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Transfus. Sci. Vol. 17, No. 4, pp. 553-558, 1996 © 1997Elsevier Science Ltd. All rights reserved Printed in Great Britain 0955-3886/96 $15.00 + .00
IVBT-documented Platelet Function Correlates with Flow Cytometric Data J. Hoffmann* G. Bonacker* V. Kretschmer* ~: T. Schulzki* J. H e i m a n n s t when marked with antibodies against GP IIIb and GP Ib than those with short OT and ST (OT < 100 s/ST < 15 rain). Similar results were obtained when evaluating the data relative to the bone marrow status: patients with < 1000 WBC//~I showed significantly less platelet fluorescence when marked with anti-GP IIIb and anti-GP Ib than thrombocytopenic patients, who had a spontaneous platelet rise beyond 30,000 platelets//~l a few days later. One day after platelet transfusion, significantly more platelets with high GP IIIb and Ib expression could be found. We were also able to document better transfusion efficacy of platelet concentrates with high GP IIIb and Ib expression. Finally, patients with high bleeding scores showed less GP Ib expression on the platelets than patients with low bleeding scores. In summary, the IVBT-documented platelet function clearly corresponded to an increased expression of the collagen receptor and the thrombin receptor of platelets. © 1997 Elsevier Science Ltd. •
• Thrombocytopenic patients with identical platelet counts often show different bleeding tendencies owing to significant differences in the platelet function. This could be demonstrated by the in vitro bleeding test (IVBT). Using flow cytometry, we tried to find characteristics of platelet antigen expression in order to explain these differences in function. Thirty patients with bone marrow hypoplasia receiving 65 platelet transfusions (mainly from a cell separator) were observed for 3 to 29 days. Size, granulation and fluorescence of platelet-rich plasma (n = 522 samples) were evaluated using monoclonal antibodies against GP IIIb (collagen receptor), GP IIb/IIIa (flbrinogen receptor) and GP Ib (thrombin receptor). We defined separate gates for each antibody using the results from 50 normals and by laying an orthograde cross over the gate to divide the gate into four equal quadrants. The platelet populations were divided into four different groups according to the occlusion time (OT) of the IVBT and the Simplate ® time (ST). The thrombocytes with the most impaired function (OT > 485 s/ST > 30 rain) had significantly less platelet fluorescence
INTRODUCTION Thrombocytopenic patients show very different bleeding tendencies,which, to some extent, obviously correlate with the platelet counts. Consequently, there is great uncertainty with regard to the indications for platelet transfusion.
"Department of Transfusion Medicine and Haemostaseology, University Hospital, Marburg, Germany. tDepartment of Internal Medicine, University Hospital, Marburg, Germany. ~:Author for correspondence at Abteilung ftir Transfusionsmedizin und Gerinnungsphysiologie am Universit~itsKlinikum Marburg, Conradistral~e, D- 35033 Marburg, Germany. 553
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Basing the decision on platelet count alone does not consider platelet function. On the other hand, easy and reliable tests permitting the determination of platelet function in thrombocytopenic patients with platelet counts below 50 x 109/L do not yet exist. Such tests should allow one to predict the real bleeding risk and the efficacy of platelet transfusions. Therefore, a Consensus Conference of NIH in 1987 concluded that one of the most important directions for future research in this context is the development of a practical test to predict the likelihood of clinical platelet-related bleeding.' Previously, our group reported that the in vitro bleeding test {IVBT1, adapted for the examination of thrombocytopenic blood, is well suited for the evaluation of the bleeding risk in chronic thrombocytopenic patientsA 3 In the present study, we additionally investigated the expression of glycoproteins on platelets of patients with bone marrow hypoplasia in order to find a specific correlate for the results with the IVBT.
MATERIALS AND METHODS Clinical Study The study included 30 patients with bone marrow hypoplasia. A primary aim of the study was to investigate the patients daily over longer periods of time 13-29 days}. The study comprised a total of 522 observation days. The patients received 65 HLA-compatible platelet concentrates {PCsl from the cell separator {mainly the Fresenius AS 104}? Platelet-rich plasma IPRP, n = 522} was analysed by flow cytometry {Cytoron, Ortho Diagnostics}. PRP was separated by gravity sedimentation {about 90 min} of 4 mL citrated blood pipetted onto 3 mL of Ficoll. Size, granulation and fluorescence were evaluated using monoclonal anti-CD36 IGP IIIb, collagen receptorl, anti-CD41a {GP IIb/IIIa, flbrinogen receptor}, and anti-CD42b
{GP Ib, thrombin receptor} as markers (Immunotech, Marseille, France}. A solution of antibody and buffer was added to 100/zl PRP {2.5 × 106 platelets} and incubated in the dark on a shaker for 30 min. Then the reaction was stopped by 500 /zL 1% phosphatebuffered paraformaldehyde solution. After another incubation of 30 min, 2 mL of phosphate buffer was added before forward measuring [detection gain 130, amplification logarithmically} and side scatter {155, log] as well as green fluorescence {120, log}. The noise threshold was set in the SSC-FSC graph with an angle of 6. We measured for 20 s or for 20,000 counts. Separate gates for each antibody were defined by the results from 50 healthy blood donors. The thrombocytopenia-adapted IVBT was performed in triplicate at the Thrombostat 4000@ {VDG, Seon, Germany~ now Dade}, using 120-/zm filters. The occlusion time lOT} was measured and the median of the three determinations calculated, s Whenever patients and clinicians agreed, the in vivo bleeding time was determined by a modified IVY bleeding time, the Simplate ® time. In order to describe the bleeding tendency we calculated a bleeding score based on Crosby's classification from daily clinical examination of the patients. 3,6
RESULTS 1. Relation between IVBT and Glycoprotein Expression There was a close inverse correlation between the expression of GP IIIb as well as GP Ib with the IVBT-OT IFig. 1}. Thrombocytopenic blood samples with a very short OT {less than 100 s, group D} showed a significantly greater percentage of platelets with high GP IIIb and GP Ib expression than those with a very long OT [group A}. No significant differences could be obtained for the GP IIb/IIIa expression.
IVBT-documented Platelet Function 555
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2. Relation between Bone Marrow Status and Glycoprotein Expression
3. Relation between Simplate ® Time and Glycoprotein Expression
The bone marrow status also seemed to play a significant role with regard to platelet function {Fig. 2). With increasing leukocyte counts, and particularly before a spontaneous platelet increase beyond 30,000//zL, the IVBT-OT became significantly shorter and the GP IIIb and Ib expression increased. Similar results were obtained for GP IIb/IIIa but did not reach significance.
Simplate ® time and IVBT correlated with each other and inversely with the GP IIIb and Ib expression CFig. 3}. Again, GP IIb/IIIa expression showed the same tendency without becoming significant. 4. Relation between Bleeding Score and Glycoprotein Expression
The bleeding score also significantly 500
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556
Transfus. Sci.
Vol. 17, N o . 4
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Platelet transfusion led to a significant shortage of the IVBT-OT, which clearly correlated with the platelet increase and rise of the percentage of platelets with high GP Ib expression {from 9% pretransfusion to 22% at the first day posttransfusion, Fig. 5). We even succeeded in demonstrating better transfusion efficacy of PCs with high GP IIIb and Ib
expression than of PCs with low GP IIIb and Ib expression {Fig. 6). Despite comparable platelet increments, the WBTOT became significantly shorter after platelet transfusion of PCs with high GP IIIb and GP Ib expression than of PCs with low GP IIIb and GP Ib expression (p < 0.05}. DISCUSSION This study again confirms the results that the thrombocytopenia-adapted WBT is better suited than other methods for 500
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IVBT-documented Platelet Function
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Figure 5. IVBT and number of platelets with different GP Ib expression before and after platelet transfusion. determining the platelet-related bleeding risk in thrombocytopenic patients? ,3 Although only weak bleeding signs were observed because of prophylactic platelet transfusions, 6 IVBT and bleeding score clearly correlated with each other. Additionally, the study showed that the IVBT reflects the efficacy of platelet transfusion and is able to replace the in vivo bleeding time, which shows too high a variation, is time-consuming, and cannot be simply applied repeatedly according to the risks of
bleeding, infection and scarification. 7,8 The results of flow cytometry help to understand why different patients with equally low platelet counts show different bleeding tendencies. Stronger expression of the glycoproteins IIIb and Ib on the platelets correlates with a lower bleeding tendency as documented by a lower bleeding score and a shorter in vivo bleeding time. The glycoprotein expression appears to correlate with the bone marrow regeneration. The IVBT documents these functional differences very
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558 Transfus. Sci. Vol. 17, No. 4 sensitively and even reflects differences in the glycoprotein expression of transfused platelets. Similar correlations could be seen between glycoprotein IIb/IIIa and IVBT, and Simplate ®time as well as bone marrow status, although no significance could be found due to the high variability. No correlations could be obtained on other flow cytometric parameters like granularity and size. In conclusion, the thrombocytopeniaadapted IVBT can help to determine the platelet-related bleeding risk in thrombocytopenic patients. It is well suited for the control of platelet transfusion efficacy. The IVBT may replace the in vivo bleeding time in most cases. Additionally, the IVBT in thrombocytopenic patients allows the ex vivo analysis of parameters (e.g. drugs) that may influence non-vascular primary haemostasis in vivo. 9 Unfortunately, the IVBT with the Thrombostat 4000 is no longer available. An improved system, the so-called "Platelet function analyser" (PFA 100, Dade) is already under clinical evaluation. However, the necessary adaption for use in thrombocytopenic patients remains to be established.
REFERENCES
1. Office of Medical Application of Research, National Institute of Health: Consensus conference on platelet trans-
fusion therapy, lAMA 1987; 257:17771780. 2. Kretschmer V, Huss B, Bonacker G, Hoffmann 1, Bewarter S, Weber S, Schulzki T, K6ppler H, Heymanns J: Thrombocytopenia-adapted In Vitro Bleeding Test assesses platelet function in thrombocytopenic patients. Seminars in Thrombosis and Hemostasis 1995; Suppl. 2:79-90. 3. Kretschmer V, Huss B, Weber S, Bewarder S, Schulzki T, K6ppler H, Heymanns J: A contribution to the indications for platelet transfusion and determination of its therapeutic efficacy. Transfus Sci 1994; 15:361-369. 4. Kretschmer V, Planitz C, Dietrich G, Pflfiger KH: Transfusion results of platelet concentrates using different cell separators. Infusionsther 1991; 18:196-198. 5. Kretschmer V, Huss B, Dietrich G, Heymanns J, PfliJger KH: Determination of bleeding risk in thrombocytopenic patients receiving platelet substitution. Transfus Sci 1993; 14:27-34. 6. Crosby WH: Wet purpura, dry purpura. JAMA 1975; 232:744-745. 7. Harker LA, Slichter SJ: The bleeding time as a screening test for evaluation of platelet function. N Engl l Med 1972; 287:155-158. 8. Poller L, Thomson JM, Thomdon JA: The bleeding time: current practice in the UK. Clin Lab Haematol 1984; 6:369-373. 9. Kratzer MAA, Negrescu EV, Hirai A, Yeo YK, Franke P, Siess W: The Thrombostat system: a useful method to test antiplatelet drugs and diets. Seminars in Thrombosis and Hemostatsis 1995; Suppl. 2:25-31.