[Rare red blood group discovered by a blood group discrepancy: a case report]. / Découverte d'un phénotype érythrocytaire rare suite à une difficulté de groupage : à propos d'un cas.

ABO typing is essential for preventing ABO incompatibility transfusion reactions. Discrepancy exists when reactions in forward grouping do not match with reverse grouping. Any discrepancies reported should be investigated so that correct blood group is reported minimizing the chances of transfusion reaction. The most common causes of ABO discrepancy are cold autoantibodies and missing serum reactivity. We report a rare alloantibody anti-PP1Pk discovered during the resolution of a grouping difficulty with a positive control. Anti-PP1Pk is associated with hemolytic transfusion reactions. In our observation, we were faced with transfusional impasse because of the unavailability of a national rare blood bank or a compatible donor on the registry of individuals with a rare blood phenotype.

Minor histocompatibility antigens HA-8 and PANE1 in the TUNISIAN population.

BACKGROUND: Minor histocompatibility antigens (mHAgs) are endogenous immunogenic peptides initially identified due to complications detected in several contexts of HLA geno-identical hematopoietic stem cell transplantation (HSCT). In this study, we chose to examine the molecular polymorphism of the mHAgs HA-8 and PANE1 in the Tunisian population. MATERIAL AND METHODS: This study was conducted on 150 healthy and unrelated individuals. The DNA extraction and Sequence-Specific Primers PCR (PCR-SSP) methods were used for the molecular genotyping of the selected SNPs: PUM3 (rs2173904) and CENPM (rs5758511). RESULTS: Our results show that, 94% of Tunisians are carriers of the PANE1R allele (immunogenic variant of the PANE1 mHAg) and 68% of Tunisians are carriers of the HA-8R allele (immunogenic variant of the HA-8 mHAg). Furthermore, this study shows that about 5% of the Tunisians are carrier of the PANE1R antigen and its HLA molecule of presentation (the PANE1R/HLA-A*0301 combination). However, only 2% of Tunisians are carrier of the HA-8R/HLA-A*0201 combination, that is, the HA8 immunogenic variant and its specific HLA molecule of presentation. CONCLUSION: Our results are close to those reported in Caucasian, Asiatic, and African populations, this may be explained by the historical events experienced by Tunisia for millennia. These results could be used for further clinical and anthropological studies.

Second Tunisian case of Anti -Tja mediated recurrent abortions.

The diagnosis and the treatment of rare phenotypes remain a problematic situation in many countries especially in Tunisia. Individuals with rare phenotype may develop clinically significant red cell antibodies directed against the high incidence Antigens they lack. A 35 years old patient was referred to our laboratory to explain a high incidence (twelve) of recurrent miscarriage during the first and second terms of pregnancy. This patient was grouped as O Rhesus: 1, -2, -3, 4, 5 K:-1. In her plasma we identified a pan-reactive anti-PP1PK antibody (anti-Tja) recognized to be responsible of spontaneous recurrent abortions. The red cell phenotype was P1 and Tja negative. More investigations concluded to the absence of auto and other allo-antibodies association. Therapeutic plasmapheresis from early stages was suggested for the future pregnancy to remove anti-public antibodies in order to maintain normal placenta functions. The Anti-Tja antibody, naturally occurring in patients with rare p phenotype, has the ability to induce recurrent spontaneous miscarriages and to cause immediate hemolytic transfusion reactions. Despite the absence of compatible donors in her family, this patient is not in an impasse situation because two donors with the same phenotype were identified when investigating a first case in 2013.

Predictors of autoimmune hemolytic anemia in beta-thalassemia patients with underlying red blood cells autoantibodies.

In beta-thalassemia patients, erythrocyte autoantibodies can remain silent or lead to Autoimmune Hemolytic Anemia (AIHA).The aim of this study was to identify predictors of AIHA in beta-thalassemia patients with positive Direct Antiglobulin Test (DAT), in Tunisia. This longitudinal prognosis study was carried out on beta-thalassemia patients with a positive confirmed DAT. Predictors of AIHA were identified the Kaplan-Meier method. A Cox model analysis was used to identify independent predictors. Among 385 beta thalassemia patients, 87 developed positive DAT (22.6%). Autoimmune hemolytic anemia was occurred in 25 patients. Multivariate analysis showed that AIHA was independently associated with beta-thalassemia intermedia and similar family history of AIHA. Splenectomy in patients with positive DAT was independently associated with an increased risk of AIHA (HR = 6.175, CI: 2.049-18.612, p < 0.001). The risk of developing AIHA was higher during the first 72 transfusions. Autoimmune hemolytic anemia was significantly associated with polyspecific DAT (anti-complement and anti-IgG), blood group AB and prior alloimmunization. Whereas transfusion by phenotypic and leukoreduced blood was a protective factor. In summary, splenectomy after autoimmunization, prior alloimmunization, DAT specificity (IgG with complement), thalassemia intermedia, AB blood group and family history of AIHA were strongly associated with AIHA. Leukoreduced blood transfusion had a proven preventive role.

Bronchiolitis obliterans after allogeneic hematopoietic stem cell transplantation: The effect of NOD2/CARD15 mutations in a Tunisian population.

Bronchiolitis obliterans (BO) is a serious lung complication that can develop after allogenic stem cell transplantation. It has been suggested that single nucleotide polymorphisms (SNPs) that affect the NOD2/CARD15 gene impair its function and result in an uncontrolled innate immune response in the recipient, thereby leading to BO. One hundred eighty-one donor-recipient pairs were analyzed for the association between NOD2 gene variants (SNP8 [Arg702Trp], SNP12 [Gly908Arg], and SNP13 [Leu1007fsinsC]) and the occurrence of BO. Ten patients (2.8%) developed this complication. The incidence of BO increases in recipient variant patient group from 4.7% to 23% in donor Wild-type group in SNP8 (p < 0.001). The incidence rose to 19% when the recipient carried the SNP12 variant (p < 0.001) in the Tunisian population. Analyses demonstrated that recipient NOD2CARD15 variants (SNP8 and SNP12) present a greater risk in developing BO than recipients without mutation. Our study demonstrated that NOD2/CARD15 typing may be useful in identifying patients at high risk for BO.

Haemostatic balance in cirrhosis.

Despite the prolongation of coagulation tests, recent studies reported an increased frequency of thromboembolic events in patients with cirrhosis. The aim of this study was to evaluate the haemostatic balance in cirrhotic patients through assessing the variation of pro- and anticoagulant factors and evaluating the in-vitro thrombin generation in patients with cirrhosis and in healthy patients. Fifty-one cirrhotic patients with or without thromboembolic events and 50 controls matched by age and sex were enrolled. Procoagulant (factors VII, II, V, VIII, and XII) and inhibitor (protein C, protein S and antithrombin) factor activities were determined. Thrombin generation was measured as endogenous thrombin potential (ETP). Haemostatic balance was assessed by means of both procoagulant to inhibitor coagulation factor ratios and ETP with to without protein C activation ratios. There were 24 males and 27 females. The mean age was 57.8 years [16-91 years]. Pro and anticoagulant factors were significantly lower in patients than in controls (P < 0.001) except for factor VIII and protein S. In fact factor VIII level was significantly higher in patients than in controls and protein S levels were not significantly different between patients and controls. Almost all the pro to anticoagulant factor ratios were higher in cirrhotics than in controls, especially the factor VIII to protein C ratios which increased significantly from Child Pugh A to C (P < 0.001), the ratio of ETP with to without protein C activator was higher in patients than in controls, but did not reach a significant level (0.8 vs. 0.52) There was no statistically significant difference between Child classes. When comparing patients with history of thrombosis (n = 7) to those matched by age and sex and without history of thrombosis (n = 14), the ratios were not statistically different between the two groups. Haemostatic changes in cirrhosis tend to rebalance the haemostatic system. This state often results in a hypercoagulable state attested by increased pro- to anticoagulant factor ratios and a normal thrombin generation.

[The JAK2 mutation in myeloproliferative disorders: A predictive factor of thrombosis]. / La mutation JAK2 dans les syndromes myéloprolifératifs BCR-ABL négatifs: facteur prédictif de thrombose.

BACKGROUND: BCR-ABL negative myeloproliferative neoplasms (MPN) include polycythemia Vera (PV), essential thrombocythemia (ET) and primitive myelofibrosis (PMF). the JAK2 V617F mutation has been introduced since 2008 as a major diagnostic criterion on the one hand and on the other hand, it would be linked to increased risk of thrombotic complications. AIM: This study aimed to evaluate the association of JAK2 mutation and thrombotic events in MPN. METHODS: A retrospective study concerning 45 BCR-ABL negative MPN patients (mean age=53 old years, sex ratio=0.8) was conducted. RESULTS: They were classified as PV (22 patients), ET (17 patients), PMF (3 patients) and atypical MPN (3 patients). The JAK2 mutation was found in 64.4% of patients: 72.7% of PV patients, 47% of ET patients and 66.7% of PMF patients. Thrombotic events were recorded in 11 patients (24.4%). Cerebral arteries and portal vein were the most frequent localizations. The JAK2 mutation was an independent risk factor of thrombotic events. CONCLUSION: Consequently, it seems that screening for JAK2 mutation in BCR-ABL negative MPN could play a role in identifying patients at high risk of vascular complications.

Do FY antigens act as minor histocompatibility antigens in the graft-versus-host disease paradigm after human leukocyte antigen-identical sibling hematopoietic stem cell transplantation?

FY antigens are candidate minor histocompatibility antigens relevant to renal allograft rejection, but no data have been reported about their role in graft-versus-host disease (GVHD) incidence after human leukocyte antigen (HLA)-identical siblings hematopoietic stem cell transplantation (HSCT). The aim of this study was to examine the effect of donor/recipient disparity at FY antigens on the incidence of GVHD in Tunisian patients receiving an HLA-identical HSCT. This work enrolled 105 Tunisian pairs of recipients and their HLA-identical sibling donors of HSCs. FY genotyping was performed with the polymerase chain reaction-sequence-specific primer method and donor/recipient disparity for these antigens was analyzed at two levels: incompatibility and nonidentity. The case-control analyses showed no significant correlation between FY disparity and the incidence of either acute or chronic GVHD. Sample size calculation showed that 572 cases and 1716 controls would be necessary to be able to detect a significant association with 80% power and two-sided type I error level of 5% (α=0.05). The lack of association in the studied cohort may be explained by the low immunogenicity of FY antigens in HSCT context, compared with other antigens such as HA-1 and CD31.

Evidence that erythrocyte DARC-positive phenotype can affect the GVHD occurrence after HLA-identical sibling HSCT.

Chemokine receptors are very important players in the pathogenesis of GVHD. The aim of this study is to test the hypothesis that the lack of expression of the DARC receptor on erythrocytes can affect the GVHD incidence. A total of 105 recipients and their 105 respective sibling donors of HSCs were enrolled in this study. All patients were evaluated for acute and chronic GVHD. The DARC genotyping assay was performed using the SSP-PCR method. The case-control analyses showed that the donor DARC 146G allele and T(-46)G(146) haplotype, coding for the FY2 version of DARC, are very significant in the GVHD paradigm because they are associated with the incidence of acute effects of this outcome in recipients (p=0.007, χ²=7.200). It seems that this version of DARC receptor is a powerful facilitator of chemokine transcytosis and subsequently leukocyte migration into GVHD target organs.

Investigation of the effect of donor platelet endothelial cell adhesion molecule 1 polymorphism on the graft-vs.-host disease occurrence in Tunisian recipients of hematopoietic stem cells.

OBJECTIVE: The aim of this study is to examine the effect of donor PECAM-1 alleles and haplotypes for the SNPs L98V, S536N, and R643G on the occurrence of GVHD in Tunisian recipients of HSCs. DESIGN AND METHODS: This study enrolled 102 patients and their 102 respective HLA-identical sibling donors of HSCs. The PECAM-1 SNPs genotyping assay was performed using sets of sequence specific primers (SSP-PCR). RESULTS: The single marker association analysis showed that the L98 allele, in a recessive genetic model, may be a potential risk factor only for acute GVHD (p=0.036, OR=2.580, 95% C.I. = 1.053-6.326). However, the haplotype analysis showed a lack of association between donor's PECAM-1 SNPs and GVHD incidence in recipient. CONCLUSION: The homozygosity state for donor PECAM-1L98 allele may be a significant risk factor for acute GVHD. This is probably due to its action on the function of donor leukocytes especially during the extravasation process.

Effect of donor CTLA-4 alleles and haplotypes on graft-versus-host disease occurrence in Tunisian patients receiving a human leukocyte antigen-identical sibling hematopoietic stem cell transplant.

The CTLA-4 genetic variation, such as single nucleotide polymorphisms (SNPs) may be critical and can affect the functional activity of cells that initiate the graft-versus-host disease (GVHD) effects. The aim of this study is to examine the effect of donor CTLA-4 alleles and haplotypes for the -318C>T and the 49A>G polymorphisms on the occurrence of GVHD in Tunisians recipients of HSCs. A total of 112 patients and their 112 respective sibling donors of HSCs were enrolled in this study. All patients had either grades 0-I or grades II-IV acute GVHD, or chronic GVHD. The SNPs genotyping assay was performed using sets of sequence specific primers (SSP-PCR). The single marker association analysis showed that the 49G allele, in a genetic recessive model, may be a potential risk factor only for the chronic GVHD (p = 0.032, odds ratio [OR] = 2.58, 95% confidence interval = 1.05-6.32). The haplotypes analyses showed that the CTLA-4 -318C49G nucleotide combination is significantly associated with the incidence of chronic GVHD (p = 0.043, χ² = 3.27). Donor CTLA-4 -318C49G haplotype may be a significant risk factor for developing chronic GVHD after allo-stem cell transplantation. We suppose that donor T cells expressing this haplotype in a homozygous state have higher proliferation than those expressing other haplotypes, especially after recognition of the recipient's minor histocompatibility antigens.

Acute graft-vs.-host disease correlates with the disparity for the PECAM-1 S536N polymorphism only in the HLA-B44-like positive Tunisian recipients of HSCs.

GVHD is the major cause of mortality after HLA-identical HSCT. Such complication has been widely linked to donor/recipient disparity for minor histocompatibility antigens (MiHAgs). PECAM-1 is one of potential human MiHAgs but its effect on the GVHD occurrence remains not clear. In order to examine such association in the Tunisian cohort of HSCs recipients, we performed a retrospective study on patients who undergone HLA-identical HSCT between 2000 and 2009. Genotyping of the three selected PECAM-1 polymorphisms (rs668, rs12953 and rs1131012) was performed with SSP-PCR method. Univariate analyses showed that grades II-IV acute GVHD were considerably linked to the non-identity for rs12953 only in HLA-B44-like positive patients (p=0.010, OR=10.000). Multivariate analysis for chronic GVHD showed that this outcome may be affected only by the adulthood and the conditioning regimen. Our findings support the previously reported data suggesting a significant association between the PECAM-1 disparity and the risk of acute GVHD.

Mismatch for the minor histocompatibility antigen HA-2 and GVHD occurrence in HLA-A*0201-positive Tunisian recipients of HSCs.

Graft-versus-Host disease (GVHD) has been widely linked to immunogenetic causes such as disparity between the recipient and its HLA geno-identical donor for some Non-HLA antigens called minor histocompatibility antigens (MiHAgs). HA-2 is one of potential human MiHAgs but its effect on the GVHD occurrence remains not clear. In order to examine such association in the Tunisian cohort of HSCs recipients, we performed a retrospective study on patients who received an HLA-identical HSCT between 2000 and 2009. The study was performed on 60 HLA-A2-positive patients who had received a haematopoietic stem cell transplant from an HLA-identical sibling. All patients received cyclosporine A and/or methotrexate for GVHD prophylaxis. HA-2 genotyping assay was performed with SSP-PCR method and HLA-A*0201 positive samples were identified mainly with Luminex HLA-Typing method. Luminex HLA-Typing assay showed that only 53 cases were positives for the HLA-A*0201 allele. Among these cases, only 3 pairs were mismatched for the MiHAg HA-2. Acute GVHD occurred in 01 HA-2-mismatched pair while chronic GVHD was detected in 02 disparate couples. Univariate and multivariate analyses showed that MiHAg HA-2 disparity does not have any significant effect on the occurrence of either acute or chronic GVHD. This last one appeared to be correlated only with the age of patient (adulthood) (p: 0.011, OR: 22.092). Our findings support the previously reported data denying the influence of the HA-2 disparity on the GVHD occurrence after HSCT.

Does minor histocompatibility antigen HA-1 disparity affect the occurrence of graft-versus-host disease in tunisian recipients of hematopoietic stem cells?

INTRODUCTION: Minor histocompatibility antigen HA-1 (MiHAg-HA-1) disparity between a patient and his or her human leukocyte antigen (HLA) genoidentical donor has been widely associated with an increased risk of graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. OBJECTIVE: To examine the effect of HA-1 disparity on the incidence of both acute and chronic graft-versus-host disease in Tunisian recipients of hematopoietic stem cells. METHODS: A total of 60 patients and their 60 respective sibling hematopoietic stem cell donors were enrolled in this study. All patients prophylactically received cyclosporine A and/or methotrexate for graft-versus-host disease. An HA-1 genotyping assay was performed with the SSP-PCR method, and HLA-A*0201- and/or HLA-A*0206-positive samples were identified using the Luminex HLA typing method. RESULTS: The Luminex HLA typing assay showed that 54 patients were positive for either the HLA-A*0201 or HLA-A*0206 alleles. Among these cases, six pairs were mismatched for MiHAg-HA-1. Both acute and chronic graft-versus-host disease occurred in four mismatched patients (Fisher's p-values were 0.044 and 0.170, respectively). A univariate logistic regression model analysis showed that only acute graft-versus-host disease may be affected by recipient MiHAg-HA-1 disparity (p: 0.041, OR: 6.727), while chronic graft-versus-host disease correlates with both age and recipient/donor sex mismatch (p: 0.014, OR: 8.556 and p: 0.033, OR: 8.664, respectively). CONCLUSION: Our findings support previously reported data suggesting a significant association between HA-1 disparity and the risk of acute graft-versus-host disease following hematopoietic stem cell transplantation.

Does minor histocompatibility antigen HA-1 disparity affect the occurrence of graft-versus-host disease in tunisian recipients of hematopoietic stem cells?

INTRODUCTION: Minor histocompatibility antigen HA-1 (MiHAg-HA-1) disparity between a patient and his or her human leukocyte antigen (HLA) genoidentical donor has been widely associated with an increased risk of graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. OBJECTIVE: To examine the effect of HA-1 disparity on the incidence of both acute and chronic graft-versus-host disease in Tunisian recipients of hematopoietic stem cells. METHODS: A total of 60 patients and their 60 respective sibling hematopoietic stem cell donors were enrolled in this study. All patients prophylactically received cyclosporine A and/or methotrexate for graft-versus-host disease. An HA-1 genotyping assay was performed with the SSP-PCR method, and HLA-A*0201- and/or HLA-A*0206-positive samples were identified using the Luminex HLA typing method. RESULTS: The Luminex HLA typing assay showed that 54 patients were positive for either the HLA-A*0201 or HLA-A*0206 alleles. Among these cases, six pairs were mismatched for MiHAg-HA-1. Both acute and chronic graft-versus-host disease occurred in four mismatched patients (Fisher's p-values were 0.044 and 0.170, respectively). A univariate logistic regression model analysis showed that only acute graft-versus-host disease may be affected by recipient MiHAg-HA-1 disparity (p: 0.041, OR: 6.727), while chronic graft-versus-host disease correlates with both age and recipient/donor sex mismatch (p: 0.014, OR: 8.556 and p: 0.033, OR: 8.664, respectively). CONCLUSION: Our findings support previously reported data suggesting a significant association between HA-1 disparity and the risk of acute graft-versus-host disease following hematopoietic stem cell transplantation.