Specific quantification of heparin-dependent antibodies for the diagnosis of heparin-associated thrombocytopenia using an enzyme-linked immunosorbent assay

THROMBOSIS RESEARCH 62; 377-387,199l

0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

SPECIFIC QUANTIFICATION OF HEPARIN-DEPENDENT ANTIBODIES FOR THE DIAGNOSIS OF HEPARIN-ASSOCIATED THROMBOCYTOPENIA USING AN ENZYME-LINKED IMMUNOSORBENT ASSAY

Yves GRUEL’, Alain RUPIN112, Luc DARNIGEl , Pascale MOALICREVERDIAU1, Pascale POUMIER-GASCHARD3, Christian BINETA, Pierre BARDOS2 and Jean LEROY1 2Laboratoire d’lmmunologie, Faculte de Medecine, 1 Laboratoire d’Hematologie, and 3CRTS, 37044 Tours Cedex, France. (Received 3.12.1990; accepted in revised form 7.2.1991 by Editor J. Soria) ABSTRACT

In order to specifically detect heparin-dependent antibodies in patients with suspected heparin-associated thrombocytopenia (HAT), bindable IgG an adapted ELISA test was developed. Serum-platelet (SPb-IgG) were measured in the absence and in the presence of heparin in the sera from a/ 25 normal controls, 25 patients treated by heparin without thrombocytopenia, 29 thrombocytopenic patients not receiving heparin and b/ 12 patients with confirmed HAT. In the absence of heparin, the 12 HAT sera showed normal or elevated SPbIgG levels (range =10.4-36 Arbitrary Units or AU) as compared to healthy controls (8-17.1 AU). After coincubation of HAT sera with heparin (0.25, 0.50, 0.75, and 1 IU/ml), SPb-IgG levels were consistently elevated (range = 22.8-150 AU), and this increase in IgG binding (equal in mean to 200 %) was always inhibited with 5 IU/ml of heparin. In contrast, a mean maximum increase in SPblgG levels of only 20 % was registered in all control groups whatever the tested heparin concentration. Thus, this ELISA allows the specific diagnosis of HAT by demonstrating a serum IgG binding on platelets only in the presence of therapeutic concentrations of heparin.

INTRODUCTION

Heparin-associated complication of heparin Key Words:

thrombocytopenia (HAT) is a rare but severe therapy due to heparin-dependent antibodies (1).

Heparin-associated

thrombocytopenia, 377

ELISA

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ELISA IN HAT

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These antibodies, having more frequently an IgG isotype, bind to the platelet membrane in the presence of heparin, and lead to cell activation and aggregation which account for the thrombotic complications observed in about 65 % of patients. Several different procedures have been developed to detect heparin-dependent antibodies in affected patients. The aggregation of normal control platelets by the patient plasma or serum in the presence of heparin is the most frequently used, but this method is not sufficiently sensitive (2). A significant improvement in sensitivity was achieved by using 14Cserotonin release tests with an increase in specificity by measuring the release at therapeutic (0.1 IU/ml) and very high (100 IU/ml) heparin concentrations (3). However, this test cannot be easily applied in every laboratory since it requires radioactive reagents. Recently, we developed an ELISA for the quantification of specific serum antiplatelet antibodies which has been applied to the study of thrombocytopenia in systemic lupus erythematosus (4). In the present work, we adapted this ELISA in order to HAT sera and we demonstrated that this method is a potentially study sensitive and specific in-vitro procedure to detect heparin-dependent antibodies, but only if samples are incubated with several concentrations of heparin corresponding to those reached during preventive or curative treatments by heparin.

MATERIALS AND METHODS Patients with Heparin-Associated Thrombocytopenia Twelve patients who had developed heparin-associated thrombocytopenia were studied. All of them fulfilled the following criteria: a/ They had a normal platelet count before heparin treatment. b/ They developed thrombocytopenia with a platelet count consistently lower than 100.109/L after 5 to 15 days of unfractionated heparin therapy. c/ The thrombocytopenia resolved within five days following unfractionated heparin discontinuation. d/ Other causes of thrombocytopenia (eg. infection, other drugs) were excluded. The clinical characteristics for these 12 cases are summarized in Table I. In 11 patients, heparin-induced aggregation tests performed as previously described (5) were found to be positive with the platelets from at least one normal donor. Five additional cases were also studied. In these patients, the diagnosis of HAT was primarily suspected but unlikely since another cause for thrombocytopenia was clearly found and/or the platelet count was not corrected after heparin discontinuation. The interval between heparin discontinuation and collection of samples varied from 12 to 72 hours. Controls The controls consisted of a/ 25 healthy and untransfused subjects, b/ 25 hospitalized patients receiving unfractionated heparin for 5 to 15 days and having a normal platelet count, and c/ 29 thrombocytopenic patients with a

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In the latter group, 15 patients had platelet count lower than 100.109/L. 14 had and thrombocytopenia autoimmune classical features of thrombocytopenia of variable origin, including hypersplenism, sepsis, disseminated intravascular coagulation and chemotherapy. Specimens Sera samples were collected into vacutainer tubes, centrifuged 15 mins), separated and kept frozen at -30°C until assays.

Clinical I

I

I

and Laboratory

1

PATIENT SEX AGE

Table I. Data for Patients

g,

with HAT.

1

I

I

I

(1,200

PLATELET THROYBOTIC DIC INTERVAL NADIR BETWEEN COMPLICATIONS ( a ) HEPAAIN AND (PER pL) THROMWCYTO -

MANAGEL~ENT

AND CLINICALOUTCOME (b)

AVK (c)-Recovery AVK-Recovery Antiplatelet

agents-Recovery

AVK-Recovery LMWH (d) then AVK-Recovery Surgery Antiplatelet

+ AVK-Amputation agents+AVK-Recovery

Surgery+Antiplatelet Recovery 9

M

67

9

23.000

DVT, PE

+

10

F

87

15

53.000

Arterial

-

I 11

I M

I 12

I M

I 81

I I 1751

I

I

11

35,000

9

I I 49,000

I

-

Arterial I

I

AVK+Antiplatelet agents-Recovery AVK + Antiplatelet agents-Death frorr cerebral thrombosis Surgery,

I None

1 -

agents

Antiplatelet

AVK, Antiplatelet Recovery agents+AVK

agents

- Recovery

(a) DIC = Disseminated intravascular coagulation. (b) All patients recovered normal platelet count after heparin discontinuation. (c) AVK = Antivitamin K agents. (d) LMWH = Low-Molecular weight heparin. (e) DVT, PE = Deep vein thrombosis, Pulmonary Embolism.

Enzvme-linked immunosorbent a= for Serum Platelet bindable IaG ISPbIaG) in the absence and in the oresence of heparin Serum platelet bindable IgG (SPb-IgG) were assayed using a recently developed procedure (4) but modified as follows: Normal platelets from at least 5 healthy donors with blood group 0 were prepared by differential centrifugation, washed three times in EDTA phosphate buffer pH 7.4 (0.14 M NaCI, 0.0264 M Na2 HP04 - 2 H20, 0.009 M EDTA), then fixed with 1% paraformaldehyde (PFA) in phosphate buffer saline (PBS). 15.106 PFA - fixed platelets were added in the wells of lanes A,C,E,G (target-wells) of a Nunclon

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microtiter plate ( Nunc, Kamstrup, Denmark). 50 pl of PBS-EDTA were placed into the wells of lanes B,D,F,H (platelet-free wells). After centrifugation 1,000 g for 5 minutes, the plates were incubated with 100 ul of 10% adult bovine serum in PBS (ABS) 1 hour at 20°C then washed 3 times with PBS 0.05% Tween 20. Each plate was kept frozen at -30°C for a period no longer than 3 months. All sera were diluted at 1:25 (50 pl) in ABS in the absence or in the presence of heparin from porcine intestinal mucosa (Sigma, St Louis, MO), at 5 different concentrations chosen on the basis of preliminary studies and corresponding to 0.25, 0.50, 0.75, 1 or 5 IU/ml for undiluted sera (final concentrations of 0.01, 0.02, 0.03, 0.04 or 0.2 IU/ml for sera diluted at 1:25). All sera were incubated for 1 hour at 37°C in duplicate both in target and platelet-free wells. For each plate, a standard curve was established using 5 serial dilutions (1:200 to 1:3,200) of an anti-Leka serum (6) which correspond with 200 to 12.5 arbitrary units (AU). After incubation of sera and 3 washings with PBS - 0.05% Tween 20, the platelet binding of IgG was revealed using a horse-radish peroxidase conjugated goat F(ab’)2 fragment anti-human IgG-Fc specific (Cappel, Westchester, USA) diluted at 1 :iO,OOO in ABS (50 pl) incubated for one hour at 37OC. After 3 washings, 50 pl of OPD (0.5 mg/ml) substrate were added and left for 10 minutes in the dark and the reaction stopped by H2SO4 2N (50 pl). The absorbances were read at 492 nm using a Tween-reader microplate spectrophotometer (Flow Laboratories, Irvin, Scotland) and data were transmitted to an IBM-PC XT computer equipped with the Tiftersoft program (Flow Laboratories). For each test, the absorbance of the platelet-free well was subtracted from the absorbance of the adjacent target-well. Mean values were then fitted on the standard curve and the results were expressed in arbitrary units (AU).

RESULTS Preliminarv tests (Figure 1) In order to define the concentrations of heparin which could allow the most accurate detection of heparin-dependent antibodies, we studied the serum from two patients suffering from severe HAT with thrombotic complications (L. and G. corresponding to patients 10 and 7 of Table I). The evolution of SPbIgG levels was evaluated without heparin and in the presence of 12 different concentrations of heparin ranging from 0.1 to 5 IU/ml of undiluted sera (i.e 0.004 to 0.2 IU/ml after dilution at 1:25). As compared to baseline values (without heparin), sera from both patients induced a dramatic increase in SPb-IgG levels (to 150 AU for L. and 105 AU for G.) when incubated with therapeutic concentrations of heparin (0.1 to 1 IU/ml). With higher concentrations (2 or. 5 IU/ml), this platelet binding of serum IgG was completely inhibited. The concentrations of heparin which achieved the higher IgG binding on platelets were not the same for both sera (0.6-l IU/ml for L. and 0.2-0.4 IU/ml for G.). Therefore, in addition with the determination of SPb-IgG level in the absence of heparin or in the presence of heparin at 5 IU/ml, we tested all further control and HAT sera with four additional heparin concentrations, ie 0.25, 0.5, 0.75, 1 IU/ml.

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Arbitrary Units

Arbitrary Units

120,

200

80 60

0 0

.l .2 .3 .4 .5 .6 .7 .8 .9

Heparin

1

2

5

Concentrations

0

.l .2 .3 .4 .5 .6 .7 .8 .9

1

2

5

(IU/ml)

Figure 1 SPb-IgG levels in HAT sera from patients L. and G. after coincubation with heparin concentrations varying from 0 to 5 IU/ml. (Presented data are means of two separate experiments).

Controls (Figure 2, Table II) In normal healthy subjects (n = 25) and in non thrombocytopenic patients receiving heparin (n = 25), identical SPb-IgG levels were measured without heparin (A@). After coincubation with heparin, the serum IgG binding on platelets was not significantly modified with a mean maximum increase jn AU value (recorded with either 0.25, 0.5, 0.75 or 1 IU/ml of heparin or AUMax) of only 20 percent in both groups (Ratio AUMax/AUO equal to 1.2). The variations in SPblgG levels were also studied with sera from 29 patients having an autoimmune thrombocytopenia (ATP, n = 15) or a low platelet count associated with various disorders (sepsis, cirrhosis, DIC or chemotherapy, n = 14). As expected, baseline SPb-IgG levels were found to be elevated with most of ATP sera (n =ll) as compared to normal controls, with AU 0 values higher than 30 in 9 cases. In thrombocytopenic patients having miscellaneous diseases, no or only a moderate increase (in 7 cases) of baseline SPb-IgG level was noted. In both thrombocytopenic groups, as in normal controls, no significant elevation in SPb-IgG levels could be demonstrated after coincubation of sera, platelets with unfractionated heparin. Thus, in all these cases, the ratio AU”ax/AUO did not reach values higher than 1.5 except for one serum from a patient receiving heparin (R =1.56) and another from a patient with ATP (R = 1.52). In addition, none of the 5 sera from patients for whom the diagnosis of HAT was excluded exhibited a significant increase in SPb-IgG level after coincubation with heparin, the ratio AlJMaX/AUO remaining lower than 1.5.

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Table SPb-IgG Levels in Control Groups and HAT Patients in the absence or in the presence of Heparin (0.25, 0.50, 0.75, 0.1, 1 .O and 5.0 IU/ml). Results are expressed in Arbitrary Units (means f 1 standard deviation) and ranges are indicated in parentheses. The R values correspond to the means of individual ratio caiculated as follows: Maximum AU recorded with heparin or AWax Baseline AU without heparin or AU0 0

Normal controls (n=30)

0.25

0.50

12.8 f 2.5

0.75

129

(8-17.1)

f 31

~ (i-19.3)

5.0

1.0

133 1

f23

i8-17)'

134f

R

32

Heparin Treatment (n=25)

11.2 f 1.7 11.7 f 2.2

Miscellaneous Thrombocytopenia (n=14)

19.6 f 6.6 18.5 f 6.8 21.3 f 8.1

Autoimmune Thrombocytopenia (n=l5)

33.5 f 14

31.2 f 12

36.4 f 16

34 f 15.5

(14.2-52)

(10.1-50)

(10.5-62)

(9.4-63)

(8.1-15)

(10-31.2)

(8.3-15)

(10-30.6) (10.4-38)

19.6f8~20.3f7.1~18.5t6.2~1.18t0.217 (10-30.8)

I

(10-31.8)

I

(10.4-36)

(10.3-70)

52.5 f 36 (13.5-1351 I

(10-28.4) I

I

I

I

I

I

I

150) (11.4-133)

I

1.20 f 0.190 (0.94-1.52)

I

I

46.6k41138t32.7121k8.312.97f1.61 (21-

(0.86-1.5) I

I

I

21.5 f 8.9 35 f 17.8

(l-1.4;)

11.5 f 1.8

(8.2-16.8)

I

ieparin-associated Thrombocytopenia (n-1 2)

1.20 f 0134

1 (i-19.6) 1

(12-38)

I (1.61-7.69)

Patients with Heparin-Associated Thrombocvtopenia (Table II, Figure 3) 12 patients who satisfied at all the criteria for the clinical diagnosis of HAT were studied (Table I). Four patients had acute arterial thrombosis together with thrombocytopenia and 3 presented either a new venous thromboembolic event or an aggravation of a previous vein thrombosis. In addition, biological signs of disseminated intravascular coagulation (with at least a fibrinogen level lower than 1 g/liter, ‘factor V less than 50 % and the presence of serum fibrin degradation products) were present in 2 cases . In the 12 patients and in the absence of heparin, a moderate elevation of the SPb-IgG level (A@) was found in 7 cases (range = 18.9-36), while the 5 others had baseline values lower than 18 AU and identical to those of normal controls. and with at least one of the tested In the presence of heparin concentrations (0.25, 0.50, 0.75, or 1 IU/ml), a significant increase in SPbIgG level was always demonstrated, the AlJMax values ranging from 22.8 to 150. According to the individual ratio AUMWAUO (or R), two different groups of patients could be separated: the first group including patients 1, 2, 4, 6, 9, and 11, exibited a significant but moderate heparin-dependent platelet IgG

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20-

70.

1% .c 3

16.

8 5 3

14.

2 E

12.

.g

10.

!z

8. ,

“1 6

8

10 AU

12

14

16

18

20

10

20

30

40

5.0

6.0

7.0

AU without Heparin

without Heparin Fiaure

2

SPb-IgG levels measured without heparin (Horizontal axis) and maximum AU values recorded with heparin (Vertical axis) in Normal controls (o), Patients receiving heparin with a normal platelet count (e), Miscellaneous thrombocytopenia ( A ) and Autoimmune thrombocytopenia ( A ). The symbols (+) correspond to the five patients for whom HAT was excluded (right).

160010

140.E 120. 2 % I 100. !z .r 3 2 8 Of .E 3 I

AU Max /AU ’

03

1 = 1.61 2 = 1.73 3 = 3.80 4 = 1.80 5 = 2.78 6 = 1.95 7 = 3.41 8 = 3.95 9 = 2.18 10 = 7.69 11 = 1.68 12 = 3.03

?? 7

60. 4 0.

Ej.gure 3 : SPb-IgG levels in HAT sera without (Horizontal axis, AU 0) and with heparin (Vertical axis, AU”ax). Each point and individual ratio AU”ax/ AU0 (Included Table) are numbered from 1 to 12, corresponding to patients’ numbers in Table I.

2 0.

OJ 0

-

. 20

-

. 40

-

. 60

-

.

.

.

80

100

120

AU without Heparin

.

. 140

1

160

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ELISA IN HAT

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binding with R values ranging from 1.61 to 2.16. In the second group (cases 3, 5, 7, 8, 10, and 12 with patient 5 having negative platelet aggregation tests), higher values of AUMax/AUo ratio (from 2.78 to 7.69) were recorded. However, no relationship between the amount of serum heparin-dependent antibodies and clinical severity of HAT (thrombosis) was apparent. With 5 IU/ml, the IgG platelet binding was completely inhibited in all patients, with SPb-IgG levels quite similar or lower to those measured without heparin. Each of these HAT sera were tested in 3 to 5 different experiments and always fulfilled the 3 criteria: AUMax > 20 AU, ratio AUMax/AUo > 1.5 and AU5 similar or lower than AU”.

DISCUSSION To exclude confirm or the diagnosis of heparin-associated thrombocytopenia (HAT), laboratory tests are often necessary, particularly in patients with evolutive thrombosis for whom the consequences of continuing or discontinuing heparin can be important. Several methods have been proposed to detect heparin-dependant antibodies in patients in whom HAT is suspected and the most specific and sensitive appears to be the measurement of serum-induced platelet 14Cserotonin release (3). Another approach, is to demonstrate directly the heparin-dependent binding of serum IgG on platelet surface using an enzyme-linked immunorbent assay (ELISA). This was investigated by Howe and Lynch by studying 5 HAT sera but these authors tested only one high heparin concentration (5 IU/ml) and the clinical usefulness of their procedure remained uncertain. To develop the ELISA presented here, we first defined the heparin concentrations which could allow the specific detection of heparin-dependent antibodies. Preliminary studies performed with the sera from two confirmed HAT clearly indicated that an increase of serum IgG binding on platelets could only be observed if heparin was coincubated at concentrations similar to those obtained during preventive or curative treatments. In contrast, this platelet IgG binding was always inhibited with higher concentrations of heparin equivalent to 5 IU/ml. This ELISA unimodal pattern of HAT IgG binding on platelets in the presence of heparin is quite similar to those described using 14CSerotonin release tests (3) and was related to a binding of heparin-IgG immune complexes to platelet Fc receptors (8). Thus, high concentrations of heparin may induce a dissociation of immune complexes Our leading to a decrease of heparin-dependent IgG - platelet interactions. preliminary tests also suggested that the heparin concentrations allowing the maximum binding of serum IgG to the platelet surface can vary widely from one sample to another. This latter observation explains why we systematically tested several heparin concentrations in our ELISA to study all further control and HAT sera. Each of the 12 patients with confirmed HAT exhibited an unimodal pattern of serum IgG binding on platelets in the presence of heparin with a/ SPb-IgG levels higher than 20 AU with at least one of the tested heparin concentrations 0.25, 0.5, 0.75, or 1 IU/ml, b/ a ratio AUMax/AUo higher than 1.5 and c/ SPb-IgG levels at 5 IU/ml of heparin similar

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385

or lower to those recorded without heparin. In contrast, none of the Sera from healthy controls had a maximum SPb-IgG level higher than 20 AU with heparin and the ratio AUMVAtJo (or R) always remained lower than 1.5. In addition, when sera from the other control groups (including the 5 patients for whom the diagnosis of HAT was excluded) were coincubated with heparin, no increase in SPb-IgG level was induced except in 2 cases: one serum from a patient receiving heparin with a normal platelet count for which the R was from a 1.56 but the maximum AU value was normal (17.6) and one thrombocytopenic patient with systemic lupus erythematosus (R = 1.52) but this heparin-induced platelet IgG binding was not inhibited with 5 IU/ml of heparin. Thus, the specificity of this ELISA test is very high, if we consider AUMax with heparin > 20 AU, ratio AUMax/AUe > 1.5 that the three criteria, and AU at 5 IU/ml similar or lower than AUo, have to be fulfilled for the diagnosis of heparin-associated thrombocytopenia.. The sensitivity of the test is more complex to evaluate since no standard for HAT is available. TO surmount this difficulty, Sheridan et al (3) categorized 28 suspected patients into 4 groups, with “definite”, “probable”, “possible” and “unlikely” HAT, and release test with then, they succeeded in correlating positive t 4Cserotonin the clinical likehood of HAT. According to their clinical criteria, all our 12 cases are definite (n = 4) or probable (n = 8) HAT and all have positive ELISA tests, suggesting that this procedure is also very sensitive. But, we were in our study cases with “possible” HAT, since we never unable to include heparin treatment nor readminister unfractionated heparin continue heparin-associated thrombocytopenia is strongly (rechallenge) when suspected. In comparison to platelet aggregation tests, our ELISA method can avoid false negative laboratory diagnosis of HAT. Indeed, one of our cases (Patient 5) had negative platelet aggregation tests and it was necessary to test 5 to 10 recently collected normal platelet rich plasma to detect the heparindependent aggregating factor in most of the others. This heterogeneity in platelet response to HAT sera or plasma was clearly shown by several investigators and may account for the low sensitivity of aggregation tests (up to 36 % for Kelton et al, 2). The ELISA procedure presented here allows the simultaneous study of several therapeutic concentrations of heparin with several different reactive donor’s platelets which can be selected on the basis of preliminary tests using well-defined HAT sera. In addition, plates coated with formaldehyde-fixed platelets can be kept frozen at least for 3 months without loss of sensitivity, and all these advantages might overcome the difficulties secondary to platelet aggregation tests. Since anti Leka serum is not available in all laboratories, results can also be expressed in fentogram per platelet as shown by Howe and Lynch (7) after establishing a standard curve with purified normal human IgG. However in this case, it remains usefull to check regularly the amount of platelets present in wells by using a monoclonal antibody specific for membrane glycoproteins such as Ilb/llla. In summary, this work indicates that ELISA measurement of serum heparin-dependent antibodies can allow the specific diagnosis of heparinassociated thrombocytopenia and can be more routinely applied in any laboratory than 1 4C-serotonin release tests. In addition this methodology,

386

ELISA IN HAT

which directly demonstrates the platelet antibodies could, together with 14Cserotonin the subsequent cell activation, be a useful physiopathology of HAT.

Acknowledoements: Wautier (Hbpital serum.

Vol. 62, No. 5 binding of heparin-dependent release tests which evaluate tool to investigate further the

The authors gratefully thank Dr B. Boizard and Pr J.L Lariboisiere, Paris, France) for the donation of anti-Leka

1. WARKENTIN, T.E., KELTON, J.G. Heparin-induced Med. 40, 31-44, 1989.

thrombocytopenia.

Ann. Rev.

2. KELTON, J.G., SHERIDAN, D., BRAIN, H., POWERS, P-J., TURPIE, A.G., CARTER, C.J. Clinical usefulness of testing for a heparin-dependent platelet aggregation factor in patients with suspected heparin-associated thrombocytopenia. J. Lab. C/in. Med. 103, 606-612, 1984. 3. SHERIDAN, D., CARTER, C., KELTON, J.G. A diagnostic induced thrombocytopenia. Blood, 67, 27-30, 1986.

test for heparin-

4. RUPIN A., GRUEL, Y., POUMIER-GASCHARD, P., CHASSAIGNE, M., LEROY, J., BARDOS, P. Thrombocytopenia in systemic lupus erythematosus: Association anticardiolipin Clin. with antiplatelet and antibodies. Immunol. Immunopathol., 55, 418-426, 1990. 5. LEROY, J., LECLERC, M.H., DElAHOUSSE, B., GUEROIS G., FOLOPPE, P., GRUEL, Y., TOULEMONDE, F. Treatment of heparin-associated thrombocytopenia and thrombosis with Low Molecular Weight Heparin (CY 216). Sem. Thrombos. Haemostas., 11, 326-329, 1985. 6. BOIZARD, B., WAUTIER, J.L. Leka, a new platelet antigen Glanzmann’s thrombasthenia. VOX Sanguinis, 46, 47-54, 1984. 7. HOWE, S.E., LYNCH, D.M. evaluation of thrombocytopenia 554-559, 1985.

absent

in

An enzyme-linked immunosorbent assay for the induced by heparin. J. Lab. C/in. Med., 105,

8. KELTON, J.G., SHERIDAN, D., SANTOS, A., SMITH, J., STEEVES, K., SMITH, C., BROWN, C., MURPHY, W.G. Heparin-induced thrombocytopenia: laboratory studies. Blood, 72, 925-930, 1988.

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9. SALEM, H.H, VAN DER WEYDEN, M.B. Heparin-induced thrombocytopenia. Variable platelet-rich plasma reactivity to heparin-dependent platelet aggregating factor. Pathology, 15, 297-299, 1983. 10. PFUELLER, S.L., DAVID, R. Different platelet specificities of heparindependent platelet aggregating factors in heparin-associated immune thrombocytopenia. &it. J. Haematol., 64, 149-l 59, 1986.