short report
Teicoplanin-dependent antibodies: detection and characterization
Stephen F. Garner,1,2 Kate Campbell,1,2 Graham Smith,1,2 Colin Hurd,1,2 Simon J. Davidson,3 Marilyn Treacy,4 John F. Burman,3 Hartmut Kroll5 and Willem H. Ouwehand1,2 1
National Blood Service, Cambridge, UK,
2
Department of Haematology, University of
Cambridge, Cambridge, UK, 3Royal Brompton Hospital, London, UK, 4Chase Farm Hospital, Enfield, UK, and 5Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany Received 6 December 2004; accepted for publication 3 February 2005 Correspondence: Stephen F. Garner, National
Summary There are only a few reports of thrombocytopenia associated with clinical doses of teicoplanin, a glycopeptide antibiotic used against Gram-positive bacteria. We investigated 39 patients receiving teicoplanin; 31 were thrombocytopenic with platelet counts between 1–105 · 109/l and 8 were not thrombocytopenic. We identified 14 thrombocytopenic cases (45%) and two (25%) non-thrombocytopenic cases with IgG teicoplanin-dependent platelet-reactive antibodies. Use of glycoprotein (GP) capture enzyme-linked immunosorbent assay with platelets and GPIIb/IIIa transfected Chinese Hamster Ovary cells as well as flow cytometry with GP-deficient platelets indicated that the GPIIb/IIIa complex is a major target antigen of these antibodies. Keywords: platelets, thrombocytopenia.
GPIIb/IIIa,
antibiotic,
teicoplanin,
drug-induced
Blood Service Cambridge and Department of Haematology, University of Cambridge, Long Road, Cambridge CB2 2PT, UK. E-mail:
[email protected]
Many drugs have been implicated as causing immune thrombocytopenia with haptenization of glycoproteins (GP) being the most common mechanism. Drug-dependent antibodies often recognize epitopes on GPIIb/IIIa or GPIb/V/IX (Rizvi et al, 1999), and occasionally on platelet endothelial cell adhesion molecule-1 (Kroll et al, 2000). Teicoplanin is a glycopeptide antibiotic that has been associated with neutropenia and haemolytic anaemia (Del Favero et al, 1989; Wilson, 1998; Coluccio et al, 2004) but only two cases of routine administration inducing thrombocytopenia have been reported. In the first case, a fall in platelet count occurred 2 d after treatment and clinical observations suggested teicoplanin induced the thrombocytopenia (Terol et al, 1993), while in the second case, teicoplanin-dependent antibodies were demonstrated, but without determining specificity (Veldman et al, 1996). We have expanded on these observations by studying the incidence of IgG teicoplanin-dependent antibodies in 39 patients during teicoplanin therapy, 31 with thrombocytopenia and eight without. Antibodies were found in 14 (45%) of the thrombocytopenic cases and two (25%) of the non-thrombocytopenic cases. Further studies showed antibody specificity for GPIIb/IIIa.
Materials and methods Thirty-one patients with suspected drug-induced thrombocytopenia and platelet counts between 1 and 105 · 109/l were investigated using samples taken at the time thrombocytopenia was identified. Most referrals (n ¼ 19) were from cardiothoracic patients, with the remainder from various clinical groups (Table I). Eight cardio-thoracic patients on teicoplanin but without severe thrombocytopenia were also tested before and after teicoplanin treatment. Within this latter group the platelet counts ranged from 144 to 355 · 109/l prior to teicoplanin treatment and 217–588 · 109/l after treatment. Ethical consent was obtained. The presence of teicoplanin-dependent antibodies was investigated by testing the patients’ sera against platelets in both the absence and presence of 100 lg/ml teicoplanin (Targocid; Marion Merrell Ltd, Uxbridge, UK). An increase in IgG antibody binding in the presence of the drug was taken as evidence for the presence of teicoplanin-dependent antibodies. Antibodies were detected by indirect platelet immunofluorescence test (PIFT) as previously described (Garner et al, 2000). Briefly, platelets were incubated with the patients’ sera at 20C then washed and resuspended with fluorescein
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 279–281
doi:10.1111/j.1365-2141.2005.05430.x
Short Report Table I. Referred cases. Case no. Clinical category Platelet count · 109/l* IgG MFI ratio Antibody-negative cases 1 Haematological malignancy 2 Cardio-thoracic 3 Cardio-thoracic 4 Haematological malignancy 5 Peritonitis 6 Cardio-thoracic 7 Cardio-thoracic 8 Haematological malignancy 9 Cardio-thoracic 10 Cardio-thoracic 11 Cardio-thoracic 12 Peritonitis 13 Cardio-thoracic 14 Cardio-thoracic 15 Cardio-thoracic 16 Cardio-thoracic 17 Orthopaedic Antibody-positive cases 18 Cardio-thoracic 19 Cardio-thoracic 20 Cardio-thoracic 21 Cardio-thoracic 22 Abscess 23 Cardio-thoracic 24 Cardio-thoracic 25 Peritonitis 26 Cardio-thoracic 27 Abscess 28 Orthopaedic 29 Cardio-thoracic 30 Pneumonia 31 Orthopaedic
Refractory
0Æ6
25 59 Refractory
0Æ9 1Æ0 1Æ0
67 4 105 Refractory
1Æ0 1Æ0 1Æ2 1Æ3
40 26 84 8 29 56 53 62 6
1Æ3 1Æ3 1Æ4 1Æ5 1Æ6 1Æ6 1Æ7 1Æ8 2Æ1
44 4 4 78 5 36 30 78 5 1 11 59 4 8
2Æ6 2Æ6 3Æ2 3Æ3 3Æ9 5Æ5 6Æ6 6Æ7 7Æ0 10Æ4 12Æ3 15Æ9 18Æ3 30Æ2
Results Antibody detection The fluorescence produced by negative control sera increased slightly in the presence of teicoplanin, with the anti-IgG value increasing a maximum of 2Æ5-fold. This value was therefore used as the cut-off for determining positivity for teiocoplanindependent antibodies. The thrombocytopenic patients were ranked from 1 to 31 according to the strength of their IgG PIFT results. The first 17 patients had anti-IgG values below the cut-off, with IgG MFI ratios between 0Æ6 and 2Æ1 (Table I). The remaining 14 cases (18–31) were considered to have teicoplanin-dependent antibodies, giving between 2Æ6 and 30Æ2-fold increase in IgG fluorescence in the presence of teicoplanin (Table I). Antibodies were detected in two of the eight non-thrombocytopenic patients on teicoplanin. These occurred after teicoplanin administration, and gave 4- and 4Æ8-fold increases in fluorescence values in the presence of teicoplanin.
Antibody characterization
*Platelet count at the time the sample was taken for antibody investigation. Ratio of the result in the presence of teicoplanin divided by the result in the absence of teicoplanin. These patients were multi-transfused and refractory to platelet transfusions. Platelet counts were therefore not relevant.
isothiocyanate (FITC)-conjugated rabbit anti-human IgG (DakoCytomation, Ely, UK). After incubation the platelets were washed and examined for bound FITC by flow cytometry. Results were expressed as a ratio of the median fluorescence intensity (MFI) in the absence and presence of the drug. In two cases with sufficient sample, antibody specificity was determined using; (i) PIFT with platelets from two GPIIb/IIIa deficient Glanzmann’s thrombasthenia patients, (ii) the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay using platelets, as previously described (Kiefel, 1992; Kroll et al, 2000) with three monoclonal 280
antibodies (moAbs) against GPIIb/IIIa, and single moAbs against GPIba, GPV, GPIa/IIa, CD31 and HLA class I, and (iii) MAIPA using Chinese Hamster Ovary (CHO) cells expressing GPIIb/IIIa (Großjohann et al, 2004). In the latter two assays, teicoplanin was included at 1 mg/ml for the sensitization stage, and subsequently at 100 lg/ml.
Sample 31 was tested by PIFT with platelets from two Glanzmann’s thrombasthenia cases and gave reduced reactivity (3Æ3 and 19-fold increases) when compared with reactivity with normal platelets (38Æ8-fold increase), implicating GPIIb/IIIa as the target antigen. This was confirmed by MAIPA assays using sera from cases 27 and 31. Both samples gave negative results with all moAbs in the absence of teicoplanin (OD £ 0Æ153). However, positive results were obtained in the presence of teicoplanin, but only with the three GPIIb/IIIa moAbs (OD 0Æ36–3Æ5). In addition, strong reactivity was obtained with GPIIb/IIIa-transfected CHO cells in the presence of teicoplanin (OD ‡ 3), but not in the absence of teicoplanin (OD £ 0Æ128).
Case reviews In the antibody-negative group, which contained all the haematological malignancy cases, there were factors other than teicoplanin therapy that could have been responsible for the thrombocytopenia. These included multiple drug treatment regimens, and the co-existence of other conditions, such as sepsis. Although such complications were also seen in most of the antibody-positive cases, there were seven instances where other underlying causes were not identified, and platelet counts suggested an association between antibodies and
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 279–281
Short Report Table II. Details of selected antibody-positive cases.
Platelet counts · 109/l Case
IgG MFI ratio*
Clinical category
22 23 26 27 28 30 31
3Æ9 5Æ5 7Æ0 10Æ4 12Æ3 18Æ3 30Æ2
Abscess Cardio-thoracic Cardio-thoracic Abscess Orthopaedic Pneumonia Orthopaedic
Heparin
Heparin antibodies
Pre
At referral
Post
Days
Yes Yes§ Yes** No Yes No Yes
Negative Negative Negative NT Negative NT Negative
250 92– 398 320 328 282 NA
2 36 3 36 11 8 8
200 Died 374 Died 196 130 373
4 13 2 2 2 11 9
NT, not tested; NA, not available. *Ratio of the result in the presence of teicoplanin divided by the result in the absence of teicoplanin. Platelet counts prior to receiving teicoplanin, at the time samples were referred for antibody testing and after teicoplanin was stopped. Days between administration of teicoplanin and identification of thrombocytopenia. §Also receiving tazocin and ranitidine. –Patient known to be antibody negative at this time. **Also receiving meropenem. Patient had received a previous course of teicoplanin.
thrombocytopenia. Five of these cases also received heparin, but none had heparin-dependent antibodies (Table II). In six of these seven cases, platelet counts prior to teicoplanin were ‡92 · 109/l, and in all seven these dropped to £36 · 109/l between 2 and 13 d after teicoplanin administration. In all but one of the five surviving cases, platelet counts returned to normal after withdrawal of teicoplanin (Table II).
Discussion We have confirmed the existence of teicoplanin-dependent antibodies in thrombocytopenic patients during teicoplanin therapy and identified GPIIb/IIIa as the target antigen. The antibodies were also detected in some patients on teicoplanin with normal platelet counts, indicating that the antibodies are not always clinically significant. Despite this, there were some cases in which the evidence suggested that the antibodies did contribute to the thrombocytopenia. In these instances, thrombocytopenia followed teicoplanin administration and was resolved when the drug was stopped. One can therefore conclude that, although mechanisms of thrombocytopenia may be multiple and complex, tests for teicoplanin-dependent antibodies may be of clinical use in cases of profound thrombocytopenia. However, prospective studies, including haematological and drug reviews, are needed to determine the clinical sensitivity and specificity of such tests.
Acknowledgements We gratefully acknowledge the hospitals for referring the samples. In particular, Dr J. K. Davies, Dr P. Coates and Dr L. R. Bindu for providing case details and Dr Jeremy Cordingley for arranging ethical approval and sample provision from nonthrombocytopenic patients. We also acknowledge the assist-
ance of the Platelet Immunology Reference Laboratory at the National Blood Service Cambridge and Patrycja Hartwich for their serological expertise, and Dr Nicholas Watkins for valuable comments during the manuscript preparation.
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