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.

[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.

[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.