Short Communication

Department of Gynaecology and Haematological Laboratory, De Wever Hospital, Heerlen, The Netherlands

Assessment of Whole-Blood Spontaneous Platelet Aggregation during Pregnancy Using an Impedance Particle Counter

Keywords

Abstract

Platelets Spontaneous platelet aggregation Impedance-based cell counter Pregnancy

The slide test method of Velaskar and Chitre for determining platelet aggregation in whole blood after induction of aggrega­ tion was modified for spontaneous platelet aggregation and evaluated. The reproducibility was satisfactory (CV 1-3%). The results obtained with this method and the method of Velaskar were not significantly different. The Spearman rank correlation was 0.75 (p < 0.0001). We established reference values for the particle counter method and Velaskar’s method in pregnant and non-pregnant women; no significant change in spontaneous platelet aggregation was seen throughout preg­ nancy. In order to estimate the clinical value of the test in pregnancy, we followed up a number of pregnant patients with primary enhanced spontaneous whole-blood platelet aggrega­ tion before and after treatment with low-dose acetylsalicylic acid. The test was found to be suited for the detection of spon­ taneous whole-blood platelet aggregation and for the follow-up after treatment with acetylsalicylic acid. Further studies are necessary, however, to assess the predictive value of an aber­ rant test result during pregnancy.

A.C. van H o f3 J.M.H. Ubachsa J. W.J. van Werschh

Introduction

Over the past 10 years, it has become clear that platelets play an important role in the pathogenesis of pregnancy-induced hyperten­

Received: April 23, 1991 Accepted in revised form: October 17, 1991

sion, hypertensive pregnancy and intra-uterine fetal growth retardation [1-3] although the precise mechanism is still not known. Ap­ parently then, alteration of the platelet behav­ iour may contribute to the vascular complica-

Dr. J.W.J. van Wersch Haematological Laboratory De Wever Hospital PO Box 4446 NL-6401 CX Heerlen (The Netherlands)

© 1992 S. Karger AG, Basel 0301-0147/92/ 0223—0160S2.75/0

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Haemostasis 1992;22:160-164

Materials, Methods and Patients Materials Blood was drawn from the antecubital vein with a Monovette needle No. 2 (21-gauge) into a plastic tube containing 0.3 ml trisodium citrate (1/10 vol) and into a plastic tube containing EDTA-K3 (final concentra­ tion 1.5 mmol/1 EDTA-K3). Within 5 min after blood collection, the first platelet counts in both samples were done. Concomitantly, a blood smear was made of the citrated blood. This procedure was repeated after 30 min. As Falcon et al. [ 13] showed that the stirring of blood influences the test results, the samples were only gently mixed just prior to the platelet count. The blood smears were stained with May-Griinwald-Giemsa so­ lution and counted microscopically for the quantifica­ tion of the platelet aggregates. An aggregate was de­ fined as a group of cells consisting of three or more platelets. The number of platelet aggregates was quan­ tified by 200-cell platelet differentiation and expressed as a percentage of the free platelets. The instrumental spontaneous platelet aggregation percentage was calcu­ lated from the measurements in citrated whole blood according to the following formula: platelet count at starting point platelet count at 30 min w ----------------------------------------------- X 100% . platelet count at starting point In the EDTA-K3 whole-blood we also assessed the following: the platelet count, mean platelet volume, platelet distribution width and plateletcrit. The plate­ let count in EDTA-K3 blood was determined simulta­ neously as a reference, in which no alteration in plate­ let count should occur [5, 13, 14], A Sysmex NE-8000 impedance-based particle counter (TOA Medicals, Kobe, Japan) was used for the determination of all platelet parameters. Patients One hundred and twenty healthy pregnant women were included. Their mean age was 30 years (range 1841 years). A second group consisted of 31 patients with small for gestational age fetuses whose estimated weights were below the P5 level according to Kloosterman [15]. The gestational age was based on the last menstrual period and ultrasound determination be­ tween 8 and 14 weeks. This patient group with small for gestational age fetuses was studied before and after treatment with low-dose acetylsalicylic acid (80 mg/ day). The control group consisted of 22 healthy female volunteers (mean age 26), all taking oral contracep­ tives.

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tions in pregnancy-induced hypertension. The intrinsic platelet activity can be assessed by measuring spontaneous platelet aggrega­ tion in whole blood [4, 5]. Several methods are available, among them optical aggregometry. This method requires the preparation of platelet-rich plasma by centrifugation, which removes not only the red and white blood cells, but also eliminates the largest (most active) platelets from the platelet population to be tested [4, 6, 7], Velaskar and Chitre [8] therefore presented a new test for determining spontaneous platelet aggregation in whole blood. Blood smears were made at specific time intervals after venepuncture and stained with May-Griinwald-Giemsa solution. The free and aggregated platelets were counted mi­ croscopically and the percent aggregation cal­ culated. Abbate et al. [4] compared optical aggregometry and whole-blood impedance aggregometry and concluded that whole blood should be used to study spontaneous platelet aggregation. We used an impedance-based particle platelet counter for the measurement of the whole-blood spontaneous platelet aggregation, by which the residual free platelets are counted so that the percentage of aggregated platelets can be precisely calculated. Blood smears were made according to the method of Velaskar and Chitre [8] as a reference method. Moreover, the reference values for spon­ taneous whole-blood platelet aggregation throughout pregnancy and for non-pregnant women have been assessed. Finally, the tests were applied in a number of pregnant patients with enhanced spontaneous whole-blood platelet aggregation and intra-uterine fetal growth retardation before and after treatment with acetylsalicylic acid, because numerous studies have shown the positive effects of antiplatelet therapy in pregnancy-induced hy­ pertension, pre-eclampsia and intra-uterine fetal growth retardation [9-11],

Table 1. Reproducibility of the method of Velaskar and of the particle counter method using citrated blood (n = 10)

Point of time

0-5 min 30 min

Method of Velaskar

Particle counter method

SD, G/l CV, %

SD, G/l CV, %

4.1 6.5

1.8 5.1

2.1 3.3

1.0 3.0

Table 2. Comparison of the spontaneous platelet aggregation percentages by the method of Velaskar and by the particle counter method

with ASA

Fig. 1. Comparison of whole-blood spontaneous platelet aggregation (%) before and after treatment with low-dose acetylsalicylic acid (ASA; 80 mg/day) in a group of 31 patients with small-for-gestational-age fetuses. Significance was tested with the paired t test: p value < 0.0001).

Statistical Analysis The influence of gestational age on the various parameters was determined by linear regression. To compare the other parameters, the Spearman rank cor­ relation test was used. The significance of differences between groups was tested with the paired Student t test. The reproducibility was calculated from determi­ nations in duplicate, as indicated by Haeckel [16].

Results

The reproducibility of the particle counter method is shown in table 1. The correlation between the method of Velaskar and the par­

162

X,% SD, % n

Particle counter method1

11.2 15.5 131

11.7 14.3 131

1 Spearman rank coefficient of correlation: r = 0.75 ( p < 0. 0001).

2 The difference between the methods was not statis­ tically significant at the 0.05 level (Mann-WhitneyWilcoxon test).

ticle counter method in establishing platelet aggregability is significant (r = 0.75 ; P < 0.0001). There is no significant difference be­ tween the two methods (table 2). Table 3 shows the reference values of whole-blood spontaneous platelet aggregation during pregnancy and in non-pregnant con­ trols. For the particle counter method, it ranged from 0 to 23% in pregnant women, from 0 to 18% in non-pregnant women. The values obtained with the Velaskar method differed only slightly from these fig­ ures. Figure 1 compares the mean percent whole-blood platelet aggregation before and

van Hof/Ubachs/van Wersch

Spontaneous Platelet Aggregation and Pregnancy

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with ASA

Method of Velaskar1

Table 3. Reference values for whole blood spontaneous platelet aggregation in healthy pregnant and non-pregnant women based on the 95-percentile method

Reference range, %

after treatment in pregnant women treated with low-dose acetylsalicylic acid (80 mg/day) for intra-uterine fetal growth retardation. It can be clearly seen that acetylsalicylic acid treatment normalizes (10.9 ± 6.1 %) the pre­ viously increased (49 ± 14.5%) spontaneous whole-blood platelet aggregation.

Discussion

The reproducibility of the methods for the measurement of spontaneous platelet aggregability was unsatisfactory and had to be im­ proved. We used the method of Velaskar and Chitre [8], designed for the measurement of induced whole-blood platelet aggregation, to measure the spontaneous whole blood platelet aggregation on an impedance-based particle counter. With coefficients of 1 and 3 % at the designated times, this method is highly repro­ ducible. Comparison of the particle counter method with the method of Velaskar demon­ strated a highly significant correlation (r = 0.75, p < 0.0001). Both aggregation tests, i.e. platelet aggregability, did not change signifi­ cantly during pregnancy. Moreover, there ap­ pears to be no significant difference between the normal values for the spontaneous wholeblood platelet aggregation in pregnant and non-pregnant women, which is an indication of the relative stability of platelet aggregation

particle counter method

0-31

0-23 0-18

0-20

n.s. n.s.

during pregnancy. The results of whole-blood spontaneous platelet aggregation in pregnan­ cies with intra-uterine fetal growth retarda­ tion before and after treatment with acetylsal­ icylic acid, suggest that whole-blood sponta­ neous platelet aggregation can really exist and can be normalized by that treatment, proba­ bly by diminishing the activity of the biologi­ cally more active platelets [17, 18], In conclusion, the use of the impedancebased particle counter for the measurement of spontaneous platelet aggregation is a quick, reliable and easy procedure, which eliminates the human error arising when counting the platelet aggregates microscopically. It might be useful for the detection of pregnancies at risk with elevated spontaneous whole-blood platelet aggregation and for the follow-up af­ ter antiplatelet treatment, but the precise im­ pact of elevated spontaneous whole-blood platelet aggregation in complicated pregnan­ cies still has to be established prospectively.

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Pregnant women (n = 120) Non-pregnant women (n = 22)

method of Veskalar

Significance

1 Wallenburg HCS, Rotmans N: En­ hanced reactivity of the platelet thromboxane pathway in normotensive and hypertensive pregnancies with insufficient fetal growth. Am J Obstet Gynecol 1982;144:523-528. 2 Morrison R, Crawford J, MacPherson M, Heptinstall S: Platelet behav­ iour in normal pregnancy, preg­ nancy complicated by essential hy­ pertension and pregnancy-induced hypertension. Thromb Haemost 1985;54:607-611. 3 Walker JJ, Cameron AD, Bjomsson S, Singer CRJ, Fraser C: Can plate­ let volume predict progressive hy­ pertensive disease in pregnancy? Am J Obstet Gynecol 1989; 161: 676-679. 4 Abbate R, Boddi M, Prisco D, Gensini GF: Ability of whole blood aggregometer to detect platelet hyperaggregability. Am J Clin Pathol 1989;91—1:159—164. 5 Splawinska B, Kuzniar J, Splawinski J: Is platelet aggregation present in whole blood in vitro? Thromb Haemost 1990;64:180. 6 Thompson CB, Jakubowski JA, Quinn PG, Deykin D, Valeri CR: Platelet size as a determinant of platelet function. J Fab Clin Med 1983; 101 —2:205—213.

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7 Riess H, Braun G, Brehm G, Hiller E: Critical evaluation of platelet ag­ gregation in whole human blood. Am J Clin Pathol 1986;85:50-56. 8 Velaskar DS, Chitre AP: A new as­ pect of platelet aggregation and a test to measure it. Am J Clin Pathol 1982;77:267-274. 9 Sweeny JD, Fabuzetta JW, Michielson CE, Fitzpatrick JE: Whole blood aggregation using impedance and particle counter methods. Am J Clin Pathol 1989;92:794-797. 10 Beaufils M, Donsimoni R, Uzam S, Colan JC: Prevention of pre­ eclampsia by early antiplatelet ther­ apy. Lancet 1985;i:840—842. 11 Wallenburg HCS, Makovitz JW, Dekker GA, Rotmans N: Low-dose aspirin prevents pregnancy-induced hypertension and preeclampsia in angiotensin sensitive primigravidae. Lancet 1986;i: 1—3. 12 Wallenburg HCS, Rotmans N: Pre­ vention of recurrent idiopathic fetal growth retardation by low dose aspi­ rin and dipyridamole. Am J Obstet Gynecol 1987;157:1230-1235.

13 Falcon C, Arnout J, Vermylen J: Platelet aggregation in whole blood - Studies with a platelet counting technique. Methodological aspects and some applications. Thromb Haemost 1989;61:423-428. 14 Fox SC, Burgess-Wilson M, Heptin­ stall S, Mitchell JRA: Platelet aggre­ gation in whole blood using the ultra-flo 100 platelet counter. Thromb Haemost 1982;48:327-329. 15 Kloosterman GJ: On intrauterine growth. Int J Gynecol Obstet 1970; 8:895. 16 Haeckel R: Qualitätssicherung im medizinischen Labor. Köln, Deut­ scher Ärzte-Verlag, 1985; p 163. 17 Whigham KAE, Howie PW, Drum­ mond AH, Prentice CRM: Abnor­ mal platelet function in pre-eclamp­ sia. Br J Obstet Gynecol 1978;85: 28-32. 18 Prentice CRM, McNicol GP: Coag­ ulation, fibrinolysis and platelets function in pre-eclampsia, essential hypertension and placental insuffi­ ciency. J Obstet Gynecol Br Commonw 1971;78:992-1003.

van Hof/Ubachs/van Wersch

Spontaneous Platelet Aggregation and Pregnancy

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References

Assessment of whole-blood spontaneous platelet aggregation during pregnancy using an impedance particle counter.

The slide test method of Velaskar and Chitre for determining platelet aggregation in whole blood after induction of aggregation was modified for spont...
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