Human leukocyte antigen immunization in transfusion-dependent Moroccan patients with beta-thalassemia major: prevalence and risk factors.

INTRODUCTION: Beta-thalassemia major patients need a regular blood transfusion to have an initial normal growth. However, these patients have an increased risk of developing alloantibodies. Our main goal was to study HLA alloimmunization in Moroccan Beta-thalassemia patients by confronting it with transfusion and demographic criteria, exploring the involvement of HLA typing profile in the development of HLA antibodies and in turn determining risk factors for their development. METHODS: The study consisted of 53 Moroccan pediatric patients with Beta-thalassemia major. Screening for HLA alloantibodies was performed using Luminex technology Whereas HLA genotyping was done with sequence-specific primers (PCR-SSP). RESULTS: In this study, 50.9% of patients have been identified as positive for HLA antibodies, with 59.3% having both HLA Class I and Class II antibodies. A significant increase frequency of DRB1*11 allele was revealed in non-immunized patients (34.6% vs. 0%, p = 0.001). Our results also revealed that the majority of our HLA immunized patients were women (72.4% vs. 27.6%, p = 0.001), and transfused with more than 300 units of RBC units (66.7% vs. 33.3%, p = 0.02). There were statistically significant differences when comparing these frequencies. CONCLUSIONS: This paper revealed that the transfusion dependent Beta-thalassemia major patients are exposed to risk of developing HLA antibodies following transfusions with leukoreduced RBC units. The HLA DRB1*11 was a protective factor against HLA alloimmunization in our beta-thalassemia major patients.

Collection, cryopreservation and thawing of stem cells for children weighing less than 25 Kg with high-risk neuroblastoma: A single center results in Morocco

ABSTRACT Introduction: An important component of the advances made in neuroblastoma treatment has been the use of peripheral blood stem cells to support high-dose chemotherapy. In this study, we report our experience on a series of small children who have undergone standard and large volume leukaphersis (LVL) procedures, provide an update on a single institution's experience with cryopreservation of autologous peripheral blood stem cells (PBSCs), using 10% dimethyl sulfoxide (DMSO) and applying post-thaw DMSO depletion and analyze a number of variables that may affect viability. Methods: A total of 36 aphereses were performed on 29 children weighing less than 25 kg between July 2016 and October 2019 at the Ibn Sina university hospital. Results: Seven females and twenty-two males, median bodyweight 14 kg (9 - 22). A single apheresis was sufficient to obtain at least 3 × 106/kg body weight (BW) of CD34+ cells in 82.8% of the cases. The LVL was performed in 22 aphereses. A median number of 5.9 × 106/ kg CD34 cells were collected per apheresis. A total of 60 PBSC samples were cryopreserved and 46 samples were infused. The mean cell viability percentage decreased from 94.75 ± 1.14% before freezing to 70.84 ± 8.6% after thawing (p < 0.001). No correlation was found between post-thaw viability and storage time (r = -0.233; p = 0.234) or number of total nucleated cells (r = 0.344; p = 0.073). Conclusion: Leukapheresis is safe and feasible in small pediatric patients if the appropriate measures are used. Cryopreservation poses numerous challenges, especially a decrease in cell viability after thawing.

Collection, cryopreservation and thawing of stem cells for children weighing less than 25 Kg with high-risk neuroblastoma: A single center results in Morocco.

INTRODUCTION: An important component of the advances made in neuroblastoma treatment has been the use of peripheral blood stem cells to support high-dose chemotherapy. In this study, we report our experience on a series of small children who have undergone standard and large volume leukaphersis (LVL) procedures, provide an update on a single institution's experience with cryopreservation of autologous peripheral blood stem cells (PBSCs), using 10% dimethyl sulfoxide (DMSO) and applying post-thaw DMSO depletion and analyze a number of variables that may affect viability. METHODS: A total of 36 aphereses were performed on 29 children weighing less than 25 kg between July 2016 and October 2019 at the Ibn Sina university hospital. RESULTS: Seven females and twenty-two males, median bodyweight 14 kg (9 - 22). A single apheresis was sufficient to obtain at least 3 × 106/kg body weight (BW) of CD34+ cells in 82.8% of the cases. The LVL was performed in 22 aphereses. A median number of 5.9 × 106/kg CD34 cells were collected per apheresis. A total of 60 PBSC samples were cryopreserved and 46 samples were infused. The mean cell viability percentage decreased from 94.75 ± 1.14% before freezing to 70.84 ± 8.6% after thawing (p < 0.001). No correlation was found between post-thaw viability and storage time (r = -0.233; p = 0.234) or number of total nucleated cells (r = 0.344; p = 0.073). CONCLUSION: Leukapheresis is safe and feasible in small pediatric patients if the appropriate measures are used. Cryopreservation poses numerous challenges, especially a decrease in cell viability after thawing.

[Husband to wife renal transplantatation and pretransplant HLA immunization: about two cases]. / Transplantation rénale entre époux et immunisation anti-HLA en prégreffe: à propos de deux observations.

Renal transplantation is the best therapeutic approach for end-stage kidney disease. Renal transplantation can be performed using living donors or brain-dead donors. Vaccination in recipients poses a real problem with the transplantation process because it is responsible for particular difficulties in choosing a donor and above all exposes to the risk of transplant rejection. We here report two cases of husband to wife renal transplantation. The recipients had low levels of antibodies against HLA antigens of donors but sex-associated differences in post-transplant results were found. Indeed, HLA immunization after pregnancy is a real obstacle to husband to wife renal transplantation. Despite the low husband-wife HLA matching (unrelated donor), renal transplantation is a good alternative for renal transplantation from brain-dead donors, who are lacking in Morocco.

Reason and Resolution of High Negative Control Beads in Solid-Phase Immunoassay.

OBJECTIVES: The Luminex technology is the most sensitive diagnostic method for HLA antibody detection and identification. However, the interpretation of immunoassays is commonly affected by the artifact, and non-specific background. Sera from some patients show high negative control bead (NC) value, which makes assessing and interpretation of HLA antibodies difficult. In this study, we evaluated the effect of Adsorb Out reagent, dithiothreitol (DTT), and Ethylenediaminetetraacetic acid (EDTA) on the NC median fluorescence intensity value by comparing treated versus untreated patient sera. In addition, we wanted to identify whether kidney disease and administered medication influenced high NC median fluorescence intensity values by comparing patient versus control results. MATERIALS AND METHODS: HLA antibody screening was performed on 3500 serum samples. Sera were analyzed using the standard protocol for Luminex antibody screening. Sera with high NC values were preincubated with Adsorb Out, DTT, and EDTA. Screening of these sera was then performed. RESULTS: We found that 4% of samples showed high NC values. Adsorb Out, DTT, and EDTA decreased the NC values at 723.5 (299.25-1443) versus 85 (34-218; P < .001), at 723.5 (299.25-1443) versus 184 (106-597; P < .001), and at 723.5 (299.25-1443) versus 455 (131-1177; P = .004). These succeeded in bringing back NC values to normal range in 69.2%, 43%, and 30% of treated sera, respectively. In addition, the differences of corticoids, immunosuppressive, and heparin drugs between patients and controls were statistically significant (P < .001, < .001, and = .043). However, presence of kidney disease was not significant between these groups. CONCLUSIONS: All pretreatments had an important effect in decreasing negative control values, with Adsorb Out having highest efficiency. Serum-specific components could contribute to high negative control bead median fluorescence intensity values. Further studies are needed to determine the adequate pretreatment of patient sera.

[Cytotoxicity of natural anti-HLA antibodies in Moroccan patients awaiting for kidney transplantation]. / Cytotoxicité des anticorps anti-HLA naturels chez des patients marocains candidats à la greffe rénale.

The presence of anti-HLA antibodies in the serum of a patient result from an immune response produced during an immunizing event as transfusion, pregnancy or graft. These antibodies can be cytotoxic by activating the complement pathway via C1q and may cause organ rejection during the transplant. Some male patients awaiting kidney transplantation are seropositive for anti-HLA antibodies when they have no immunizing antecedent event. These antibodies are qualified as natural antibodies. Our work is to assess the cytotoxicity of natural anti-HLA antibodies in patients followed at the immunology laboratory of the blood transfusion service and hemovigilance (STSH) as part of the kidney transplant. PATIENTS AND METHODS: We evaluated the cytotoxicity of HLA antibodies detected in male Moroccan patients without immunization history using C1qScreen One Lambda reagent for Luminex™. RESULTS: Non-immunized men were positive for HLA antibodies screening in 25.4%. These antibodies are not cytotoxic. CONCLUSION: Our study showed a positivity rate of natural HLA antibody low than the literature (25.4% against 63%). It appears that these natural antibodies are not cytotoxic and their involvement in renal transplant remains to be determined.

HLA Class II with Lupus Nephritis in Moroccan Patients.

Lupus nephritis (LN) is a disease with a poor prognosis. The association between LN and the Human leukocyte antigen (HLA) genes has never been studied on a Moroccan population. The aim of this work was to evaluate the distribution of the HLA class II alleles in patients with LN and to determine susceptible and protective HLA alleles/haplotypes in LN. The association between these alleles, disease severity of LN, and age at onset were also investigated. Seventy-five patients with LN were compared with 169 healthy unrelated controls. HLA class II alleles typing was performed by polymerase chain reaction-sequence-specific primers (PCR-SSP). A significant increase of HLA-DRB1*15 allele frequency (p = 0.001) and a significant decrease of the HLA-DRB1*04 allele (p = 0.04) were observed in LN patients. The frequency of HLA-DRB1*15-DQB1*06 haplotype (p = 0.003) was increased in the patients while that of HLA-DRB1*04-DQB1*03 (p = 0.027) was decreased. A significant increase of HLA-DRB1*15 allele frequency (p = 0.0001) and HLA-DRB1*15-DQB1*06 haplotype (p = 0.002) was observed in patients with class IV LN. In the Moroccan population we demonstrated the positive association of HLA class II alleles and haplotypes with LN and with a severe form of nephritis. HLA-DRB1*15 allele does not determine the age of disease onset in LN.

[Serological tests for celiac disease in Moroccan patients with type 1 diabetes]. / Dépistage sérologique de la maladie cœliaque chez des patients marocains atteints de diabète type 1.

Celiac disease (CD) is an autoimmune disease frequently associated with type 1 diabetes (T1D). The prevalence of CD in patients with T1D varies from 3 to 6%. The clinical manifestation of CD in patients with T1D is classified as asymptomatic in about half of cases. Our study aims to determine the frequency of anti-tissue transglutaminase autoantibodies (IgA-tTG) and anti-gliadin antibodies (AGA) in patients with type 1 diabetes in order to early recommend jejunal biopsy and establish a gluten-free diet before the onset of clinical signs and complications of celiac disease. Subjects included in this study were patients with T1D and untreated CD who showed no signs of this disease. The detection of IgG tTG, IgG IgA and IgG AAG was performed using Luminex technology. We enrolled 31 patients. The study involved 16 men and 15 women. IgA AAG were positive in 4(13%) patients and IgG were positive in 7(22,5%) patients. IgA tTG were positive in 3(10%) patients and IgG was positive in one (3%) patient. In our study the association of diabetes type 1 with biomarkers of CD is not uncommon hence the importance of systematic screening for type 1 diabetes. The diagnosis of this atypical and silent CD form is important given the risk of serious complications such as malabsorption and gastrointestinal cancers.

High background in Luminex® assay for HLA antibody screening: Interest of Adsorb Out™.

INTRODUCTION: The Luminex® technology has become an integral component of clinical decision-making and diagnosis of transplanted organ rejection. Despite the superior sensibility of this technology, it is not completely problem free. We have observed in these bead-based assays that sera of some patients give a high negative control bead (NC) value which makes assessing HLA antibodies difficult. Treatment of sera by the Adsorb Out™ reagent may reduce the high background. In this study, we want to evaluate the effect of the Adsorb Out™ on the NC's MFI value by comparing treated and untreated patients' sera. METHODS: HLA antibody screening was performed on 3011 sera. These sera came from patients awaiting and undergoing renal transplant from different Moroccan hospitals. The sera were analyzed using the standard protocol for Luminex® antibody screening. Sera with high NC's value has been pre-incubated by the Adsorb Out™, and analyzed on Luminex®. RESULTS: 3% of studied samples have high NC's value. The Adsorb Out™ decreases the NC's value and brings it back to a normal range in 62.2% treated sera. It has no effect in 12.3%. The Adsorb Out™ effect depends only of NC's value, independently to age, storage date, sex and immunization. CONCLUSION: The Adsorb Out™ reagent has an important effect in decreasing NC value of sera. However, it has no effect in some patient's sera. In these cases we could try another treatment, as EDTA, DTT. The non-specific binding may be caused by multiple patient-specific factors, it would be important to search correlation between them and NC's values.

Moroccan study of HLA (-A, -B, -C, -DR, -DQ) polymorphism in 647 unrelated controls: Updating data.

The scope of this study is to investigate the HLA (Human Leukocyte Antigen) distribution and polymorphism in a large sample of healthy Moroccans in order to extend and update the available data. 647 unrelated Moroccan controls originating from diverse regions of the country were typed using microlymphocytotoxicity for HLA-A and -B, sequence-specific-primer amplification for -C, -DR, and -DQ and Luminex HD for specific -DR. The most frequent allele groups detected were HLA-A2 (19.2%), -B44 (12.4%), -C*07 (24.4%), -DRB1*03 (16.9%), -DRB1*04 (18.4%), -DQB1*02 (28.7%) and -DQB1*03 (27.8%). The most predominant specific alleles found for DRB1 were: *03:01, *04:02, *04:05, *07:01, *11:01, *13:02 and *15:01. Rare allelic variants were detected, for the first time in Moroccan population, at the DRB1*03 (*03:52, *03:54, *03:56), DRB1*07 (*07:07, *07:11, *07:16) and DRB1*11 (*11:70) locus. The most frequent haplotypes were: A2-B44, A30-B18, A2-C*16, A30-C*06, B14-C*08, B58-C*07, B45-C*06, DRB1*03-DQB1*02, DRB1*04-DQB1*03, DRB1*07-DQB1*02 and DRB1*15-DQB1*06. Comparison of genetic distances and haplotypes with other populations shows that the Moroccans are genetically closer to North Africans and Europeans than to sub-Saharan Africans. Our results reflect the high degree of HLA polymorphism in the Moroccan population and provide a useful baseline of healthy Moroccan controls for disease association and anthropological studies.

[Detection of HLA antibodies by Luminex and kidney transplantation decision making: a case report]. / Détection des anticorps anti-HLA par Luminex et décision de transplantation rénale : à propos d'un cas.

We report the case of a patient planned for living-related donor kidney transplantation. HLA antibodies research, performed by the Luminex method, is positive for all the available sera. Cross match was performed by complement-dependent lymphocytotoxicity technique with three family members. The first donor, her husband, was excluded due to the positivity of the cross-match and the presence of donor-specific anti HLA antibody (DSA). The second one, her daughter, was immediately excluded because the recipient also presented DSA. Despite the negativity of the cross match, the decision to transplant the patient with the third donor, her son, was difficult to take because of the presence of DSA identified by the Luminex method. Consequently, the patient could not be transplanted.

[Evaluation of high resolution Luminex technology for specific HLA-DRB1* typing in the Moroccan population]. / Évaluation de la technologie Luminex de haute résolution pour le typage HLA-DRB1* spécifique dans la population marocaine.

The aim of this study is to evaluate the possibility of using routinely Luminex high definition technology for the specific HLA typing of donors and recipients of hematopoietic stem cells. 340 HLA-DRB1 *, all from Moroccan individuals were first tested at the generic level and then at the specific level by PCR-SSO Luminex high definition. Alleles identified correspond to those originally found with the generic typing. The specificity could be determined only in 41.5% of cases. The percentage of specific alleles identified for DRB1 * 04 was 78.7% and varies between 17.6% and 34.6% for other alleles. Of the eight haplotypes tested, blanks obtained by PCR-SSP were all resolved. Our results confirm the reliability of the method, since we confirm the alleles identified at the generic level. Luminex high definition technology can be used for HLA-DRB1 * 04 typing, and for the resolution of ambiguities and blanks. However, it must be completed by other techniques (PCR-SSP, SBT) in the context of hematopoietic stem cells.

Remission in early rheumatoid arthritis treated with conventional DMARDs. Results of a two-year follow-up study of El Ayachi Moroccan cohort.

OBJECTIVE: This study aimed to evaluate remission in patients with early RA treated by conventional DMARDs and to identify its possible predictor factors. METHODS: Patients with early RA (<12 months) were enrolled in a 2-year follow-up study. Standard evaluation completed at baseline and at 24 months included clinical, laboratory, functional and structural assessment. Clinical remission after 2 years of follow-up was defined when DAS28 was less than 2.6. Possible predictor factors for remission were analyzed. RESULTS: Fifty-one patients (88.2% women, mean age of 46.9 [24-72] years, mean disease duration of 24 [6-48] weeks) were enrolled in this study. The delay in referral for specialist care was 140 [7-420] days. Rheumatoid factor, anti-CCP, HLA-DRB1*01 and DRB1*04 alleles were present respectively in 62.5, 56.6, 11.8, and 45.1% of patients. At 24 months, 77.2% received a median dose of 5 (0-8) mg/day of prednisone and 65.2% was taking methotrexate (MTX). 13.6% of patients had stopped their DMARD because of socioeconomic difficulties. At 24 months, we noted a significant improvement of morning stiffness, pain score, swollen joint count, ESR, CRP, DAS28 and HAQ scores. Remission at 2 years was noted in 34.8% of patients and was significantly associated in univariate but not in multivariate analysis to male sex (P=0.02) and to short delay in referral for specialist (P=0.03). CONCLUSION: In this cohort of early RA patients treated with conventional DMARDs, especially with methotrexate in monotherapy, remission at 2-year of follow-up was obtained in one third of patients. No predictor factors of remission were found out. These results should be verified by further studies.

Profile and course of early rheumatoid arthritis in Morocco: a two-year follow-up study.

BACKGROUND: This study aimed to establish the profile and the evolution of an early Rheumatoid arthritis (RA) cohort in the Moroccan population and also to search possible predictor factors of structural progression. METHODS: Patients with early RA (< 12 months) were enrolled in a 2-year follow-up study. Clinical, biological, immunogenetic, and radiographical data were analyzed at study entry and at 24 months. Presence of radiographic progression was retained when the total score was superior to the smallest detectable difference (SDD) calculated to be 5.4 according the Sharp/van der Heijde (SVDH) method. RESULTS: Fifty one patients (88.8% women, mean age of 46.9 [ 24-72 ] ± 10.8 years, mean disease duration of 24 [ 6-48 ] ± 13.9 weeks) were enrolled in this study. 68.6% were illiterate and 19.6% reported at least one comorbid condition. The mean delay in referral for specialist care was 140 [ 7-420 ] ± 43 days.Thirteen patients (62.5%) were IgM or IgA RF positive. HLA-DRB1*01 and DRB1*04 alleles were present respectively in 11.8% and 45.1% of patients.At baseline, 35.3% patients were taking corticosteroids and 7.8% were under conventional DMARDs.At 24 months, 77.2% received a median dose of 5 mg/day of prednisone. Methotrexate (MTX) was the most frequently prescribed DMARD, being taken by 65.2% of patients. 13.6% of patients had stopped their DMARD because of socioeconomic difficulties.Comparison of clinical and biologic parameters between baseline and 24 months thereafter revealed a significant global improvement of the disease status including morning stiffness, pain score, swollen joint count, DAS 28 and HAQ scores, ESR and CRP.Sixteen patients (34.8%) were in remission at 2 years versus no patients at baseline; P < 0.001.Forteen patients (27.5%) had at least one erosion at baseline. Radiographic progression occurred in 33.3% of patients and was associated in univariate analysis to swollen joint count (p = 0.03), total SVDH score (P = 0.04) and joint space narrowing score (P = 0.03). No independent factors of radiographic progression were shown by logistic regression. CONCLUSIONS: These study reports, provided for the first time in Morocco, a developing African country, a large amount of information concerning the profile and the course of early RA.Patients who were receiving, for most of them, Methotrexate in monotherapy and low doses of corticosteroids, showed an improvement of all clinic and biologic disease parameters. Moreover, DAS remission was obtained in one third of patients and two thirds of the cohort had no radiographic progression at 2 years. No predictor factors of radiographic progression were found out.These results should be confirmed or not by a large unbiased RA cohort which will give more relevant information about early RA characteristics and its course and will constitute a major keystone of its management.

[Rejection of kidney graft: mechanism and prevention]. / Rejet de la greffe rénale: mécanisme et prévention.

Rejection occurs after the introduction of a genetically different graft, in a recipient. Nowadays, it is still a major obstacle in renal transplantation and reflects a normal protective immune response of a recipient against a foreign antigen. Involving many mechanisms of the innate and adaptive immunity, this reaction results in renal parenchymal lesions witch may progress to graft destruction and loss of its function. Several ways are currently used to reduce the action of the immune system and consequently reduce the risk of rejection. After a presentation of the main actors and the sequence of events leading to rejection, we will describe the strategy used by antirejection teams' transplantation. We will successively consider the prevention (pre-transplant immunological assessment, preventive immunosuppressive therapy), the monitoring (search for antibodies, biopsies) and the treatment.