External proficiency testing for histocompatibility and immunogenetics in today and future.

The Histocompatibility and Immunogenetics laboratories provide disease association and pharmacogenetic analyses as well as the tests required for transplantation immunology and transfusion medicine. They perform Human Leukocyte Antigen (HLA) genotyping in patients/recipients and potential donor candidates for solid organ and stem cell transplants using various molecular methods, and determine mismatches. In addition, they also perform HLA antibody tests to detect anti-HLA antibodies in patients and flow cross-matches to evaluate donor-recipient compatibility. Evidence-based clinical guidelines have emphasized the importance of laboratory tests in clinical practices for a long time. Understanding the principles of Quality Control and External Quality Assurance is a fundamental requirement for the effective management of Tissue Typing laboratories. When these processes are effectively implemented, errors in routine assays for transplantation are reduced and quality is improved. In this review, the importance of Quality Assurance, Quality control and proficiency testing in Histocompatibility and Immunogenetic testing, the necessity of external proficiency testing (EPT) for accreditation, and existing and potential EPT programmes will be reviewed and evaluated in the light of the literature.

Can novel methods replace the gold standard chimerism method after allogeneic hematopoietic stem cell transplantation?

After hematopoietic stem cell transplantation, chimerism assay is a useful approach to monitor the success of the transplant and to select the appropriate treatment strategy, such as donor leukocyte infusion or immunosuppressive drug dosage. Short tandem repeat PCR is the method that has been accepted as the gold standard for chimerism. However, it has not yet been sufficient to detect mixed chimerism in patients with minimal residual disease. Simultaneously, recent years have been marked by developing sensitive, high-throughput, and accurate molecular genetic assays. These novel methods have subsequently been adapted for the analysis of post-transplant chimerism. In this review, we discuss the technical features of both novel and conventional gold standard chimerism assays. We also discuss their advantages and disadvantages.

The correlation of results of panel reactive antibody, identification, and single antigen beads in detection of anti-HLA antibodies: Istanbul Faculty of Medicine, tissue typing laboratory experience.

BACKGROUND: We have performed a retrospective analysis of anti-HLA class I MHC and class II MHC antibodies measured using a single antigen bead (SAB) assay and a panel reactive antibody (PRA) assay. MATERIAL AND METHODS: A group of 256 patients with end-stage renal disease (ESRD) was tested for anti-HLA antibodies in the tissue typing laboratory between 2017 and 2020. In the cohort, the serum samples of patients waiting for transplantation were tested. Both the PRA and SAB tests of these patients were analyzed using the Luminex (Immucor) method. The threshold of positivity was accepted as median fluorescence intensities (MFI) ≥1000 for PRA screening and MFI ≥750 for SAB screening. RESULTS: Overall, antibodies to HLA antigens were detected in 202 (78.9%) out of 256 patients in the PRA study. Antibodies against both class I/II antigens were detected only in 15.6% of these patients, whereas antibodies against only against class I HLA in 31.3% and only against class II HLA in 32.0%. By comparison, the SAB study found that 66.8% of patients were positive for HLA antigens. Furthermore, donor-specific antibodies (DSA) were detected in 52.0% of PRA-positive patients and 52.6% of SAB-positive patients. It was shown that 168 patients (83.2%) out of 202 PRA-positive patients were found to be SAB-positive. In addition, 51 patients negative in the SAB assay (94.4%) were also negative in the PRA assay. Statistical analysis established a significant correlation between the PRA and SAB positivity (p > 0.001). It was also shown that MFI ≥3000 PRA positivity for class I HLA antigens (p = 0.049) and MFI ≥5000 PRA positivity for class II antigens (p < 0.001) correlated with the SAB positivity in patients. CONCLUSION: Our results showed the importance of both PRA and SAB assays to define the status of sensitization in patients.

COSMC expression as a predictor of remission in IgA nephropathy.

PURPOSE: The impact of core 1,3-galactosyltransferase-specific molecular chaperon (COSMC) gene expression and methylation profile on clinical progression of IgA nephropathy (IgAN) is unclear. The aim of this study was to determine the clinical significance and the relation of the COSMC gene expression and methylation pattern with the progression of IgAN. METHODS: Thirty-nine biopsy-confirmed IgAN patients, 11 healthy relatives and 20 healthy controls were recruited. The COSMC mRNA levels and methylation profile of COSMC gene promoter were measured using the quantitative real-time PCR. The galactose-deficient IgA1 (Gd-IgA1) levels were measured using ELISA in serum and cell culture supernatant. The effect of IL-4 and AZA on COSMC expression and methylation and the correlation of COSMC gene expression and methylation levels with baseline kidney function tests, histology and long-term outcomes were examined. RESULTS: The mean COSMC mRNA level was significantly lower, and serum Gd-IgA1 level was higher in IgAN patients compared with the control groups (p < 0.001, and p = < 0.001, respectively). The COSMC mRNA levels were correlated with intensity of hematuria (r = - 0.41, p = 0.009), serum creatinine level (r = - 0.37, p = 0.002) and eGFR (r = 0.36, p = 0.002). The COSMC methylation levels were correlated with age (r = 0.25, p = 0.04) and baseline eGFR (r = - 0.326, p = 0.006). Twenty IgAN patients (51.3%) reached to complete (5, 12.8%) or partial remission (15, 38.5%) after a median of 34.5 months (IQR, 13.75-71). In multivariable Cox regression analysis, COSMC mRNA expression (adjusted HR (aHR) 1.871, 95% CI 1.287-2.722, p = 0.001) and Oxford T score (aHR 0.355, 95% CI 0.146-0.859, p = 0.022) predicted the remission. CONCLUSION: COSMC mRNA level is a novel biomarker candidate to predict the remission in IgAN patients.

Immune modulation as a consequence of SARS-CoV-2 infection.

Erroneous immune responses in COVID-19 could have detrimental effects, which makes investigation of immune network underlying COVID-19 pathogenesis a requisite. This study aimed to investigate COVID-19 related alterations within the frame of innate and adaptive immunity. Thirty-four patients clinically diagnosed with mild, moderate and severe COVID-19 disease were enrolled in this study. Decreased ILC1 and increased ILC2 subsets were detected in mild and moderate patients compared to healthy controls. NK cell subsets and cytotoxic capacity of NK cells were decreased in severe patients. Moreover, CD3+ T cells were reduced in severe patients and a negative correlation was found between CD3+ T cells and D-dimer levels. Likewise, moderate and severe patients showed diminished CD3+CD8+ T cells. Unlike T and NK cells, plasmablast and plasma cells were elevated in patients and IgG and IgA levels were particularly increased in severe patients. Severe patients also showed elevated serum levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-8, reduced intracellular IFN-γ and increased intracellular IL-10 levels. Our findings emphasize that SARS-CoV-2 infection significantly alters immune responses and innate and acquired immunity are differentially modulated in line with the clinical severity of the disease. Elevation of IL-10 levels in NK cells and reduction of CD3+ and CD8+ T cells in severe patients might be considered as a protective response against the harmful effect of cytokine storm seen in COVID-19.

Regenerative Medicine Application of Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC) has recently generated interest in regenerative medicine. For the definition of MSC, three criteria have been proposed - plastic adherent property, specific surface antigens, and multipotent differentiation capacity. MSC exists in almost all tissues such as synovium, fat, liver, dental pulp, cord blood, Wharton's jelly, and also differentiates into osteoblast, chondrocyte, adipocyte, epithelial, and neuron cells originating from three germ layers. The use of different MSCs for regenerative therapies has been studied over the years as a promising option for treatment of tissue damages and various diseases. Here, the most frequently applied and newly developed stem cell-based techniques are designated, and recent MSC applications knowledge for regenerative medicine in the field are explained.

Recombination frequencies of human leukocyte antigen loci in hematological malignancies among Turkish population.

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (HSCT) is a treatment option with growing performance for leukaemia, aplastic anaemia and genetic disorders. The frequency of MHC (Major Histocompatibility Complex) gene locus recombination is increased at loci close to the telomeres and in the female gender. The aim of the present study is to document the recombination events by pedigree diagrams with the primary goal to determine the frequency of recombination in a different ethnic population from mostly reported studies. METHODS: Altogether 9545 allogeneic HSCT recipients and their family-based potential donors (n:36231) were included in this retrospective study. RESULTS: Recombinations were determined in 118 (F/M:50/68) out of 9545 families enrolled on the study. These were present in 40 of the patients and 78 of healthy donors. The frequency of recombinations was 0.42% and 0.22%, in patients and donors, respectively. Of the 118 recombinations, 60 were detected in A locus (13 inpatients), 14 in B locus (3 inpatients) and 42 in DR locus (22 inpatients). In our study, due to recombinations in HLA (Human Leukocyte Antigen)-A,-B,-DR loci, we found that some patient-donor pairs became 6/5 matched instead of 6/6 (n:45), eliminating the allogeneic HSCT possibility for the patients from the full-matched siblings. CONCLUSION: To our knowledge, this is the first study reporting the recombination frequencies in HLA loci among Turkish population and thus, providing informative data to the clinicians regarding the cross-over possibilities in Turkish patients with haematological malignancies.

Frequency of RHD variants in serologically weak D Turkish blood donors.

BACKGROUND: RhD typing has remained of primary importance, as being the leading cause of hemolytic disease of the newborn. Among Rh system's 55 blood group antigens, RhD is the most immunogenic. We aimed with this study to determine weak D/partial D variant frequency in blood donors who were admitted to our blood center and have serologically designated blood group weak D. MATERIALS AND METHODS: We screened blood donors who admitted between 2011 and 2017 to our blood center. Sixty-seven serologically weak D phenotyped donors have participated in the study. These donors' samples were studied further by Polymerase Chain Reaction Sequence- Specific Primers (PCR-SSP) for determining D variants. RESULTS: Weak D phenotype was detected in 228(0.12 %) out of 177,554 donors. Sixty-seven of them agreed to take part in the study. The frequency of weak D and partial D was 68.7 % (n = 46), and 22.4 % (n = 15), in order. The most encountered weak D and partial D variant was type 15 and DFR type, respectively. CONCLUSIONS: The prevalence of serologically weak D phenotypes varies by race and ethnicity. Turkey is a country covering a mixture of European and Asian DNA with different ethnic groups. Thus, our research as giving the overall distribution of RHD variants from the largest city of Turkey, which may reflect the general ethnic background of the country, would help to the establishment of a databank for blood banking. This paper is the first molecular study on RHD variants in Turkey. New molecular research would be more reliable and precise.

Can Direct-Acting Antiviral Treatment Change the Immunologic Risk Profile in Patients Infected with Hepatitis C Virus Who Are on the Cadaveric Waiting List?

BACKGROUND: In patients with hepatitis C virus (HCV) infection, the activation of the immune system by the virus or viral proteins leads to the production of numerous autoantibodies and clinical manifestations. The objectives of this study were to investigate the relationship between HCV and anti-HLA antibodies, as well as the effect of viremia on the antibody response and of direct-acting antivirals (DAAs) on anti-HLA antibody persistence in patients on the waiting list for a cadaveric kidney transplant. METHODS: A total of 395 patients from the cadaveric renal transplant waiting list were included in the study. The patients were grouped according to the presence of HCV infection, and patients with HCV positivity were further divided into a spontaneous clearance group and a persistent group. Anti-HLA antibodies were examined before and after treatment of the patients in the persistent group. The One Lambda Luminex method (Thermo Fisher Scientific, Waltham, MA, United States) was used to assess both HLA class I and II alleles and the anti-HLA antibody profile. RESULTS: Anti-HLA class I and II antibodies were detected in 48.2% and 55.1%, respectively, of the patients infected with HCV and in 21.8% and 20.4%, respectively, of the patients who were not infected. The level of anti-HLA A3, A11, B72, B52, Cw6, Cw16, DR3, and DQ4 antibodies was significantly higher in the patients infected with HCV. There was no statistically significant difference in class I and II antibody titration between the HCV-infected spontaneous clearance group and the persistent group (class I mean fluorescence intensity [MFI] ± SD: 13,583 ± 6224, 13,450 ± 9540, P = .808; Class II MFI ± SD: 13,000 ± 8673, 8440 ± 8302, P = .317, respectively). There was no significant difference in the class I and class II anti-HLA antibody profile and titration in the persistent group after treatment with DAAs (P > .05). CONCLUSIONS: The results of this study demonstrated that hepatitis C DAA treatment did not change the anti-HLA antibody profile and titration.

EFI region 8 Balkan external proficiency testing for HLA typing: Results of 15 years.

Istanbul Medical Faculty, Department of Medical Biology, as the first EFI-accredited HLA laboratory in Turkey since 1999 has been organizing both national and international quality control tests for HLA typing under the banner of the "Balkan external proficiency testing (BEPT)" encompassing countries from EFI regions 8. The first round of BEPT in 2004 was organized for low-resolution HLA-A,-B typing by molecular methods and 12 centres participated in the exercise. In 2005, low-resolution HLA-DR typing was added, and in 2007, low-resolution HLA-C and DQ typing were added to the exercise and 28 centres participated. In 2015, high-resolution (four digits or higher typing) HLA-A,-B,-C,-DR and -DQ typing added to the exercise and 40 centres participated. In the last 2 years, 2017 and 2018, the number of participating centres increased to 48 and 52, respectively. When the distribution of low-resolution typing methods applied in the exercises were investigated, it was found that 82% of the centres in the first round of BEPT in 2004 used PCR-SSP, whereas in the last round in 2018, 26% of the centres preferred SSP, while the rest used SSO (50%) or SSP and SSO (24%) methods. Methods for high-resolution typing were SBT (41%), NGS (18%), SSO (18%), SSP (6%), SBT + NGS (6%) and SBT + SSP (11%). In 15th trial for BEPT, nomenclature mistake rate was 12%, and erroneous result rate was 6% for HLA samples. The most common mistakes in the exercises were nomenclature mistakes. The EFI Standards Version 7.0 is stated "HLA type can be reported using a hyphen if homozygosity is not proven by family studies." In order to provide standard results among HLA laboratories and since accurate HLA typing leads to significant increase of graft and patient survival, quality control exercises should be performed in all HLA-based tests periodically.

Infectious Complications of Induction Therapies in Kidney Transplantation.

BACKGROUND Cytomegalovirus (CMV) and BK virus (BKV) are post-transplant opportunistic viral infections that affect patient and graft survival. This study was designed to evaluate the risk of BKV nephropathy and CMV disease in kidney transplant recipients who received induction therapy with ATG or basiliximab. MATERIAL AND METHODS We retrospectively analyzed information on 257 adult patients who underwent kidney transplantation between January 2007 and 2017. Patients were categorized into 3 groups according to the induction therapies. The primary endpoint was the onset of CMV disease or biopsy-confirmed BKV nephropathy. The secondary endpoints were biopsy-proven rejection episodes, graft loss, loss to follow-up, and death. RESULTS We followed 257 patients for a median of 55.5 months. The incidence of CMV disease was significantly higher in the only ATG group compared to the group without induction treatment (p<0.001). There was no significant difference in the incidence of BKV nephropathy among groups (p>0.05). The dosage of ATG (OR, 10.685; 95% CI, 1.343 5 to 85.009; P=0.025) was independent risk factor for death. CONCLUSIONS This study demonstrated that a higher dosage of ATG in high-risk patients is associated with an increased risk of CMV disease and patient death, also, reducing the dosage may be a rational strategy for increasing graft and patient's survival.

Isolated extramedullary cutaneous relapse despite concomitant severe graft-vs.-host disease and tissue chimerism analysis in a patient with acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation: A case report.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with acute lymphoblastic leukemia (ALL). The curative potential of allo-HSCT for ALL is, in part, due to the graft-vs.-leukemia (GVL) effect, in addition to the intensive conditioning chemo-radiotherapy. However, relapse remains the major cause of treatment failure following allo-HSCT for ALL. In the allo-HSCT setting, testing for genetic markers of hematopoietic chimerism has become a part of the routine diagnostic program. Routine chimerism analysis is usually performed in peripheral blood or bone marrow; in fact, little is known about the value of tissue chimerism in patients with extramedullary relapse (EMR) after the allo-HSCT setting. The present study reports on, a case of a patient with ALL who experienced isolated cutaneous EMR despite ongoing graft-vs.-host disease (GVHD), and the results of peripheral blood and skin tissue chimerism studies using multiplex polymerase chain reaction (PCR) of short tandem repeats (STR-PCR). The present case demonstrates that, although complete remission and/or chimerism may be achieved in the bone marrow, chimerism achieved at the tissue level, and the subsequent GVL effect, may be limited, despite concomitant severe GVHD following allo-HSCT. Our tissue chimerism analysis results provide a good example of how skin tissue may be a 'sanctuary' site for effector cells of GVL, despite active GVHD and complete hematopoetic chimerism.

Do the aryl hydrocarbon receptor interacting protein variants (Q228K and Q307R) play a role in patients with familial and sporadic hormone-secreting pituitary adenomas?

AIM: The aim of this study was to examine mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene by sequence analysis of exons 1-6 using leukocyte genomic DNA obtained from a cohort of familial isolated pituitary adenoma (FIPA) and apparent sporadic functional pituitary adenoma Turkish patients. METHODS: Fourteen FIPA and 90 sporadic pituitary adenoma (somatotrophinoma, prolactinoma, and corticotrophinoma) patients, 1 sporadic gigantism case, and 70 healthy controls were included in the study. RESULTS: We did not detect AIP mutations in patients with FIPAs or sporadic pituitary adenomas, including the gigantism case. Only two exonic homozygous missense single-nucleotide polymorphisms (rs641081 [Q228K] and rs4930195 [Q307R]) were identified in the AIP locus. Minor allele frequencies of the Q307R and Q228K variants were significantly higher in FIPA patients compared to controls. In addition, the minor allele frequency of the Q228K variant was significantly increased in patients with sporadic somatotrophinomas compared to controls, whereas the minor allele frequency of the Q307R variant was significantly increased in corticotrophinoma patients compared to controls. Conversely, the minor allele frequencies of Q228R and Q307R variants were similar between patients with prolactinomas and controls. No AIP gene mutation or variant was observed in the sporadic gigantism patient. These results suggest that Q228K and Q307R variants in the AIP gene might be involved in the genetic susceptibility to familial and sporadic pituitary adenomas (somatotrophinoma and corticotrophinoma) in the Turkish population.

Prognostic importance of single-nucleotide polymorphisms in IL-6, IL-10, TGF-ß1, IFN-γ, and TNF-α genes in chronic phase chronic myeloid leukemia.

The aim of this study was to explore the association between polymorphisms of five cytokine genes and clinical parameters in patients with Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) treated with imatinib. We analyzed five cytokine genes (interleukin [IL]-6, IL-10, gamma interferon [IFN-γ], transforming growth factor beta-1 [TGF-ß1], and tumor necrosis factor-alpha [TNF-α]) in 60 cases with Ph+ CML and 74 healthy controls. Cytokine genotyping was performed by the polymerase chain reaction-sequence-specific primer. All data were analyzed using the de Finetti program and SPSS version 14.0 for Windows. No significant differences were detected between the CML group and healthy controls with respect to the distributions and numbers of genotypes and alleles in TNF-α, TGF-ß1, IL-10, and IFN-γ. However, the GG genotype associated with high expression in IL-6 was found to be significantly more frequent in CML as compared to controls (p=0.010). The median follow-up time was 49.3 months (range 6.1-168.4) and the median duration of imatinib treatment was 39.5 months (range 5.2-103.4) for these patients. On multivariateanalysis, only IL-10 GCC/GCC highly produced haplotypes were significantly associated with a shorter event-free survival. The relationship between cytokine genotypes/haplotypes and clinical parameters in CML has not been investigated before. Our results suggest that IL-10 may be a useful marker for CML prognosis and theGG genotype of the IL-6 gene may be associated with susceptibility.

Association of HLA phenotypes of end-stage renal disease patients preparing for first transplantation with anti-HLA antibody status.

Patients with pre-transplantation high levels of panel reactive antibody (PRA) have an increased risk of graft failure, and renal transplantation in sensitized patients remains a highly significant challenge worldwide. The influence of anti-human leukocyte antigen (HLA) antibodies on the development of rejection episodes depends on patient-specific clinical factors and differs from patient to patient. The HLA typing of the recipient might influence the development of anti-HLA antibodies. Some HLA antigens appear to be more immunogenic than others. The aim of this study is to demonstrate the distribution of HLA phenotypes in PRA-positive and PRA-negative end-stage renal disease (ESRD) patients on the basis of having sensitizing events or not. Our study included 642 (mean age: 41.54; female/male: 310/332) ESRD patients preparing for the first transplantation and who are on the cadaveric kidney transplantation waiting list of Istanbul Medical Faculty in 2008-2009. Class I HLA-A,B typing was performed by complement-dependent cytotoxicity (CDC) method, whereas class II HLA-DRB1 typing was performed by low-resolution polymerase chain reaction (PCR)-sequence-specific primer (SSP). All serum samples were screened for the presence of IgG type of anti-HLA class I- and II-specific antibodies by enzyme-linked-immunosorbent assay (ELISA). PRA-negative group consisted of 558 (86.9%) and PRA-positive group included 84 (13.1%) patients. We have found statistically significant frequency of HLA-A3 (p=0.018), HLA-A66 (p=0.04), and HLA-B18 (p=0.006) antigens in PRA-positive patients and DRB1*07 (p=0.02) having the highest frequency in patients with sensitizing event history but no anti-HLA development suggesting that DRB1*07 might be associated with low risk of anti-HLA antibody formation.

Impact of HLA on the underlying primary diseases in Turkish patients with end-stage renal disease.

The number of patients with end stage renal disease (ESRD) is increasing faster than the number of renal transplantations performed per year worldwide. Of the primary diseases leading to ESRD, diabetic nephropathy is the leading cause. The purpose of the present study is to investigate the association of HLA with the primary diseases leading to ESRD in Turkish patients. A total of 3230 individuals comprising 587 ESRD patients and 2643 healthy controls were enrolled into the study. Class I HLA-A, -B typing was performed by CDC method, while class II HLA-DRB1 typing was performed by low resolution PCR-SSP. We found a significant negative association between almost all A locus antigens and primary disease groups classified as chronic glomerulonephritis and hypertensive nephrosclerosis (p < 0.05). HLA-B58 and HLA-DRB1*03 significantly correlated with amyloidosis and diabetic nephropathy, respectively. Determination of HLAs as risk factors for primary diseases leading to ESRD might be beneficial in preventing progression to ESRD and recurrence of the primary disease post-transplantation.

Tissue chimerism analysis after donor lymphocyte infusion: a case of deceptive evidence.

A 31-year-old female with refractory extramedullary myeloid leukemia relapse underwent peripheral blood stem-cell transplantation from her HLA-identical brother. Hematopoietic recovery followed disappearance of the lesions. Studies three-months post-transplant showed complete chimerism (CC). Fourteen months after transplantation, the patient presented with an increasing number of extramedullary sites of biopsy-proven disease relapse (as nodular skin lesions). Bone marrow was in remission with maintenance of CC. However, tissue chimerism analysis showed full recipient-cell population. After one course of conventional dose chemotherapy followed by mobilized donor-lymphocyte infusions (DLI), a complete response was achieved. DLI continued monthly but she developed new skin lesions accompanied by multiple cervical masses. Bone marrow and tissue chimerism revealed both recipient and donor cell population. We conclude that tissue chimerism analysis after DLI may not accurately document the cell origin.

HLA B-27 subtypes in Turkish patients with spondyloarthropathy and healthy controls.

The frequency and the distribution of HLA-B27 subtypes in spondylarthropathy (SpA) patients and controls were investigated in a sample Turkish population. B27 subtyping was performed by PCR-SSP method in two groups: 49 unrelated HLA-B27 positive Turkish patients with the diagnosis of SpA according to the European Spondyloarthropathy Study Group Criteria, and 55 HLA-B27 positive healthy controls. The frequency of HLA-B*27 was 2.6% in the Turkish population, and B*2705 was the predominant allele among patients with SpA. The difference was mainly between male patients and male controls The proportion of B*2705 among B27-positive patients and controls was significantly different (P=0.02). Our study supports other reports from different populations which showed that B*2705 and B*2702 were more frequent in Caucasian patients with SpA.