Platelets (X. 10"IL)
31 32 23 34 14
23 11 11 16 5
8 21 12 18 9
64 (47-85) 53(27-72) 53 (23-77) 52 (20-83) 46(19-65)
14.9(1.9-49.0) 9.3 (4.9-35.3) 16.3 (7.3-56.0) 72.6 (4.5-227.2) 8.8(4.7-18.9)
11.6(7.5-16.1) 13.1 (7.7-15.2) 17.4(14.0-23.1) 11.4(9.3-14.6) 12.6(11.3-15.8)
406(143-1,325) 838(618-1,900) 559(158-1,080) 491 (147-1,720) 670(468-1,697)
IM » idiopathic myelofibrosis; ET = essential thrombocytosis; PV = polycythemia vera; CML chronic myelogenous leukemia; RT = reactive thrombocytosis. • SI unit = g/dL X 10 - g/L.
each agonist was classified as hypo-, hyper-, and normoaggregation when the responses to at least two of the three agonist concentrations were lower than, higher than, or within the reference range. Reference values were based on results obtained in the study of 40 healthy control subjects. SPA was defined as a spontaneous decrease of optical density of PRP of more than 20% or an increase of impedance superior to 0.5 ohm within 15 minutes from the beginning of stirring. When SPA was present, the effect of preincubation of samples (5 minutes at 37 °C, without stirring) with acetyl-salycilic acid (2 g/L) and apyrase (100 mg/L) (Sigma Chemical Company) was evaluated. Preliminary experiments demonstrated that this amount of apyrase completely inhibited platelet aggregation in WB that was exposed to 1 /imol/L ADP. RESULTS Tables 2 and 3 describe the occurrence of SPA in PRP and WB both in the native and normalized preparations. SPA often was observed in WB samples with high platelet
TABLE 2. SPONTANEOUS PLATELET AGGREGATION IN PRP IN PATIENTS WITH MYELOPROLIFERATIVE DISORDERS AND REACTIVE THROMBOCYTOSIS (IN PARENTHESES NUMBER OF PATIENTS WITH SPA/NUMBER OF PATIENTS STUDIED) % of Patients with SPA
All patients (native PRP)* Patients with pit > 300 X lO'/L. (native PRP)* Patients with pit > 300 X 109/L. (normalized PRP)t Patients with pit < 300 X 109/L (native PRP)*
IM
ET
PV
CML
RT
13 (4/31) 19 (3/16) 0 (0/16) 7 (1/15)
28 (9/32) 28 (9/32) 3 (1/32)
9 (2/23) 13 (2/15) 0 (0/15) 0 (0/8)
3 (1/34) 8 (1/13) 0 (0/13) 0 (0/21)
28 (4/14) 28 (4/14) 0 (0/14) 0 (0/0)
—
(0/0)
IM = idiopathic myelofibrosis; ET = essential thrombocytosis; PV = polycythemia vera; CML = chronic myelogenous leukemia; RT = reactive thrombocytosis. * PRP with platelet concentration equal to that one observed in each patient, t PRP containing 250 X 109 platelets/L.
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coagulant, 9/1). PRP and platelet poor plasma (PPP) were obtained by standard methods as previously reported.6 PRP was adjusted with PPP to 250 X 109 platelets/L (250 X 103/mm3) (normalized PRP) and to a concentration of platelets equal to that observed in each patient (native PRP). Platelet aggregation in PRP was studied by the optical method of Born7 with a whole blood lumiaggregometer model 500 (Chrono-Log Corporation, Havertown, PA). Platelet aggregation in WB was studied by the impedenzometric method of Cardinal and Flower8 with the same instrument. Platelet function was investigated in unmodified samples (native WB) and in samples reconstituted, mixing appropriate amounts of packed red cells, PRP, and PPP from each patient to obtain 0.40 (40%) red blood cell Hct and 250 X 109 platelets/L (250 X 103/ mm 3 ) (normalized WB).3 In both PRP and WB, platelet aggregation was stimulated by the addition of ADP (1, 5, and 20 /tmol/L, final concentration) (Sigma Chemical Company, St Louis, MO), epinephrine (1, 5, and 10 junol/L), and collagen ( 1 , 5 , and 20 mg/L) (Mascia Brunelli, Milan, Italy). Platelet aggregation stimulated by
84
COAGULATION AND TRANSFUSION MEDICINE Original Article TABLE 3. SPONTANEOUS PLATELET AGGREGATION IN WHOLE BLOOD IN PATIENTS WITH MYELOPROLIFERATIVE DISORDERS AND REACTIVE THROMBOCYTOSIS (IN PARENTHESES NUMBER OF PATIENTS WITH SPA/NUMBER OF PATIENTS STUDIED) % of Patients with SPA
All patients (native WB)* Patients with pit > 300 X 109/L (native WB)* Patients with pit > 300 X 10'/L (normalized WB)f Patients with pit < 300 X 109/L (native WB)*
IM
ET
PV
CML
RT
45 (14/31) 50 (8/16) 37 (6/16) 40 (6/15)
59 (19/32) 59 (19/32) 9 (3/32)
26 (6/23) 40 (6/15) 20 (3/15) 0 (0/8)
26 (9/34) 53 (7/13) 23 (3/13) 9 (2/21)
64 (9/14) 64 (9/14) 0 (0/14)
—
(0/0)
— (0/0)
1M = idiopathic myelofibrosis: ET = essential thrombocytosis: PV = polycythemia vera; CML = chronic myelogenous leukemia; RT = reactive thrombocytosis. * Unmodified whole blood. t Whole blood containing 250 X 10' platelets/L and 40% red blood cell Ht.
In the patients and in the experimental conditions in which SPA was not present, platelet aggregation induced by ADP, collagen, and epinephrine was studied. Table 4 describes the results we obtained. Platelet aggregation in response to epinephrine was reduced in the majority of the patients both in WB and PRP. On the contrary, platelet response to collagen and ADP was different in PRP with respect to WB. PRP from the majority of patients had normal or reduced aggregation, and few showed increased aggregation. Using normalized WB, the percentage of subjects with platelet hyper-function was comparatively greater, while the majority of patients had hyper-aggregation when native WB was used. The only exceptions were the patients with PV: in this pathological condition, although the majority of subjects had normal platelet aggregation in PRP, reduced platelet function in native WB was commonly observed.
DISCUSSION In vivo platelet aggregation occurs in the presence of red cells and at different platelet concentrations in each subject. Despite this, the study of in vitro platelet aggregation usually is performed in PRP with fixed platelet count because the densitometric method of Born7 is not suitable for the study of platelet function under conditions that do not allow the passage of light (presence of red cells, very high platelet concentration). Fortunately, recent methodologic improvements have allowed the study of platelet function in WB. Investigations with the impedenzometric method of Cardinal and Flower8 and the single platelet counting method 9 demonstrated that erythrocytes can interfere with platelet aggregation with both stimulatory 410 " and inhibitory mechanisms. 2312 Moreover, in some pathologic conditions characterized by a thrombotic tendency, WB studies revealed platelet hyper-function where PRP studies showed normal or reduced platelet aggregation.1314 In a previous investigation, we compared platelet aggregation in PRP (studied by the method of Born) with that in WB (studied by the impedenzometric method) in 18 patients with IM. Our results demonstrated normal or reduced platelet function in PRP and platelet hyper-function in WB in most patients.5 The present study of additional 31 subjects with IM confirmed these findings; in addition, it demonstrated that similar discrepancies between WB and PRP platelet aggregation may occur in CML, ET, and PV. The high incidence of SPA in native WB from patients with myeloproliferative disorders seems to have been partially dependent on the high platelet concentration because spontaneous platelet clumping often was observed in reactive thrombocytosis. However, the
A.J.C.P. • January 1991
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count from both myeloproliferative disorders and RT. In contrast, when WB samples contained a normal platelet concentration, SPA was observed only in myeloproliferative disorders. Combining the results obtained in native WB from patients with 300 X 109 platelets/L (300 X 103/ mm 3 ) or less and in normalized WB from patients with more than 300 X 109 platelets/L, SPA was observed in 39% of cases of IM, 9% of ET, 13% of PV, 15% of CML, and 0% of RT. SPA was observed in PRP samples from a consistent number of subjects with high platelet count, whereas it was exceptionally rare in samples with normal platelet concentration. The addition of aspirin or apyrase to the samples of native WB before stirring inhibited SPA in most cases: SPA was always prevented or reduced by aspirin, whereas apyrase inhibited or reduced SPA in 100% of patients with CML and PV, 78% of IM, 75% of ET, and 80% of RT.
85
BALDUINI ET AL.
Platelet Aggregation in PRP and WB TABLE 4. PLATELET AGGREGATION IN RESPONSE TO COLLAGEN, ADP, AND EPINEPHRINE IN NORMALIZED PRP, NORMALIZED WB, AND NATIVE WB. VALUES REPRESENT % OF PATIENTS WITH NORMAL (N), REDUCED (R), OR INCREASED (I) PLATELET AGGREGATION Normalized PRP* ADP
Collagen
IM CML ET PV
Epinephrine
N
R
/
N
R
I
N
R
/
50 56 77 74
50 35 19 22
0 9 4 4
43 53 71 61
50 29 29 22
7 18 0 17
33 38 29 9
63 62 71 87
4 0 0 4
Normalized WB} ADP
Collagen N
R
/
N
R
/
N
R
/
63 41 41 35
16 17 14 15
21 42 45 50
26 28 24 30
37 45 45 40 Native WB%
37 27 31 30
22 7 14 0
47 76 76 90
31 17 10 10
ADP
Collagen
IM CML ET PV 3
Epinephrine
N
R
/
N
R
/
N
R
/
12 28 8 29
6 8 0 41
82 64 92 30
6 24 15 6
18 24 8 76
76 52 77 18
12 12 23 6
76 68 53 88
12 20 24 6
IM = idiopathic myelofibrosis; ET = essential thrombocytosis; PV = polycythemia vera; CML chronic myelogenous leukemia; RT = reactive thrombocytosis. • PRP containing 250 X 10' platelets/L
high platelet concentration was not the only factor responsible for SPA in myeloproliferative disorders; this phenomenon also was observed in samples with normal platelet concentration. SPA in WB preparations containing 300 X 109 platelets/L (300 X 103/mm3) or less was more frequent in IM than in ET, PV, and CML, and it never occurred in RT. The inhibitory effect of aspirin and apyrase on SPA suggests that extracellular ADP and a cyclooxygenase-dependent mechanism were responsible for this phenomenon. Our previous observation—that red cells from patients with IM were unable to induce SPA in normal platelets, whereas normal red cells induced SPA in pathologic platelets—supports the hypothesis that the presence of erythrocytes, through chemical and/or physical mechanisms, reveals an intrinsic platelet abnormality. The comparison of platelet aggregation in PRP with that in WB after collagen and ADP stimulation confirms that, red blood cells may exert a pro-aggregating effect in myeloproliferative disorders. This effect is not a constant feature; in some subjects, platelet aggregation was unchanged or reduced in the presence of erythrocytes. Finally, our
t Whole blood containing 250 X 10' plate!ets/L and 40% red blood cell Ht. X Unmodified whole blood.
results confirmed that platelet hypo-function in PRP after stimulation with epinephrine is a feature that distinguishes all myeloproliferative disorders,1 while revealing that it also occurs when WB is used. In conclusion, our investigation demonstrated that in myeloproliferative disorders the study of platelet aggregation in WB reveals platelet hyper-function, whereas the optical method of Born in PRP demonstrates normal or reduced platelet aggregation. This difference is more often observed in IM than in ET, PV, or CML and seems to be dependent on an intrinsic abnormality of platelets that may be activated by stirring in the presence of red cells.
REFERENCES 1. Schafer AI. Bleeding and thrombosis in the myeloproliferative disorders. Blood 1984;64:1-22. 2. Abbate R, Favilla S, Boddi M, Costanzo G, Prisco D. Factors influencing platelet aggregation in whole blood. Am J Clin Pathol 1986;86:91-96. 3. Balduini CL, Bertolino G, Noris P, Ascari E. The effect of red cells on platelet aggregation. A study with the electronic whole blood aggregometer. Scand J Clin Lab Invest 1988;48:337-340.
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IM CML ET PV
Epinephrine
86
COAGULATION AND TRANSFUSION MEDICINE Original Article
4. Reimers RC, Sutera SP, Joist JH. Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms. Blood 1984;64:1200-1206. 5. Balduini CL, Bertolino G, Gamba G, et al. Platelet aggregation in platelet rich plasma and whole blood in 18 patients affected by idiopathic myelofibrosis. Eur J Haematol 1988;41:267-272. 6. Balduini CL, Sinigaglia F, Damiani G, Bertolino G, Bisio A, Zocchi E. Effect of a monoclonal antibody against GPs Hb-IIIa on platelet aggregation and ATP secretion. Exp Hematol 1986;13:338-342. 7. Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962;194:927-929. 8. Cardinal DC, Flower RJ. The electronic aggregometer. A novel device for assessing platelet behaviour in blood. J Pharmacol Methods 1980;3:135-138. 9. Butchers J, Humphrey PPA, Hyde JJ, Lumley P, Spurting NW. The evaluation of a new electronic counting technique for the measurement of platelet aggregation in human whole blood in vitro. Br J Pharmacol 1980;70:160-165.
10. Riess H, Braun G, Brehm G, Hiller E. Critical evaluation of platelet aggregation in whole human blood. Am J Clin Pathol 1986;85: 50-56. 11. Saniabadi AR, Lowe GDO, Barbenel JC, Forbes CD. A comparison of spontaneous platelet aggregation: relative importance of clinical and physical mechanisms. Thromb Haemost 1984;51:115-118. 12. Galvez A, Badimon L, Badimon JJ, Fuster V. Electrical aggregometry in whole blood from human, pig and rabbit. Thromb Res 1986;56: 128-132. 13. Gordge A, Dodd NJ, Rylance PB, Weston MJ. An assessment of whole blood impedance aggregometry using blood from normal subjects and haemodialysis patients. Thromb Res 1984;36:1727. 14. Juhan-Vague I, Chignard M, Poisson C, et al. Possible participation of adenosine 5-diphosphate, arachidonic acid and PAF-acether in the platelet proaggregating effect of uncontrolled insulin-depenent diabetic erythrocytes in vitro. Thromb Res 1985;38:8389.
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A.J.C.P. • January 1991
Platelet Aggregation in Platelet-Rich Plasma and Whole Blood in 120 Patients with Myeloproliferative Disorders CARLO L. BALDUINI, M.D., GIAMPIERA BERTOLINO, M.D., PATRIZIA NORIS, M.D., AND GIAN CARLO PILETTA, M.D.
platelet count because it also occurred in samples with normal platelet content (at variance with 13 subjects with reactive thrombocytosis, in which SPA was observed only in samples with high platelet concentration). Platelets from patients with idiopathic myelofibrosis had the highest tendency to undergo SPA. (Key words: Platelet aggregation; Idiopathic myelofibrosis; Essential thrombocytosis; Polycythemia vera; Chronic myelogenous leukemia; Myeloproliferative disorders) Am J Clin Pathol 1991;95:82-86
Platelets in myeloproliferative disorders are abnormal in many ways. One common abnormality is a reduction of platelet aggregation in platelet-rich plasma (PRP) after stimulation with many physiological stimuli.1 Such evidence has been obtained with the optical method of Born, which assesses in vitro platelet function in experimental conditions quite different from those occurring in vivo. One of the most relevant differences is the absence of red cells, which are known to interfere in many ways with platelet aggregation.2"4 For this reason, we previously studied platelet function in whole blood (WB) in 18 myelofibrotic patients by the impedenzometric method of Cardinal and Flower and compared these results with those obtained in PRP by the optical method of Born.5 While PRP studies revealed that 33% and 66% of patients were hypo-aggregating and normo-aggregating, respectively, WB studies demonstrated spontaneous platelet aggregation (SPA) in 55% of cases. On the basis of these
preliminary data, we extended our study to include a larger number of patients with myeloproliferative disorders. MATERIALS AND METHODS Patients
One hundred twenty consecutive subjects with myeloproliferative disorders observed in our department from January 1987 to December 1989 were investigated. Thirtyone had idiopathic myelofibrosis (IM), 32 had essential thrombocytosis (ET), 23 had polycythemia vera (PV), and 34 had Phi-positive chronic myelogenous leukemia (CML) in chronic phase. Thirteen subjects with reactive thrombocytosis (RT) after withdrawal of cytotoxic drug therapy or splenectomy also were investigated. Table 1 describes some characteristics of these patients. All patients underwent bone marrow trephine biopsy of the posterior iliac crest. Red cell mass was measured in subjects with ET and PV. Any medication interfering with platelet function was withdrawn at least one week before From the Institute of Clinical Medicine 2, Department of Interna! the study of platelet function. Medicine, University of Pavia-IRCCS S. Matteo, Pavia. Italy. Received January 8, 1990; received revised manuscript and accepted for publication April 9, 1990. Supported by grants from MPI and CNR, Italy. Address reprint requests to Dr. Balduini: Clinica Medica II, IRCSS S. Matteo, Piazzale Giolgi, 27100 Pavia, Italy. 82
Platelet
Aggregation
Blood obtained by venipuncture of the antecubital vein was mixed with 3.8 g/dL trisodium citrate (blood/anti-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
In vitro platelet aggregation in platelet-rich plasma (PRP) and in whole blood (WB) was assessed in 31 patients with idiopathic myelofibrosis, 32 with essential thrombocytosis, 23 with polycythemia vera, and 34 with chronic myelogenous leukemia. In PRP most subjects showed normal or reduced platelet aggregation, whereas in WB the majority of patients showed increased platelet function. Spontaneous platelet aggregation (SPA) was observed frequently in WB, whereas it was seldom observed in PRP. SPA in WB was inhibited by in vitro addition of aspirin and apyrase, and SPA was only partially dependent on high
83
BALDUINI ET AL. Platelet Aggregation in PRP and WB TABLE 1. CLINICAL AND LABORATORY DATA OF THE PATIENTS WITH MYELOPROLIFERATIVE DISORDERS AND REACTIVE THROMBOCYTOSIS Sex
IM ET PV CML RT 3
No. of Patients
M
F
Age (yr)
WBC (X 10'IL)
Hb* (gldL)
Platelets (X. 10"IL)
31 32 23 34 14
23 11 11 16 5
8 21 12 18 9
64 (47-85) 53(27-72) 53 (23-77) 52 (20-83) 46(19-65)
14.9(1.9-49.0) 9.3 (4.9-35.3) 16.3 (7.3-56.0) 72.6 (4.5-227.2) 8.8(4.7-18.9)
11.6(7.5-16.1) 13.1 (7.7-15.2) 17.4(14.0-23.1) 11.4(9.3-14.6) 12.6(11.3-15.8)
406(143-1,325) 838(618-1,900) 559(158-1,080) 491 (147-1,720) 670(468-1,697)
IM » idiopathic myelofibrosis; ET = essential thrombocytosis; PV = polycythemia vera; CML chronic myelogenous leukemia; RT = reactive thrombocytosis. • SI unit = g/dL X 10 - g/L.
each agonist was classified as hypo-, hyper-, and normoaggregation when the responses to at least two of the three agonist concentrations were lower than, higher than, or within the reference range. Reference values were based on results obtained in the study of 40 healthy control subjects. SPA was defined as a spontaneous decrease of optical density of PRP of more than 20% or an increase of impedance superior to 0.5 ohm within 15 minutes from the beginning of stirring. When SPA was present, the effect of preincubation of samples (5 minutes at 37 °C, without stirring) with acetyl-salycilic acid (2 g/L) and apyrase (100 mg/L) (Sigma Chemical Company) was evaluated. Preliminary experiments demonstrated that this amount of apyrase completely inhibited platelet aggregation in WB that was exposed to 1 /imol/L ADP. RESULTS Tables 2 and 3 describe the occurrence of SPA in PRP and WB both in the native and normalized preparations. SPA often was observed in WB samples with high platelet
TABLE 2. SPONTANEOUS PLATELET AGGREGATION IN PRP IN PATIENTS WITH MYELOPROLIFERATIVE DISORDERS AND REACTIVE THROMBOCYTOSIS (IN PARENTHESES NUMBER OF PATIENTS WITH SPA/NUMBER OF PATIENTS STUDIED) % of Patients with SPA
All patients (native PRP)* Patients with pit > 300 X lO'/L. (native PRP)* Patients with pit > 300 X 109/L. (normalized PRP)t Patients with pit < 300 X 109/L (native PRP)*
IM
ET
PV
CML
RT
13 (4/31) 19 (3/16) 0 (0/16) 7 (1/15)
28 (9/32) 28 (9/32) 3 (1/32)
9 (2/23) 13 (2/15) 0 (0/15) 0 (0/8)
3 (1/34) 8 (1/13) 0 (0/13) 0 (0/21)
28 (4/14) 28 (4/14) 0 (0/14) 0 (0/0)
—
(0/0)
IM = idiopathic myelofibrosis; ET = essential thrombocytosis; PV = polycythemia vera; CML = chronic myelogenous leukemia; RT = reactive thrombocytosis. * PRP with platelet concentration equal to that one observed in each patient, t PRP containing 250 X 109 platelets/L.
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coagulant, 9/1). PRP and platelet poor plasma (PPP) were obtained by standard methods as previously reported.6 PRP was adjusted with PPP to 250 X 109 platelets/L (250 X 103/mm3) (normalized PRP) and to a concentration of platelets equal to that observed in each patient (native PRP). Platelet aggregation in PRP was studied by the optical method of Born7 with a whole blood lumiaggregometer model 500 (Chrono-Log Corporation, Havertown, PA). Platelet aggregation in WB was studied by the impedenzometric method of Cardinal and Flower8 with the same instrument. Platelet function was investigated in unmodified samples (native WB) and in samples reconstituted, mixing appropriate amounts of packed red cells, PRP, and PPP from each patient to obtain 0.40 (40%) red blood cell Hct and 250 X 109 platelets/L (250 X 103/ mm 3 ) (normalized WB).3 In both PRP and WB, platelet aggregation was stimulated by the addition of ADP (1, 5, and 20 /tmol/L, final concentration) (Sigma Chemical Company, St Louis, MO), epinephrine (1, 5, and 10 junol/L), and collagen ( 1 , 5 , and 20 mg/L) (Mascia Brunelli, Milan, Italy). Platelet aggregation stimulated by
84
COAGULATION AND TRANSFUSION MEDICINE Original Article TABLE 3. SPONTANEOUS PLATELET AGGREGATION IN WHOLE BLOOD IN PATIENTS WITH MYELOPROLIFERATIVE DISORDERS AND REACTIVE THROMBOCYTOSIS (IN PARENTHESES NUMBER OF PATIENTS WITH SPA/NUMBER OF PATIENTS STUDIED) % of Patients with SPA
All patients (native WB)* Patients with pit > 300 X 109/L (native WB)* Patients with pit > 300 X 10'/L (normalized WB)f Patients with pit < 300 X 109/L (native WB)*
IM
ET
PV
CML
RT
45 (14/31) 50 (8/16) 37 (6/16) 40 (6/15)
59 (19/32) 59 (19/32) 9 (3/32)
26 (6/23) 40 (6/15) 20 (3/15) 0 (0/8)
26 (9/34) 53 (7/13) 23 (3/13) 9 (2/21)
64 (9/14) 64 (9/14) 0 (0/14)
—
(0/0)
— (0/0)
1M = idiopathic myelofibrosis: ET = essential thrombocytosis: PV = polycythemia vera; CML = chronic myelogenous leukemia; RT = reactive thrombocytosis. * Unmodified whole blood. t Whole blood containing 250 X 10' platelets/L and 40% red blood cell Ht.
In the patients and in the experimental conditions in which SPA was not present, platelet aggregation induced by ADP, collagen, and epinephrine was studied. Table 4 describes the results we obtained. Platelet aggregation in response to epinephrine was reduced in the majority of the patients both in WB and PRP. On the contrary, platelet response to collagen and ADP was different in PRP with respect to WB. PRP from the majority of patients had normal or reduced aggregation, and few showed increased aggregation. Using normalized WB, the percentage of subjects with platelet hyper-function was comparatively greater, while the majority of patients had hyper-aggregation when native WB was used. The only exceptions were the patients with PV: in this pathological condition, although the majority of subjects had normal platelet aggregation in PRP, reduced platelet function in native WB was commonly observed.
DISCUSSION In vivo platelet aggregation occurs in the presence of red cells and at different platelet concentrations in each subject. Despite this, the study of in vitro platelet aggregation usually is performed in PRP with fixed platelet count because the densitometric method of Born7 is not suitable for the study of platelet function under conditions that do not allow the passage of light (presence of red cells, very high platelet concentration). Fortunately, recent methodologic improvements have allowed the study of platelet function in WB. Investigations with the impedenzometric method of Cardinal and Flower8 and the single platelet counting method 9 demonstrated that erythrocytes can interfere with platelet aggregation with both stimulatory 410 " and inhibitory mechanisms. 2312 Moreover, in some pathologic conditions characterized by a thrombotic tendency, WB studies revealed platelet hyper-function where PRP studies showed normal or reduced platelet aggregation.1314 In a previous investigation, we compared platelet aggregation in PRP (studied by the method of Born) with that in WB (studied by the impedenzometric method) in 18 patients with IM. Our results demonstrated normal or reduced platelet function in PRP and platelet hyper-function in WB in most patients.5 The present study of additional 31 subjects with IM confirmed these findings; in addition, it demonstrated that similar discrepancies between WB and PRP platelet aggregation may occur in CML, ET, and PV. The high incidence of SPA in native WB from patients with myeloproliferative disorders seems to have been partially dependent on the high platelet concentration because spontaneous platelet clumping often was observed in reactive thrombocytosis. However, the
A.J.C.P. • January 1991
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count from both myeloproliferative disorders and RT. In contrast, when WB samples contained a normal platelet concentration, SPA was observed only in myeloproliferative disorders. Combining the results obtained in native WB from patients with 300 X 109 platelets/L (300 X 103/ mm 3 ) or less and in normalized WB from patients with more than 300 X 109 platelets/L, SPA was observed in 39% of cases of IM, 9% of ET, 13% of PV, 15% of CML, and 0% of RT. SPA was observed in PRP samples from a consistent number of subjects with high platelet count, whereas it was exceptionally rare in samples with normal platelet concentration. The addition of aspirin or apyrase to the samples of native WB before stirring inhibited SPA in most cases: SPA was always prevented or reduced by aspirin, whereas apyrase inhibited or reduced SPA in 100% of patients with CML and PV, 78% of IM, 75% of ET, and 80% of RT.
85
BALDUINI ET AL.
Platelet Aggregation in PRP and WB TABLE 4. PLATELET AGGREGATION IN RESPONSE TO COLLAGEN, ADP, AND EPINEPHRINE IN NORMALIZED PRP, NORMALIZED WB, AND NATIVE WB. VALUES REPRESENT % OF PATIENTS WITH NORMAL (N), REDUCED (R), OR INCREASED (I) PLATELET AGGREGATION Normalized PRP* ADP
Collagen
IM CML ET PV
Epinephrine
N
R
/
N
R
I
N
R
/
50 56 77 74
50 35 19 22
0 9 4 4
43 53 71 61
50 29 29 22
7 18 0 17
33 38 29 9
63 62 71 87
4 0 0 4
Normalized WB} ADP
Collagen N
R
/
N
R
/
N
R
/
63 41 41 35
16 17 14 15
21 42 45 50
26 28 24 30
37 45 45 40 Native WB%
37 27 31 30
22 7 14 0
47 76 76 90
31 17 10 10
ADP
Collagen
IM CML ET PV 3
Epinephrine
N
R
/
N
R
/
N
R
/
12 28 8 29
6 8 0 41
82 64 92 30
6 24 15 6
18 24 8 76
76 52 77 18
12 12 23 6
76 68 53 88
12 20 24 6
IM = idiopathic myelofibrosis; ET = essential thrombocytosis; PV = polycythemia vera; CML chronic myelogenous leukemia; RT = reactive thrombocytosis. • PRP containing 250 X 10' platelets/L
high platelet concentration was not the only factor responsible for SPA in myeloproliferative disorders; this phenomenon also was observed in samples with normal platelet concentration. SPA in WB preparations containing 300 X 109 platelets/L (300 X 103/mm3) or less was more frequent in IM than in ET, PV, and CML, and it never occurred in RT. The inhibitory effect of aspirin and apyrase on SPA suggests that extracellular ADP and a cyclooxygenase-dependent mechanism were responsible for this phenomenon. Our previous observation—that red cells from patients with IM were unable to induce SPA in normal platelets, whereas normal red cells induced SPA in pathologic platelets—supports the hypothesis that the presence of erythrocytes, through chemical and/or physical mechanisms, reveals an intrinsic platelet abnormality. The comparison of platelet aggregation in PRP with that in WB after collagen and ADP stimulation confirms that, red blood cells may exert a pro-aggregating effect in myeloproliferative disorders. This effect is not a constant feature; in some subjects, platelet aggregation was unchanged or reduced in the presence of erythrocytes. Finally, our
t Whole blood containing 250 X 10' plate!ets/L and 40% red blood cell Ht. X Unmodified whole blood.
results confirmed that platelet hypo-function in PRP after stimulation with epinephrine is a feature that distinguishes all myeloproliferative disorders,1 while revealing that it also occurs when WB is used. In conclusion, our investigation demonstrated that in myeloproliferative disorders the study of platelet aggregation in WB reveals platelet hyper-function, whereas the optical method of Born in PRP demonstrates normal or reduced platelet aggregation. This difference is more often observed in IM than in ET, PV, or CML and seems to be dependent on an intrinsic abnormality of platelets that may be activated by stirring in the presence of red cells.
REFERENCES 1. Schafer AI. Bleeding and thrombosis in the myeloproliferative disorders. Blood 1984;64:1-22. 2. Abbate R, Favilla S, Boddi M, Costanzo G, Prisco D. Factors influencing platelet aggregation in whole blood. Am J Clin Pathol 1986;86:91-96. 3. Balduini CL, Bertolino G, Noris P, Ascari E. The effect of red cells on platelet aggregation. A study with the electronic whole blood aggregometer. Scand J Clin Lab Invest 1988;48:337-340.
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IM CML ET PV
Epinephrine
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COAGULATION AND TRANSFUSION MEDICINE Original Article
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A.J.C.P. • January 1991
