- Email: [email protected]
Demonstration of a coagulation inhibitor in patients with recurrent spontaneous abortion
358
HAEMATOLOGY A N D BLOOD SOCIETY OF AUSTRALIA
sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme ininiunoassay procedures exhibited greater sensitivity and a clearer separation between normal and abnormal samples, in patients with disseminated intravascular coagulation, than did a latex assay or the staphylococcal clumping test. Preliminary clinical evaluation indicates advantages in monitoring consumption coagulopathies secondary t o disseminated intravascular coagulation and in detecting other forms of intravascular coagulation. These procedures can also detect soluble fibrin complexes (presumably crosslinked) in plasma. A protocol using both plasma and serum assays is being evaluated in the diagnosis and monitoring of intravascular coagulation.
HIGH-DOSE IgG FOR IMMUNE THROMBOCYTOPENICPURPURA (ITP): CLINICAL RESPONSE AND LYMPHOCYTE SUBSET CHANGES R. L. BREARI-EY,B. ROWBOTHAM & R . COLLINS*Divisions of Haematology & immunology, * Royal Brisbane Hospital, Qld Sixteen adults with ITP received 1.5-2.0 g/kg i-v human IgG over 3-5 consecutive days. Ten patients were within 16 w k of presentation and 6 had chronic thrombocyropenia. Peripheral blood lymphocytes were examined before and at days 8 and 14 post IgG with the OKT series of monoclonal antibodies. Fluorescence was detected visually and with a Fluorescence Activated Cell Sorter. Eleven of 12 patient, who had previously received 1-2 mg/kg prednisone for at least 6 wk had a rapid response compared to none of 4 who received IgG as their initial treatment (p = ,005). Five patients with acute ITP relapsed (platelets less than 50 x 109/1)after a mean 23 d , but 6 (3 acute, 3 chronic) are in unmaintained remission a mean 120 d post infusion. No significant correlation was found between acute changes in total lymphocyte counts, absolute numbers of OKT4 (helper, inducer) or OKT8 (suppressor, cytotoxic), T-lymphocytes and response to IgG. There was a trend (0.5 > p > O . l ) for the OKT4/8 ratio t o increase at day 8 in long-term responders compared to treatment failures. The ratio had returned to pre-treatment levels at day 14. High-dose IgG does not appear to be indicated as initial treatment for ITP, and long-term responses cannot reliably be predicted by shortterm changes in OKT4/8 lymphocyte populations.
APPLICATION OF A SENSITIVE ELISA TECHNIQUE IN THE MEASUREMENT OF PLATELET-BINDABLE IgG, IgA and IgM IN AUTO- AND ALLO-IMMUNE THROMBOCYTOPENIAS S. L. PFwi.1 E.K& D. UKB!ANICO\ADeparrment of Medicine, Monash University Medical School, A (fred Hospital, Melbourne, Vic Most methods for detecting antiplatelet antibodies arising in immune thrombocytopenias are either relatively insensitive, cumbersome or require the use of radioisotopes. A simple, rapid and quantitative enzyme-linked immunosorbent assay (ELISA) has been established to measure platelet-bindable (PB) immunoglobulins, PB-IgG, PB-IgA and PB-IgM. The wells of microtitre plates are coated with washed, pooled O + normal platelets and then incubated with dilutions of either normal or patient serum. The plates are washed and then incubated with alkaline-phosphatase-linked antibodies raised in goats against human IgG, IgA or IgM. When the plates are again washed and subsequently incubated with p-nitrophenol phosphate, the rate of colour development (A405 is proportional to serum immunoglobulin bound to the platelets. Values for patient sera were considered positive if they exceeded by more than 2 SD the mean of the values obtained from 20 normal sera. Sera from 4 patients with post-transfusion thrombocytopenia were studied: all were positive, 3 at titres of > 1/2048 for both IgG and IgA. Serum from the mother of a child with neonatal thrombocytopenia due to PIAJ and HLA incompatibility showed titres of PB-lgG, IgA and IgM > 1/2048. Of 14 patients with idiopathic thrombocytopenia serum
Pathology (1984), 16, July from 80% contained raised levels of PB-IgG; the majority also had raised PB-IgA. All 4 patients with systemic lupus erythematosus studied so far had increased levels of PB-IgC; most also had increased PB-IgA. The frequent finding of PB-IgA in serum from patients with thrombocytopenia may help explain some of the difficulties in correlating the extent of thrombocytopenia with platelet antibody levels detected by previously available methods. Whether the PB-immunoglobulins are platelet-specific antibodies or immune complexes has not yet been established. Drug-dependent antiplatelet antibodies could also be detected by the assay by measuring the increase in immunoglobulin binding in the presence of the drug. Increases greater than 2 SD above the mean obtained with 10 normal sera were considered positive. Of 29 patients with thrombocytopenia following ingestion of Quinine or Quinidine, 22 showed increased PB-IgC. In some, drug-dependent increases in PB-IgA and PB-IgM were also observed. This procedure is thus able to detect different types of antiplatelet antibody and comparing platelets from different donors is applicable to platelet cross-matching procedures.
DEMONSTRATION OF A COAGULATIONINHIBITOR IN PATIENTS WITH RECURRENT SPONTANEOUS ABORTION SIEWCHOONG,M. BROADWAY, B. G. F I R K I N& M. A. HOWARD Department of Medicine, Monash Medical School, Alfred Hospital, Melbourne, Vic A correlation has previously been noted between the presence of the lupus inhibitor and multiple spontaneous abortions. The current investigation was undertaken t o extend ,these observations t o include a group of women with recurrent spontaneous abortions (RSA) without an underlying cause. A coagulation profile including the partial thromboplastin time with kaolin (PTTK) and a dilute simplastin time test (DSTT) was carried out on 2 groups of patients. Ten patients with the lupus inhibitor and defined systemic lupus erythematosus and 24 women with RSA were investigated. All patients with the lupus inhibitor had a prolonged PTTK which failed to correct when mixed with an equal volume of fresh normal plasma. Correction was achieved in each instance by substitution of activated platelets for the phospholipid in the PTTK. Using the DSTT 8/10 of these patients showed a significant prolongation at a 1/200 dilution of simplastin which also failed to correct on mixing with fresh normal plasma. 18/24 patients from the RSA group had a normal coagulation profile. The 5 patients from the RSA group showed a prolonged DSTT as an isolated coagulation abnormality and one patient had both a prolonged PTTK and DSTT. Correction by fresh normal plasma was not achieved indicating the presence of an inhibitor of the intrinsic clotting mechanism of these patients. Thus it is possible to identify 2 forms of the lupus inhibitor: one is identified by a prolonged PTTK and is bypassed by activated platelets; a second inhibitor is recognized by the DSTT and appears to be associated with recurrent spontaneous abortion.
MOLECULAR CHARACTERIZATION OF DRUG-DEPENDENT ANTIBODY-PLATELET INTERACTION USING MONOCLONAL ANTIBODIES MICHAEL C. BERNDT,H . A. BULL,B. H . CHONG,H. ZOLA*& P . A. CASTALDIDepartment of Medicine, University of Sydney, Westmead Centre, Westmead, NS W, and *Department of Immunology, Flinders Medical Centre, Adelaide, SA Recent data from our laboratory have provided the first definitive evidence that quinine/quinidine drug-dependent antiplatelet antibodies interact with the platelet membrane glycoprotein Ib “complex”. The G P Ib “complex” consists of G P Ib (2 disulfide-linked, glycosylated
HAEMATOLOGY A N D BLOOD SOCIETY OF AUSTRALIA
sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme ininiunoassay procedures exhibited greater sensitivity and a clearer separation between normal and abnormal samples, in patients with disseminated intravascular coagulation, than did a latex assay or the staphylococcal clumping test. Preliminary clinical evaluation indicates advantages in monitoring consumption coagulopathies secondary t o disseminated intravascular coagulation and in detecting other forms of intravascular coagulation. These procedures can also detect soluble fibrin complexes (presumably crosslinked) in plasma. A protocol using both plasma and serum assays is being evaluated in the diagnosis and monitoring of intravascular coagulation.
HIGH-DOSE IgG FOR IMMUNE THROMBOCYTOPENICPURPURA (ITP): CLINICAL RESPONSE AND LYMPHOCYTE SUBSET CHANGES R. L. BREARI-EY,B. ROWBOTHAM & R . COLLINS*Divisions of Haematology & immunology, * Royal Brisbane Hospital, Qld Sixteen adults with ITP received 1.5-2.0 g/kg i-v human IgG over 3-5 consecutive days. Ten patients were within 16 w k of presentation and 6 had chronic thrombocyropenia. Peripheral blood lymphocytes were examined before and at days 8 and 14 post IgG with the OKT series of monoclonal antibodies. Fluorescence was detected visually and with a Fluorescence Activated Cell Sorter. Eleven of 12 patient, who had previously received 1-2 mg/kg prednisone for at least 6 wk had a rapid response compared to none of 4 who received IgG as their initial treatment (p = ,005). Five patients with acute ITP relapsed (platelets less than 50 x 109/1)after a mean 23 d , but 6 (3 acute, 3 chronic) are in unmaintained remission a mean 120 d post infusion. No significant correlation was found between acute changes in total lymphocyte counts, absolute numbers of OKT4 (helper, inducer) or OKT8 (suppressor, cytotoxic), T-lymphocytes and response to IgG. There was a trend (0.5 > p > O . l ) for the OKT4/8 ratio t o increase at day 8 in long-term responders compared to treatment failures. The ratio had returned to pre-treatment levels at day 14. High-dose IgG does not appear to be indicated as initial treatment for ITP, and long-term responses cannot reliably be predicted by shortterm changes in OKT4/8 lymphocyte populations.
APPLICATION OF A SENSITIVE ELISA TECHNIQUE IN THE MEASUREMENT OF PLATELET-BINDABLE IgG, IgA and IgM IN AUTO- AND ALLO-IMMUNE THROMBOCYTOPENIAS S. L. PFwi.1 E.K& D. UKB!ANICO\ADeparrment of Medicine, Monash University Medical School, A (fred Hospital, Melbourne, Vic Most methods for detecting antiplatelet antibodies arising in immune thrombocytopenias are either relatively insensitive, cumbersome or require the use of radioisotopes. A simple, rapid and quantitative enzyme-linked immunosorbent assay (ELISA) has been established to measure platelet-bindable (PB) immunoglobulins, PB-IgG, PB-IgA and PB-IgM. The wells of microtitre plates are coated with washed, pooled O + normal platelets and then incubated with dilutions of either normal or patient serum. The plates are washed and then incubated with alkaline-phosphatase-linked antibodies raised in goats against human IgG, IgA or IgM. When the plates are again washed and subsequently incubated with p-nitrophenol phosphate, the rate of colour development (A405 is proportional to serum immunoglobulin bound to the platelets. Values for patient sera were considered positive if they exceeded by more than 2 SD the mean of the values obtained from 20 normal sera. Sera from 4 patients with post-transfusion thrombocytopenia were studied: all were positive, 3 at titres of > 1/2048 for both IgG and IgA. Serum from the mother of a child with neonatal thrombocytopenia due to PIAJ and HLA incompatibility showed titres of PB-lgG, IgA and IgM > 1/2048. Of 14 patients with idiopathic thrombocytopenia serum
Pathology (1984), 16, July from 80% contained raised levels of PB-IgG; the majority also had raised PB-IgA. All 4 patients with systemic lupus erythematosus studied so far had increased levels of PB-IgC; most also had increased PB-IgA. The frequent finding of PB-IgA in serum from patients with thrombocytopenia may help explain some of the difficulties in correlating the extent of thrombocytopenia with platelet antibody levels detected by previously available methods. Whether the PB-immunoglobulins are platelet-specific antibodies or immune complexes has not yet been established. Drug-dependent antiplatelet antibodies could also be detected by the assay by measuring the increase in immunoglobulin binding in the presence of the drug. Increases greater than 2 SD above the mean obtained with 10 normal sera were considered positive. Of 29 patients with thrombocytopenia following ingestion of Quinine or Quinidine, 22 showed increased PB-IgC. In some, drug-dependent increases in PB-IgA and PB-IgM were also observed. This procedure is thus able to detect different types of antiplatelet antibody and comparing platelets from different donors is applicable to platelet cross-matching procedures.
DEMONSTRATION OF A COAGULATIONINHIBITOR IN PATIENTS WITH RECURRENT SPONTANEOUS ABORTION SIEWCHOONG,M. BROADWAY, B. G. F I R K I N& M. A. HOWARD Department of Medicine, Monash Medical School, Alfred Hospital, Melbourne, Vic A correlation has previously been noted between the presence of the lupus inhibitor and multiple spontaneous abortions. The current investigation was undertaken t o extend ,these observations t o include a group of women with recurrent spontaneous abortions (RSA) without an underlying cause. A coagulation profile including the partial thromboplastin time with kaolin (PTTK) and a dilute simplastin time test (DSTT) was carried out on 2 groups of patients. Ten patients with the lupus inhibitor and defined systemic lupus erythematosus and 24 women with RSA were investigated. All patients with the lupus inhibitor had a prolonged PTTK which failed to correct when mixed with an equal volume of fresh normal plasma. Correction was achieved in each instance by substitution of activated platelets for the phospholipid in the PTTK. Using the DSTT 8/10 of these patients showed a significant prolongation at a 1/200 dilution of simplastin which also failed to correct on mixing with fresh normal plasma. 18/24 patients from the RSA group had a normal coagulation profile. The 5 patients from the RSA group showed a prolonged DSTT as an isolated coagulation abnormality and one patient had both a prolonged PTTK and DSTT. Correction by fresh normal plasma was not achieved indicating the presence of an inhibitor of the intrinsic clotting mechanism of these patients. Thus it is possible to identify 2 forms of the lupus inhibitor: one is identified by a prolonged PTTK and is bypassed by activated platelets; a second inhibitor is recognized by the DSTT and appears to be associated with recurrent spontaneous abortion.
MOLECULAR CHARACTERIZATION OF DRUG-DEPENDENT ANTIBODY-PLATELET INTERACTION USING MONOCLONAL ANTIBODIES MICHAEL C. BERNDT,H . A. BULL,B. H . CHONG,H. ZOLA*& P . A. CASTALDIDepartment of Medicine, University of Sydney, Westmead Centre, Westmead, NS W, and *Department of Immunology, Flinders Medical Centre, Adelaide, SA Recent data from our laboratory have provided the first definitive evidence that quinine/quinidine drug-dependent antiplatelet antibodies interact with the platelet membrane glycoprotein Ib “complex”. The G P Ib “complex” consists of G P Ib (2 disulfide-linked, glycosylated