A patient with oxaliplatin immune-induced syndrome (OIIS) who also developed leucovorin and palonosetron-associated thrombocytopenia.

OBJECTIVES: We report a case of an 83 year old man who developed oxaliplatin immune-induced syndrome (OIIS) after his 19th cycle of FOLFOX (5FU, leucovorin, oxaliplatin). When oxaliplatin was omitted from his next cycle of chemotherapy he continues to show signs of drug-induced immune thrombocytopenia (DITP) and was found to have drug-dependent, platelet-reactive antibodies (DDPA) to leucovorin and palonosetron as well as oxaliplatin. METHODS: The patient was admitted for monitoring but required no transfusions and thrombocytopenia resolved without treatment during his first admission. Drug-dependent antibody testing was performed on his blood by the Blood Center of Wisconsin (Diagnostic Laboratories; Milwaukee, WI). RESULTS: No RBC or platelet IgG or IgM antibodies were detected in the absence of any drugs, but upon addition of palonosetron, leucovorin, or oxaliplatin, the tests became strongly positive for anti-RBC IgG and anti-platelet IgG antibodies. DISCUSSION: Repeated administration of oxaliplatin can result in drug-induced immune thrombocytopenia (DITP) or autoimmune hemolytic anemia (AIHA). This phenomenon has recently been termed OIIS and may additionally include Evan's syndrome or thrombotic microangiopathy (TMA). Here we describe a patient who developed OIIS with drug-dependent, platelet-reactive antibodies (DDPA) to leucovorin and palonosetron. To our knowledge, these two drugs have never been described in the literature as a cause of DDPA. We suggest that OIIS in addition to oxaliplatin-induced thrombocytopenia may be associated with the development of DDPAs to other drugs causing clinically significant thrombocytopenia which is important to recognize and manage with discontinuation of provoking agents.

Human platelet antigens - 2013.

To date, 33 human platelet alloantigens (HPAs) have been identified on six functionally important platelet glycoprotein (GP) complexes and have been implicated in alloimmune platelet disorders including foetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP) and multitransfusion platelet refractoriness (MPR). The greatest number of recognized HPA (20 of 33) resides on the GPIIb/IIIa complex, which serves as the receptor for ligands important in mediating haemostasis and inflammation. These include HPA-1a, the most commonly implicated HPA in FNAIT and PTP in Caucasian populations. Other platelet GP complexes, GPIb/V/IX, GPIa/IIa and CD109, express the remaining 13 HPAs. Of the recognized HPAs, 12 occur as six serologically and genetically defined biallelic 'systems' where the -a form designates the higher frequency allele and the -b form, the lower. Twenty-one other HPAs are low-frequency or rare antigens for which postulated higher frequency -a alleles have not yet been identified as antibody specificities. In addition to the HPA markers, platelets also express ABO and human leucocyte antigen (HLA) antigens; antibodies directed at the former are occasionally important in FNAIT, and to the latter, in MPR.

Report on the 14th International Society of Blood Transfusion Platelet Immunology Workshop.

BACKGROUND AND OBJECTIVES: The aims of the 14th ISBT Platelet Immunology Workshop were to evaluate in-house methods for detection of antibodies to human platelet antigens, to compare the sensitivity and specificity of antibody detection using a panel of monoclonal antibodies and to evaluate genotyping methods and establish procedures for drug-dependent antibody detection. MATERIALS AND METHODS: Forty-two laboratories from 23 countries participated. Samples and reagents provided for the five different exercises. RESULTS: The ability of participating laboratories to correctly identify the HPA antibody specificity in the nine samples ranged from 20% to 97%. The greatest difficulty was observed with samples that contained antibodies against HPA-3b and GPIV. The significant differences in optical density values by monoclonal antibody of immobilization of platelet antigens (MAIPA) assay were observed when testing the same platelet-specific antibodies. HPA genotyping of DNA with novel mutations did not significantly affect the results. The overall average discrepancy rate was 2·15% for genotyping of 10 DNA samples from well-characterized Epstein­Barr virus transformed cell lines. For detection of drug-dependent antibodies, excellent results for specificity and sensitivity were obtained by the laboratories using the MAIPA and flow cytometry. CONCLUSIONS: Most laboratories were able to identify the majority of HPA antibodies; however, significant disparities were observed in proficiency testing. MAIPA assay sensitivity is influenced by the monoclonal antibody clone used. DNA with new mutations and EBV cell lines are valuable samples to ensure accurate genotyping. A sensitive and specific drug-dependent antibody assay performed well in the hands of participants.

Drug-induced immune thrombocytopenia: pathogenesis, diagnosis, and management.

Drug-induced immune thrombocytopenia (DITP) can be triggered by a wide range of medications. Although many cases of DITP are mild, some are characterized by life-threatening bleeding symptoms. The pathogenesis of DITP is complex, in that at least six different mechanisms have been proposed by which drug-induced antibodies can promote platelet destruction. It is possible in many cases to identify antibodies that react with platelets in the presence of the sensitizing drug, but the required testing is technically demanding and not widely available. Therefore, a decision on whether to discontinue an implicated medication in a patient suspected of having DITP must be made on clinical grounds. An algorithm is available that can be helpful in assessing the likelihood that a particular drug caused thrombocytopenia, but the most important aspects of patient management are a high index of suspicion and a careful history of drug exposure in an individual who presents with acute, often severe thrombocytopenia of unknown etiology. How drugs induce platelet-reactive antibodies and how, once formed, the antibodies cause platelet destruction following exposure to the drug is poorly understood. Further studies to address these issues and characterize more completely the range of drugs and drug metabolites that can cause DITP are needed.

Detection and identification of platelet antibodies and antigens in the clinical laboratory.

As a result of the unique functional properties of platelets, more-robust methods were required for detection of antibodies raised against them. Immunofluorescence detection by flow cytometry, solid-phase red cell adherence, and antigen capture ELISAs are some of the current tests that have been developed to meet the challenges of platelet antibody detection and identification and antigen phenotyping. Recently developed protein liquid bead arrays are becoming the next-generation platelet antibody tests. Fueled by development of PCR and determination of the molecular basis of the PlA1 human platelet antigen (HPA), serologic platelet typing has now been replaced by genotyping of DNA. Allele-specific PCR, melting curve analysis, and 5'-nuclease assays are now evolving into more high-throughput molecular tests. Laboratory testing for the diagnosis of immune platelet disorders has advanced considerably from its humble beginnings.

Prospective evaluation of a new platelet glycoprotein (GP)-specific assay (PakAuto) in the diagnosis of autoimmune thrombocytopenia (AITP).

Assays measuring platelet-associated immunoglobulin G (PAIgG), while highly sensitive, lack specificity in diagnosing autoimmune thrombocytopenia (AITP). We prospectively evaluated a new commercially available glycoprotein (GP)-specific assay, the PakAuto (GTI, Brookfield, WI), for its clinical usefulness in distinguishing immune from nonimmune thrombocytopenia (TP), in 216 patients with autoimmune TP (both primary "idiopathic" and "secondary") and 46 patients with TP due to other causes. This assay is designed to detect both platelet-associated (direct assay) and plasma (indirect assay) antiplatelet antibodies specific for GPs IIb/IIIa, Ib/IX, and Ia/IIa. The mean platelet counts of the immune (79 +/- 7 x 10(9)/L) and nonimmune groups (78 +/- 7 x 10(9)/L), were similar (P=0.95). The direct assay was positive in 114/216 patients with AITP (53%), and 13/46 with nonimmune TP (28%). Among the AITP group, the majority (61%) of patients with positive test results had autoantibodies reactive against all three GP targets. The sensitivity, specificity, positive, and negative predictive values for the direct PakAuto were 53%, 72%, 90%, and 24%, respectively, comparable to previously published experience of GP-specific assays. However, in some cases of TP due to nonimmune cause, the PakAuto was highly specific. Only 3 of 22 patients with gestational and 1 of 8 with familial/congenital TP had a positive direct assay, indicating that the test may be particularly useful for excluding an immune etiology for TP in certain patient subgroups.

Delayed thrombocytopenia after treatment with abciximab: a distinct clinical entity associated with the immune response to the drug.

BACKGROUND: Acute thrombocytopenia is a recognized side-effect of treatment with the fibrinogen receptor antagonist, abciximab, a chimeric (human/mouse) Fab fragment. The etiology of this complication is not fully understood. Generally, abciximab-induced thrombocytopenia occurs within a few hours of starting treatment with the drug. We have characterized a group of 13 patients who first developed thrombocytopenia 3-6 days after abciximab was discontinued. OBJECTIVE: To characterize clinical and serological aspects of this newly recognized clinical entity. PATIENTS AND METHODS: Clinical information was obtained from attending physicians and review of hospital records. Antibodies reactive with abciximab-coated platelets were characterized by flow cytometry. RESULTS: In each patient, IgG and/or IgM antibodies reactive with abciximab-coated platelets were identified. These antibodies could be distinguished from similar antibodies present in many normal persons by two criteria-they were relatively resistant to inhibition by normal Fab fragments, and they reacted preferentially with platelets coated with 7E3, the murine monoclonal antibody from which peptide sequences in abciximab are derived. Antibodies with these characteristics were not found in pretreatment serum from three of the thrombocytopenic patients or in patients given abciximab who did not develop thrombocytopenia. CONCLUSIONS: 'Delayed thrombocytopenia' after treatment with abciximab is caused by antibodies produced in response to the drug. These antibodies may be specific for murine peptide sequences in abciximab but could recognize other target epitopes on abciximab-coated platelets. Physicians administering abciximab should be aware of this potential complication of treatment, which usually occurs after discharge from hospital.

Severe neonatal alloimmune thrombocytopenia caused by antibodies to human platelet antigen 3a (Baka) detectable only in whole platelet assays.

BACKGROUND: Maternal antibodies that cause neonatal alloimmune thrombocytopenia are commonly identified by solid-phase assays that detect the causative antibodies on the basis of their reactions with specific PLT glycoproteins. Two cases of severe neonatal alloimmune thrombocytopenia caused by maternal antibodies specific for human PLT antigen 3a (HPA-3a [Baka]) that failed to give the expected reactions in some solid-phase assays were recently encountered. STUDY DESIGN AND METHODS: PLT-reactive antibodies were characterized by three different solid-phase assays and by flow cytometry. RESULTS: The two maternal antibodies gave negative reactions in the antigen capture ELISA, modified antigen capture ELISA, and MoAb immobilization of PLT antigens tests but reacted strongly in flow cytometry with intact PLTs that were HPA-3a+. Other sera samples specific for HPA-3a reacted equally well in all assays. CONCLUSIONS: The two antibodies appear to recognize an epitope on the HPA-3a+ form of glycoprotein IIb that is lost when PLTs are solubilized in detergent, as required for solid-phase assays. The diagnosis was made in these cases because no HLA antibodies were present, allowing an HPA-3a-specific reaction to be identified with intact PLTs as targets. Such antibodies are likely to be overlooked when HLA antibodies are also present.

Serologically documented loracarbef (Lorabid)-induced immune thrombocytopenia.

We report here the first case of severe immune thrombocytopenia induced by a second-generation cephalosporin antibiotic, Loracarbef. A 56-year old white female developed acute severe thrombocytopenia associated with acute respiratory symptoms following administration of Loracarbef. She responded to Loracarbef withdrawal and systemic corticosteroid administration. Loracarbef-dependent platelet-reactive antibodies were demonstrable in her serum by flow cytometry.

TRALI due to granulocyte-agglutinating human neutrophil antigen-3a (5b) alloantibodies in donor plasma: a report of 2 fatalities.

BACKGROUND: TRALI is usually an immunologic reaction to WBC antibodies in infused plasma and ranks second only to ABO mismatch as a cause of transfusion-associated death. Implicated donors are usually multiparous women (>/=3 pregnancies). STUDY DESIGN AND METHODS: Two fatal cases of TRALI were evaluated by reviewing clinical and laboratory findings and characterizing alloantibodies present in donor plasma. Investigation for WBC antibodies was by lymphocytotoxicity (LCT), FlowPRA (FlowPRA, One Lambda, Inc.) and granulocyte immunofluorescence and agglutination assays. Patient 1 was a 62-year-old man with chronic T-cell lymphocytic leukemia, and Patient 2 was a 54-year-old woman undergoing a cadaveric kidney transplant. Both patients developed acute respiratory distress and hypotension during (Patient 1) and approximately 30 minutes after (Patient 2) transfusion. Fulminant pulmonary edema ensued in both cases necessitating mechanical ventilation and both patients died within 24 hours of the onset of respiratory complications. RESULTS: The donors of the implicated blood components were women with a history of two pregnancies but no blood transfusions. Weak apparently panreactive granulocyte antibodies were detected with flow cytometry. However, in the granulocyte agglutination test, strong antibodies specific for human neutrophil antigen (HNA)-3a (5b) were identified in both donors. CONCLUSION: It is concluded that female blood donors with only two previous pregnancies can form clinically important granulocyte-reactive alloantibodies leading to fatal TRALI reactions in recipients. The sometimes devastating consequences of TRALI should prompt the development of strategies to prevent or reduce its incidence. Further research is warranted to investigate recipient and donor factors responsible for TRALI, including whether 5b (HNA-3a) alloantibodies are especially prone to cause severe reactions, and to better characterize the HNA-3a (5b) antigen, particularly at the molecular level.

Blood group A and B antigens are strongly expressed on platelets of some individuals.

It is widely thought that expression of ABH antigens on platelets is insufficient to materially affect the survival of ABH-incompatible platelets in transfusion recipients, but anecdotal reports of poor survival of A and B mismatched platelets suggest that this is not always the case. The A and B antigen expression on platelets of 100 group A(1) and group B blood donors was measured, and 7% and 4%, respectively, had platelets whose A and B antigen levels consistently exceeded the mean plus 2 SD. On the basis of flow cytometric and statistical analysis, donors whose platelets contained higher than normal levels of A antigen were subdivided into 2 groups, designated Type I and Type II ("high expressers"). Serum A(1)- and B-glycosyltransferase levels of A and B high expressers were significantly higher than those of group A(1) and B individuals with normal expression. H antigen levels were low on the red cells of high expressers, indicating that the anomaly affects other cell lineages. Immunochemical studies demonstrated high levels of A antigen on various glycoproteins (GPs) from high-expresser platelets, especially GPIIb and PECAM (CD31). The A(1) Type II high-expresser phenotype was inherited as an autosomal dominant trait in one family. The sequences of exons 5, 6, and 7 of the A(1)-transferase gene of one Type II A(1) high expresser and exon 7 from 3 other genes were identical to the reported normal sequences. Further studies are needed to define the molecular basis for the high-expresser trait and to characterize its clinical implications. (Blood. 2000;96:1574-1581)

Immunoglobulin isotype identification in red cell antibodies using flow cytometry.

BACKGROUND: Identifying the isotype of an immunoglobulin (IgM vs. IgG) detected in a patient sample is especially important in anticipating the risk of hemolytic disease of the newborn. Currently, 2-mercaptoethanol (2-ME) treatment of a sample is used in the authors' laboratory to degrade IgM, and this is followed by retesting. This method has multiple drawbacks. The purpose of this study was to develop a flow cytometry (FC) assay that would replace the 2-ME treatment protocol (2-ME treatment). STUDY DESIGN AND METHODS: A preliminary FC assay was developed, modified, and refined through the use of stock antibodies. Then, 10 samples containing antibodies were tested in parallel by the FC assay and 2-ME treatment. RESULTS: When a 10-unit mean channel fluorescence change was used as an index of a positive result, the FC assay detected all isotypes identified by 2-ME treatment. The FC assay was also able to identify mixtures of isotypes. One antibody that had not reacted in conventional agglutination testing was detected by the FC assay. The amount of fluorescence and the agglutinating strength of the antibody did not parallel each other. In one case, this discrepancy may have reflected an antibody that was primarily IgA. CONCLUSIONS: The FC assay appears to be as accurate as 2-ME treatment in differentiating IgG from IgM. The FC assay produces a positive endpoint for both isotypes, will identify IgA, requires less sample, and has no odor.

Clinical significance of increased density in the proximal femoral marrow detected by visual inspection on abdominopelvic postcontrast CT examination.

PURPOSE: The purpose of this study was to ascertain the clinical significance of increased density of the proximal femoral diaphyseal marrow when incidentally detected on postcontrast abdominopelvic CT examinations. METHOD: The proximal femoral marrow of 63 patients was classified as normal or abnormal based on visual inspection by three attending radiologists. Abnormal density was defined as attenuation greater than that of adjacent musculature. The attenuation of the marrow was also measured. All patient medical records were reviewed for pertinent laboratory and clinical data. RESULTS: Increased marrow density had a low sensitivity for anemia (28%) but a high specificity and positive predictive value (100%) for anemia. Three of these patients had unilaterally increased attenuation associated with local pathology. Visual inspection was adequate for identifying abnormalities in instances of underlying malignancy. CONCLUSION: Increased density of the proximal femoral diaphysis is a highly specific finding for a marrow replacement process. Anemia was the most common clinical diagnosis in our series of patients with abnormal marrow density. Although the sensitivity for increased bone marrow attenuation is low, the extremely high specificity and positive predictive value of this finding for marrow reconversion and/or replacement suggest that even if detected incidentally, it should not be disregarded and, in the absence of a preexisting causative diagnosis, warrants further evaluation. The specific pattern of marrow abnormality may be helpful in differentiating localized processes from other processes that may affect hematopoietic function in a more widespread or global distribution. In particular, visual inspection of marrow density was more specific for identifying asymmetric marrow density in localized processes than was a quantitative measure of difference between the two femurs (>20 HU).

An antibody from a patient with ranitidine-induced thrombocytopenia recognizes a site on glycoprotein IX that is a favored target for drug-induced antibodies.

Although thrombocytopenia associated with the use of histamine H2 receptor (H2R) antagonists has been described, a drug-dependent, platelet-reactive antibody has not previously been identified in such cases. We studied serum from a patient who developed acute, severe thrombocytopenia after exposure to the H2 receptor antagonist, ranitidine, and identified an antibody that reacted with normal platelets in the presence of this drug at pharmacologic concentrations. In flow cytometric and immunoprecipitation studies, the antibody was shown to be specific for the glycoprotein Ib/IX complex (GPIb/IX). From the pattern of monoclonal antibody (MoAb) inhibition and the reactions of antibody with Chinese hamster ovary (CHO) cells transfected with GPIX and GPIbbeta, we found that the patient's antibody is specific for an epitope on GPIX close to, or identical with a site recognized by the MoAb SZ1 that is a common target for antibodies induced by quinine and quinidine, drugs structurally unrelated to ranitidine. These findings provide evidence that immune thrombocytopenia can be caused by sensitivity to an H2 R antagonist and suggest that the SZ1 binding site on GPIX may be a common target for drug-induced antibodies. Further studies of the epitope for which SZ1 is specific may provide clues to the mechanism(s) by which drugs promote tight binding of antibody to a membrane glycoprotein and cause platelet destruction in patients with drug sensitivity.

Posterior inferior junction line and left pleuroesophageal stripe: their association with emphysema.

In an effort to define the posterior inferior junction line (PIJL) and its clinical associations more precisely, 64 posteroanterior radiographs demonstrating the PIJL or left pleuroesophageal stripe (LPES) were analyzed for the presence of emphysema, kyphosis, air-filled esophagus, and/or tortuous aorta. Pursuant to the possible association of a PIJL or LPES with an air-filled esophagus, posteroanterior radiographs of 66 patients with achalasia were evaluated for the presence of a PIJL or LPES. To determine the components of the PIJL or LPES, 50 randomly selected computed tomographs (CT) of the chest were reviewed. Finally, 118 posteroanterior radiographs of patients with emphysema were analyzed for the presence of a PIJL and/or LPES to determine the sensitivity of the line/stripe for emphysema. The finding of a PIJL and/or LPES had a combined sensitivity of 23% for emphysema. Although certain other anatomic constructs lead to the presence of a line or stripe, emphysema is the most commonly associated clinical entity with a positive predictive value of 65.8%. The line and/or stripe is formed by interfaces between lung/lung, lung/esophagus, or both at different levels.