Original Paper Vox Sang 1992;63:119-121
R. W.A . M. Kuijpersa W. H . Ouwehand’ W. Peelen J. J. Michielsd C. P. Engelfriet a A . E. G. Kr. von dem Borne“.‘ Departement of Immunological Haematology, Central Laboratory of the Netherlands and Laboratory Redfor Cross Experimental Transfusion and Service Clinical Immunology, University of Amsterdam, The Netherlands; Departement of Haematological Medicine, University of Cambridge, UK; St. Jansdal Hospital, Harderwijk, Academic Hospital Dijkzigt, University of Rotterdam; ‘ Department of Haematology, Academic Medical Centre and Centre of Blood Cell Research, Amsterdam, The Netherlands
Thrombocytopenia Due to Platelet Glycoprotein IIbAlla-Reactive Autoantibodies Non-Reactive with Platelets from EDTA Blood ................................................................................................. Abstract We describe 2 patients with thrombocytopenia and autoantibodies in the blood against platelet membrane glycoprotein (GP) IIb/IIIa, in whom reactivity of the antibodies with their antigen was greatly diminished in the presence of sodium EDTA. One patient also showed a marked thrombocytopathy, suggesting a functional blocking of G P IIb/IIIa by these autoantibodies. These observations show that some immunologically important autoantibodies can easily be missed in routine serological investigations in which EDTA blood is used, obscuring the autoimmune nature of a thrombocytopenia and possibly of a thrombopathy as well.
...........
Introduction
Autoimmune thrombocytopenia is caused by circulating platelet autoantibodies. In routine serological tests for the diagnosis of autoimmune thrombocytopenia, platelets from EDTA-anticoagulated blood (EDTA platelets) are used in most laboratories. As is well known, Ca2+chelation by EDTA induces conformational changes in the glycoprotein (GP) complex G P IIb/IIIa which can lead to the binding of so-called crypt-antigen antibodies which may result in ‘pseudothrombocytopenia’ [l,21. Here, we describe that a conformational change of G P IIb/IIIa induced by EDTA may lead to the loss of the binding of some autoantibodies to platelets. Such antibodies react only weakly with platelets in the presence of EDTA, but strongly with platelets isolated from citrate blood. They were detected in the blood of 2 patients with
Received: Octoher 16. l9YO Revised manuscript received: January 3. 1992 Accepted: January 8. I992
idiopathic thrombocytopenia and a severe haemorrhagic diathesis and were directed against an epitope on G P IIW IIIa on the patients’ own platelets.
Case Histories Cuse I
A 28-year-old man presented with thrombocytopenia and purpura for which he underwent a splenectomy. Five years later, a recurrence of the haemorrhagic diathesis occurred which presented with petechiae and haematomas. The severity of the presentingsymptoms increased over 3 months and resulted in frequent bleeding from the nose and gums. On admission to the hospital, blood haemoglobin level was 6.1 mmol/l and platelet count 57x1OY/1.The bleeding time (Ivy) was >15 min. A direct immunofluorescence test with platelets was not performed. Several blood transfusions were given for the anaemia. After 6 days, the platelet count was 60X1Oy//l. Administration of random donor platelet concentrate did not give an in-
Dr. A.E.G. Kr. von dem Borne do Puhlication Secretariat Central Laboratory of the Netherlands Red Cross Blood Transfusion Service PO Box 9406. NL-1006 AK Amsterdam (The Netherlands)
0 1992 S. Karger AG. Basel 0042-9()07/Y2/0!6324)119 $2.75/0
Table 1. EDTA and citrate platelet tests ’
Serum negative control patient 1 patient 2
crement. The bleeding time remained >15 min. The patient became increasingly comatose. A CT scan showed a large intracerebral density in the left hemisphere with displacement to the right. The patient died from this cerebral haemorrhage despite high-dose intravenous gammaglobulin (IVIg) and platelet transfusions. Post-mortem bone marrow showed a reactive megakaryocytic hyperplasia. At autopsy, there was a frontoparietal brain haemorrhage. Case 2 A 16-month-old girl was admitted to the hospital because of extensive purpura which developed 1day after a common cold and 2 weeks after vaccination against mumps, measles and rubella. On admission, the platelet count was 4x1Oy/1and the bleeding time >10 min (Ivy). Because there was no improvement in the platelet count at the end of the first week, a 5-day course of IVIg was started (0.4 g IVIglkg body weightlday). The platelet count began to rise after 1day and reached 3O9x1Oy/1on day 3 and 649x1O9/1 on day 5. After 2 months, the platelet count was 283x1O9/1.
Materials and Methods Of patient 1only serum was available for investigation, whereasof patient 2, platelets could be studied after a remission was induced. Blood was drawn by venepuncture with either potassium EDTA or sodium citrate as an anticoagulant, or was allowed to clot. Immunofluorescence on paraformaldehyde-treated platelets (PIFT) was performed asdescribedbyvon demBorneet al. [3]. ‘Citrateplatelets’ were washed troughout the procedure with 0.195 mM phosphatebuffered saline (0.9% w/v) with 0.1% bovine serum albumin (w/v); EDTA platelets were washed with the same buffer in the presence of 9 mM Na2 EDTA. Fluorescein isothiocyanate-labelled polyclonal rabbit immunoglobulin (Ig) conjugates were used directed against human Ig, IgG, IgA or IgM. The monoclonal antibody immobilization of platelet antigen assay (MAIPA) was performed as described by Kiefel et al. [4]. Monoclonal antibodies (mAbs) used were CLB-CD61 (CLB-thromb/l-C17) against GP IIb/IIIa, CLB-CD42b against GPIb and CLB-CD49b (CLB-thromb/4-1OG11)against VLAa 2chain.
Results
The serum of patient 1, which was taken before the blood transfusions, gave weakly positive reactions with platelets from 3 out of 6 donors, using EDTAplatelets and
120
EDTA platelets
Citrate platelets
Ig
IgG
IgA
IgM
Ig
IgG
IgA
(+)/-
-
-
-
-
-
-
-
-
(+I -
(+)
++ ++ +++ +++
IgM
an anti-Igconjugate. No direct test was perfomed. Citrate platelets from all 6 donors gave strong reactions, caused by antibodies of the IgG class. The serum did not react with EDTA or citrate platelets from a patient with Glanzmann’s disease. No further tests could be performed because no more serum was available. Serum from the second patient, obtained during the thrombocytopenic phase was only weakly reactive with EDTA platelets in the PIFT, and with anti-Ig serum only. The tests with citrate platelets was strongly positive both with an anti-Ig and an anti-IgG conjugate, but negative with an anti-IgA and anti-IgM conjugate (results are summarized in table 1). Serum and EDTA as well as citrate-anticoagulated blood from the patient were again tested 4 days after the platelet count had returned to normal. The direct PIFT with the EDTA and citrate platelets was negative, and the serum reacted very weakly with citrate donor platelets. However, after incubation of the patients’ own platelets with the serum sample taken during the thrombocytopenic phase, the PIFT was weakly positive with EDTA platelets, but strongly positive with the citrate platelets, proving the autoimmune nature of the antibodies. To identify the antigen recognized by the autoantibodies, the serum was examined in the MAIPA. Only with mAbs directed against GP IIb/IIIa (mAb C17) and with donor platelets in the absence of EDTA, results were strongly positive.
Discussion
In 2 patients with severe idiopathic thrombocytopenia, platelet autoantibodies were detectable which reacted far more strongly with donor platelets isolated from citrate than with platelets from EDTA blood. By binding Ca2+ ions, EDTA is known to affect the conformation of the GP IIb/IIIa complex [ 5 ] ,while a total dissociation of the complex can be induced at 37°C in the presence of EDTA [6]. EDTA-dependent platelet autoantibodies are directed
Kuijpers/Ouwehand/Peelen/MichieIs/ Engelfriethon dem Borne
Antigen-Conformation-Dependent Platelet Autoantibodies
against epitopes on GP IIb/IIIa, which apparently are exposed by EDTA. Such antibodies are associated with ‘pseudothrombocytopenia’ by causing an ex-vivo aggregation of the platelets. These antibodies are often of the IgM class [2]. The autoantibodies of the above 2 patients were also directed against an epitope on the GP IIb/IIIa complex. However, the expression of this epitope was strongly diminished by exposure to EDTA. Moreover, the antibodies of the patients tested were of the IgG class and apparently capable of inducing a severe haemorrhagic diathesis. Despite the relatively high platelet count of the first patient, there was a very severe bleeding tendency. This suggests that the antibodies were also capable of inducing a functional defect of the platelets, as has been described before for GP IIb/IIIa-reactive autoantibodies [7]. The platelets from neither of the 2 patients could be examined during the thrombocytopenic phase, but the
autoimmune nature of the antibodies of the second patient was made plausible with her own platelets when her platelet count had normalized. Direct proof for the presence of platelet-bound autoantibodies in vivo was not available. Unfortunately, a direct test and an eluate test in the acute phase was not possible. However, the co-occurrence of the autoreactive antibodies and the thrombocytopenia in this case makes the causative nature of these antibodies likely. In conclusion, we describe platelet autoantibodies recongnizing epitopes on G P IIWIIIA that react preferentially with platelets prepared from citrate blood, but only very weakly with EDTA platelets. These antibodies could be easily missed in routine investigations in which mostly EDTA blood is used.
................................................................................................................................................... References 1 Pegels JG, Bruynes ECE, Engelfriet CP, et al: Pseudothrombocytopenia: An immunologic study on platelet antibodies dependent on ethylene diamine tetra acetate. Blood 1982; 59:157-161. 2 von dem Borne AEG Kr, van der Lelie J , Vos JJE, et al: Antibodies against crypt-antigensof platelets; in D&caryF, Rock GA (eds): Platelet Serology, Current Studies in Haematology and Blood Transfusion. Basel, Karger, 1986, pp 3346.
3 von dem Borne AEG Kr, Verheugt FWA, Oosterhof F: A simple immunofluorescence test for the detection of platelet antibodies. Br J Haematol 1978;61:374-375. 4 Kiefel V, Santoso S, Weisheit M, et al: Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): A new tool for the identification of platelet-reactive antibodies. Blood 1987;70:1722-1726.
5 Jennings LK, Phillips DR: Purification of glycoprotein IIb and IIIa from human platelet plasma membranes and characterization of a calcium-dependent-glycoproteinIIb-IIIa complex. J Biol Chem 1982;257:10458-10466. 6 Pidard D , Dirdry D , Kunicky TJ, Nurden A T Temperature dependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. Blood 1986;57:604. 7 Niessner H, Clemetson KJ, Panzer S , et al: Acquired thrombasthenia due to GP IIblIIIaspecific platelet autoantibodies. Blood 1986; 681571-576.
121
R. W.A . M. Kuijpersa W. H . Ouwehand’ W. Peelen J. J. Michielsd C. P. Engelfriet a A . E. G. Kr. von dem Borne“.‘ Departement of Immunological Haematology, Central Laboratory of the Netherlands and Laboratory Redfor Cross Experimental Transfusion and Service Clinical Immunology, University of Amsterdam, The Netherlands; Departement of Haematological Medicine, University of Cambridge, UK; St. Jansdal Hospital, Harderwijk, Academic Hospital Dijkzigt, University of Rotterdam; ‘ Department of Haematology, Academic Medical Centre and Centre of Blood Cell Research, Amsterdam, The Netherlands
Thrombocytopenia Due to Platelet Glycoprotein IIbAlla-Reactive Autoantibodies Non-Reactive with Platelets from EDTA Blood ................................................................................................. Abstract We describe 2 patients with thrombocytopenia and autoantibodies in the blood against platelet membrane glycoprotein (GP) IIb/IIIa, in whom reactivity of the antibodies with their antigen was greatly diminished in the presence of sodium EDTA. One patient also showed a marked thrombocytopathy, suggesting a functional blocking of G P IIb/IIIa by these autoantibodies. These observations show that some immunologically important autoantibodies can easily be missed in routine serological investigations in which EDTA blood is used, obscuring the autoimmune nature of a thrombocytopenia and possibly of a thrombopathy as well.
...........
Introduction
Autoimmune thrombocytopenia is caused by circulating platelet autoantibodies. In routine serological tests for the diagnosis of autoimmune thrombocytopenia, platelets from EDTA-anticoagulated blood (EDTA platelets) are used in most laboratories. As is well known, Ca2+chelation by EDTA induces conformational changes in the glycoprotein (GP) complex G P IIb/IIIa which can lead to the binding of so-called crypt-antigen antibodies which may result in ‘pseudothrombocytopenia’ [l,21. Here, we describe that a conformational change of G P IIb/IIIa induced by EDTA may lead to the loss of the binding of some autoantibodies to platelets. Such antibodies react only weakly with platelets in the presence of EDTA, but strongly with platelets isolated from citrate blood. They were detected in the blood of 2 patients with
Received: Octoher 16. l9YO Revised manuscript received: January 3. 1992 Accepted: January 8. I992
idiopathic thrombocytopenia and a severe haemorrhagic diathesis and were directed against an epitope on G P IIW IIIa on the patients’ own platelets.
Case Histories Cuse I
A 28-year-old man presented with thrombocytopenia and purpura for which he underwent a splenectomy. Five years later, a recurrence of the haemorrhagic diathesis occurred which presented with petechiae and haematomas. The severity of the presentingsymptoms increased over 3 months and resulted in frequent bleeding from the nose and gums. On admission to the hospital, blood haemoglobin level was 6.1 mmol/l and platelet count 57x1OY/1.The bleeding time (Ivy) was >15 min. A direct immunofluorescence test with platelets was not performed. Several blood transfusions were given for the anaemia. After 6 days, the platelet count was 60X1Oy//l. Administration of random donor platelet concentrate did not give an in-
Dr. A.E.G. Kr. von dem Borne do Puhlication Secretariat Central Laboratory of the Netherlands Red Cross Blood Transfusion Service PO Box 9406. NL-1006 AK Amsterdam (The Netherlands)
0 1992 S. Karger AG. Basel 0042-9()07/Y2/0!6324)119 $2.75/0
Table 1. EDTA and citrate platelet tests ’
Serum negative control patient 1 patient 2
crement. The bleeding time remained >15 min. The patient became increasingly comatose. A CT scan showed a large intracerebral density in the left hemisphere with displacement to the right. The patient died from this cerebral haemorrhage despite high-dose intravenous gammaglobulin (IVIg) and platelet transfusions. Post-mortem bone marrow showed a reactive megakaryocytic hyperplasia. At autopsy, there was a frontoparietal brain haemorrhage. Case 2 A 16-month-old girl was admitted to the hospital because of extensive purpura which developed 1day after a common cold and 2 weeks after vaccination against mumps, measles and rubella. On admission, the platelet count was 4x1Oy/1and the bleeding time >10 min (Ivy). Because there was no improvement in the platelet count at the end of the first week, a 5-day course of IVIg was started (0.4 g IVIglkg body weightlday). The platelet count began to rise after 1day and reached 3O9x1Oy/1on day 3 and 649x1O9/1 on day 5. After 2 months, the platelet count was 283x1O9/1.
Materials and Methods Of patient 1only serum was available for investigation, whereasof patient 2, platelets could be studied after a remission was induced. Blood was drawn by venepuncture with either potassium EDTA or sodium citrate as an anticoagulant, or was allowed to clot. Immunofluorescence on paraformaldehyde-treated platelets (PIFT) was performed asdescribedbyvon demBorneet al. [3]. ‘Citrateplatelets’ were washed troughout the procedure with 0.195 mM phosphatebuffered saline (0.9% w/v) with 0.1% bovine serum albumin (w/v); EDTA platelets were washed with the same buffer in the presence of 9 mM Na2 EDTA. Fluorescein isothiocyanate-labelled polyclonal rabbit immunoglobulin (Ig) conjugates were used directed against human Ig, IgG, IgA or IgM. The monoclonal antibody immobilization of platelet antigen assay (MAIPA) was performed as described by Kiefel et al. [4]. Monoclonal antibodies (mAbs) used were CLB-CD61 (CLB-thromb/l-C17) against GP IIb/IIIa, CLB-CD42b against GPIb and CLB-CD49b (CLB-thromb/4-1OG11)against VLAa 2chain.
Results
The serum of patient 1, which was taken before the blood transfusions, gave weakly positive reactions with platelets from 3 out of 6 donors, using EDTAplatelets and
120
EDTA platelets
Citrate platelets
Ig
IgG
IgA
IgM
Ig
IgG
IgA
(+)/-
-
-
-
-
-
-
-
-
(+I -
(+)
++ ++ +++ +++
IgM
an anti-Igconjugate. No direct test was perfomed. Citrate platelets from all 6 donors gave strong reactions, caused by antibodies of the IgG class. The serum did not react with EDTA or citrate platelets from a patient with Glanzmann’s disease. No further tests could be performed because no more serum was available. Serum from the second patient, obtained during the thrombocytopenic phase was only weakly reactive with EDTA platelets in the PIFT, and with anti-Ig serum only. The tests with citrate platelets was strongly positive both with an anti-Ig and an anti-IgG conjugate, but negative with an anti-IgA and anti-IgM conjugate (results are summarized in table 1). Serum and EDTA as well as citrate-anticoagulated blood from the patient were again tested 4 days after the platelet count had returned to normal. The direct PIFT with the EDTA and citrate platelets was negative, and the serum reacted very weakly with citrate donor platelets. However, after incubation of the patients’ own platelets with the serum sample taken during the thrombocytopenic phase, the PIFT was weakly positive with EDTA platelets, but strongly positive with the citrate platelets, proving the autoimmune nature of the antibodies. To identify the antigen recognized by the autoantibodies, the serum was examined in the MAIPA. Only with mAbs directed against GP IIb/IIIa (mAb C17) and with donor platelets in the absence of EDTA, results were strongly positive.
Discussion
In 2 patients with severe idiopathic thrombocytopenia, platelet autoantibodies were detectable which reacted far more strongly with donor platelets isolated from citrate than with platelets from EDTA blood. By binding Ca2+ ions, EDTA is known to affect the conformation of the GP IIb/IIIa complex [ 5 ] ,while a total dissociation of the complex can be induced at 37°C in the presence of EDTA [6]. EDTA-dependent platelet autoantibodies are directed
Kuijpers/Ouwehand/Peelen/MichieIs/ Engelfriethon dem Borne
Antigen-Conformation-Dependent Platelet Autoantibodies
against epitopes on GP IIb/IIIa, which apparently are exposed by EDTA. Such antibodies are associated with ‘pseudothrombocytopenia’ by causing an ex-vivo aggregation of the platelets. These antibodies are often of the IgM class [2]. The autoantibodies of the above 2 patients were also directed against an epitope on the GP IIb/IIIa complex. However, the expression of this epitope was strongly diminished by exposure to EDTA. Moreover, the antibodies of the patients tested were of the IgG class and apparently capable of inducing a severe haemorrhagic diathesis. Despite the relatively high platelet count of the first patient, there was a very severe bleeding tendency. This suggests that the antibodies were also capable of inducing a functional defect of the platelets, as has been described before for GP IIb/IIIa-reactive autoantibodies [7]. The platelets from neither of the 2 patients could be examined during the thrombocytopenic phase, but the
autoimmune nature of the antibodies of the second patient was made plausible with her own platelets when her platelet count had normalized. Direct proof for the presence of platelet-bound autoantibodies in vivo was not available. Unfortunately, a direct test and an eluate test in the acute phase was not possible. However, the co-occurrence of the autoreactive antibodies and the thrombocytopenia in this case makes the causative nature of these antibodies likely. In conclusion, we describe platelet autoantibodies recongnizing epitopes on G P IIWIIIA that react preferentially with platelets prepared from citrate blood, but only very weakly with EDTA platelets. These antibodies could be easily missed in routine investigations in which mostly EDTA blood is used.
................................................................................................................................................... References 1 Pegels JG, Bruynes ECE, Engelfriet CP, et al: Pseudothrombocytopenia: An immunologic study on platelet antibodies dependent on ethylene diamine tetra acetate. Blood 1982; 59:157-161. 2 von dem Borne AEG Kr, van der Lelie J , Vos JJE, et al: Antibodies against crypt-antigensof platelets; in D&caryF, Rock GA (eds): Platelet Serology, Current Studies in Haematology and Blood Transfusion. Basel, Karger, 1986, pp 3346.
3 von dem Borne AEG Kr, Verheugt FWA, Oosterhof F: A simple immunofluorescence test for the detection of platelet antibodies. Br J Haematol 1978;61:374-375. 4 Kiefel V, Santoso S, Weisheit M, et al: Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): A new tool for the identification of platelet-reactive antibodies. Blood 1987;70:1722-1726.
5 Jennings LK, Phillips DR: Purification of glycoprotein IIb and IIIa from human platelet plasma membranes and characterization of a calcium-dependent-glycoproteinIIb-IIIa complex. J Biol Chem 1982;257:10458-10466. 6 Pidard D , Dirdry D , Kunicky TJ, Nurden A T Temperature dependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. Blood 1986;57:604. 7 Niessner H, Clemetson KJ, Panzer S , et al: Acquired thrombasthenia due to GP IIblIIIaspecific platelet autoantibodies. Blood 1986; 681571-576.
121
