Journal of Thrombosis and Haemostasis, 13: 1–3
DOI: 10.1111/jth.12852
RECOMMENDATIONS AND GUIDELINE
Recommendations for standardization of laboratory testing for drug-induced immune thrombocytopenia: communication from the SSC of the ISTH D. M. ARNOLD,* B. R. CURTIS† and T. BAKCHOUL,‡ FOR THE SUBCOMMITTEE ON PLATELET IMMUNOLOGY *Department of Medicine, McMaster University, Canadian Blood Services, Hamilton, ON, Canada; †Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI, USA; and ‡Institute for Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany
To cite this article: Arnold DM, Curtis BR, Bakchoul T, for the Subcommittee on Platelet Immunology. Recommendations for standardization of laboratory testing for drug-induced immune thrombocytopenia: communication from the SSC of the ISTH. J Thromb Haemost 2015; DOI: 10.1111/jth.12852.
Introduction Drug-induced immune thrombocytopenia (DITP) is a lifethreatening clinical syndrome that is underrecognized and difficult to diagnose [1]. Clinical criteria can help determine the likelihood that a drug was the cause of thrombocytopenia, but confirmation of the diagnosis requires laboratory testing to demonstrate the presence of drugdependent platelet antibodies. Test methods for these assays must be able to show (i) drug-dependence, (ii) immunoglobulin binding to platelets, and (iii) platelet specificity [2] and ideally should be reproducible across laboratories [3]. Current test methods are limited by the lack of standardization. There is a need to improve accessibility of testing for the most commonly implicated drugs, which means that the methods of testing including sample collection, handling of target platelets, drug concentrations, and the type of immunoassay should be standardized. Establishing uniform test methods and procedures will improve the reproducibility of results across laboratories and enable reliable comparisons. This ISTH recommendation document was developed to improve the standardization of laboratory testing for drugdependent platelet-associated antibodies with the most freCorrespondence: Donald M Arnold, McMaster University, 1280 Main Street West Room 3V-50, Hamilton, Ontario L8S 4K1, Canada. Tel.: +1 905 521 2100 X76300; fax: +1 905 521 4971. E-mail: [email protected]
quently implicated prescription drugs: quinine, vancomycin, sulfamethoxazole/trimethoprim, and pipericillin/ tazobactam. Recommendations were based on experience from three reference laboratories in the United States (Blood Center of Wisconsin), Canada (McMaster University), and Germany (Ernst-Moritz-Arndt-University Greifswald) and informed by discussions with members of the Platelet Immunology Scientific Subcommittee of ISTH.
Recommendations Sample collection for DITP testing
Timing of sample collection Blood samples for DITP testing should be collected during the acute episode of thrombocytopenia as soon as DITP is suspected. If thrombocytopenia has resolved, we recommended retrieving a stored sample collected during the time of the acute episode, if available. Testing for DITP may be performed on a sample collected up to 3 weeks after the acute event, even if the thrombocytopenia has resolved (or is resolving) by that time. Longer delays should be avoided because of reduced sensitivity due to disappearance of the antibody. Anticoagulant in the sample Clotted serum samples and citrate samples are acceptable for DITP testing. EDTA should be avoided because it can cause glycoprotein complexes, including GPIIb/IIIa, to dissociate, thus impairing the binding of complex-dependent antibodies. Stored EDTA samples may be made suitable for DITP testing by recalcification. Preparation of test platelets for the assay
Received 15 October 2014 Manuscript handled by: W. Ageno Final decision: P. H. Reitsma, 21 December 2014 © 2015 International Society on Thrombosis and Haemostasis
Target platelets are required for DITP testing. This material provides the substrate for binding of drug-dependent
2 D. M. Arnold et al
platelet antibodies. Purified platelet glycoproteins are not a suitable alternative because they do not retain their native confirmation in such antigen binding assays. Either freshly isolated platelets from volunteer donors or stored platelets (0.1% sodium azide) can be used for DITP investigations. For fresh platelets, blood should be collected from the donor using gentle suction (rather than vacuum suction) through a 21-gauge needle with the donor in a sitting position. Tourniquets should be avoided or only lightly applied to the arm during sample collection to avoid in vivo platelet activation. Donors should be in good health without concomitant medical illnesses. They should not have ingested medications that may interfere with platelet function for at least 7 days, including non-steroid anti-inflammatory medications. Donors should be blood group O and express the HPA1a antigen to allow for the use of anti–HPA-1a antibodies as a positive control (see ‘positive control sera’). Donor blood samples for platelet isolation should be collected into citrate-containing tubes (e.g. citrate phosphate dextrose, anticoagulant citrate dextrose solution A or B). Platelet isolation should be performed using gentle centrifugation (120 9 g, 20 min, room temperature), and the platelet pellet should be washed twice with phosphatebuffered saline (PBS). The final platelet count should be adjusted to 500 9 106 mL 1 using PBS. Test methods
Both flow cytometry and enzyme immunoassays (EIAs) can be used to investigate for the presence of drug-dependent antibodies. Flow cytometry Healthy donor platelets are isolated and incubated with test serum in the presence and absence of drug [4]. Platelets are incubated with serum and drug and washed with buffer containing drug at the same concentration (see below). After washing of the platelets, drugdependent platelet-associated immunoglobulins are detected by flow cytometry using florescent-labeled antihuman IgG and anti-human IgM [5]. The test is performed in parallel in the absence of drug and washed in drug-free buffers to ensure that the reaction is drug dependent. Enzyme immunoassay (EIA) Platelets isolated from healthy donors are added to wells in a microtiter plate [6]. Patient serum is incubated with platelets in the presence of the drug or buffer. After washing, drug-dependent platelet-bound antibodies are detected using an enzymelabeled goat anti-human IgG and IgM. Patient samples and controls
Patient samples and positive and negative controls must be tested with and without drug added in the same concentrations as the test sample.
Drug concentration Although therapeutic concentrations can be used for many drugs when performing the assay (Table 1), 1 mg mL 1 is a good starting concentration [7]. While supratherapeutic concentrations (1 mg mL 1) of vancomycin in testing may give false-positive reactions with normal serum controls, the use of these concentrations can help get water-insoluble drugs in solutions, like sulfamethoxazole (B. Curtis, D. Bougie, R. Aster, unpublished data). The drug must also be present in all washing buffers at the same concentration to prevent drug-dependent antibodies from dissociating during the washing steps. Drug solubility The solubility of some drugs may limit the ability to test drugs in the assay. For example, sulfamethoxazole and trimethoprim are only slightly soluble in aqueous solutions at neutral pH. Drugs dissolved in alcohol and then diluted in buffer or drug dissolved in buffer with stirring and addition of 0.1 mmol L 1 NaOH or HCl to maintain pH can sometimes be used to solubilize drugs at higher concentrations. An effective method to increase the solubility of sulfamethoxazole and achieve a concentration of 1–2 mg mL 1 is to dissolve the drug in buffer containing 5% bovine serum albumin [4]. Positive control sera The ideal positive control is serum from a patient with known drug-dependent platelet antibodies to the drug in question. Recognizing that this is rarely available, suitable alternatives include serum from a patient with a drug-dependent antibody to another drug, or serum containing anti-HPA-1a (WHO standard 106/05, or in-house validated platelet antibody positive control) to ensure at a minimum that the assay can detect antibody bound to platelets. If anti-HPA-1a serum is used, platelets from HPA-1a-positive donors is necessary to avoid false-negative results. Negative control sera The ideal negative control for DITP testing is a serum sample from a patient who received the drug in question but did not develop thrombocytopenia. Recognizing that this is rarely available, a suitable negative control is serum from a healthy donor. In addition, testing patient sera without drug is an important control step to confirm drug-dependency. Table 1 Therapeutic concentrations of the most commonly implicated drugs in DITP reactions Drug
Therapeutic concentration*
Solubility
Quinine Vancomycin Sulfamethoxazole/ trimethoprim Pipericillin/ tazobactam
8–15 lg mL 15–30 lg mL 1 Sulfa: 40 lg mL 1 Trimethoprim: 2 lg mL 1 Pipericilliin: 0.9–298 lg mL Tazobactam: 0.5–34 lg mL
0.5 mg mL 1 ≥ 100 mg mL 0.5 mg mL 1
1
1
50 mg mL
1
1
1
*Concentration depends on time, dosage, and route of administration. © 2015 International Society on Thrombosis and Haemostasis
Standardization of laboratory testing for DITP 3
Interpretation of results
The test for drug-dependent platelet antibodies is considered positive when the patient sample demonstrates antibody binding to platelets in the presence but not in the absence of the drug. This means that the test sample has a mean fluorescence intensity (by flow cytometry) or optical density reading (by EIA) of at least 2 standard deviations above that obtained without drug present. Another approach is to estimate the fluorescence or optical density ratio by dividing the result with drug by the result without drug. A fluorescence ratio above 1.5 could be used as a positive cut-off [8]. However, each lab should establish their own cut-offs, by testing 10–20 sera from normal, healthy subjects and performing statistical analysis. A positive test has a high specificity for drug-dependent platelet antibodies; thus, patients with a positive test should be advised to avoid future drug exposures. A negative test does not rule out the syndrome, given the low sensitivity of the test with specific drugs. Possible reasons for ‘false-negative’ results include (i) a metabolite of the drug, rather than the drug itself was the cause of DITP; (ii) poor solubility of certain drugs and inability to prepare drug solutions for the assay; and (iii) improper timing of sample collection until after the thrombocytopenia resolved (and antibody levels have reduced or disappeared). For patients with a negative test in whom DITP is strongly suspected, a carefully supervised drug rechallenge can be considered. This may be useful when the implicated drug is particularly common (e.g. acetaminophen) or when no alternative drug is available. Conclusion Although the syndrome of DITP is relatively uncommon, it can be associated with severe morbidity and mortality. In addition, a confirmed diagnosis of DITP has important implications for patients with respect to future drug avoidance. While testing for most drugs will require referral to a reference laboratory, the recommendations proposed here provide suggestions for the methodology and drug-specific requirements to allow hospital laboratories to perform initial testing for the most commonly implicated prescription drugs. Prospective evaluation of these recommendations is needed. Addendum T. Bakchoul designed the study, analyzed the data, critically wrote the manuscript, and revised the intellectual
© 2015 International Society on Thrombosis and Haemostasis
content of the manuscript. B. Curtis analyzed the data and critically wrote the manuscript. D. Arnold designed the study, critically wrote the manuscript, and revised the intellectual content of the manuscript. All authors approved the version to be published of the manuscript. Acknowledgements This study was supported by a grant from the German Society for Research (Deutsche Forschungsgemeinschaft DFG) to T. B. (BA-5158/1-1). Disclosure of Conflict of Interest T. Bakchoul reports grants from DFG during the conduct of the study. B. Curtis reports personal fees from GTIImmucor outside the submitted work. B. Curtis has a patent on ‘Method of detecting cytophenia that is mediated by drug-dependent antibody binding to blood cells’ issued and a patent on ‘Method of simultaneous detection of heparin-dependent immunoglobulins types G, A, and M’ pending. D. Arnold has nothing to disclose. References 1 Aster RH, Curtis BR, McFarland JG, Bougie DW. Drug-induced immune thrombocytopenia: pathogenesis, diagnosis, and management. J Thromb Haemost 2009; 7: 911–8. 2 Arnold DM, Kukaswadia S, Nazi I, Esmail A, Dewar L, Smith JW, Warkentin TE, Kelton JG. A systematic evaluation of laboratory testing for drug-induced immune thrombocytopenia. J Thromb Haemost 2013; 11: 169–76. 3 Arnold DM, Nazi I, Warkentin TE, Smith JW, Toltl LJ, George JN, Kelton JG. Approach to the diagnosis and management of drug-induced immune thrombocytopenia. Transfus Med Rev 2013; 27: 137–45. 4 Curtis BR, McFarland JG, Wu GG, Visentin GP, Aster RH. Antibodies in sulfonamide-induced immune thrombocytopenia recognize calcium-dependent epitopes on the glycoprotein IIb/IIIa complex. Blood 1994; 84: 176–83. 5 Curtis BR, McFarland JG. Detection and identification of platelet antibodies and antigens in the clinical laboratory. Immunohematology 2009; 25: 125–35. 6 Grossjohann B, Eichler P, Greinacher A, Santoso S, Kroll H. Ceftriaxone causes drug-induced immune thrombocytopenia and hemolytic anemia: characterization of targets on platelets and red blood cells. Transfusion 2004; 44: 1033–40. 7 Meyer O, Hoffmann T, Aslan T, Ahrens N, Kiesewetter H, Salama A. Diclofenac-induced antibodies against RBCs and platelets: two case reports and a concise review. Transfusion 2003; 43: 345–9. 8 Curtis BR. Drug-induced immune thrombocytopenia: incidence, clinical features, laboratory testing, and pathogenic mechanisms. Immunohematology 2014; 30: 55–65.
DOI: 10.1111/jth.12852
RECOMMENDATIONS AND GUIDELINE
Recommendations for standardization of laboratory testing for drug-induced immune thrombocytopenia: communication from the SSC of the ISTH D. M. ARNOLD,* B. R. CURTIS† and T. BAKCHOUL,‡ FOR THE SUBCOMMITTEE ON PLATELET IMMUNOLOGY *Department of Medicine, McMaster University, Canadian Blood Services, Hamilton, ON, Canada; †Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI, USA; and ‡Institute for Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany
To cite this article: Arnold DM, Curtis BR, Bakchoul T, for the Subcommittee on Platelet Immunology. Recommendations for standardization of laboratory testing for drug-induced immune thrombocytopenia: communication from the SSC of the ISTH. J Thromb Haemost 2015; DOI: 10.1111/jth.12852.
Introduction Drug-induced immune thrombocytopenia (DITP) is a lifethreatening clinical syndrome that is underrecognized and difficult to diagnose [1]. Clinical criteria can help determine the likelihood that a drug was the cause of thrombocytopenia, but confirmation of the diagnosis requires laboratory testing to demonstrate the presence of drugdependent platelet antibodies. Test methods for these assays must be able to show (i) drug-dependence, (ii) immunoglobulin binding to platelets, and (iii) platelet specificity [2] and ideally should be reproducible across laboratories [3]. Current test methods are limited by the lack of standardization. There is a need to improve accessibility of testing for the most commonly implicated drugs, which means that the methods of testing including sample collection, handling of target platelets, drug concentrations, and the type of immunoassay should be standardized. Establishing uniform test methods and procedures will improve the reproducibility of results across laboratories and enable reliable comparisons. This ISTH recommendation document was developed to improve the standardization of laboratory testing for drugdependent platelet-associated antibodies with the most freCorrespondence: Donald M Arnold, McMaster University, 1280 Main Street West Room 3V-50, Hamilton, Ontario L8S 4K1, Canada. Tel.: +1 905 521 2100 X76300; fax: +1 905 521 4971. E-mail: [email protected]
quently implicated prescription drugs: quinine, vancomycin, sulfamethoxazole/trimethoprim, and pipericillin/ tazobactam. Recommendations were based on experience from three reference laboratories in the United States (Blood Center of Wisconsin), Canada (McMaster University), and Germany (Ernst-Moritz-Arndt-University Greifswald) and informed by discussions with members of the Platelet Immunology Scientific Subcommittee of ISTH.
Recommendations Sample collection for DITP testing
Timing of sample collection Blood samples for DITP testing should be collected during the acute episode of thrombocytopenia as soon as DITP is suspected. If thrombocytopenia has resolved, we recommended retrieving a stored sample collected during the time of the acute episode, if available. Testing for DITP may be performed on a sample collected up to 3 weeks after the acute event, even if the thrombocytopenia has resolved (or is resolving) by that time. Longer delays should be avoided because of reduced sensitivity due to disappearance of the antibody. Anticoagulant in the sample Clotted serum samples and citrate samples are acceptable for DITP testing. EDTA should be avoided because it can cause glycoprotein complexes, including GPIIb/IIIa, to dissociate, thus impairing the binding of complex-dependent antibodies. Stored EDTA samples may be made suitable for DITP testing by recalcification. Preparation of test platelets for the assay
Received 15 October 2014 Manuscript handled by: W. Ageno Final decision: P. H. Reitsma, 21 December 2014 © 2015 International Society on Thrombosis and Haemostasis
Target platelets are required for DITP testing. This material provides the substrate for binding of drug-dependent
2 D. M. Arnold et al
platelet antibodies. Purified platelet glycoproteins are not a suitable alternative because they do not retain their native confirmation in such antigen binding assays. Either freshly isolated platelets from volunteer donors or stored platelets (0.1% sodium azide) can be used for DITP investigations. For fresh platelets, blood should be collected from the donor using gentle suction (rather than vacuum suction) through a 21-gauge needle with the donor in a sitting position. Tourniquets should be avoided or only lightly applied to the arm during sample collection to avoid in vivo platelet activation. Donors should be in good health without concomitant medical illnesses. They should not have ingested medications that may interfere with platelet function for at least 7 days, including non-steroid anti-inflammatory medications. Donors should be blood group O and express the HPA1a antigen to allow for the use of anti–HPA-1a antibodies as a positive control (see ‘positive control sera’). Donor blood samples for platelet isolation should be collected into citrate-containing tubes (e.g. citrate phosphate dextrose, anticoagulant citrate dextrose solution A or B). Platelet isolation should be performed using gentle centrifugation (120 9 g, 20 min, room temperature), and the platelet pellet should be washed twice with phosphatebuffered saline (PBS). The final platelet count should be adjusted to 500 9 106 mL 1 using PBS. Test methods
Both flow cytometry and enzyme immunoassays (EIAs) can be used to investigate for the presence of drug-dependent antibodies. Flow cytometry Healthy donor platelets are isolated and incubated with test serum in the presence and absence of drug [4]. Platelets are incubated with serum and drug and washed with buffer containing drug at the same concentration (see below). After washing of the platelets, drugdependent platelet-associated immunoglobulins are detected by flow cytometry using florescent-labeled antihuman IgG and anti-human IgM [5]. The test is performed in parallel in the absence of drug and washed in drug-free buffers to ensure that the reaction is drug dependent. Enzyme immunoassay (EIA) Platelets isolated from healthy donors are added to wells in a microtiter plate [6]. Patient serum is incubated with platelets in the presence of the drug or buffer. After washing, drug-dependent platelet-bound antibodies are detected using an enzymelabeled goat anti-human IgG and IgM. Patient samples and controls
Patient samples and positive and negative controls must be tested with and without drug added in the same concentrations as the test sample.
Drug concentration Although therapeutic concentrations can be used for many drugs when performing the assay (Table 1), 1 mg mL 1 is a good starting concentration [7]. While supratherapeutic concentrations (1 mg mL 1) of vancomycin in testing may give false-positive reactions with normal serum controls, the use of these concentrations can help get water-insoluble drugs in solutions, like sulfamethoxazole (B. Curtis, D. Bougie, R. Aster, unpublished data). The drug must also be present in all washing buffers at the same concentration to prevent drug-dependent antibodies from dissociating during the washing steps. Drug solubility The solubility of some drugs may limit the ability to test drugs in the assay. For example, sulfamethoxazole and trimethoprim are only slightly soluble in aqueous solutions at neutral pH. Drugs dissolved in alcohol and then diluted in buffer or drug dissolved in buffer with stirring and addition of 0.1 mmol L 1 NaOH or HCl to maintain pH can sometimes be used to solubilize drugs at higher concentrations. An effective method to increase the solubility of sulfamethoxazole and achieve a concentration of 1–2 mg mL 1 is to dissolve the drug in buffer containing 5% bovine serum albumin [4]. Positive control sera The ideal positive control is serum from a patient with known drug-dependent platelet antibodies to the drug in question. Recognizing that this is rarely available, suitable alternatives include serum from a patient with a drug-dependent antibody to another drug, or serum containing anti-HPA-1a (WHO standard 106/05, or in-house validated platelet antibody positive control) to ensure at a minimum that the assay can detect antibody bound to platelets. If anti-HPA-1a serum is used, platelets from HPA-1a-positive donors is necessary to avoid false-negative results. Negative control sera The ideal negative control for DITP testing is a serum sample from a patient who received the drug in question but did not develop thrombocytopenia. Recognizing that this is rarely available, a suitable negative control is serum from a healthy donor. In addition, testing patient sera without drug is an important control step to confirm drug-dependency. Table 1 Therapeutic concentrations of the most commonly implicated drugs in DITP reactions Drug
Therapeutic concentration*
Solubility
Quinine Vancomycin Sulfamethoxazole/ trimethoprim Pipericillin/ tazobactam
8–15 lg mL 15–30 lg mL 1 Sulfa: 40 lg mL 1 Trimethoprim: 2 lg mL 1 Pipericilliin: 0.9–298 lg mL Tazobactam: 0.5–34 lg mL
0.5 mg mL 1 ≥ 100 mg mL 0.5 mg mL 1
1
1
50 mg mL
1
1
1
*Concentration depends on time, dosage, and route of administration. © 2015 International Society on Thrombosis and Haemostasis
Standardization of laboratory testing for DITP 3
Interpretation of results
The test for drug-dependent platelet antibodies is considered positive when the patient sample demonstrates antibody binding to platelets in the presence but not in the absence of the drug. This means that the test sample has a mean fluorescence intensity (by flow cytometry) or optical density reading (by EIA) of at least 2 standard deviations above that obtained without drug present. Another approach is to estimate the fluorescence or optical density ratio by dividing the result with drug by the result without drug. A fluorescence ratio above 1.5 could be used as a positive cut-off [8]. However, each lab should establish their own cut-offs, by testing 10–20 sera from normal, healthy subjects and performing statistical analysis. A positive test has a high specificity for drug-dependent platelet antibodies; thus, patients with a positive test should be advised to avoid future drug exposures. A negative test does not rule out the syndrome, given the low sensitivity of the test with specific drugs. Possible reasons for ‘false-negative’ results include (i) a metabolite of the drug, rather than the drug itself was the cause of DITP; (ii) poor solubility of certain drugs and inability to prepare drug solutions for the assay; and (iii) improper timing of sample collection until after the thrombocytopenia resolved (and antibody levels have reduced or disappeared). For patients with a negative test in whom DITP is strongly suspected, a carefully supervised drug rechallenge can be considered. This may be useful when the implicated drug is particularly common (e.g. acetaminophen) or when no alternative drug is available. Conclusion Although the syndrome of DITP is relatively uncommon, it can be associated with severe morbidity and mortality. In addition, a confirmed diagnosis of DITP has important implications for patients with respect to future drug avoidance. While testing for most drugs will require referral to a reference laboratory, the recommendations proposed here provide suggestions for the methodology and drug-specific requirements to allow hospital laboratories to perform initial testing for the most commonly implicated prescription drugs. Prospective evaluation of these recommendations is needed. Addendum T. Bakchoul designed the study, analyzed the data, critically wrote the manuscript, and revised the intellectual
© 2015 International Society on Thrombosis and Haemostasis
content of the manuscript. B. Curtis analyzed the data and critically wrote the manuscript. D. Arnold designed the study, critically wrote the manuscript, and revised the intellectual content of the manuscript. All authors approved the version to be published of the manuscript. Acknowledgements This study was supported by a grant from the German Society for Research (Deutsche Forschungsgemeinschaft DFG) to T. B. (BA-5158/1-1). Disclosure of Conflict of Interest T. Bakchoul reports grants from DFG during the conduct of the study. B. Curtis reports personal fees from GTIImmucor outside the submitted work. B. Curtis has a patent on ‘Method of detecting cytophenia that is mediated by drug-dependent antibody binding to blood cells’ issued and a patent on ‘Method of simultaneous detection of heparin-dependent immunoglobulins types G, A, and M’ pending. D. Arnold has nothing to disclose. References 1 Aster RH, Curtis BR, McFarland JG, Bougie DW. Drug-induced immune thrombocytopenia: pathogenesis, diagnosis, and management. J Thromb Haemost 2009; 7: 911–8. 2 Arnold DM, Kukaswadia S, Nazi I, Esmail A, Dewar L, Smith JW, Warkentin TE, Kelton JG. A systematic evaluation of laboratory testing for drug-induced immune thrombocytopenia. J Thromb Haemost 2013; 11: 169–76. 3 Arnold DM, Nazi I, Warkentin TE, Smith JW, Toltl LJ, George JN, Kelton JG. Approach to the diagnosis and management of drug-induced immune thrombocytopenia. Transfus Med Rev 2013; 27: 137–45. 4 Curtis BR, McFarland JG, Wu GG, Visentin GP, Aster RH. Antibodies in sulfonamide-induced immune thrombocytopenia recognize calcium-dependent epitopes on the glycoprotein IIb/IIIa complex. Blood 1994; 84: 176–83. 5 Curtis BR, McFarland JG. Detection and identification of platelet antibodies and antigens in the clinical laboratory. Immunohematology 2009; 25: 125–35. 6 Grossjohann B, Eichler P, Greinacher A, Santoso S, Kroll H. Ceftriaxone causes drug-induced immune thrombocytopenia and hemolytic anemia: characterization of targets on platelets and red blood cells. Transfusion 2004; 44: 1033–40. 7 Meyer O, Hoffmann T, Aslan T, Ahrens N, Kiesewetter H, Salama A. Diclofenac-induced antibodies against RBCs and platelets: two case reports and a concise review. Transfusion 2003; 43: 345–9. 8 Curtis BR. Drug-induced immune thrombocytopenia: incidence, clinical features, laboratory testing, and pathogenic mechanisms. Immunohematology 2014; 30: 55–65.
