Severe thrombocytopenia in a patient following liver transplantation caused by HPA‐1a antibodies produced by the liver donor
Authors: Paul F. Lindholm, Hau C. Kwaan, Glenn Ramsey, Brian R. Curtis, Jonathan Fryer
Journal: American Journal of Hematology (2017)

The patient was a 59 y/o male with HCV-associated cirrhosis and HIV, who received a living donor liver transplant in 2016. The patient's blood type was group A, Rh (D) positive, with a negative RBC antibody screen. In the perioperative period, the patient received 7 units RBC, 9 units plasma, 7 units platelets and 4 pools of cryoprecipitate. He received an additional 1 RBC unit, 8 units of plasma, 2 units of platelets and 1 unit of cryoprecipitate in the postoperative period. Nine days after transplantation, he was readmitted with ecchymosis on his right upper inner thigh and a right arm hematoma and found to have acute kidney injury, edema, ascites drainage and severe thrombocytopenia. The patient's blood counts included: platelets 7000/μL; WBC 13 300/μL and hemoglobin 16.5 g/dL. The blood smear showed thrombocytopenia and a normochromic, normocytic anemia with increased polychromasia and increased anisocytosis (Figure 1). There was no morphologic evidence of microangiopathic process. The prothrombin time was 12.6 seconds (normal range 9.2 to 13.0 seconds); INR 1.2; aPTT 24.3 seconds (normal 24.6 to 37.4 seconds). The patient's platelet count was in the 20 000–40 000/μL range at original presentation and in the immediate post-transplant period. The HIV viral load was reported to be less than 20 copies/mL by HIV quantitative real-time PCR. His medications included: methylprednisolone, mycophenolate mofetil, prednisone, tacrolimus, emtricitabine, raltegravir, valganciclovir, piperacillin-tazobactam, and sulfamethoxazole-trimethoprim. Peripheral blood smear from the second hospital day of readmission (tenth day following transplantation) for thrombocytopenia which showed thrombocytopenia and a normochromic, normocytic anemia with increased polychromasia and increased anisocytosis (Photomicrograph courtesy of Dr. Yihua Chen, Northwestern University) [Color figure can be viewed at wileyonlinelibrary.com] In the post-transplant setting, severe thrombocytopenia can be caused by myelosuppression due to medications, idiopathic immune thrombocytopenia, immune thrombocytopenia associated with HCV and HIV infection and hypersplenism. When there is an acute and severe fall in the platelet count, post-transfusion purpura should be considered. Thrombocytopenia seen in hemolytic-uremic syndrome, myelosuppression, and disseminated intravascular coagulation can be ruled out by clinical history and laboratory findings. The platelet count reached a nadir of 3000/μL on the tenth day and was less than 10 000/μL for the next 4 days (Figure 2). Platelet transfusion yielded very low and transient platelet increments during this time. Clinical course of the patient's platelet count following liver transplantation. The platelet count started at 45 000/μL prior to the procedure (day 0a) and was 120 000/μL following transfusion of 7 units of platelets by the end of the procedure (day 0b). After the transplantation procedure, the first platelet count of the day is shown. The platelets fell to 7000 platelets per μL on day 9 after allogeneic liver transplantation when the patient presented with severe thrombocytopenia. On day 10, the platelet count reached a nadir of 3000/μL. The patient received several platelet transfusions on days 9–13 with modest platelet increments. The numbers above the arrows indicate the number of platelet units that were transfused at the indicated times. The patient received intravenous gamma globulin on days 10–13, followed by methylprednisolone on days 13–17. The platelet count gradually improved and returned to the normal range on the 28th postoperative day When the platelet count is responding poorly to repeated transfusions, immune thrombocytopenia should be considered. Among treatments for immune thrombocytopenia, intravenous immune globulin (IVIG) most frequently gives a rapid response. The patient was given 4 doses of IVIG, 0.5 g/kg IV daily (on days 10–13), with no response in his platelet counts (Figure 2). Immune suppressive measures include use of high dose corticosteroids. He was then given a 5 day course of methylprednisolone, 125 mg on days 13–17. The patient's platelet count recovered steadily and returned to normal by the 28th postoperative day. The resolution of thrombocytopenia with immune suppressive therapy lead to an investigation into the immune nature of the severe thrombocytopenia. Because the patient presented with severe thrombocytopenia 9 days after transplantation and blood product exposure, post-transfusion purpura was considered and laboratory tests were ordered. The solid phase platelet antibody screen (Capture-P, Immucor, Inc., Norcross, GA) and the HLA antibody screen were negative on the second day of admission (day 10 after transplantation) (Figure 2). Also, no platelet-reactive antibodies were detected by flow cytometry. The HLA antibody screen performed by Luminex FlowPRA® Class I screening test was negative. A Platelet Antibody Bead Array (PABA) assay was performed on the patient's sera collected postoperative days 2, 12, 13, and 28 and tested negative (results not shown). Platelet antibodies were not detected in the patient's serum, most likely because the anti-HPA-1a was on the autologous platelets and not of sufficient level in the plasma for detection. However, serum from the liver donor showed strong (24x normal) HPA-1a specific platelet antibodies detected by flow cytometry and confirmed by PABA (Table 1). A later sample showed that the patient's platelets were sensitized with antibody to GPIIbIIIa (see below). Detection and identification of platelet-reactive antibodies was performed at the Platelet & Neutrophil Immunology International Reference Lab, BloodCenter of Wisconsin (BCW), Milwaukee using both a flow cytometry test with intact target platelets,1 and a highly sensitive and specific Platelet Antibody Bead Array (PABA)2 for detection and identification of platelet specific antibodies against human platelet alloantigens (HPA). Flow cytometry testing was performed as previously described.3 Briefly, after incubation of serum with group O target platelets having known HPA types, platelets were then washed and incubated with fluorescent-labeled anti-human IgG and IgM and platelet-bound fluorescence measured by FACSCanto II flow cytometer. For the PABA test, the patient's or donor's serum were separately incubated with each of 5 different platelet targets with various HPA types that enabled detection of alloantibodies against HPA-1, −2, −3, −4, and −5. Platelets were washed, and then lysed in detergent to release antibody-GP complexes, and platelet lysates were incubated with a mixture of polystyrene beads coated with monoclonal antibodies for capture of the platelet antibody-GP complexes. A mixture of five different beads was used and each of the 5 beads had a different monoclonal attached (Bead 1: GPIIb/IIIa, 2: GPIa/IIa, 3: GPIb/IX, 4: GPIV, 5: class I HLA) (Table 1). Beads were washed 3 times and incubated with fluorescent anti-human IgG (Jackson ImmunoResearch Labs, West Grove, PA) for detection of patient HPA antibodies. Bead fluorescence intensity was measured on a Luminex 200 (Luminex Corporation) and results determined using an in-house developed software program to call HPA antibody specificity.2 Because flow cytometry and PABA tests showed that the donor had anti-HPA-1a specific platelet antibodies, a direct test for platelet-associated immunoglobulins on patient's platelets was performed at BCW using the PAKAuto ELISA kit (Immucor, Inc., Norcross, GA), as previously described.4 Antibodies were eluted from the patient's platelets using a low pH buffer. An eluate of the patient's platelets collected on postoperative day 28 demonstrated antibodies against GPIIb/IIIa (the HPA-1 antigen is an epitope on the GPIIb/IIIa glycoprotein complex) (results not shown). Testing of the patient's platelets was not performed prior to postoperative day 28 because patient's platelets samples were not available. Taken together, the results show that the donor had HPA-1a platelet specific antibodies and the patient's platelets were sensitized with GPIIb/IIa reactive antibodies, indicating the severe thrombocytopenia was most likely due to passenger lymphocyte syndrome with HPA-1a specific antibodies. Drug-Induced Immune Thrombocytopenia (DIIT) was ruled out with negative tests for sulfamethoxazole-trimethoprim and piperacillin-tazobactam drug-dependent antibodies (results not shown). Additional investigation indicated that the unrelated liver donor was a 34 year old female with blood group A, Rh (D) positive RBCs with a negative antibody screen with no history of receiving blood transfusions. She had five pregnancies with three miscarriages from unknown causes and two normal term deliveries. There was no known bleeding disorder or thrombocytopenia reported for any of her offspring. The donor's HPA-1 genotype was 1b/1b confirming her ability to produce HPA-1a antibodies. The patient's genotype was HPA-1a/a, - 2a/b, −3a/b, -4a/a, -5a/a, −6a/a, −9a/a, and −15b/b. HPA genotyping of DNA for human-platelet-antigens (HPA) was performed at BCW by real-time PCR with HPA-specific hydrolysis probes as described previously.2, 5 Thrombocytopenia in liver disease may result from portal hypertension, bleeding, platelet consumption and impaired platelet production due to reduced thrombopoietin levels. Myelosuppression can also result from HCV infection, ethanol consumption, folic acid deficiency and immunosuppressive therapy. In the post-transplantation setting hemolytic uremic syndrome (HUS) and disseminated intravascular coagulation also should be considered. Severe, isolated thrombocytopenia which develops after liver transplantation is unusual. Causes of severe thrombocytopenia in the post-transplant setting include post-transfusion purpura (PTP) and immune thrombocytopenia (ITP) and Drug-Induced Immune Thrombocytopenia (DIIT). In addition, there are rare isolated case reports of severe thrombocytopenia in patients with the passenger lymphocyte syndrome.6, 7 Here, we report a patient who developed severe thrombocytopenia on the ninth day following living unrelated donor partial liver transplantation. The patient was exposed to multiple blood products and investigation for post-transfusion purpura (PTP) and alloimmune thrombocytopenia were performed. In PTP, increased platelet clearance and thrombocytopenia occur when a HPA-1a negative recipient develops anti-HPA-1a alloantibodies 7–10 days after receiving a blood product containing platelets or platelet membranes from a HPA-1a positive donor.8, 9 It is interesting that platelet clearance in PTP involves autologous as well as transfused platelets and is caused by autoantibodies that form concomitantly with the potent HPA-1a alloantibodies.8 In post-transfusion purpura, the patient develops alloantibodies specific for one or more human platelet alloantigens (most commonly HPA-1a) to which the patient was exposed to by transfusion.8 The HPA-specific alloantibodies are thought to be pathogenic and cause the thrombocytopenia. This may occur by antibodies binding to transfused alloantigens bound to autologous platelets. An alternate theory posits that an autoantibody also specific for HPA-1a may develop in parallel to the alloantibody and bind to the autologous platelets. The thrombocytopenia usually resolves with a decrease in the autoantibody levels. In contrast, in the postallogeneic transplant setting, alloantibodies produced by donor lymphocytes reactive with patient platelet specific GPIIIa (HPA-1a) alloantigens are pathogenic in the passenger lymphocyte syndrome.6, 7 This scenario is rare, but appears analogous to passenger lymphocyte syndrome in the setting of ABO mismatch leading to RBC hemolysis. Passenger lymphocyte syndrome is generally self-limited, due to the brief survival of passively transferred lymphocytes and the short half-life of the resulting alloantibodies. The immunologic testing revealed that the liver donor had strong HPA-1a specific antibodies and the liver recipient's platelets were sensitized with antibodies directed toward GPIIbIIIa carrying the HPA-1a antigen. Furthermore, the recipient HPA genotype was HPA-1a/a and the donor's genotype was HPA-1b/1b confirming her ability to produce HPA-1a antibodies. Therefore, the severe thrombocytopenia and immune clearance of the patient's platelets was most likely due to liver donor passenger lymphocytes producing HPA-1a specific antibodies which sensitized the patient's HPA-1a positive platelets. Antibodies could not be detected in the patient's serum, probably because serum HPA-1a antibody levels were too low for detection since the vast majority of antibody was bound to the patient's platelets. Following treatment with high dose corticosteroids, intravenous gamma globulin and transplant immunosuppression regimen including mycophenolate mofetil, prednisone and tacrolimus, the platelet counts returned to near normal range on the 28th postoperative day. The clinical picture and laboratory findings lead us to conclude that this patient developed thrombocytopenia likely caused by donor lymphocytes from the liver allograft, leading to alloimmune platelet destruction and thrombocytopenia. Once it is established that the cause of thrombocytopenia is due to the presence of HPA-1a antibodies alloreactive with the recipient's platelets, then HPA-Ibb matched platelets should be used for transfusion. Approximately 2 percent of donors have this phenotype. Although passenger lymphocyte syndrome is generally self-limited as discussed above, some benefit may be obtained by providing washed RBC units to avoid exposure to platelet membranes and recurrence of thrombocytopenia. Severe thrombocytopenia with platelets being the target of antibodies produced in passenger lymphocyte syndrome have been described rarely.6, 7 Following liver transplantation, one patient developed severe, life-threatening thrombocytopenia from a donor who had idiopathic thrombocytopenic purpura.6 The liver donor had alloantibodies and autoantibodies directed against GPIIb/IIIa in the serum which were also identified in the recipient after transplantation and platelet destruction was most likely due to transfer of platelet anti-GPIIb/IIIa antibody-producing lymphocytes from the liver allograft. The platelet antibodies and thrombocytopenia resolved after treatment with steroids, gamma globulin, plasma exchange and a second liver transplant with recovery of platelet counts. In a second report, severe thrombocytopenia caused by HPA-1a antibodies developed in two kidney recipients and a liver recipient from a multiparous donor who died of a cerebrovascular accident.7 The three separate recipients were later found to be mismatched with the donor for HPA-1a. One of the kidney recipient's thrombocytopenia resolved following a HPA-1a negative platelet transfusion and splenectomy. Thrombocytopenia in the liver recipient improved following treatment with anti-thymocyte globulin. The other kidney recipient died of multiorgan failure 21 days after transplant. We describe a rarely recognized severe thrombocytopenia that developed in a patient following liver transplantation from a multiparous donor later discovered to have alloreactive passenger lymphocytes producing HPA-1a antibodies against the patient's platelets. The patient's thrombocytopenia resolved following platelet transfusions, intravenous gamma globulin and methylprednisolone treatment. Laboratory investigation of the organ donor and recipient are critical to diagnose the etiology of the thrombocytopenia and to select compatible platelets for transfusion.