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Background: primary graft failure is a devastating complication after allogeneic transplant. Donor specific antibodies has been reported as a major risk factor contributing to graft failure. We retrospectively analyzed our haploidentical transplant registry to report the incidence and impact of DSA and anti-HLA on engraftment in a cohort of malignant hematologic disease conditioned with myeloablative regimens. Methods: retrospective analysis of consecutive cases with malignant hematologic disorders who received a myeloablative haplo-HSCT at a single center. Results: 107 patients were identified with a median recipient age of 22 and a median donor age of 31. Sixty-two patients had AML (58%), 29 had ALL (27%), and 16 (15%) had other malignancies. Sixty-one recipients (57%) had positive anti-HLA, 56 of them had the DSA results available, of these 17 patients had DSAs (15% of the total number of patients, or 28% of patients who have anti-HLA antibodies). The median cumulative MFI was 2062 (IQR: 1038 - 6500). Sixty three percent of the DSA were against class II HLA antigens, while 37% were against class I HLA antigens. The OS, CIR, aGvHD, and cGvHD did not differ between patients with and without anti-HLA antibodies, nor between patients with and without DSA. The gender of the recipient and donor, as well as the gender mismatch between recipient and donor, were statistically associated with the incidence of anti-HLA antibodies, but not with DSA. Three patients only developed GF (2.8%), one was primary (0.9%) and the other two had secondary GF (1.9%). None of the GF cases was in patients with anti-HLA antibodies or DSA. Conclusion: In our cohort, the presence of anti-HLA or DSAs did not affect the outcomes including the incidence of PGF following myeloablative haplo-HSCT for malignant hematologic disorders.

Saudi Arabia is the largest of the Arabian Gulf countries with a total population of 33.41 million as of 2017. This report summarizes the experience from four leading tertiary care hematopoietic stem cell transplantation (HSCT) centers in Saudi Arabia representing more than 90% of all HSCTs performed in the country. Between 1984 and 2016, a total of 6,184 HSCTs were performed. Of these, 3,586 HSCTs were performed in adults and 2,598 HSCTs were performed in pediatric patients. Malignancy was the main indication for transplantation (47%). While most transplants were performed from an identical sibling donor, HSCTs from cord blood, unrelated and, more recently, haploidentical donors have also been performed. Relative shortage of HSCT bed capacity is perceived to be a limiting factor in Saudi Arabia. Lately, more HSCT centers are emerging with rapid growth, which may significantly improve the access to HSCT in the country in the near future.

Background Our study was designed to investigate the frequencies and distributions of familial hemophagocytic lymphohistiocytosis (FHL) associated genes in Saudi patients. Methods FHL associated gene screening was performed on 87 Saudi patients who were diagnosed with hemophagocytic lymphohistiocytosis (HLH) between 1995 and 2014. The clinical and biochemical profiles were also retrospectively captured and analyzed. Results Homozygous mutations and mono-allelic variants were identified in 66 (75.9%) and 3 (3.5%) of the study participants, respectively. STXBP2 was the most frequently mutated gene (36% of patients) and mutations in STXBP2 and STX11 accounted for 58% of all FHL cases and demonstrated a specific geographical pattern. Patients in the FHL group presented at a significantly younger age than those belonging to the unknown-genetics group (median, 3.9 vs. 9.4 mo; P=0.005). The presenting clinical features were similar among the various genetic groups and the 5-year overall survival (OS) was 55.4% with a 5.6 year median follow-up. Patients with PRF1 mutations had a significantly poorer 5-year OS (21.4%, P=0.008) and patients undergoing hematopoietic stem cell transplant (72.4%) had a significantly better 5-year OS (66.5% vs. 0%, P=0.001). Conclusion Our study revealed the predominance of the STXBP2 mutations in Saudi patients with FHL. A genetic diagnosis was possible in 80% of the cohort and our data showed improved survival in FHL patients who underwent hematopoietic stem cell transplant.

Background: In response to the global Mpox outbreaks, this survey aimed to assess the knowledge, perceptions, and advocacy of Mpox vaccines among solid organ transplant healthcare workers (HCWs) in Saudi Arabia. Methods: A cross-sectional survey was conducted among solid organ transplant HCWs in Saudi Arabia from 15 August to 5 September 2022. A total of 199 responses were received from participants primarily working in the kidney (54.8%) and liver (14.6%) transplant units. Results: The survey found that most participants were aware of the 2022 Mpox outbreak, but the majority were more concerned about COVID-19 than Mpox. While the majority of participants thought laboratory personnel and HCWs in direct contact with Mpox patients should receive the vaccine, less than 60% believed that all HCWs should be vaccinated. Additionally, over half of the participants lacked knowledge of animal–human transmission of the virus. Conclusion: The results highlight the need for increased education on Mpox among transplant HCWs in Saudi Arabia, particularly regarding the virus’s transmission dynamics and vaccines. This education is crucial to improve HCWs’ understanding of this emerging disease, especially given their vulnerability during the COVID-19 pandemic.

Histological evaluation of the pancreas graft is usually done on demand resulting in significant delays. This analysis reports on endoscopic protocol duodenal graft biopsies at regular intervals to determine feasibility, safety, and monitoring benefits.Protocol duodenal graft biopsies in 27 consecutive pancreas transplants (10 simultaneous pancreas kidney [SPK], 17 pancreas after kidney [PAK]) with a follow-up of a minimum of 12 months were performed at days 14, 30, 90, 180, 360, 430. University of Pittsburgh Medical Center classification for intestinal rejection was used. C4d staining was performed when antibody-mediated rejection was suspected.Overall patient and pancreas graft survival was 100% and 93% at a mean follow-up of 2.8 years. One hundred sixty-seven endoscopic biopsy procedures were performed in 27 grafts without any complication. Biopsies revealed rejection in 3 (30%) SPK recipients and in 15 (82%) of PAK recipients as early as 14 days posttransplant. Two patients underwent PAK retransplantation diagnosed with acute rejection at day 180. All except 1 recipient being treated for rejection, showed histological improvement following antirejection treatment. Following transient treatment success, a total of 3 pancreas grafts were lost for immunological reason. One loss was immediate despite antirejection treatment, 1 secondary to nonresolving rejection at 7 months and the third due to recurrent rejection 15 months posttransplantation. Additionally, biopsies detected vascular (venous thrombosis) and overimmunosuppression (cytomegalovirus infection) complications.Protocol graft duodenal biopsies detect complications after whole-organ pancreas transplantation, are useful in guiding therapy, and carry potential for improving outcome.

We estimated HLA allele and haplotype frequencies of the Saudi Arabian population from a sample of 45,457 registered stem cell donors. The most frequent HLA alleles were A*02:01g (18.5%), C*06:02g (16.1%), B*51:01g (14.1%), DRB1*07:01g (16.2%), DQB1*02:01g (30.5%), and DPB1*04:01g (33.6%). The most frequent 5-locus haplotypes were A*02:05g~C*06:02g~B*50:01g~DRB1*07:01g~DQB1*02:01g (1.73%), A*02:01g~C*06:02g~B*50:01g~DRB1*07:01g~DQB1*02:01g (1.66%), and A*26:01g~C*07:02g~B*08:01g~DRB1*03:01g~DQB1*02:01g (1.38%). Furthermore, we used the calculated haplotype frequencies to estimate stem cell donor matching probabilities for Saudi Arabian donor and patient populations under various matching requirements. These results are relevant for strategic donor registry planning in the Kingdom of Saudi Arabia.

The prevalence of HLA-B27 in the general population and in axial spondyloarthritis (axSpA) patients in Saudi Arabia is unknown. The aim of this study was to evaluate the prevalence of HLA-B27 in these two populations and describe the delay in diagnosis of axSpA patients. The prevalence of HLA-B27 in the general population was evaluated using cord blood and healthy organ transplant donor databases. Data from patients with axSpA were collected retrospectively from five centers. Ankylosing spondylitis (AS) was diagnosed based on a positive X-ray, as evaluated by two independent readers. Patients with inflammatory bowel disease and psoriasis were excluded. A total of 134 axSpA patients were included, of whom 107 (79.9%) had AS, and most (67.2%) were males. HLA-B27 was positive in 60.4, 69, and 25.9% of patients with axSpA, AS, and non-radiographic axSpA (nr-axSpA), respectively. The median and interquartile range (IQR) ages at symptom onset and disease diagnosis were 26 (20–33) and 30 (25–38) years, respectively. The median delay to diagnosis was 3 (1–6) years. There was a negative correlation between the time of onset of symptoms and the delay in diagnosis (r = −0.587). Male gender and HLA-B27 positivity were associated with a younger age at symptom onset/diagnosis (p

Hematopoietic cell transplantation (HCT) activity is increasing at an unprecedented pace with > 50,000 allogeneic transplants occurring annually worldwide. Establishing a functional HCT donor registry can be very challenging with respect to ethnicities, financial, technical, and geopolitical issues. Extensive planning steps are essential to overcome the expected challenges while establishing the registry, and to maintain its functionality. A few strategies can help move past those challenges and push the development of such registries forward. Authorities involved in HCT donor registry establishment will have to balance the advantages and costs of such a project and accommodate the emerging alternatives such as cord blood or related haploidentical transplants. Miscalculations and incomplete understanding of the various aspects of the process can have tremendous impact on the optimization of a HCT donor registry especially in developing countries. Herein we present some challenges in establishing such a registry and present potential solutions.

Introduction: Fanconi Anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure (BMF), constitutional anomalies and high risk of developing cancer. Distinguishing FA from severe aplastic anemia (SAA) can be challenging especially in asyndromic patients. We undertook a clinical and laboratory cohort study of adolescent and young adult (AYA) patients with a diagnosis of FA treated at our institution to characterize the clinical features in our population, and conducted a prospective translational study to explore integration of a genomic and proteomic approach for improved diagnosis and molecular characterization of FA. Methods: Data on FA patients was obtained from an institutionally approved BMF database and hematopoietic stem cell transplant (HSCT) database. Further data was obtained from a register of chromosomal breakage (CB) analysis results. Index cases were identified if they were older than 14 years of age at the time of diagnosis or under the care of adult hematology with a clinical diagnosis of FA based on the presence of BMF and abnormal CB, or clinical phenotype with the presence of homozygous FA related genes. Family pedigrees were constructed based on history. In addition, patients presenting with BMF were enrolled onto an institutionally approved study investigating proteomic biomarkers and genomics of BMF syndromes. Consented peripheral blood samples and/or extracted DNA were subject to either panel based next generation sequencing (NGS) testing as part of the Saudi Genome Project or subjected to whole exome sequencing (WES) by external lab. For proteomic analysis, peripheral blood plasma (PBP) samples from 6 patients with FA, 10 SAA patients and 7 normal controls were subjected to expression proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). Result: Patients and clinical features: 55 patients (26 M, 29 F) in 30 families were identified. While 18 patients (32%) were referred with a diagnosis/suspicion of FA, in 26 (47%) FA was diagnosed at our institution. The most frequent anomaly was short stature (14 patients, 25%), skin changes (7, 12%), urogenital abnormalities (7, 12%), dysmorphism/craniofacial abnormalities (7, 12%), hands anomalies (4, 7%); 12 (22%) had no recorded anomalies. 18 patients (33%) developed a malignancy either before or after diagnosis of FA: solid tumors in 5 (9%), AML and/or MDS in 15 (27%); 3 (5%) of these patients had both solid tumors and AML/MDS. Diagnostic Tests: 35 patients (63.6%) had a positive CB analysis with diepoxybutane (DEB) or mitomycin-C (MMC) testing; in 5 patients (9%) DEB testing was borderline and 3 (5%) had a normal CBA but had a diagnostic phenotype+/- family history and presence of a homozygous mutation in a known FA related gene. 14 patients had cytogenetic abnormalities and abnormalities involving chromosome 1 were the most frequent (50%). Mutation Analysis: Mutational analysis was available for 12 (22%) cases; homozygous mutations in FA genes were identified in 10 patients (18%) in 7 families (23% of families): FANCA (5 patients/3 families); BRIP1 (2/2); FANCP (1/1); FANCD2 (2/1). In one case, post matched sibling (HSCT) blood sample revealed a known pathogenic heterozygous c.2632G>C,p.Glu878Gln mutation in FANCA, suggesting a carrier donor. Proteomic analysis: Over 1650 unique PBP protein species were identified of which 605 were significantly differentially expressed (≥ 2 to ∞ - fold change & p < 0.001) between SAA /FA/ normal control subjects (Fig 1a). DNMT3A, Kinase Insert Domain Receptor (KDR) and TGFB-1 was found to be highly expressed in SAA versus FA, while ATM and APOB were highly expressed in FA versus SAA (Fig.1b). Treatment outcomes: 36 out of 55 patients (65%) received HSCT. Actuarial survival of HSCT (n=37) and non-HSCT (n=14) patients was 70% and 77%, respectively. Treatment details were not available on 6 (11%). CONCLUSION: We report the first characterization of AYA patients with FA in Saudi Arabia. Our report emphasizes the need for a high index of suspicion of a diagnosis of FA in BMFs. CB may be falsely negative in cases, and panel based and/or WES based NGS testing increases diagnostic accuracy; in this cohort, mutations in FANCA were the most frequent (50%). Occurrence of hematological and solid tumors is a significant risk in these AYA patients. We also report proteomic panels as potential biomarkers that distinguish FA from SAA and may provide mechanistic insights. Disclosures No relevant conflicts of interest to declare.

Rheumatoid arthritis (RA) is a complex autoimmune disease with a poorly understood pathogenesis. The disease is associated with polyclonal B cell activation and the production of autoantibodies (autoAbs), but there is a longstanding controversy as to whether such Abs contribute to, or are secondary to, the pathogenesis of RA. To address the potential pathogenicity of human RA–associated Abs, we developed a passive transfer model involving mice deficient in the low-affinity inhibitory Fc receptor, FcγRIIB. We report that plasma or serum from patients with active RA can induce inflammation and histological lesions in FcγRIIB−/− mice consistent with arthritis, and that this pathogenic activity is caused by the immunoglobulin G–rich fraction. Our results suggest that humoral autoimmunity can contribute directly to autoimmune arthritis, and that FcγRIIB−/− mice are a promising model to evaluate the arthritogenic potential of human autoAbs.

Aim Testing for IgM anti-HLA antibodies is not routinely tested in the majority of the HLA laboratories, despite the harm that they might cause. We report here a case of 69 years old male with End Stage Renal Disease (ESRD) who received a kidney from deceased donor and developed de novo Donor Specific Antibody dnDSA with IgM isotype. Methods Pre-transplant, patient was flow cytometry crossmatched (FCXM) with the potential deceased donor for T and B cells. Screening for the presence of anti-HLA antibodies was performed using the LabScreen Single Antigen Beads (SAB) for IgG, IgM and C1q assay (One Lambda Thermo-Fisher). Results Pre-transplant FCXM and SAB testing were negative. After two weeks of transplant, creatinine was elevated to 467umol/L and the Kidney biopsy confirmed BANFF III rejection. SAB testing was negative for IgG isotype, on the other hand it was positive for IgM with anti-A1 and anti-DQ5 DSA. Interestingly, the C1q-assays fail to detect the IgM dnDSA!? The rejection was successfully treated with steroid, IVIG and thymoglobulin. Subsequently, the creatinine dropped down and the SAB testing became negative for HLA antibodies. Conclusions This case re-emphasize the challenge that might be caused by DSA of an IgM isotype. Furthermore, the same patient serum that was tested IgG-negative, C1q-negative was tested IgM-single antigen positive, it that means when C1q assay is negative, we cannot rule out IgM antibodies? Further testing/studies is needed to answer this question.

Introduction Sensitization to HLA antibodies has been associated with worse outcome in lung transplant recipients. Many lung transplant centers avoid transplanting patients with positive flow cytometry crossmatch (FCXM). However, delaying the transplant in these patients may increase their mortality on the waiting list. We are reporting two cases of crossmatch positive lung transplantation. Case scenario A 37-year old lady diagnosed with pulmonary fibrosis. Patient sensitized to HLA class I and II at time of transplant.The FCXM was positive T-cell (+78 MCS) and B-cell (+165 MCS). She had multiple DSA: Anti A2 (2795 MFI), Anti DR51 (2215 MFI), Anti DR52 (2088 MFI), Anti DQB1:05:02 (655 MFI), Anti DR14 (1411 MFI), anti DP2 (813 MFI). Second case is a 58-year old lady with pulmonary fibrosis secondary to Sjogren’s syndrome. Patient sensitized to HLA class II only at the time of transplant. The T-cell crossmatch was negative while, B-cell was positive (+318 MCS). She had multiple DSA: Anti DRB1∗10:01(14776 MFI), Anti-DR53 (4796 MFI), Anti-DP1 (1942 MFI), Anti- DQB1∗05:01(3384 MFI), Anti-DP2 (790 MFI). In view of patients’ deterioration, lung transplantation was preceded despite of positive crossmatch results, using combination of plasmapheresis, Intravenous immunoglobulin (IVIG) and Thymoglobulin. Both patients did well with no evidence of graft rejection. However, the repeated HLA antibodies screening during follow up showed persistent DSA. in the first patient: Anti-A2(1377 MFI), Anti-DRB1∗14 (4864 MFI), Anti-DR52 (3044 MFI), Anti-DR51 (1708 MFI), Anti-DP2 (1993 MFI). In the second patient: Anti-A1 (3561 MFI), AntiA24 (712 MFI), Anti-DQB1∗05:01(5837 MFI), Anti-DP1 (692 MFI), Anti-DRB1∗10:01 (643 I). Conclusion Lung transplantation is possible in patients with crossmatch positive, using perioperative desensitization protocol. Desensitization treatment did neither eliminate DSA nor prevent de novo DSA formation. However, follow up of these patients showed no evidence of graft rejection. Long-term data is needed.

Aim Sensitization events such as blood product transfusion, pregnancy and transplantation can change the HLA antibody profile of patients in its specificity and strength. Here we report a case of HLA class I and II negative antibody screened patient who received a kidney and within only 10 days of transplant was tested crossmatch positive with pancreas potential donor. Methods The antibody profile was evaluated using both LABScreen PRA and LABScreen single antigens (SA) beads (One Lambda, Inc.). The crossmatch was performed using pronase treated T cell and B cell IgG flow cytometry crossmatch (FCXM). FCXM was consider positive when the T cell and B cell were more than 38 and more than 50 MCS respectively. Results Historical serum,five months before Kidney transplant was positive for HLA class I only and the highest MFI was 2083 MFI with no DSA against the received kidney.Patient serum at the time of Kidney transplant was negative for HLA class I and II. Patients serum tested ten days post kidney transplant (at the time of pancreas offer) was positive for HLA class I only with anti B13 (10574 MFI) DSA against the offered pancreas and not DSA to the transplanted kidney. The anti B13 DSA was also present in the historical serum (five months before Kidney transplant) with only 855 MFI. FCXM with the potential pancreas donor was positive for T-cell (+336 MCS) and B (+246 MCS) for the current serum and completely negative for the previous serum which was ten days earlier. Conclusion This case highlights the strength of the immune memory cells and the need to always consider the historical antibody profile of the patients in risk assessment of the transplant. Furthermore, transplanting patients who are fit for simultaneous pancreas-kidney transplantation with kidney than pancreas may trigger immune response that ultimately hindered pancreas transplant difficult.

Aim Luminex single antigen technology for antibody identification has enabled laboratories to provide highly accurate virtual crossmatch results prior to transplantation. Both manufacturers, One Lambda (OL) and Immucor (IC) single antigen reagent panels differ in the HLA alleles composite and density. We report here two cases that single antigen panel for each behave significantly different. Method Serum of Patient (RH) and (AM) were tested with OL and IC single antigen panels. Flow cytometry and CDC crossmatchs of both patients were performed with their corresponding donors. Results Serum of (RH) and (AM) tested with OL single antigen panel that showed the following DSA’s, anti DQB1*06:03 (2510 MFI) and anti A2 (891 MFI), B41 (1631 MFI) respectively. CDC and flow crossmatch were highly positive for both sera despite low Median Fluorescent Intensity (MFI) of the identified DSA. The same sera were tested with IC single antigen panel; patients (RH) and (AM) showed anti DQB1*06:03 (17946 MFI) and anti A2 (10500 MFI) respectively. Furthermore, both sera were diluted 1:4 and re-tested with OL single antigen panel. The identified DSA strength in the diluted sera for both patients showed higher MFI values for A2 (3048 MFI), B41 (2863 MFI) for AM patient and DQB1*06:03 (10503 MFI) for RH. Conclusion Significantly, higher prozone effect was observed with OL single antigen panel than in IC. Serum to bead-volume ratio may have contributed to the lower correlation between OL versus IC. Testing patients sera by more than one manufacturer is an advantageous in determination of antibody strength and improves correlation with the crossmatch results.

Aim Positive Complement Dependent Cytotoxicity (CDC) and Flow Cytometry Crossmatch (FCXM) usually correlate with high titer of Donor Specific Antibodies (DSA). Now a days assignment of relevant DSA is based on solid phase assay (SPA), in which false low/negative results can be observed due to prozone effect. Here we present three cases underwent a workup for living related kidney transplant that their crossmatch data does not correlate with the single antigen (SA) Median Fluorescent Intensity (MFI) value. Method B cell and/or T cell AHG-CDC, FCXM and antibody identification by SPA were performed for three living related pairs. Results Two patients show positive T cell AHG-CDC crossmatch and one shows positive B cell CDC crossmatch with 8 reaction strength at 1:8 dilution and positive FCXM with very high Median Channel Shift (MCS). However the DSA result was negative for one patient and the other two patients showed A2 (891 MFI) and DQ6 (1299 MFI) respectively. The SA testing indicated possible skewed MFI value, therefore all serum were diluted 1:4 to overcome a prozone effect if present. Conclusion MFI value of the SPA was not correlated with the high MCS and positive CDC crossmatch in undiluted serum due prozone phenomena of high antibody concentration. Therefore serum dilution should be considered if strong crossmatch reactivity observed.

In 2013, one laboratory from Saudi Arabia reported a rare allele of DQB1 locus to IMGT using two different cells. At our laboratory, we observed four unrelated individuals typed for DQB1 locus by RSSO as DQB1∗06:29/123. Since DQB1∗06:29/123 has the same beads reaction by RSSO, Sequence based typing for the four individuals confirmed the typing to be DQB1∗06:123. In addition, the four unrelated individuals shared the same haplotype (A∗02; B∗50; C∗06; DRB1∗15:01; DQB1∗06:123). This particular allele may be included in the common and well-documented (CWD) alleles. As per the 2012 published CWD HLA alleles an allele that is detected three times or more in unrelated individuals with a specific haplotype as discussed above should be included in the well-documented category.

Aim Recent studies have indicated that the presence of HLA donor specific antibodies (DSA) in patients undergoing mismatched hematopoietic stem cell transplantation (HSCT) represent a considerable risk factor for graft failure. Therefore, the role of pre-existing anti-HLA antibodies in HSCT has drawn increasing attention due to increased number of patients who receive partially HLA mismatched, HLA haploidentical and cord blood HSCT. The aim of this study is to determine the frequency of the presence of anti-HLA antibodies in our adult HSCT program. Method The presence of anti-HLA antibodies before transplantation was determined prospectively in 200 HSCT adult recipients at King Faisal Specialist Hospital and Research Center, using mixed screen beads in a solid phase assay Results Anti-HLA antibodies were detected in 127/200 of the patients (63%). Among those sensitized, 34/127 (27%) were sensitized to HLA class I only and 13/127 (10%) sensitized to HLA class II only and 80/127 (63%) sensitized to both HLA class I and HLA class II. Conclusion Our result has shown a higher level of HLA sensitization (64%) compared to several published studies that reported up to 40% HLA sensitization. Since our patient pool shows a higher prevalence of HLA sensitization, this further emphasizes the need to accurately identify the presence or absence of DSA; which if present poses a significant risk of delayed or complete graft failure.

Introduction: Various forms of aplastic anemia (AA)/bone marrow failure syndromes (IBMFs) show significant clinical and molecular heterogeneity with significant clinical overlap and are often diagnosed based on established clinical and pathological criteria. While >70 genes have been identified in patients with AA/IBMFs, most cases have are labelled as idiopathic with no identifiable genetic abnormlaity found. Precise detection of genetic abnormalities in these patients may assist in more accurate molecular diagnosis in these patients, proper counseling, cancer surveillance and personalized clinical intervention. Method: As part of the Saudi Human Genome Project, we developed a comprehensive 405 gene panel encompassing all known Mendelian hematological disorders (hemolytic anemias, aplastic anemias/bone marrow failure syndromes, coagulation disorders) using the Ion Torrent AmpliSeq technology. Patients who met the clinical diagnosis of aplastic anemia/bone marrow failure syndrome were enrolled into this study. Peripheral blood samples were subjected to this next-generation sequencing analysis. Results: We validated the Saudi Mendeliome assay using 642 samples with known mutations across various medical specialties. We then tested 37 patients with AA/IBMFS using this Proton-Ion sequencing platform. Mutations were identified in 7/37 (19%) of patients, followed by whole exome sequencing (WES) in those patients without identifiable mutations. Conclusion: Compared with clinical WES and/or whole genome sequencing (WGS), which are still expensive, time consuming and difficult to interpret, this novel and comprehensive targeted gene panel is more economical (< $150), faster (3-4 weeks), upgradable (by spiking in newly identified AA/IBMFs genes) and can be used to genotype patients with acquired aplastic anemia/bone marrow failure syndromes and guide their management. Second tier testing using WES/WGS is recommended for cases without identifiable mutations. Disclosures No relevant conflicts of interest to declare.

Background An ABO incompatible non sensitized male renal transplant patient was worked-up with his full HLA matched brother. The patient was desensitized with rituximab (500 mg 2 weeks prior to transplant) and four sessions of plasmapheresis and small dose intravenous immunoglobulin (100 mg/kg after each plasma session). The crossmatch included both current serum (the day before transplant) and previous serum (before the start of desensitization). Method A standard three color flow cytometry crossmatch was performed against the potential donor (1 mg/ml pronase treated cells) and the serum was tested by Luminex PRA screen (One Lambda Inc.). An evaluation of the pronase effectiveness was assessed using anti- CD20 monoclonal antibodies. Results The patient PRA screen was negative for HLA class I and II and the crossmatch results were negative for T cells and positive B cell IgG (133 MCS) for the current serum and negative T and B cell IgG for the previous serum. Staining the B cells used in the crossmatch with anti-CD20 demonstrated the complete absence of the CD20 receptors. The auto B cell IgG flow crossmatch was not assessed due to absence of patients’ B cells as a result of rituximab effect. Conclusion This case demonstrates that the desensitization using rituximab, plasmapheresis, and intravenous immunoglobulin for depleting antibodies in ABO incompatible renal transplant patients prior to transplantation can cause B cell nonspecific binding despite treating the cells with pronase. This nonspecific binding results in positive B cell IgG flow crossmatch. Using different anti-CD20 monoclonal antibodies for the evaluation efficacy of pronase activity is suggested to confirm complete cleavage of the CD20 receptors.

Aim Patients anti HLA antibody profile could change overtime in its specificity, strength and/or behavior. Here we are presenting a case for a patient with end stage renal disease (ESRD) workup for living related kidney transplant, in which antibody profile changed between the initial and final work-up (the day before transplant). Methods HLA Class I highly sensitized patient with anti A26 and anti B50 Donor Specific Antibody (DSA) with 2904, 12168 Median fluorescent Intensity (MFI) respectively, underwent desensitization protocol with Rituximab, high dose IVIG and Plasma Apheresis. One day before transplantation, final Complement Dependent Cytotoxicity crossmatch (CDCXM) as well as Flow Cytometry Crossmatch (FCXM) were performed utilizing historical serum “initial work-up sera prior to desensitization” and current serum post desensitization. Results As can be seen in the table below, the characteristics of the patients’ DSA changed between the initial and final work-up. The DSA changed from complement fixing antibody with a positive CDCXM crossmatch to non-complement fixing antibodies with negative CDCXM crossmatch. Conclusion This case highlights the relevant of frequent testing of anti-HLA antibodies in renal transplant candidates. As seen above, the antibody Isotype switched from fixing to none complement fixing. Thereafter, such conversion could modify the treatment modality. Result. Initial work-up Final work-up CDCXM Positive Negative FCXM Positive Positive Undiluted sera A26 (2904 MFI), B50 (12168 MFI) A26 (12421 MFI), B50 (984 MFI) Diluted sera (1:4) A26 (16680 MFI), B50 (6247 MFI) A26 (5936 MFI), B50 (Negative) C1q Positive Negative

Aim The method of choice in most of the HLA laboratories for the identification of anti HLA antibodies is the solid phase assay Luminex technology. This method is very sensitive and it is being used for virtual crossmatching as well as for paired kidney exchange (PKE) programs. The most challenging factor for the paired kidney exchange programs is the high sensitization level of the listed couples determined by Luminex-IgG MFI. Now it is increasingly believed that the complement fixing donor specific antibodies (DSA) are the more clinically relevant than non-complement fixing antibodies. In this study we determined impact of using Luminex C1q instead of Luminex-IgG in the pool of the couples listed in our PKE program. Methods A cohort of 12 patients was chosen randomly from the total of 28 couples constituting our HLA incompatible PKE program. All samples were tested by Luminex-IgG and Luminex-C1q (One Lambda) for HLA class I and II antibodies. The unacceptable antigens listed in the PKE are those with more than 6000 MFI for HLA-A,B,DR and DQ. For HLA-C it was 10000 MFI. Results The percentage of complement fixing antibodies detected for HLA class I and II were 36% and 35% respectively. When the unacceptable antigens entered in the PKE program were those Luminex-IgG positive, there were no exchangeable pairs. However when the unacceptable antigens entered in the PKE program were only those C1q positive, we found four one-way exchangeable pairs. Conclusions Our data suggest that we should consider listing unacceptable antigens in the PKE program using the Luminex-C1q positive instead of Luminex-IgG positive. Listing unacceptable antigen in the PKE program solely based on Luminex-IgG will deprive many patients from being transplanted with compatible pairs.

Aim The HLA haplotype frequencies for Middle Eastern populations are not well known relative to the rest of the world. At The King Faisal Specialist Hospital and Research Center in Riyadh we have collected HLA typing information for thousands of donors and cord blood units from years of stem-cell and renal transplants with the goal of establishing a stem cell registry in the Kingdom of Saudi Arabia. We aim in this study to use these HLA typings to estimate Saudi population haplotype frequencies and in follow-on work to project HLA match rates for the Saudi population as the registry expands. Methods We calculated HLA A∼C∼B∼DRB1∼DQB1 haplotype frequencies from 2,723 Saudi donors and cord blood units typed by DNA methods, resolving phase and allelic ambiguity using the Expectation Maximization algorithm. Haplotype frequencies were estimated to the allele family level for class I loci and to high resolution at class II loci. Results The most common Saudi HLA haplotype was A∗02∼C∗06∼B∗50∼DRB1∗07:01∼DQB1∗02:02 at a frequency of 4.5%. This haplotype is also the 32nd most common haplotype in the US Caucasian population at a frequency of 0.38%. The 2nd most common Saudi haplotype was A∗02∼C∗07∼B∗07∼DRB1∗15:01∼DQB1∗06:02 at 1.7% frequency, which is also the 4th most common haplotype among US Caucasians, and within Europe is most common in Scandinavia. The 3rd most common Saudi haplotype, A∗26∼C∗07∼B∗08∼DRB1∗03:01∼DQB1∗02:01 at 1.6% frequency, is the 14th most common haplotype in US Asian Pacific Islanders at 0.64%, but is quite rare in US Caucasians (rank 466; 0.0036%). Conclusions his pilot haplotype frequency study and the match rate projection to follow will help to form a data-driven approach to the establishment of a robust stem cell registry of adult donors and cord blood units that will aid the Saudi population in need of transplant. Collection of more detailed ancestry information on Saudi donors has the potential to improve match estimates and ensure all subpopulations are well-served.

Aim In Saudi the chance of finding matched sibling is around 70% which is higher than many other countries, due to large family size. For the remaining 30% of the patients, the probability of finding a match in the international unrelated bone marrow registries is slim to null. The emergence of the cord blood transplantation that allows less than perfect HLA matching between patient and cord blood unit (CBU), gave a chance to more patients to be transplanted. In this study we analyzed the chance of finding a suitable CBU, the level of HLA ABDR matching and the cost effectiveness of the cord blood bank at King Faisal Specialist Hospital and Research Center. Methods A total of 266 consecutive searches (216 for pediatrics and 50 for adult patients) were evaluated within the 3440 CBU inventory at KFSH & RC cord blood bank that resulted in 69 transplants between Oct-2008 to Dec-2011. The CBU was typed at the low resolution level for HLA-A, HLA-B and at the high resolution level for HLA-DRB1 alleles. Results 89.1% of the patients found at least one CBU equal or greater than 4/6 HLA match with total nucleated (TNC) cell ⩾3.7 × 10 7 /kg. 50.2 of the patients had 4/6 HLA match, 42.2% of the patients had 5/6 HLA match and 7.6% had 6/6 HLA match CBU. 10.9% of the patients did not find 4/6 or better match. As the numbers of CBU is adding up, the need to imported CBU is decreased. In 2009, 44% of the donor CBU were imported, in 2010 16.6% and in 2011 only 9% were imported. In addition to less time from search until the unit ready for transplant, the cost of the CBU from our cord blood bank is only 21% of the cost of the imported CBU. Conclusions This study clearly indicates the cost effectiveness of the institution cord blood bank and the needs to increase the inventory size to improve the probability of finding 5/6 or full house 6/6 CBU.

Aim Proper selection of positive control (PC) serum in flow cytometry (FC) crossmatch is essential in validation the procedure. In our laboratory we developed a selection method of the optimal pooled PC serum dilution that assures the sensitivity superiority of flow crossmatch over anti-human globulin enhanced complement dependent cytotoxicity (CDC-AHG). Methods Highly and broadly sensitized patients with >10000 MFI were pooled together. The pooled PC is serially diluted (1:2-1:256) and CDC-AHG crossmatched against target cells in which the end point was determined (highest dilution giving positive reaction). Thereafter, we FC crossmatched target cells with the further diluted PC serum than end point observed in the CDC-AHG crossmatch. Results Positive reactions were seen at 1:8 dilutions in CDC-AHG crossmatch, however; they were positive up to 1:128 in FC crossmatch. Therefore, we selected the highest dilution of pooled PC serum that shows positive flow cytometry crossmatch. Conclusions In flow cytometry crossmatch, using optimized dilution of the positive control rather than extremely positive control serum, will assure the flow cytometry crossmatch sensitivity superiority over CDC-AHG crossmatch.

Aim When doing search for matched unrelated donor (MUD) in the international bone marrow donor registries, it is recommended to start the search with HLA class I and II high resolution typing of the patient. However, performing high resolution typing specially for HLA class I; is not available in many laboratories in contrast to HLA class II high resolution, due to cost of equipment, reagent and technical expertise and TAT. The Aim of this study was to determine the correlation between the results of HLA class I typing obtained by One Lambda rSSO Lab Type method (suggested allele level typing) and high resolution HLA sequence based typing (SBT). Methods A total of 50 samples previously SBT typed specimen were also typed by rSSO Lab Type (One Lambda, Inc.). The correlation was made of the ability of the rSSO Lab Type low resolution typing suggested first or second allele choice in each locus to predict the correct HLA allele. Results See [ Table 1 ]. Conclusions When we obtain the potential matched donors list for the search that was performed with low resolution HLA class I and high resolution HLA class II typing of the patient, in our hand we found the Lab Type rSSO method to be fast and reliable for screening (inclusion/exclusion) of possible match donor. Once a potential matched donor is identified based on the alleles identified by the Lab Type rSSO. An actual high resolution SBT typing would have been ready for confirmation of the matching level.

Studies both in mice and humans have argued that immunodominant MHAg eliciting CD8 T cells cause increased risk for GVHD. We have shown that the H60 MHAg is remarkably immunodominant in that an overwhelmingly predominant CD8 T cell response developed after the transfer of T cells and bone marrow (BM) into MHAg-mismatched lethally irradiated recipients is directed against H60. Here we address whether H60 is a barrier to lethal (L) GVHD. Matching of donor cells and recipients for H60 while remaining disparate for all other MHAg failed to result in improved survival, indicating that H60 is not a barrier for LGVHD. Expression of H60 is normally limited to hematopoietic tissues, possibly explaining H60’s inertness in LGVHD. To broaden its expression, we produced transgenic mice expressing H60 driven by a tissue-ubiquitous cassette. We then evaluated whether H60 expressed broadly in recipients resulted in GVHD. Naïve B6 T cells transferred into B6-H60 transgenic or congenic mice failed to show any evidence of GVHD regardless of a robust anti-H60 CD8 T cell response. Augmentation of the response to H60 by preimmunization, resulting on over 50% of the CD8 T cell compartment being H60-specific in GVHD target organs, an failed to result in LGVHD. The overall results are inconsistent with a recent report (B. Kappel, et al. Blood, 2006.107:2045) and clearly indicate that potent CD8 T cell responses to MHAg are remarkably inert in GVHD.