Single-donor Platelet Units Research Articles

Assessment of Platelet Crossmatch Results by Solid Phase Red Cell Adherence Assay among Pediatric Hemato-oncology Patients in a Tertiary Care Oncology Center

ABSTRACT Background and Objectives: This study employed platelets cross-matched by the solid-phase red cell adherence (SPRCA) method and oc-related it with posttransfusion platelet count increment in terms of corrected count increment (CCI) and percent platelet recovery (PPR) among pediatric hemato-oncology patients. It did not study subgroups of patients nor did it have a control group. This was an observational study. The objective of the study was to assess platelet crossmatch results by the SPRCA method and find its correlation with posttransfusion platelet count increment among pediatric hemato-oncology patients in a tertiary care oncology center. Methods: This was a prospective observational study performed on pediatric patients with hematological malignancies requiring platelet transfusions during August 2019–January 2020. An assessment of the response to platelet transfusion on the participants was analyzed against the crossmatch results using SPRCA to measure the response in terms of CCI and PPR. Results: A total of 80 pediatric multiply transfused patients of hematological malignancies were transfused with ABO-identical single donor platelet unit which was subsequently tested for compatibility SPRCA assay. Among a total of 80 platelet crossmatches, 86% (69/80) were found to be compatible. Among 69 patients with compatible platelet crossmatches, 91% (63/69) had adequate CCI (>7500). Among 11 patients with incompatible crossmatches, 73% (8/11) had adequate CCI, and 27% (3/11) had inadequate CCI. Statistically significant association was found between crossmatch compatibility and PPR. Conclusions: Transfusion of crossmatched platelets from the available inventory to multiply transfused patients of hematological malignancies can be an option. It is very important to assess the posttransfusion platelet responses in such patients in terms of CCI as well as PPR. The SPRCA assay is a rapid and effective method to select the compatible unit from the inventory and is feasible to implement in an oncology setup to cater the requirement of multiple platelet transfusions to patients with hematological malignancies.

Alloantibody Levels, Immunoglobulin Degradation Products, and Platelet Transfusion Response over Successful Desensitization of Severe Alloimmune Platelet Refractoriness with a Novel IgG-Targeted Enzyme Therapeutic

INTRODUCTION Alloimmune platelet refractoriness (APR), an insufficient response to platelet transfusion due to anti-HLA alloantibodies, affects ~25% of acute myeloid leukemia (AML) patients. In APR, HLA- or crossmatch-compatible platelet units may not be identifiable, precluding transfusion and increasing hemorrhage risk. Single antigen bead testing (SAB) semi-quantitates antibody levels against cognate HLA antigens in units of mean fluorescence intensity (MFI). MFI >3,000 suggests an incompatible platelet antigen for transfusion. Imlifidase, an IgG-specific cysteine protease, cleaves IgG first into single-cleaved IgG (scIgG) with limited Fc-mediated function, then into inactive F(ab') fragments 1. We previously reported successful desensitization and provision of permissive platelet transfusions in an APR patient following desensitization treatment with imlifidase. Here we report pre- and post-desensitization alloantibody (PDAb) levels in correlation with IgG degradation products and response to single donor platelet transfusion. CASE As described previously 2, a 43-year-old highly sensitized (calculated PRA [cPRA] 99.9%) woman with AML and history of life-threatening thrombocytopenic hemorrhage secondary to APR presented for matched-unrelated donor stem cell transplantation. Nationwide, few compatible platelet units were identified to support her through the anticipated platelet nadir. Following conventional desensitization, cPRA did not change significantly. Following imlifidase-desensitization [0.25 mg/kg IV (D-1 and D1)], cPRA decreased significantly. 7 single-donor, antigen-avoidance platelet units produced corrected count increments (CCI) [median (interquartile range) of 6,000 (500-7500)]. The patient experienced no hemorrhage and was discharged stable, post-platelet engraftment on D+19. METHODS With IRB approval, patient data and samples (D-16; D-1 post-conventional desensitization with rituximab and therapeutic plasma exchange; D0 post-imlifidase dose 1; D2 post-imlifidase dose 2; and D7, D14 and D29 follow-up samples) were collected. Serial dilutions of patient D-1 serum were tested with SAB (LABScreen single antigen, One Lambda/ThermoFisher, West Hills, California). SDS-PAGE (gradient gel, non-reduced conditions), Western Blot (antiF(ab') detection), and SAB were performed and acceptable antigens (to molecular specificity, targeted by MFI ≤3,000) were compared over the clinical course. Summative MFIs targeting transfused single donor platelet units were compared with 1-hour CCI. Wilcoxon matched-pairs signed rank for matched, nonparametric values and Chi-squared or Fisher's exact for categorical variables were used. P <0.05 was significant. RESULTS/DISCUSSION The D-1 sample (post-conventional desensitization, pre-imlifidase) showed exquisite HLA sensitization. The highest Class A and B antibodies demonstrated an effective MFI of >4,000,000, each (Fig. A). PDAb and the number of unacceptable antigens decreased significantly in D0 (post-imlifidase) vs. D-1 (pre-imlifidase) samples; Intact IgG and scIgG were eliminated from the D0 and D2 samples (Fig. B). Of 7 single donor platelet transfusions, the 5 producing adequate response were each targeted by a summative MFI ≤4,000 (median 4,000 MFI, range 300-4,000). The 2 units producing inadequate CCI response were targeted by summative MFI >4,000 (5,050, 4,900-5,200), suggesting a threshold summative MFI precluding adequate response. CONCLUSIONS Here we show that the patient's pre-desensitization alloantibody levels significantly exceeded the analytical range of the SAB; PDAb levels declined significantly from baseline, correlating with lack of intact IgG and scIgG following imlifidase; and permissive transfusions (CCI >5,000) were seen to platelets targeted by ≤4,000 summative MFI. These results suggest imlifidase is capable of effective desensitization despite profound baseline sensitization, with rapid cleavage of intact IgG to F(ab'), resulting in the temporary reduction of unacceptable antigens. Larger scale investigation of imlifidase in desensitization of APR patients requiring significant transfusion over a limited period of time, along with investigation into transfusion strategies in desensitized APR, are warranted. REFERENCES Am J Transpl. 2022;22(3):691-697.Blood. 2022;140 (Suppl. 1):8562-3. .

Vaginal Delivery in a Primipara with Glanzmann Thrombasthenia

Vaginal Delivery in a Primipara with Glanzmann Thrombasthenia

FREQUENCY OF THE APPROPRIATE USE OF PLATELET CONCENTRATES IN A TERTIARY CARE HOSPITAL

Objective: The objective of the study was to determine frequency of the appropriate use of platelet concentrates in a tertiary care hospital. Material and Methods: It was a descriptive cross-sectional study that was conducted in Department of Pathology, Pakistan Atomic Energy Commission (PAEC) General Hospital, Islamabad for a period of six months from 1st January to 30th June 2021. Patients of both genders, above five years of age receiving platelets transfusions during the study period were included in this study. An informed consent was obtained from the patients. The data was collected on a predesigned form that included patient demographic and clinical details. All patients were categorized whether the platelet transfusion was appropriate or inappropriate according to the mentioned definitions. To control confounding effects of various factors and to avoid bias in the study, the exclusion criteria were strictly followed. t test was applied to find statistical difference between appropriate and inappropriate transfusions. Results: Out of total 331 patients, 169 (51%) were females while 162 (49%) were males, male: female ratio being 1: 1.04. Mean age was 41.75 + 23.1 years with a range of 6-92 years. Maximum patients were in 31-40 years age group. Single donor platelet units were transfused to 55(16.6%) patients while rest 276 (83.4%) patients received random donor platelets. 301 (90.9%) had appropriate transfusion while 30 (9.1%) patients received blood transfusion due to inappropriate indications. (p <0.0001). Conclusion: From this study it was concluded that a significant number of patients in hospital setting receive inappropriate platelet transfusions. Key Words: Appropriate, Platelet concentrates, Transfusion

Comparison of Three Methods for Enumeration of Residual White Blood Cells in Single Donor Apheresis Platelets

Background and Objectives: The aim of this study was to compare three platforms side-by-side: Nageotte hemocytometer, flow cytometry (FC), and standard hematology analyzer for enumeration of residual white blood cells (rWBCs) in single donor platelets (SDPs) apheresis. Materials and Methods: This was a prospective observational study conducted in a tertiary care oncology center by evaluating 36 units of SDP that were collected and tested in parallel by three different methods for the enumeration of rWBCs. All tests were performed within 24 h of collection according to the manufacturers' recommended methods. Counting by the Nageotte hemocytometer was done by: rWBC/μl = (cells counted × dilution/gridded area volume [50 μl]) and FC calculation was performed by: rWBC/μl = (total beads × total number of leukocyte events/total beads acquired × total sample volume). Results: The number of rWBCs detected by FC was between 1 white blood cell (WBC)/μl and 8 WBCs/μl; whereas, those detected by Nageotte chamber were between 3 WBC/μl and 6 WBCs/μl. The range of rWBC detected by hematology analyzer was 100 WBC/μl to 270 WBCs/μl. There was no correlation observed between the results obtained in standard hematology analyzer with any of the other two methods. The concordance correlation coefficient was measured by kappa analysis and found to be 0.71 between hemocytometry and FC. Linear regression analysis also showed a moderate correlation (R 2 = 0.42) between the two methods. However, the coefficient of variation was found to be 49.32% in Nageotte method compared to the 17.55% in FC (P < 0.001). Conclusion: FC followed by Nageotte chamber counting is a better method compared to the standard hematology analyzer for the enumeration of rWBCs. To overcome the cost of FC it is high time to explore the scope and feasibility of a centralized quality monitoring system in India for all leukoreduced blood components.

Qualitative Evaluation of Platelet Concentrates Prepared with Different Methods

Introduction: One of the most important responsibility of Blood Bank is to ensure uninterrupted supply of quality blood products, namely Platelet Concentrate (PC), which are in high demand particularly during pandemic. In India, PCs are of three types prepared by different methods like Platelet Rich Plasma-Platelet Concentrate (PRP-PC), Buffy Coat Platelet Concentrates (BC- PC), and Single Donor Platelets (SDP) units. Hence, establishing optimum quality standards across all the three types of PC remains a challenge in order to maximum benefit to recipients. Aim: To assess and compare the quality parameters of platelet concentrate prepared by PRP-PC, BC-PC and apheresis methods (SDP). Materials and Methods: This laboratory based observational study was conducted at rural, tertiary care, postgraduate, teaching hospital located at South India from December 2018 to November 2019. A total of 156 PCs were studied with 52 units belonging to PRP method, 52 units BC-PC method and the remaining were 52 units of SDP prepared by apheresis method. Quality parameters include volume, pH, swirling, White Blood Cell (WBC) count/bag, platelet count/bag and platelet indices. Platelet indices include Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), and Platelet Large Cell Ratio (PLCR). Arterial Blood Gas (ABG) analysis was also done to assess (pH, partial pressure of Carbon Dioxide (pCO2), plasma bicarbonate (pHCO3-)). The p-value less than 0.05 were considered statistically significant. Statistical Package for the Social Sciences (SPSS) software version 23.0 used for statistical analysis. Results: All the 156 PC units were analysed as per the Director General of Health Services (DGHS) India, criteria. Among the 156 platelet concentrates scored as per DGHS criteria, a score of 5 was obtained for 34.61% [18/52], 30.76 [16/52] and 11.5% [6/52] SDP, BC-PC and PRP-PC units, respectively. All the PC units were sterile and Red Blood Cell (RBC) contamination was within acceptable limits. Conclusion: In this study, quality parameters of SDP was better than BC-PC and PRP-PC but with improvement and more standardisation of procedures and storage, BC-PC units can give a yield similar to that of SDP units.

Factors influencing transfusion‐associated HLA sensitization in patients bridged to heart transplantation using ventricular assist device

Bridging heart failure patients with mechanical ventricular assist devices (VAD) enables access to transplantation. However, VAD is associated with increased risk for anti-HLA antibodies associated with rejection of subsequent allografts. Factors determining alloantibody formation in these patients remain undefined. We performed a single-center retrospective cohort study of 164 patients undergoing heart transplantation from 2014 to 2017. Medical records including use of VAD, transfused blood products, anti-HLA antibody testing, crossmatch, and time to transplant were evaluated. Patients received an average of 13.8 red blood cell and 1.9 single-donor platelet units associated with VAD. There was a 28.7% increase in the incidence of anti-HLA antibodies after VAD. Development of anti-HLA antibodies did not correlate with volume or type of blood products, but with pre-VAD HLA sensitization status; relative risk of new alloantibodies in patients with pre-VAD antibodies was 3.5-fold higher than those without prior antibodies (P=.008). Development of new anti-HLA antibodies was associated with an increased time to transplant (169 vs 330days, P=.013). Our findings indicate that the presence of anti-HLA antibodies pre-VAD was the most significant risk factor for developing additional antibodies post-VAD, suggesting that a subset of patients may be predisposed to alloantibody formation.

A Case Report of Refractory Immune Thrombocytopenia (ITP) Following Reduced Intensity Conditioning (RIC) Hematopoietic Cell Transplantation (HCT) for Myelodysplastic Syndrome (MDS) Successfully Treated with Off-Label Use of Daratumumab

Background:A source of treatment refractoriness in immune cytopenias appears to be residual CD138/38 positive lymphocyte populations (Audia S et al, Blood 118:4394-400,2011; Mahevas M et al, J Clin Invest 123:432-442, 2013). Persistence of recipient's plasma cells can lead to prolonged refractory thrombocytopenia following RIC-HCT. (Fasano RM et al, Br J Haematol 166(3):425-34, 2014). Daratumumab was effective in the treatment of a child with refractory autoimmune hemolytic anemia after HCT (Tolbert et al, Blood 128:4819, 2016).Case Report:The patient is a 60-year-old man with intermediate-high risk MDS who underwent RIC-HCT with total lymphoid irradiation and antithymocyte globulin with peripheral blood graft from a fully matched unrelated male donor. The patient had mild thrombocytopenia prior to HCT consistent with MDS and had not received platelet transfusions. He had not received any prior therapy for MDS. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mycophenolate mofetil. Cytomegalovirus (CMV) serologic testing for exposure was negative for the recipient and positive for the donor. Both the patient and the donor had evidence for prior exposure to Epstein-Barr virus (EBV). He achieved engraftment on day +12. His peripheral blood chimerism on day + 30 showed full donor origin (WB 98%,CD3 96%,CD15 95%, CD19 98%, CD56 95%) and has been maintained to date. Acute skin GVHD responded to corticosteroids. While on corticosteroid therapy, he developed an abrupt decline in platelet count from 156,000/mcl on day +152 to 9, 000/mcl on Day + 166 without evidence for recurrent or active GVHD. While this was initially attributed to simultaneous EBV and CMV reactivations, severe thrombocytopenia persisted after viral clearance. An extensive work up for other etiologies of thrombocytopenia was negative. Repeated bone marrow biopsies were normal, including adequate megakaryocytosis and no MDS recurrence. Platelet associated antibody testing and platelet antigen genotyping were not conclusive for autoimmune versus alloimmune etiology. Testing for platelet HLA antibodies showed calculated Panel Reactive Antibody of 31% and unsatisfactory corrected count increment after transfusion of HLA compatible platelets units. The patient experienced prolonged severe thrombocytopenia for over 26 weeks with platelet count less than 5000/mcl for 22 weeks and only above 10,000 /mcl on 6 occasions. Potentially responsible medications were discontinued serially, but testing for drug inducted ITP was not conducted. Therapy included high dose corticosteroids, high dose immune globulin, rituximab, plasma exchange, splenectomy, romiplostim 10 mcg/kg/week, eltrombopag 100 mg to 150 mg daily for over 24 weeks, and low dose danazol. Fostamatinib was not available. Prednisone dose was tapered over many weeks to 5 mg daily. The patient experienced recurrent life-threatening and vision-threatening bleeding. Cumulative transfusions following Day + 166 were 133 single donor platelet units and 42 red cell units. All products were from CMV negative donors. Eltrombopag and danazol were deemed ineffective and tapered to discontinuation. CD38 positive cells were present in spleen and marrow by immunohistochemistry. Daratumumab 1300 mg was infused weekly x 4. Four weeks after the last dose of daratumumab, his platelet count increased to 91,000/mcl. Platelet count normalized to 150,000/mcl in week 5 or HCT Day + 383. Hypogammaglobulinemia has been the only detectable toxicity. Testing to determine autoimmune versus alloimmune origin is ongoing.Conclusion: Clinical trials of daratumumab for the treatment of severe refractory ITP are indicated. DisclosuresNo relevant conflicts of interest to declare.

α-Synuclein concentration increases over time in plasma supernatant of single donor platelets.

In platelets, α-synuclein is important in calcium-dependent granule release. Notably, cells release α-synuclein in setting of cell damage or death. Therefore, we investigated α-synuclein levels in plasma of single donor platelet (SDP) units during storage. Aliquots were obtained from same SDP units for 7days from day of donation. Additionally, randomly sampled SDP units at same storage time points were also assayed by enzyme-linked immunosorbent assay. α-Synuclein in SDP plasma increased continuously over time at each assayed time point. Significant increases were measured on day 3 (11.7±9.6ng/mL, P=0.025), day 5 (15.3±5.9ng/mL, P=0.002), and highest on day 7 (23.7±5.6ng/mL, P<0.0001) compared to day 0 (1.1±0.8ng/mL). Similar significant results were obtained in randomly sampled SDP units at same corresponding time points. Flow cytometry showed that platelets had strong expression of α-synuclein and lacked expression of other synucleins. Increases of α-synuclein during SDP storage is a steady and continuous process that increases with time. Our findings indicate that α-synuclein may represent a biomarker of platelet biological state during storage. Further research will be needed to determine how α-synuclein increases correlate with platelets' function.

Adding to platelet safety and life: Platelet additive solutions.

BACKGROUND:Platelet additive solutions (PAS) are crystalloid nutrient media used in place of plasma for platelet storage. They replace 60%–70% of plasma in platelet components, so the amount of storage plasma can be decreased. Platelets in PAS have lower risk for allergic transfusion reactions with equivalent clinical efficacy for controlling bleeding.AIM:The aim of this study is to evaluate the clinical and laboratory efficacy of PAS-platelets.MATERIALS AND METHODS:A total of 1674 single donor platelet (SDP) were collected in PAS in the month of June to September 2016 by different apheresis systems. The quality control tests were done on 356 units in 4 months. Total number of SDP were processed with Amicus device (n = 232), Trima Accel (n = 84), and MCS+ (n = 40). The parameters analyzed were antibody titer of anti-A and anti-B, volume, platelet count, pH, bacterial contamination, and reporting of adverse transfusion reaction. Antibody titers were checked by tube technique, and platelet counts were checked by hematology analyzer Sysmex poch 100i. The swirling was checked manually, and pH was checked with pH strips.RESULTS:Out of 356, 164 units were O group, 113 units were B group, 68 units were of A group, and the remaining 11 units were of AB Group. Anti-A and anti-B titer was significantly reduced in PAS-SDP and found 1:32 or less for all the units. All the units found negative for bacterial contamination. No transfusion reaction was reported of the units transfused. All other quality parameters for platelets also found satisfactory after implementing the additive solution.CONCLUSION:The ABO antibody titers were significantly reduced after addition of PAS. This facilitates the ABO incompatible SDP transfusion and helps in inventory management. The risk of allergic transfusion reaction decreases after reducing the amount of plasma from SDP units. Using PAS-SDP certainly improve the inventory management for platelets with no compromise on clinical and laboratory efficacy.

OR34 Platelet refractoriness in a patient with 0% PRA due to a rare anti-CD36(NAKa) antibody

Platelet transfusion refractoriness in patients with hematological malignancies ranges from 7% to 34%. The etiology of platelet refractoriness in this patient population is typically attributable to due to formation of HLA antibodies (Abs) from pregnancy or previous transfusion. We report platelet refractoriness in the setting of induction chemotherapy for AML from allo-Abs to NAK a antigen/CD36/glycoprotein (GP) IV. A 66yo AAF presented with transfusion dependent AML with 70% circulating peripheral blasts and started on 7 + 3 regimen. She developed profound thrombocytopenia and her platelet count was 0 by day 13. As part of her evaluation for refractoriness, HLA antibody screening was done, but was negative. An Immucor ELISA assay was then performed and revealed positive Abs to GPIV (NAK a antigen/CD36). Abs against GPIIb/IIIa, GPIa/IIa, GPIb/IX, and HLA Class I were all negative. GPIV Abs were confirmed by Blood Center of Wisconsin using monoclonal Ab MBC 131.7 against CD36. Cross-matching was performed at our institution with multiple single donor apheresis platelets, all showing strong positive reactivity. Because CD36 in these units may be soluble, an incompatible leukoreduced, irradiated and washed single donor platelet unit was given which was tolerated well with a resultant increase to 14,000/ μ L. Despite strong reactivity of this unit in the crossmatch, the patient’s platelet count increased and 1 week later was 16,000/μL despite no transfusion support. Platelet refractoriness in the setting of hematological malignancy is well documented. Abs develop from prior pregnancy and transfusion in the setting of prolonged pancytopenia in hematological malignancies. Typically, allo-Abs develop against platelet membrane structures including human platelet antigens, HLA antigens, and rarely NAK a antigen on the CD36 glycoprotein molecule. CD36 is expressed in almost 100% of white Europeans and is not detectably expressed by 2% of sub-Saharan Africans and 10% of Asians. CD36 is a soluble antigen and is expressed on various cells such as erythroblasts and monocytes. We propose that washing platelets may be a useful strategy in treating platelet refractoriness in patients with abs to NAK a antigen as finding CD36 negative donors is extremely difficult and few blood banks in North America test donors for this antigen.

Platelet Refractoriness Due to Anti-CD-36(NAKa) Ameliorated By Platelet Washing

▪Introduction:Platelet transfusion refractoriness in patients with hematological malignancies ranges from 7-34% (Blood 2015; 126:3484). The etiology of platelet refractoriness in this patient population is typically due to formation of HLA antibodies from pregnancy or previous transfusion. Although these patients receive leukoreduced products, alloimmunization is not completely prevented. We report platelet refractoriness in the setting of induction chemotherapy for acute myeloid leukemia (AML) from alloantibodies to NAKa antigen/CD-36/glycoprotein (GP) IV. We propose that washing platelets may be a potential strategy for treating platelet refractoriness in patients with NAKaantibodies as finding CD36 negative donors is extremely difficult and few blood banks in North America test donors for this antigen.Case:A 66 year old African American female presented with pancytopenia requiring transfusions. She was later diagnosed with AML with 70% circulating peripheral blasts and started on 7+3 regimen (Cytarabine for 7 days and Idarubicin for 3 days). She developed profound thrombocytopenia by day 7 and her platelet count was 0 by day 13. She was refractory to platelet transfusion from the outset. An Immucor ELISA assay was positive for anti-GPIV (NAKaantigen/CD-36). No antibody to GPIIb/IIIa, GPIa/IIa, GPIb/IX, and HLA Class I was present. Anti-GPIV was confirmed by Blood Center of Wisconsin utilizing monoclonal antibody MBC 131.7 (produced by Blood Center of Wisconsin) directed against CD36. Cross-matching was performed at our institution with multiple single donor apheresis platelets, all showing strong positive reactivity. Given the patient’s low platelet count and onset of urinary tract bleeding, an incompatible leukoreduced, irradiated, and washed single donor platelet unit was given. The patient tolerated the transfusion with no adverse events noted during or after transfusion. The patient’s platelet count rose from < 1 to 14,000/µL when measured 4 hours after the transfusion, and her count was sustained through the following day. Despite the strong, positive reactivity of this unit at crossmatch testing, this was the first successful response to multiple platelet transfusions. Unfortunately, her percentage of blasts in the peripheral blood increased and she maintained an absolute neutrophil count of 0. The patient and hematology team decided to switch to hospice as her AML was resistant to therapy and unfit for any other myelotoxic regimens. Her platelet count was checked the following week and it was 16,000/µL despite no transfusion support.Discussion:Platelet refractoriness in the setting of hematological malignancy has been well documented. Alloantibodies develop from prior pregnancy, prior transfusion or multiple transfusions in the setting of prolonged pancytopenia in hematological malignancies, especially AML. Typically, antibody develops against platelet membrane polymorphic structures including human platelet, ABO, HLA, and very rarely NAKa antigens on the CD36 glycoprotein molecule. CD36 is expressed in almost 100% of white Europeans and is not expressed by 2% of sub-Saharan Africans and 10% of Asians (Blood Transfus 2015: 13: 380). In screening of 871 US blood donors, Yamamoto et al. identified three platelet CD36 deficient individuals. Thus finding a CD36 negative platelet donor will most likely take an extended amount of time, which could be a challenge in these patients as bleeding risk is high during or after induction chemotherapy. CD36 is a soluble antigen and is expressed on various cells such as erythroblasts and monocytes. It has been proposed that when ABO mismatched platelets are transfused, circulating immune complexes may form between the soluble ABH antigens and the transfused antibody. Platelets may then be destroyed by binding of immune complexes to the Fc and C1q receptors. We speculate that a similar pathophysiology may occur in CD36 deficient patients as circulating anti-CD36 forms immune complexes with donor soluble CD36 antigen. Washing will remove most soluble CD36 and potentially mitigate the effect of immune complex formation. Based upon the limited experience with this patient, we suggest that washing in this setting may be beneficial and yield a sustained, incremental response in the platelet count in patients with platelet refractorinesssecondary to anti-NAKa. DisclosuresBlumberg:Biomet/Citra Labs: Consultancy, Honoraria.

Intravenous Anti-D immunoglobulin for control of life threatening bleed in dengue hemorrhagic fever with severe thrombocytopenia

We report an eight-year-old girl with Dengue Hemorrhagic Fever, shock and a life-threatening upper gastrointestinal and pulmonary bleed with refractory thrombocytopenia. She received 6 units of random donor platelets, 1 unit of single donor platelets, 1 unit of packed red cells and 4 units of fresh frozen plasma to control bleeding but had no response. Finally, 36 hour after onset of bleed intravenous Anti-D 75 μg/kg was given and she had a dramatic increase in platelet counts and cessation of all clinical bleed. This case report highlights that intravenous Anti-D can be a treatment option in Dengue Hemorrhagic Fever with severe refractory thrombocytopenia and massive bleeding.

Alveolar rhabdomyosarcoma with massive disseminated intravascular coagulopathy treated with systemic chemotherapy.

It is uncommon for pediatric patients with rhabdomyosarcoma to present with clinical and/or laboratory features of disseminated intravascular coagulation (DIC). We report a case of metastatic alveolar rhabdomyosarcoma with severe bleeding because of DIC in a 13-year-old boy. He experienced persistent oozing at the site of a previous operation, gross hematuria, and massive epistaxis. Two weeks after initiating combination chemotherapy consisting of vincristine, doxorubicin, and cyclophosphamide, the patients' laboratory indications of DIC began to resolve. During this period, the patient received massive blood transfusion of a total of 311 units (26 units of red blood cells, 26 units of fresh frozen plasma, 74 units of platelet concentrates, 17 units of single donor platelets, and 168 units of cryoprecipitate), antithrombin-III and a synthetic protease inhibitor. Despite chemotherapy and radiation therapy, he died 1 year later because of disease progression. In children with metastatic rhabdomyosarcoma and massive DIC, prompt chemotherapy and aggressive supportive care is important to decrease malignancy-triggered procoagulant activities.

Retrospective Review of Platelet Transfusion Practices during 2013 Dengue Epidemic of Delhi, India

Background: Dengue infection is a major public health problem. During explosive outbreaks, there is sudden surge in demands of platelet products. The present study was carried out in order to review platelet transfusion practices during the epidemic of dengue. Methods: We retrospectively reviewed the clinical details including the platelet counts and haemorrhagic tendencies of dengue patients as well as the transfusion requirements of diagnosed dengue cases admitted at our centre. Results: A total of 1,750 random donor platelet and 114 single donor platelet units were transfused to 531 patients. 23.2% platelet transfusions were found to be inappropriate Mean dosage of platelets transfused was 2 × 10¹¹ platelets per patient. A total of 347 (65.3%) patients had bleeding diathesis at the time of presentation. Skin and the oropharynx were the most common bleeding sites. Major bleeding was seen in 119 (34.3%) patients, whereas 228 (65.7%) patients had minor bleeding episodes. Conclusion: The study emphasises the need for minimising unnecessary transfusions and for using this scarce resource judiciously, which can be achieved by strict adherence to evidence-based transfusion guidelines and regular review of the on-going transfusion practices.

Continuous Platelet Transfusion Increases Platelet Increment in Refractory Hemato-Oncological Patients – a Single Center Experience

▪Background: Refractoriness to platelet transfusion is prevalent among 15-25% of hemato-oncology patients. Refractoriness has been linked to inferior clinical outcomes, including bleeding and mortality, as well as higher health care costs. Suggested etiologies to refractoriness include both non-immune and immune causes. Methods to manage refractoriness include leuko-reduction, HLA- and HPA- matched platelets, use of ABO compatible transfusions and platelet cross-matching. Leuko-reduction has been proven to decrease the rates of allo-immunization and is widely used in hemato-oncology units. Other methods have demonstrated modest effectiveness in some studies, but some of these (HLA- and HPA- matched platelets) are not widely available. There are only a few reports in the literature of continuous platelet transfusion (CPT) as an alternative method for increasing post-transfusion platelet increments. Those reports prompted the current analysis of CPT in platelet refractory patients.Aim: To evaluate the effectiveness of CPT in producing satisfactory platelet increments in refractory patients compared to the standard care of routinely prepared single donor platelet transfusions.Patients and Methods: Patients included in this study were treated for hematological malignancies in our Institution between January 2007 and December 2013. A retrospective analysis of the increment of platelets achieved in refractory patients was performed. Refractoriness was defined as a corrected calculated increment (CCI) less than 10,000 platelets per micro-liter following two successive platelet transfusions. The CCI was calculated as PPI (post-transfusion platelet count minus pre-transfusion platelet count) x BSA (body surface area measured in square meters m2) x 1011/number of platelets transfused. All refractory patients included in this analysis received single donor platelet transfusions. The practice of CPT was adopted by us in March 2008. All refractory patients who received CPT between March 2008 and December 2013 were included. These patients received a continuous 24 hour transfusion of single donor platelet units, each dose given over 4 to 6 hours, comprising of 1.5 x 1011 platelets and equaling to half a single donor platelet unit. Their outcome was compared with that of our refractory patients treated with single donor transfusions in the routine manner, between January 2007 and March 2008 (i.e. before the introduction of CPT). The CCI was used to monitor the effectiveness of each platelet unit at 12 and 24 hours post-transfusion intervals. To account for prior antigen-exposure of the patients, we chose to include for each patient the 11th consecutive platelet transfusion. Factors known to contribute to the development of refractoriness, such as infection, disseminated intravascular coagulation (DIC) and splenomegaly were evaluated for impact on the CCI. The statistical analysis was generated using SAS Software, Version 9.4. Continuous variables were presented as mean ± STD, Categorical variables were presented by (N, %). t-tests and non-parametric Wilcoxon tests were used to compare the value of continuous variables between study groups.Results: Twenty eight patients with hematologic malignancies received at least eleven single-donor platelet transfusions during 2007-2013. Twenty one of the 28 patients received CPT due to refractoriness and seven patients were treated before the introduction of this approach. CPT resulted in a significantly higher post-transfusion mean increment at 24 hours (1.16 vs. 0.37, p<0.05). Increments were higher, albeit not significantly, at 12 hours post-transfusion in the CPT group (2.45 vs. 0.36, p=0.058). Patient’s gender, age (younger than 35 years old versus older), renal failure, high grade fever, infection and DIC, splenomegaly or donor-recipient ABO compatibility were not found to significantly influence the increment.Conclusions: CPT results in significantly higher increments at 24 hours post-transfusion, and therefore suggest an effective approach to the treatment of platelet refractoriness in patients treated for hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

Aplastic anemia in pregnancy.

Although aplastic anemia was first recognized by Ehrlich in 1888, the pathogenesis of aplastic anemia has remained elusive. The prevalence of aplastic anemia in pregnancy is rare. Aplastic anemia is a subtype of anemia characterized by pancytopenia and a hypocellular bone marrow. This condition can be due to chemicals, drugs, infections, irradiation, leukemia, and inherited disorders. The treatment involves immunosuppressive therapy with antithymocyte globulin and cyclosporine and bone narrow transplantation [1]. The relationship between pregnancy and aplastic anemia remains controversial. There is universal agreement that pregnancy complicated by aplastic anemia is a serious condition [2]. The risk to the mother is mainly in the form of hemorrhage and sepsis, while the fetus may suffer from growth restriction and even intrauterine death. Hemorrhage and sepsis are responsible for more than 90 % of maternal mortality [2]. Most of the fetal complications are due to maternal anemia. All along with these, maternal infections may lead to the development of chorioamnionitis and resultant preterm labor and birth [3]. In the literature, fetal thrombocytopenia, placentomegaly, and severe oligohydramnios have also been reported. We here present two cases of pregnancy complicated by aplastic anemia, which were seen within a span of 1 year at our hospital. This high incidence is because the hospital is a tertiary care referral unit with good hematology and blood bank support. Case A A 27-year-old primigravida, at 28 weeks of gestation, was found to have a hemoglobin level of 5.6 g/dL, during regular antenatal checkup, and was transfused 2 U of packed cells (PC). She did not have any medical or surgical problems in the past, and her antenatal investigations had been normal. Subsequently, at 30 weeks of gestation, she developed spontaneous bruising, bleeding gums, epistaxis when a complete blood count revealed a hemoglobin level of 7.3 gm/dL, white blood cell count (WBC) of 4.0 × 109/L [N13L84], and platelet count of 3.8 × 109/L and reticulocyte at 0.2 %. Bone marrow biopsy was suggestive of aplastic anemia. She was advised to take cyclosporine 5 mg/kg/day (a total dose of 300 mg), which she took for 7 days and then defaulted. When she reported to our emergency department at 32 weeks of gestation with complaints of gum bleeding and purpuric rash, her hemoglobin was 8 gm/dL, and platelets 1 × 109/L. She was transfused 2 U of platelet-rich concentrates (PRC). She developed severe gestational hypertension and was started on oral alpha methyl dopa 500 mg tds. Injection dexamethasone was given for fetal lung maturation. At 33 weeks of gestation, scan showed IUGR, and with umbilical artery, Doppler study found absent end-diastolic flow. A multidisciplinary team consisting of obstetricians, anesthesiologists, hematologists, and neonatologists planned on offering an elective cesarean section under general anesthesia for severe IUGR and fetal compromise. Eventually, at 33 weeks and 5 days of gestation, she underwent elective cesarean section delivering a healthy preterm female baby of 1,300 g with Apgar scores of 9 and 9, at 1 and 5 min, respectively. Pre-operatively, her hemoglobin was 8.4 g%, and she was transfused six PRCs, intraoperatively she was given six PRCs, 1 U of PC, and 1 U of factor-7 (2.4 mg). There were no abnormal intraoperative findings, and surgery was uneventful. She was monitored in high dependency unit, received four PRCs and one single donor platelet unit, following which her platelet count was 10 × 109/L, and hemoglobin was 10 g%. Post-operatively, she developed fever, was started on cefotaxime and gentamicin, progressing on to imipenem and teicoplanin, which was changed to amphotericin and voriconazole because of mild infusional toxicity. During the ward stay, she was supported with 52 U of PRCs and 9 U of PCs. Definitive treatments of aplastic anemia like allogenic bone marrow transplant, Antithymocyte globulin (ATGAM), and Cyclosporin were offered to the patient, but she could not avail these because of financial constraints. She was discharged on request after 49 days of hospital stay and expired after 6 days of discharge. The baby, born at 33 weeks and 5 days of gestation, weighing 1,300 g was managed uneventfully in level 2 neonatal ICU for 22 days till it attained a weight of 1,780 g. The baby was discharged back home in a good condition.

Sequential Second Free Flap for Head and Neck Reconstruction in a Patient with Fanconi Anemia and Metachronous Squamous Cell Carcinoma

Sir: Fanconi anemia is a rare genetic disease characterized by chromosomal instability, bone marrow failure, congenital malformations, and increased susceptibility to both solid and leukemic malignancies. The incidence of solid malignancies approaches 28 percent by 40 years of age, with a high proportion of these being head and neck tumors.1 In a series of 754 patients with Fanconi anemia, Kutler et al. reported that 3 percent developed head and neck squamous cell carcinoma, with a 50 percent relapse rate by the age of 40.2 Although Fanconi anemia patients may develop multiple tumors over a lifetime, repeated operations/ reconstructions are rarely seen because of their shortened life expectancies. Furthermore, surgeons may be hesitant to treat patients with Fanconi anemia because of an increased risk of complications, particularly if treatment involves extensive resection or possible free tissue transfer. We have previously described a successful mandibular reconstruction with a free fibula free flap in a patient with Fanconi anemia and squamous cell carcinoma of the mandible.3 Unfortunately, the patient developed squamous cell carcinoma of the pharynx 18 months later. We now report subsequent pharyngeal reconstruction with a metachronous free flap. Initially, a 43-year-old woman with Fanconi anemia was referred to our institution with an erosive floor-of-mouth mass ultimately diagnosed as squamous cell carcinoma. She underwent hemimandibulectomy and bilateral neck dissection followed by immediate reconstruction with a right fibular osteomyocutaneous flap. The patient recovered from surgery without complications and underwent adjuvant radiation therapy. Her postoperative result was satisfactory. Eighteen months later, she was found to have squamous cell carcinoma of the hypopharynx. She then underwent total laryngopharyngectomy and construction of a neopharynx using a tubed anterolateral thigh flap. The soft tissues of the right neck were resurfaced with a separate skin paddle based off of a second perforator; the left lateral neck received a split-thickness skin graft. The patient recovered without any hematologic complications but did suffer partial skin graft loss and a small pharyngocutaneous fistula. A modified barium swallow performed 6 weeks postoperatively demonstrated a small leak that resolved 1 month later, resulting in satisfactory cosmetic and functional outcomes. As recommended by our hematologists, during both hospitalizations, the patient's perioperative hemoglobin level and platelet count were maintained above 7 g/dl and 50,000 cells/ml, respectively, by means of hematopoiesis-stimulating agents and blood product transfusions. A total of 42 units of single-donor platelets and 13 units of packed red blood cells were administered while the patient was hospitalized after the second free flap. In conclusion, patients with Fanconi anemia are at increased risk for perioperative hematologic complications. There are two published reports that describe successful free tissue transfer in Fanconi anemia patients.3,4 To our knowledge, this is the first reported case of a patient with Fanconi anemia to undergo a second sequential free flap operation for metachronous head and neck cancer reconstruction. As this case demonstrates, such large-scale reconstructions can be successfully undertaken with careful perioperative management of patients with this disease. Karly A. Kaplan, M.D. Alyssa J. Reiffel, M.D. David I. Kutler, M.D. Christine H. Rohde, M.D. Jason A. Spector, M.D.

Intravenous Busulphan in Combination with ETOPOSIDE and MELPHALAN Used as Conditioning Regimen for Autologous Hematopoietic Cell Transplantation in Lymphomas: Evaluation of Toxicity and Efficacy

AbstractAbstract 1324The standard conditioning regimen used in autologous hematopoietic cell transplantation (autoHCT) for lymphomas is BEAM. During the last year, due to Carmustine (BCNU) unavailability, a new alternative conditioning regimen was designed in our unit consisting of intravenous Busulphan (Busilvex) (3.2mg/kg/day for 3 days), Etoposide (400mg/m̂2/day for 2 days) and Melphalan (140mg/m̂2) (BEM). We retrospectively evaluated the safety and efficacy of the above regimen in 25 patients, aged 30 (16- 65) years, transplanted for Hodgkin (14) and non Hodgkin Lymphomas (DLBCL 7; large B cell mediastinal 1; T-anaplastic 2; MCL 1) between May 2009 and July 2010. The patients received a median of 3 (2- 8) lines of treatment before autoHCT. Disease was chemosensitive to pretransplantation salvage treatment in 17 [6/25 achieved complete remission (24%)], stable in 2 and refractory in 6 patients. According to the Hematopoietic cell transplantation specific comorbidity index, 20 were low (score 0) and 5 intermediate (score 1) risk. Fifteen patients received palifermin and all received cotrimoxazole, antifungal, antiviral and antibiotics as prophylaxis for at least 3 months. The median CD34+ cell dose was 5.59 (2.1- 19.59) ×106̂/Kg and patients received GCSF for a median of 10 (7- 53) days. Prompt engraftment was achieved in 23/25 patients. The median time for neutrophil engraftment (> 500/mm̂3) was day +11 (9- 29), +14 (9- 150) for platelet (>20000/mm̂3) and +11 (8- 43) for erythroid (red blood cell transfusion independency) in 24/25 patients. A second transplantation with double cord blood was offered in one patient because of engraftment failure. In one patient stable engraftment was achieved after a second infusion of autologous cells due to delayed engraftment. The median number of red blood cell transfusions were 3 (0- 15), median single donor platelet units were 4 (0- 36). No red blood cell and no platelet transfusions were required in 4 and 2 patients respectively. Infection (grade II-III) during neutropenia developed in 19 patients (bacteremias mostly; no fungal or respiratory infections). Patients were febrile for a median of 3 (1- 43) days. Twenty-four patients developed mucositis (grade I 6, II 8, III 10) and 21 received parenteral nutrition for 5 (1- 12) days. Nineteen developed liver and gastrointestinal toxicity (elevation of transaminases, nausea/vomiting) grade I- III (grade I 4, grade II 11, grade III 4). The median day of discharge was +15 (10- 32) in 24/25 patients. The patient allografted post autoHCT died at day +63 due to refractory disease and multiple infections. The median follow-up was 7.1 (3-16) months. Eleven patients (44%) were in complete remission (CR) at disease evaluation by CTs one month post autoHCT, without any further treatment. All of them had chemosensitive disease. At last follow up 22 patients are alive and 17 in CR. Three patients succumbed to their disease. The estimated 2-year overall survival (OS) is 90% and disease free survival (DFS) is 60% (95% for patients with chemosensitive disease and 15% for refractory, p<0.05). Despite of the limitations of this study with a small number of patients and short follow up period, the toxicity of the conditioning regimen (BEM) seems to be acceptable and comparable to widely used regimens and offers promising results. For patients with chemosensitive disease BEM seems to be efficacious providing high rates of OS and DFS. Disclosures:No relevant conflicts of interest to declare.

Intranasal Desmopressin Versus Blood Transfusion in Cirrhotic Patients With Coagulopathy Undergoing Dental Extraction: A Randomized Controlled Trial

Cirrhotic patients waiting for liver transplantation who need dental extractions are given fresh frozen plasma and/or platelets to correct coagulopathy. This is costly and may be associated with transfusion reactions and fluid overload. We evaluated the efficacy of intranasal desmopressin as an alternative to transfusion to correct the coagulopathy of cirrhotic patients undergoing dental extraction. Cirrhotic patients with platelet counts of 30,000 to 50,000/microL and/or international normalized ratio (INR) 2.0 to 3.0 were enrolled in a prospective, controlled, randomized clinical trial. Blood transfusion (fresh frozen plasma 10 mL/kg and/or 1 unit of single donor platelets, respectively) or intranasal desmopressin (300 microg) were given before dental extraction. A standard oral and maxillofacial surgical treatment protocol was performed by the same surgeon. Patients were followed for postextraction bleeding and side-effects over the next 24 to 48 hours. No significant differences were noted between the 2 groups in gender, age, INR, platelet count, creatinine, total bilirubin, ALT, albumin, MELD score, or number of teeth removed (median 3 vs 4). The number of teeth removed ranged between 1 and 31 in the desmopressin group and 1 and 22 in the transfusion group. No patients in desmopressin group required rescue blood transfusion after extraction. One patient in the transfusion group had bleeding after the procedure and required an additional transfusion. Another patient experienced an allergic reaction at the end of transfusion, which was effectively treated with diphenhydramine. Treatment associated average costs were lower for desmopressin ($700/patient) compared with transfusion ($1,173/patient). Intranasal desmopressin was as effective as blood transfusion in achieving hemostasis in cirrhotic patients with moderate coagulopathy undergoing dental extraction. Intranasal desmopressin was much more convenient, less expensive, and well tolerated.