HLA-matched Platelets Research Articles

P131 : PLATELET SUPPORT FOLLOWING HAPLOIDENTICAL STEM CELL TRANSPLANT: BLURRED LINES BETWEEN SELF AND NONSELF

As mismatched hematopoietic stem cell transplant (HSCT) becomes more common for treatment of hematologic malignancies, interpretation of HLA sensitization has become more complex. The patient is a 19 year old male with persistent, refractory Acute Myeloid Leukemia (AML), and a history of thrombocytopenia since one week of age. He was found to have a RUNX1 mutation, and a diagnosis of Familial Thrombocytopenia with Predisposition to AML (FT-AML). The patient was referred to St. Jude for haploidentical HSCT and NK cell therapy, using his mother as the donor. Although refractory to platelets, he was negative for anti-HLA antibodies prior to transplant. On day 62 post-HSCT, the patient had engrafted and his peripheral blood chimerism was 100% donor. However, platelet consumption was suspected, and a serum sample at that time was clearly positive for antibodies to B42, 82, 7, 81, 55, 54, 27, 56 (B7 CREG), B49, 62, 50 (B5 CREG), plus B67 and A36. The patient also had antibody to Cw8, and to 5 different DR4 specificities (DRB1 ∗ 04:01, 04:02, 04:03, 04:04, and 04:05). HLA Class I Cw and class II antigens are not expressed on platelets, but may be important in the setting of current or future HSCT. Importantly, the patient’s father has HLA-A1, B56, and DR4. The patient did not have antibodies to his own HLA antigens, or the unshared antigens from his donor, illustrating tolerance to those “self” antigens. The treatment team desired HLA-matched platelets, but these were unavailable, based on the patient’s original HLA type. Compatible platelets were selected based on his original HLA type and excluding donors with HLA antigens to which he had demonstrated antibody. Once HLA-compatible platelets were selected, the patient was no longer refractory and his condition improved. Questions arose during this process regarding which antigens should or should not be considered “self,” considering that this patient was mismatched with his donor at HLA-A, B, DR, and DQ loci. Selection of HLA-compatible platelets in the context of haploidentical HSCT is complex, given that a patient will potentially have multiple unshared donor HLA antigens expressed on blood-derived cells and his original HLA antigens on other body cells. Traditional algorithms for searching platelet donor registries may not be adequate, and may require additional manual review.

Differential Diagnosis of Thrombocytopenia in Hematopoietic Stem Cell Transplant Patients

Thrombocytopenia has remained a major concern in post-haematopoietic stem cell transplant patients. It is a strong negative indicator for survival and is often seen in patients with advanced acute and chronic Graft versus host disease (GVHD). The pathogenesis of thrombocytopenia varies from impaired thrombopoiesis to increased turnover of platelets. The dose of CD34+ cells and that of megakaryocyte specific lineage cells (CD34+ /CD41+ and CD34+ / CD61+ ) has a positive effect on thrombopoiesis. Conditions like bone marrow fibrosis, CMV infection and ganciclovir administration affect the bone marrow environment leading to ineffective thrombopoiesis. Increased destruction of platelets is seen with GVHD, drugs, infections, thrombotic microangiopathy and platelet refractoriness due to alloimmunization. The management of thrombocytopenia includes treating the underlying conditions and supportive care with platelet transfusion. Platelet refractoriness is managed using ABO matched platelets, HLA-matched platelets or cross-matched platelets for transfusion depending on the underlying cause. Some of the recent studies have concentrated on the effect on thromobopoiesis due to maturation of megakaryocytes or ploidy and the effect of cytokines on bone marrow microenvironment. Further studies are needed to incorporate these parameters as part of routine laboratory investigation in thromobocytopenic patients along with estimation of anti-platelet antibodies for better diagnosis and management of these patients.

The role of alloantibodies against human platelet antigen‐15 in multiply platelet transfused patients

Several studies have documented the role of antibodies against human platelet (PLT) antigen (HPA)-15 in alloimmune-mediated thrombocytopenia including neonatal alloimmune thrombocytopenia, PLT transfusion refractoriness (PTR), and posttransfusion purpura in Caucasian persons. However, the relevance of anti-HPA-15 in PTR among the Japanese population is still unclear. The sera of 305 multiply PLT transfused (MPT) patients, previously investigated for the presence of human leukocyte antigen (HLA) and HPA antibodies by mixed passive hemagglutination, were reexamined for the presence of HPA-15 alloantibodies, using the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) technique. Among the 305 MPT samples, antibodies against HPA-15 alloantigen was detected in seven (2.3%), two (0.66%) being anti-HPA-15a and five (1.64%) being anti-HPA-15b. Additionally, one case of CD109 panreactive antibody was found (0.33%). Among them, one aplastic anemia patient with blood group O developed multispecific anti-HLA and anti-HPA-15b alloantibody after MPTs. However, transfusion with HLA-matched PLTs of blood group AB did not result in adequate PLT count increment. Analysis of the possible influence of immune anti-A and anti-B by the MAIPA assay resulted negative, indicating that anti-HPA-15b is responsible for the refractory state in this patient. In this study, we found alloimmunization against HPA-15a and -15b in Japanese populations and demonstrated the relevance of these antibodies in a patient with PTR.

Successful Engraftment in HLA-Mismatched Bone Marrow Transplantation Despite the Persistence of High-Level Donor-Specific Anti-HLA-DR Antibody

Successful Engraftment in HLA-Mismatched Bone Marrow Transplantation Despite the Persistence of High-Level Donor-Specific Anti-HLA-DR Antibody

Open aortic valve replacement in a patient with Glanzmann's thrombasthenia: a multidisciplinary strategy to minimize perioperative bleeding

Glanzmann thrombasthenia (GT) is an autosomal recessive disorder in which the platelet (PLT) glycoprotein IIb/IIIa complex is either deficient or dysfunctional. In its most severe form, GT may result in spontaneous bleeding, although most cases are first detected in the setting of an invasive procedure. A 59-year-old male with Type I GT and a history of transfusion reactions to PLT infusions developed severe aortic stenosis secondary to bicuspid valve disease. He successfully underwent open aortic valve replacement with cardiopulmonary bypass without perioperative bleeding complications. A multidisciplinary team (anesthesia, hematology, cardiac surgery, and transfusion medicine) was established to optimize perioperative hematologic management. Bleeding risk was assessed given the patient's prior history and a dosing timeline for administration of blood products and recombinant clotting factors was established. Successful management was achieved during the operation by prophylactic administration of HLA-matched PLTs and Factor VIIa. Prophylactic PLT administration was continued through the immediate postoperative period and no bleeding complications occurred. Thromboelastograms (TEGs) were used in conjunction with traditional hematologic laboratory analysis to optimize clinical management. Patients with GT requiring cardiac surgical procedures are at high risk for perioperative bleeding complications. This case report illustrates the importance of multidisciplinary planning, TEG analysis, and the judicious use of recombinant factors to minimize operative bleeding risk.

Efficacy of HLA‐matched platelet transfusions for patients with hypoproliferative thrombocytopenia: a systematic review

HLA-matched platelets (PLTs) are widely used to transfuse patients but the effectiveness of HLA matching has not been well defined and the cost is approximately five times the cost of preparing the random-donor PLTs. The objective of this systematic review was to determine whether HLA-matched PLTs lead to a reduction in mortality; reduction in frequency or severity of hemorrhage; reduction in HLA alloimmunization, refractoriness, or PLT utilization; or improvement in PLT count increment in patients with hypoproliferative thrombocytopenia. We conducted a literature search of MEDLINE, Cochrane Controlled Register of Clinical Trials, EMBASE, and PubMed databases to April 2012. A total of 788 citations were reviewed and 30 reports were included in the analysis. Most studies did not include technologies currently in use for HLA typing or detection of HLA antibodies as 75% were conducted before the year 2000. None of the studies were adequately powered to detect an effect on mortality or hemorrhage. HLA-matched PLTs did not reduce alloimmunization and refractoriness rates beyond that offered by leukoreduction, and utilization was not consistently improved. HLA-matched PLTs led to better 1-hour posttransfusion count increments and percentage of PLT recovery in refractory patients; however, the effect at 24 hours was inconsistent. The correlation of the PLT increment with other clinical outcomes and the effect of leukoreduction on HLA-matched PLT transfusion could not be determined. Prospective studies utilizing current technology and examining clinical outcomes are necessary to demonstrate the effectiveness of HLA-matched PLT transfusion.

The Mean Fluorescence Intensities of Anti-HLA Antibodies Detected Using Micro-Bead Flow Cytometry Predict the Risk of Platelet Transfusion Refractoriness.

AbstractAbstract 2285 Background:HLA allo-immunized patients often receive matched platelets only after demonstrating platelet transfusion refractoriness (PTR). If further risk stratification was possible, high risk patients could be considered for pre-emptive HLA-matched platelets, cryopreserved autologous platelets, or possibly thrombopoietin analogues. Micro-bead flow cytometry is widely used to detect anti-HLA antibodies, and mean fluorescence intensities (MFI) obtained from these assays correlate with antibody titers. We asked whether MFIs could be used to stratify the risk of PTR among allo-immunized patients. Study design:We retrospectively identified 387 patients who received an autologous stem cell transplant or induction therapy for acute leukemia, between January 2005 and March 2012. All patients had a serum sample taken for HLA antibody assay within 6 weeks of commencing cellular blood product transfusions. No patient was scheduled to receive prophylactic HLA matched platelets. The primary endpoint was the development of PTR. To minimize the influence of sensitization occurring after screening, only outcomes during the first 2 weeks from commencing cellular blood product transfusions were considered. PTR was defined as having received ≥ 2 consecutive RDPLT transfusions associated with an 18–24h corrected count increment of < 2.5 at 18 – 24 hours.Antibody testing was performed using a micro-bead flow cytometry assay (Lifecodes LifeScreen Deluxe, with positive results confirmed by Lifecodes Class I ID assay, Gen-Probe Transplant Diagnostics, Stamford, CT) either during the treatment period, or on serum samples stored at −30°C. Mean fluorescence intensities (MFI) were acquired using a Luminex 100 analyzer (Luminex Corporation, Austin, TX), and analyzed using Lifecodes Quicktype v2.5.5 (Gen-Probe Transplant Diagnostics, Stamford, CT). We defined the predictor variable avgMFI to be the average MFI of the 7 individual beads in the assay, weighted by whether the presence of antibodies was confirmed or not: [Display omitted] where w = 1 if the presence of antibodies is confirmed, and 0 otherwise; and subscript i refers to the ith class I bead. Results:Antibodies were detected in 57 (14.7%) patients of whom 45 (78.9%) were female. A total of 1443 random donor platelet (RDPLT) transfusions (mean platelet count 2.4×1011/unit) were studied. Sixty six (17%) patients developed PTR, of whom 28 had detectable antibodies; 29 of 321 patients who did not develop PTR also tested positive. Among antibody positive patients, median avgMFI for refractory patients was 4589 versus 349 for patients who were not, Wilcoxon rank sum test P< 0.0001. (Figure 1). The area under the receiver operating characteristic curve for avgMFI as a predictor of PTR was 0.8633 (95% confidence interval: 0.7664 – 0.9602). [Display omitted] [Display omitted] Higher avgMFIs also correlated with a broader range of target antigens, likely due to increasingly avid binding to cross-reactive epitopes. (Spearman’s r = 0.7736 for correlation between avgMFI and panel reactive antibody percentages (cPRA), calculated in reference to the general American population, and used here as a surrogate for the range of antibody specificities). cPRA was >80% in 25/27 patients with avgMFI>1000, suggesting poor ability to discriminate among patients with moderate to high antibody titers, and was not an independent predictor of PTR. Hence, while the increased probability of encountering a cognate antigen on a RDPLT may partly explain the correlation between avgMFI and PTR, the avidity of binding, represented in vitro by the MFIs, appears to be a more significant determinant of risk.In conclusion, we provide evidence for the concept that PTR risk due to HLA allo-immunization is usefully predicted by the MFIs of antibodies detected using micro-bead flow cytometry. Our model allows cut-offs for identifying high risk patients to be based on the degree of risk acceptable in a given clinical situation. This should enable hematology units to develop risk-adapted strategies for supporting allo-immunized thrombocytopenic patients. Disclosures:No relevant conflicts of interest to declare.

Longitudinal management with crossmatch‐compatible platelets for refractory patients: alloimmunization, response to transfusion, and clinical outcomes (CME)

The use of crossmatch-compatible platelets (PLTs) improves posttransfusion corrected count increments (CCIs) in patients with alloimmune PLT refractoriness. However, few reports address the efficacy of utilizing this strategy for patients requiring intensive PLT transfusion therapy lasting several weeks to months. Medical records of patients with two or more PLT crossmatch assays performed between 2002 and 2010 were reviewed. All patients were refractory to random single-donor apheresis PLT units, defined as two consecutive 1-hour posttransfusion CCIs of less than 7500. A commercial solid-phase adherence assay was used for crossmatching. Seventy-one patients were included. A median of four crossmatch assays were performed per patient (range, 2-17). Mean percent reactivity in initial (58.6%) versus last (55.3%) crossmatch assay for each patient demonstrated no trend toward progressive alloimmunization (p = NS). A total of 738 crossmatched PLT units were administered with a mean ± standard deviation CCI of 7000 ± 7900 (n = 443 units with adequate 1-hr posttransfusion counts), a significant improvement over random PLTs (p < 0.001). Patients with an initial crossmatch reactivity of greater than 66% were significantly more likely to demonstrate at least one panreactive crossmatch assay, impacting the availability of compatible PLTs for optimum transfusion support. One patient (1.4%) developed WHO Grade IV bleeding. Progressive alloimmunization to mismatched antigens does not impact medium-term transfusion support with crossmatched PLTs. Increased reactivity in the initial crossmatch assay can serve as a trigger to initiate workup for HLA-matched PLTs as a second-line approach. However, for most patients, medium-term transfusion support with crossmatched PLTs offers an effective and rapid first-line approach to management of PLT transfusion refractoriness.

Living donor liver transplantation for patients immunized against human leukocyte antigen

The clinical features and perioperative management of liver transplant recipients who are already sensitized against human leukocyte antigen (HLA) prior to transplantation are not yet clear. Medical records of living donor liver transplant recipients were reviewed and clinical features of the patients possessing anti-HLA antibodies were studied. Among the 470 consecutive living donor liver transplant recipients, 6 patients (1.3%) had preformed anti-HLA antibodies. A review of the postoperative courses of these patients revealed that the problems included platelet transfusion refractoriness (PTR) due to immune-mediated destruction of platelet and thrombotic microangiopathy (TMA). PTR was observed in patients with anti-HLA class I antibodies and only HLA-matched platelet concentrate (HLA-matched PC) relieved thrombocytopenia. Intravenous gammaglobulin had an additive effect to HLA-matched PC in some cases, and platelet transfusion from close relatives might be a substitute for HLA-matched PC in life-threatening situations. Although the etiology of TMA is unremarkable, the incidence was high (67%, 4/6) compared with that in patients who were not sensitized against HLA (5.6%, 26/464; p < 0.01). Of the four patients, three were complicated with late-onset TMA. Considering these clinical features, careful preparation and postoperative management are needed for liver transplant candidates with anti-HLA antibodies.

Preparation of blood products for transfusion: Is there a best method?

There are a number of possible methods for the preparation of blood components. These vary with respect to risks and benefits to the blood system operation, to the donor and to the recipient. An understanding of these risks is necessary when deciding what component production strategies to pursue. This manuscript reviews the elements that should be considered when choosing which components to produce. The two broad schemas for component production are apheresis and the production of components from whole blood donations. The development of apheresis technologies brought significant benefits to the patient including improving the access to specialty products such as HLA-matched platelets. Benefits to blood centers include the collection of multiple transfusion doses from a single collection procedure. The challenges include the cost of the technology and a small increased risk to donors. Whole blood component preparation offers two main forms of technology: platelet rich plasma, and buffy coat, both of which are driven by platelet production. The two have different risk profiles, with overall benefits being more favorable to buffy coat production methods particularly with respect to improved process control. Issues such as donor variability affect all production methods and require further research.

Transfusion of Cryopreserved Autologous Platelets to Overcome HLA Alloimmunization in Patients with Hematological Malignancies,

Abstract 3375Alloimmunization to platelet antigens occurs in up to 25% of mainly female patients with hematologic disorders receiving intensive chemotherapies. It leads to platelet transfusion refractoriness, increased bleeding risk and often compromises further treatment. Once alloimmunization is established, only HLA-matched platelets improve platelet count increments. However, in patients with rare HLA phenotypes, the identification of HLA matched platelets may be difficult. In this situation, transfusion of autologous platelets is a clinical option, but cryopreservation of platelets has been notoriously difficult. Here, we present our clinical experience with transfusion of cryopreserved autologous platelets in patients with hematological malignancies. The platelets were collected after recovery from chemotherapy. Per patient, 2.5 (1–5) platelet aphereses were performed collecting 298 (190–598) ml per apheresis resulting in 6 (4–8) concentrates. All platelet concentrates were irradiated with 50 Gy, cryopreserved with 10% DMSO and RPMI-1640 without Phenol Red as freezing medium. Platelets were frozen by computer-controlled rate freezing and afterwards kept in liquid nitrogen. For transfusion, platelet concentrates were thawed at bedside in a warming bath (37°C) and transfused immediately without removal of DMSO. Nine patients received 36 cryopreserved autologous platelet transfusions. All patients were female and received intensive chemotherapies for acute leukemias (8) or NHL (1). The median age at diagnosis was 50 (range 30–69) years and the patients had 2 (1–4) prior pregnancies. Alloimmunization occurred 25 (15–458) days after diagnosis with a panel reactivity of 73 (14–93)%. The platelets were transfused 26 (9–133) days after cryopreservation. The platelet count before transfusion was 7 (2–16) G/L and the platelets were transfused prophylactically (32) or therapeutically (4). The median number of platelets per concentrate was 1.7 (0.8–2.9) ×1011. The corrected count increment after 1 hour (1h-CCI) was 5.2 (−1.8–20.2) and after 18–24 hours 4.7 (−5.8–25.2). There was no DMSO toxicity after infusion. The phenotype and function of the cryopreserved platelets were further analyzed in preliminary in vitro experiments by flow-cytometry. As compared to fresh platelets, cryopreservation resulted in a reduction of the size and cytoplasmic complexity. The surface glycoproteins (gp) IIa and IIIb remained unchanged while the expression of the gp Ib was reduced to 54 (47–61)%. The cryopreserved platelets were partially activated, as demonstrated by the expression of negatively charged surface phospholipids (median 73%, normal <5%) and P-selectin (33%, normal <5%). However, the fibrinogen receptor was not activated, as demonstrated by PAC-1 binding (4%, normal <5%). Stimulation with strong platelet agonists (thrombin 0.5 U/ml, convulxin 50 ng/ml) induced full activation of about 1/3 of the cryopreserved platelets, as demonstrated by PAC-1 positivity (increased from 4% to 35%), and further degranulation of the alpha granula (increased from 33% to 55%) and expression of negatively charged phospholipids (increased from 73 to 99%). In conclusion, transfusion of cryopreserved autologous platelets is a feasible alternative to HLA-matched platelets offering an important clinical option for patients with HLA alloimmunization and rare HLA phenotypes. The experimental data demonstrate that the platelets are partially stimulated by the cryopreservation, but can be further activated in vitro by strong agonists, suggesting their functionality. Disclosures:No relevant conflicts of interest to declare.

HLA- and HPA-Alloimmunization Rate In the Setting of Refractoriness to Platelet Transfusion: A Single Center Study,

Abstract 3366 Background:Refractoriness to platelet transfusion is a common clinical problem occurring in up to 34% of hematology/oncology patients (Hod and Schwartz 2008). While often non-immunologic in etiology, alloimmunization to human leukocyte antigens (HLA) or human platelet antigens (HPA) may contribute to this phenomenon. If patients are refractory to pooled, random donor platelets, most institutions have an explicit algorithm which facilitates a supply of single-donor (SD) apheresis and/or HLA-matched platelets. At our institution, platelet refractoriness is defined as a platelet count rise of <10×109/L measured within 24 hours post-transfusion of ABO-matched single-donor platelets on at least two occasions. In this circumstance, HLA matched platelets have been provided regardless of the HLA/HPA alloimmunization status. The goal of this study was to measure the HLA- and HPA-alloimmunization rates in adult patients at our institution who were refractory to single donor platelet transfusions. Methods:All patients at our centre who were refractory to single donor platelet transfusion between January 2006 and June 2011 were included. Patients were tested for the presence of HLA and HPA antibodies at the Canadian Blood Services Platelet Immunology Laboratory. HLA typing was performed by molecular SSO/SSP method and antibody identification was achieved using Luminex single antigen assay following an initial screening test. HPA antibody determination was performed by ELISA. For patients with HLA antibodies, panel reactive antibody (PRA), which estimates the percentage of potential reactivity against donors in the population, was calculated using cPRA software from the Organ Procurement Transplantation Network (OPTN). Results:Thirty-three patients (18 male and 15 female) were tested for HLA and HPA antibodies in the setting of platelet transfusion refractoriness. All patients received multiple blood transfusions prior to becoming refractory to platelets. Most patients (85%; 28/33) had a hematologic malignancy (leukemia (19), lymphoma (5), myelodysplastic syndrome (2), myelofibrosis (1), and amyloidosis (1)) while five patients had non-malignant hematological disorders (aplastic anemia (3), antiphospholipid syndrome (1), and congenital pancytopenia (1)). No HPA antibodies were identified in any patient. HLA antibodies were found in 42% of patients (14/33) and was different according to gender: 22% for males (4/18) vs 67% for females (10/15). Males (n=4) were found to be mildly to moderately sensitized to HLA with an average PRA of 33% (range 11–62%). Nine out of 10 females were highly sensitized to HLA with PRA values between 95 and 100%. There was no association between the presence of HLA antibodies and either patient diagnosis or a history of allogeneic stem cell transplantation. Conclusion:We have demonstrated a relatively high rate of HLA alloimmunization (42%) among patients refractory to single donor platelets compared to previously published reports. This may be due to differences in testing methodology and/or stringency of the definition of platelet transfusion refractoriness. Similar to other studies, we found no significant contribution of HPA antibodies to platelet refractoriness in our patient population. Patient diagnosis and/or history of allogeneic stem cell transplantation did not appear to influence the rate of HLA alloimmunization. Importantly, we found that the majority of refractory patients with HLA-antibodies and high level of HLA sensitization (PRA ≥ 95%) were female (64%; 9/14), implicating pregnancy as a possible contributor to immunologically-based platelet transfusion refractoriness. The finding of high PRA values (≥ 60%) in the majority of HLA-sensitized patients (71%, 10/14) argues against the utility of platelet crossmatching for identification of HLA compatible platelets. In addition, the marked differences in the incidence and the extent of HLA sensitization between male and female patients suggests that gender should be added as an important factor in HLA-matching algorithms for the purposes of platelet transfusion support in refractory patients to optimize the use of HLA-matched platelet donors. Disclosures:No relevant conflicts of interest to declare.

The Analysis of Patients with Refractoriness to Platelet Transfusion Due to Anti-HLA Antibodies During the Treatment of Acute Leukemia

Abstract 4339 Background and aim:Refractoriness to platelet transfusion is one of complications during the treatment of acute leukemia. The major reason is destruction of transfused platelets by anti-human leukocyte antigen (HLA) antibodies to HLA on donor's platelets. We investigated retrospectively the incidence and characteristics of patients with refractoriness to platelet transfusion due to anti-HLA antibodies during the treatment of acute leukemia. Method:From 2000 to 2010, 348 acute leukemia patients underwent chemotherapy at our hospital. Among them, we surveyed 325 patients who received platelet transfusions more than five times. Examination of anti-HLA antibodies was performed when clinically an attending physician suspected that platelet transfusion was not effective. The existence of anti-HLA antibodies were tested both lymphocyte cytotoxicity test (LCT) and anti human immunoglobulin-LCT (AHG-LCT). Result:Of the 325 patients, males were 186, and female were 139. Two hundred sixty-three patients and 62 patients suffered from acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL), respectively. The median age of all patients was 57 (15–86) years. All patients had received red blood cell and platelet products from which leukocytes were removed by filtration. Of all patients, 23 (7.1%) were positive for anti-HLA antibodies. The median age of patients with anti-HLA antibodies was 57(24–76) years. The number of male and female were 2(8.7%) and 21 (91.3 %), respectively. All female patients with anti-HLA antibody had a history of delivery. 8.3 % of AML patients and 1.6% of ALL patients were positive for anti-HAL antibodies.The median number of platelet transfusions until the presence of anti-HLA antibodies was 11 (4–48). The median date of anti-HLA antibodies detection was 27(8–263) days after first platelet transfusion. After detection of anti-HLA antibodies, all patients with anti-HLA antibodies received HLA-matched platelets, and fatal bleeding was not seen. History of delivery (p<0.001), AML (p=0.0147) and age over 50 years (p=0.0490) were risk factors for the presence of anti-HLA antibodies. The presence of anti-HLA antibodies had no impact for the survival (p=0.6033). Conclusion:During chemotherapy for the treatment of acute leukemia, 7% of patients suffered from refractoriness to platelet transfusion due to ant-HLA antibodies. The frequency was low compared with other blood disorders. Elderly AML patients with a history of delivery tended to produce anti-HLA antibodies. Patients with anti-HLA antibodies could obtain similar outcomes by using HLA-matched platelets, comparing with patients without anti-HLA antibodies. Disclosures:No relevant conflicts of interest to declare.

Perioperative management of a patient with Bernard–Soulier syndrome that has a refractoriness to platelet transfusion

A patient with Bernard–Soulier syndrome (BSS), characterized by extremely large platelets, thrombocytopenia and prolonged bleeding time underwent general anesthesia for platelet adhesion aberration. This patient has developed a refractoriness to platelet transfusion due to a frequent transfusion of platelets; however the operation was completed without abnormal bleeding by transfusing HLA-matched platelets during the procedure. BSS is a very rare syndrome so there are no well-defined protocols for the management of perioperative bleeding associated with this syndrome. In this report, we present the detail of the perioperative management for these patients with BSS who has a refractoriness to platelet transfusion.

Molecular analysis of a patient with type I Glanzmann thrombasthenia and clinical impact of the presence of anti-αIIbβ3 alloantibodies

The occurrence of transfusion-related alloimmunization against αIIbβ3 is a major concern in patients with Glanzmann thrombasthenia (GT). However, few data are available about molecular defects of GT patients with anti-αIIbβ3 alloantibodies as well as clinical impact of these antibodies on platelet transfusion. Here, we report a case of type I GT with anti-HLA and anti-αIIbβ3 alloantibodies, who underwent laparoscopic total gastrectomy due to gastric cancer. We found a novel β3 nonsense mutation, 892C > T (Arg272X), and the patient was homozygous for the mutation. Laparoscopic gastrectomy was successfully performed with continuous infusion of HLA-matched platelet concentrates and bolus injection of recombinant factor VIIa at 2 h intervals. Total bleeding was 370 mL and no red-cell transfusion was necessary. Flow cytometric analysis employing anti-αIIbβ3 monoclonal antibody revealed that the transfused platelet count was maintained around 20-30 × 10⁹/L during the operation and 10 × 10⁹/L on the following day. Flow cytometric analysis also showed that transfused platelets retained normal reactivity to ADP stimulation. These results indicate that flow cytometry is useful to assess survival and function of transfused platelets in GT patients with anti-αIIbβ3 antibodies.

Two cases of platelet transfusion refractoriness associated with anti‐CD36

Antibodies to platelet (PLT) glycoprotein (GP) IV (CD36) have been implicated in rare cases of PLT refractoriness, particularly in non-Caucasians. We report two cases of PLT transfusion refractoriness linked to anti-CD36. A 5-year-old female of Lebanese descent and a 70-year-old male of Chinese descent both failed to respond to HLA-matched PLT transfusions during acute myelogenous leukemia induction therapy. Antibody screening was performed using a PLT antibody solid-phase kit (PAKPLUS, GTI Diagnostics), followed by the monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test and, for the second case, the modified antigen capture enzyme-linked immunosorbent assay (MACE). Both patients demonstrated antibody to GP IV (CD36) on the PAKPLUS assay. On MAIPA testing, both phenotyped as CD36 negative. Anti-CD36 was demonstrated by MAIPA in the first case. In the second case, antibodies were not detected by MAIPA and variably detectable by MACE, depending on the mouse monoclonal antibody (MoAb) used. Because no Canadian CD36-negative donors were available, antigen-negative plateletpheresis units from the BloodCenter of Wisconsin were successfully transfused. Two cases of clinically significant CD36 antibodies are reported. Investigation of one case was complicated by steric inhibition of binding in the MAIPA and MACE assays with certain MoAbs. The cases demonstrate the importance of maintaining an ethnically diverse pool of rare donors and the value of international cooperation in the management of these patients.

HLA Alloantibody Persistence and De Novo Production of HLA Alloantibodies of Donor Origin Following Reduced Intensity Allogeneic Hematopoietic Stem Cell Transplantation.

AbstractAbstract 1109▪ Background:Platelet refractoriness as a consequence of HLA alloimmunization complicates allogeneic hematopoietic stem cell transplantation (HSCT). Little is known regarding the affects of reduced intensity HSCT on the incidence and duration of HLA alloimmune-mediated platelet refractoriness following reduced intensity HSCT. Methods:We retrospectively studied HLA alloimmunization in 16 patients with malignant and non-malignant hematological disorders, who underwent an allogeneic HSCT from an HLA matched relative using reduced intensity, non-myeloablative conditioning. All patients received a G-CSF mobilized, lymphocyte replete, peripheral blood stem cell (PBSC) allograft following cyclophosphamide (120 mg/kg) and fludarabine (125 mg/m2) based conditioning (+/− equine ATG 40 mg/kg × 4 days) with cyclosporine (CSA; beginning on day −4) and IV methotrexate (5 mg/m2 × 3 days) given as GVHD prophylaxis. Eight patients known to be HLA alloimmunized pre-transplant were compared to 8 control patients who were HLA alloantibody negative pre-transplant. Stored patients' serum samples from pre-transplant (day -30) and post-transplant intervals (days +30, +60, +100, +180, and ≥ 365) were analyzed for the presence of IgG antibodies to HLA class I antigens using a membrane-independent solid phase assay involving color-coded microbeads coated with HLA antigens and analysis with a flow analyzer (LABScan 100; One Lambda, Canoga Park, CA). Panel reactive antibody (PRA) and mean fluorescent intensity (MFI) were analyzed for all samples to measure HLA antibody strength and specificity, respectively. HLA alloantibodies were analyzed and compared with the degree of donor myeloid and T-cell engraftment measured on post-transplant blood samples by PCR of short tandem repeats (STRs). Results:Among the 8 alloimmunized patients who required HLA matched platelets pre-transplant, the median time until HLA antibodies disappeared was 100 days post HSCT. Remarkably, among these patients, 3/8 (37%) had HLA class I antibodies detectable for more than 100 days post-transplant including one patient who continued to show high level alloreactivity greater than 1 year after HSCT. Among the 8 control patients who tested negative for HLA alloantibodies pre-transplant, 3 acquired HLA alloantibodies after HSCT which were first detected at day +30 in all 3 cases. In 2 of the 3 cases, the donors for these patients were found to have pre-existing HLA antibodies of equivalent specificity to those found in the patient post-transplant suggesting patient acquisition of HLA antibodies was mediated by passenger donor lymphocytes that were transplanted in the allograft. Overall, HLA antibodies were detectable for more than 100 days after transplantation in 6/16 (37%) patients analyzed with 3/16 (18%) having alloantibodies detectable for more than 1 year post-transplant despite chimerism being 100% donor in myeloid and T cell lineages. Conclusions:Prolonged production of HLA alloantibodies leading to platelet refractoriness can occur following reduced intensity allogeneic HSCT and may persist even after full donor myeloid and T-cell chimerism have been achieved. Remarkably, we observed that transplantation of passenger donor lymphocytes can result in de novo HLA alloimmunization, complicating post-transplant transfusion management. Screening patients and their donors for HLA antibodies before HSCT would identify the majority of subjects at risk for alloimmune-mediated platelet refractoriness after transplantation. This new screening strategy would not only assist in transfusion support after allogeneic HSCT but could also play a role in the decision analysis for selecting optimal stem cell donors. Disclosures:No relevant conflicts of interest to declare.

Platelet transfusion refractoriness responding preferentially to single donor aphaeresis platelets compatible for both ABO and HLA

Platelet transfusion refractoriness is challenging to manage. When human leucocyte antigens (HLA)-sensitized patients fail to respond to HLA-matched (HLA-m) platelets, non-immune destruction may be assumed, and collections of HLA-m platelets abandoned. We report cases of highly HLA-sensitized patients whose only satisfactory platelet transfusion responses were consistently associated with products compatible for both HLA- and ABO-matched (HLA-m/ABO-m) platelets, and in whom unsatisfactory increments occurred if either form of major incompatibility was permitted (HLA-u or ABO-u). Absolute platelet increments (APIs) were measured and classified as satisfactory if ≥10 and unsatisfactory if <10. Patient 1, age 59 years, group O, with myelodysplastic syndrome/acute myelogenous leukemia (MDS/AML), was unresponsive to either fresh ABO-m or HLA-m platelets. Of 17 HLA-m platelets, satisfactory responses occurred for 75% of HLA-m/ABO-m units, and failures for 100% of HLA-m/ABO-u, with mean API differing significantly (14·1 vs 1·1, P = 0·0059). Of 36 HLA-m platelets given to patient 2, age 49 years, group O, Gravida 2 Para 2, with severe aplastic anaemia, a satisfactory response occurred with 75% of HLA-m/ABO-m units, and failures for 63% of the HLA-m/ABO-u (mean API 26·7 vs 7·6, P = 0·008). Increment failures from HLA-m platelets need not imply intractable refractoriness. If resources permit, selection of HLA-m/ABO-m platelets may optimise the incremental response.

Transfusion Associated Graft Versus Host Disease Following Whole Blood Transfusion from an Unrelated Donor in an Immunocompetent Patient

Graft-versus-host disease (GVHD) is a well-known complication of allogeneic bone marrow transplantation. Transfusion associated graft-versus-host disease (TA-GVHD) is much less common and nearly uniformly fatal complication of blood transfusion. The risk factors underlying the development of TA- GVHD are incompletely defined, but it is commonly seen in individuals with congenital or acquired immunodeficiency, transfusions from blood relatives, intrauterine transfusions and HLA-matched platelet transfusions. Diagnosis of TA-GVHD may be difficult at a time due to rarity in occurrence and overlapping clinical features with various infections and drug reactions. We describe a case of transfusion-associated GVHD that occurred after transfusion of whole blood from unrelated donor in an immunocompetent patient.

Diagnostic and Therapeutic Considerations on Inherited Platelet Disorders in Neonates and Children

Inherited disorders of platelets constitute a group of rare diseases that give rise to bleeding syndromes of variety severity, with more severe cases being first diagnosed during infancy and childhood. To appropriate diagnose a platelet function disorder during early childhood the knowledge of the physiological characteristics of platelets in the paediatric population is mandatory. Apart from thrombocytopenia which is quite common in neonates and children the present overview is aimed to focus on inherited platelet function disorders. Furthermore, knowledge on platelet maturation and reference values according to age are given, and a diagnostic strategy specifically adapted to a pediatric population is presented on the bases of plasmatic and molecular laboratory methodologies. Finally, therapeutic approaches are briefly summarized (antifibrinolytic agents, Desmopressin, HLA-matched platelets, recombinant factor VIIa).