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.

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.

HLA polymorphism and early rheumatoid arthritis in the Moroccan population.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune multifactorial disease which has a great socio-economic impact in Morocco. The association of HLA genes with RA was studied in various ethnic groups but not in the Moroccan population. Our study focused on evaluating the distribution of class I and class II HLA genes among Moroccan patients presenting early signs of RA. METHODS: Forty nine patients diagnosed with early RA were compared to a group of healthy controls matched by age, sex, and ethnic origin. Among the patient group, 34 were seropositive (presence of the rheumatoid factor). HLA typing of the patients and the controls was performed using microlymphocytotoxicity for class I (A and B) and PCR-SSP for class II (DR and DQ). RESULTS: We found a significant increase of the frequency of the HLA-A24 antigen (p=0.03), the DRB1*04 (p=0.004) and DQB1*03 (p=0.03) alleles and a significant decrease of the DRB1*07 allele (p=0.03) in seropositive patients. The analysis of the frequency of the DRB1*01, DRB1*10, and DRB1*14 alleles did not show any difference between the RA patients and the controls. The frequency of DR4-DQ2 and DR4-DQ4 haplotypes was increased in the patients compared to the controls while that of DR7-DQ2 and DR13-DQ6 was decreased. CONCLUSIONS: Our study suggests that DRB1*04 predisposes to RA while DRB1*07 seems protective for the Moroccan patients population. In addition we show the influence of some haplotypes DR-DQ in the susceptibility and protection against the disease.