Comparison of Single Antigen Bead-Based, Cell-Based, and Solid Phase-Based Crossmatch Methods.

AIM: Antibody assessment during pretransplantation term is important to detect donor specific antibodies. These donor-specific antibodies are determined by various crossmatch methods (flow cytometric [FCXM], complement-dependent cytotoxic [CDCXM], and Luminex [LMXM]). Recently, single antigen bead (SAB) assays have been used for the assessment of hypersensitized patients. The aim of this study was to compare sensitivity and specificity of the 3 crossmatch methods in reference to the SAB method. METHOD: In this study, 69 hypersensitized patients with high class I and/or class II panel reactive antibodies were tested using the flow cytometric SAB method. Serum samples were cross-matched by 3 crossmatch methods with the cells of a volunteer healthy individual, and the results were evaluated according to HLA and cross-reactive epitope groups (CREGs). RESULTS: Sensitivity was found to be better with T FCXM (0.91) and class I LMXM (0.87). Specificity of peripheral blood lymphocyte CDCXM method (1.0) was found to be better than the other 2 methods (0.33 and 0.57, respectively). Sensitivity of class II LMXM (0.88) was found to be better than the others (0.42 for B CDCXM and 0.82 for B FCXM, respectively). The specificity of the B CDCXM, B FCXM, and class II LMXM was similar (0.44, 0.44, and 0.33, respectively). CREGs results were similar to HLA results. CONCLUSION: Although CDCXM has high specificity for the detection of anti-HLA antibodies, it has low sensitivity. To increase sensitivity, FCXM or LMXM methods may be used with the CDCXM test. These results will be beneficial for laboratories and clinicians during graft survival and patient health assessment.

Investigation of Anti-HLA Antibodies of Highly Sensitized Patients by Single Antigen Bead and C1q Tests.

PURPOSE: The single antigen bead (SAB) test contributes to conventional cellular and solid phase crossmatch tests in renal transplantation. However, the determination of anti-HLA antibodies of the patients may not reflect the pathologic features of these antibodies. Highly sensitized patients produce antibodies against a number of HLAs; therefore, their transplantation chance decreases. In this study, we aimed to evaluate SAB and C1q test results of highly sensitized patients. METHOD: In this study, 33 end-stage renal failure patients with >80% panel reactive antibody were included. Of the patients, 58% (n = 19) were female, and 42% (n = 14) were male. The mean age was 46.2 ± 12.4. All of the serum samples were inactivated by heat before use. SAB and C1q tests were performed according to the manufacturer's instructions. RESULTS: We obtained statistically significant results between the positive bead counts and raw mean fluorescence intensity (MFI) values of 2 tests (P < .01 for class I and II). There was a statistically significant difference between the 2 tests in terms HLA-A, -C, -DR, and -DP MFI values, whereas HLA-B and -DQ MFI values were similar for the 2 tests. CONCLUSION: The difference of raw MFI values between the 2 tests may be due to the fact that the C1q test detects only IgG1 and IgG3 antibodies, whereas the SAB test can detect all IgG subtypes. We considered that anti-HLA-B and -DQ antibodies have high complement-fixing features; these antibodies should be investigated selectively due to the similarity of anti-HLA-B and -DQ antibody MFI values in the 2 tests.

A Comparative Study: Flow Cytometry, Complement-dependent Cytotoxicity, and Luminex Methods.

AIM: The cell-based flow cytometric and bead-based Luminex crossmatch methods have been used alongside the standard complement-dependent cytotoxic crossmatch (CDCXM) test to detect donor specific anti-HLA antibodies. In this study, it was aimed to compare flow cytometric crossmatch (FCXM), CDCXM, and Luminex donor-specific crossmatch (LM-XM) tests for pre-transplant assessment of patients who applied to Tepecik Education and Research Hospital for kidney transplantation from related or deceased donors. METHOD: HLA tissue typing of 1120 patients were tested using the sequence specific oligonucleotide probe method with low resolution. FCXM and LM-XM were performed according to the manufacturer's instructions. The CDCXM test was performed according to the standard procedure. The results were analyzed using SPSS version 21.0 software (IBM, Armonk, NY, United States). P < .05 was accepted as statistically significant. RESULTS: FCXM, CDCXM, and LM-XM tests were performed on 58.2% (n = 652), 91% (n = 1019), and 55.4% (n = 620) of 1120 patients. There were statistically significant differences between FCXM/CDCXM, LM-XM/CDCXM, and FCXM/LM-XM (P < .0001), although there was also a moderate correlation between them (for class I, r = .599, r = .693, and r = .507; for class II, r = .546, r = .471, and r = .495, respectively). The results obtained according to donor type were compatible with the total study group. CONCLUSION: The utilization of FCXM and/or LM-XM tests together with the CDCXM method before kidney transplantations from related and/or deceased donor may facilitate the determination of target cells of donor-specific antibodies or their antibody class, which may increase the success of transplantation.

Assessment of Interleukin 2 Cytokine Expression Levels After Renal Transplantation.

AIM: End-stage renal disease is a disease in which the kidney is not able to perform its functions. Kidney transplantation is the most effective treatment and cost-effective modality of renal replacement therapy for patients with end-stage renal disease. However, the most important problem in end-stage renal disease patients is the unpredictability of immunologic response after transplants. In this study, it was aimed to investigate the possible association between the interleukin 2 (IL-2) expression level and an organ rejection or rejection episode. MATERIALS AND METHODS: Lymphocytes were isolated from peripheral blood obtained from 21 end-stage renal disease-diagnosed patients prior to transplant and at the sixth month after transplant. CD4+ T cells were separated from lymphocytes by the magnetic cell-sorting method. The purity of these cells were controlled by a flow cytometer. After total RNA isolation from CD4+ T cells, IL-2 was examined by the real-time polymerase chain reaction (RT-PCR) method. RESULTS: Among nonrejection patients (n = 18), the IL-2 expression level decreased in 12 patients in post-transplant time, and 3 of these were statistically significant (P < .05). The level was the same in 1 of 18 patients; it increased in 5 patients, and 1 of them was significant (P < .05). The IL-2 expression level also increased in 3 patients who had a rejection episode, and the increase was statistically significant in 2 samples (P < .05). CONCLUSION: When the patients were evaluated individually, it was observed that there might be a relationship between IL-2 expression levels in CD4+ T cells and rejection episodes. The clinical data of the patients, the immunosuppressive therapies, and post-transplant evaluation of cytokines should be considered together.

Comparison of Anti-HLA Antibody Production According to Gestational Periods in Pregnant Women.

BACKGROUND: To evaluate paternal anti-HLA antibody profiles, sera samples were collected from pregnant women in different trimesters and the panel-reactive antibody (PRA) specificities were identified. METHODS: From 2013 to 2015, serum samples were obtained from 41 pregnant women who had registered at the Izmir Tepecik Education and Research Hospital Gynecology Clinic. Anti-HLA antibodies were screened by using the panel reactive antibody screening and identification tests. Sera samples were obtained at the first, second, and third trimesters. The primary outcome was to determine the anti-HLA antibody production term during pregnancy; the secondary outcome was identification of anti-HLA antibodies. RESULTS: None of the women had a sensitization history except during pregnancy. We observed that 54% of the women produced paternal antibodies, either class I or II. Class I PRA positivity of the women who had a first or second pregnancy was the same in all 3 trimesters, whereas class II positivity was increased in the third trimester. Class II and both class I and II positivity increased in the third trimester; class I positivity was decreased in the third trimester. PRA positivity could be affected by the history of pregnancy and could be raised, but no impact was observed from the history of abortion and miscarriage (odds ratios, 1.9, 0.4, and 0.5 [95% confidence intervals, 0.5-7.8, 0.1-2.0, and 0.3-0.7], respectively; P > .05). The most frequently detected antibodies were A2, B7, DR7, DR4, DR11, DR13, DQ2, and DQ8. CONCLUSIONS: Anti-HLA antibodies against paternal HLA antigens were detected more in multiparous women than in primiparous women. Anti-HLA antibody detection ratios did not change until the third trimester and were followed by a specific increase in class II anti-HLA antibody production.

Evaluation of epithelial chimerism after bone marrow mesenchymal stromal cell infusion in intestinal transplant patients.

Intestinal transplantation is the most effective treatment for patients with short bowel syndrome and small bowel insufficiencies. We evaluated epithelial chimerism after infusion of autologous bone marrow mesenchymal stromal cells (BMSCs) in patients undergoing cadaveric donor isolated intestinal transplantation (I-ITx). BMSCs were isolated from patients' bone marrow via iliac puncture and expanded in vitro prior to infusion. Two out of the 3 patients were infused with autologous BMSCs, and small intestine tissue biopsies collected post-operatively were analyzed for epithelial chimerism using XY fluorescent in situ hybridization and short tandem repeat polymerase chain reaction. We observed epithelial chimeric effect in conditions both with and without BMSC infusion. Although our results suggest a higher epithelial chimerism effect with autologous BMSC infusion in I-ITx, the measurements in multiple biopsies at different time points that demonstrate the reproducibility of this finding and its stability or changes in the level over time would be beneficial. These approaches may have potential implications for improved graft survival, lower immunosuppressant doses, superior engraftment of the transplanted tissue, and higher success rates in I-ITx.

Comparision of anti-HLA antibodies of kidney transplant candidates with chronic renal failure by two different methods: Flow-PRA and Luminex PRA.

OBJECTIVE: The aim of the study was to compare anti-HLA antibodies examined as panel-reactive antibody (PRA) in kidney transplant candidates with chronic renal failure (CRF) with the use of 2 methods: Flow-PRA and Luminex-PRA. METHODS: CRF patients displaying class I PRA (n = 34) and/or class II PRA (n = 41) were tested by the 2 different methods from April 2012 to September 2012, using antigen-coated beads. RESULTS: Eleven (32.3%) 34 patients tested for class I PRA were female and 23 (67.7%) male; 17 (41.5%) 41 patients tested for class II PRA were female and 24 (58.5%) male. Only 2 patients were preemptive, the others had been subjected to dialysis. The concordance ratio of class I PRA test results between Flow-PRA and Luminex PRA was 67.6%. Whereas 13 samples (38.2%) were positive by Flow-PRA, 22 (64.7%) were positive by Luminex-PRA. Two of the 3 patients not previously immunized were found to be positive only by Luminex PRA; 1 was noted to be positive only by Flow-PRA. Regarding class II PRA screening, the concordance between Flow-PRA and Luminex PRA was 70.7%. Whereas 14 (34.1%) samples were positive for Flow-PRA; 24 (58.5%) were positive for Luminex-PRA. The 2 patients not previously immunized were positive only in Luminex PRA. CONCLUSIONS: We speculated that the reason for the low concordance ratios was due to the use of sera that had been previously found to be indeterminate in PRA tests. We also speculated that the low concordance ratios were due to the coating procedure for the beads, which may cause changes in antigenic epitopes and decrease concordance between Flow-PRA and Luminex-PRA.

Comparison of complement-dependent cytotoxic and flow-cytometry crossmatch results before cadaveric kidney transplantation.

AIM: The presence of HLA donor-specific antibodies (DSA) before kidney transplantation decreases graft survival. In this study, we compared crossmatch results of kidney transplantation candidates, for cadaveric renal donation between March 10, 2012, and September 7, 2012. MATERIAL AND METHOD: The 47 kidney transplantation candidates tested for crossmatch included 10 for cadaveric donor organs. Two crossmatch methods were performed: complement-dependent cytotoxic crossmatch (CDCXM) and flow cytometry crossmatch (FCXM). Spleen cells were used as the source of lymphocytes for all crossmatch tests. RESULTS: The T and B cell ratios isolated from spleen were 38.8% and 34.8%, respectively. The concordance ratio of the two methods was 76.6% with 23.4% discordant results. Regarding the discordant results, 4.2% were positive CDCXM but negative FCXM; 191%, negative CDCXM but positive FCXM. All patients displaying positive crossmatches had a previous immunization history. As a result, we speculated that the positive CDCXM but negative FCXM results were due to the washing procedures in the FCXM disturbing antigen-antibody complexes. We suggest at least two different methods to be performed for crossmatch tests before kidney transplantation. CDCXM detects immunoglobulin G1 (IgG1) and IgG3, which are critical for rejection. FCXM is able to detect all IgG subgroups because of its high sensivity. As a result we suggest that both CDCXM and FCXM are preferrable strategies to detect DSAs.

Genetic alterations in gastric precancerous lesions.

To investigate the occurrence of 17p (p53) loss of heterozygosity (LOH) and increased 4N or aneuploidy in gastric precancerous lesions (GPL), and their association with Helicobacter pylori (H pylori) infection. A total of 78 gastric mucosal biopsy specimens, including 10 normal mucosa and 68 gastric precancerous lesions [chronic atrophic gastritis (CAG, n = 20), intestinal metaplasia (IM, n = 12), low grade dysplasia (LGD, n = 15), and high grade dysplasia (HGD, n = 21)] were studied using PCR and flow cytometry. A modified Giemsa staining technique was used to detect H pylori. The study was performed in Erzurum Numune Hospital between 2007 and 2009. 17p (p53) LOH was observed in (1/20) 5% of CAG, in (2/12) 16% of IM, in (3/15) 20% of LGD and in (11/21) 53% of HGD. There was correlation between prevalence of 17p (p53) LOH and histological type of GPL (P = 0.004). Similarly, increased 4N or aneuploidy was detected in (1/20) 5% of CAG, in (1/12) 8% of IM, in (2/15) 13% of LGD and in (9/21) 43% of HGD. The correlation was found between aneuploidy and histological type of GPL (P = 0.009). However, there was no correlation between presence of H pylori infection in histological type of GPL (P = 0.921). On the other hand, a significant association was found between increased 4N or aneuploidy and 17p (p53) LOH in all of GPL (P = 0.0001). However, there was no statistically significant association between H pylori infection and 17p (p53) LOH or increased 4N/aneuplody in GPL. 17p (p53) LOH and increased 4N or aneuploidy are closely related to the early stages of gastric carcinogenesis.

The alteration of sister chromatid exchange frequencies in Behçet's disease with and without HLA-B51.

BACKGROUND: The analysis of sister chromatid exchange (SCE) is a cytogenetic technique used to show DNA damage as a result of an exchange of DNA fragments between sister chromatids. It is known that there is an increased SCE frequency in Behçet's disease (BD). OBJECTIVE: To investigate whether human leucocyte antigen (HLA)-B51-positive patients with Behçet's disease exhibit higher SCE frequencies than those without HLA-B51. METHODS: Lymphocytes from 75 patients (38 women, 37 men) and from 50 controls (28 women, 22 men) were cultured in darkness for 72 h in the presence of bromodeoxyuridine. Metaphase chromosomes were stained with a fluorescence plus Giemsa technique after a standard harvest procedure. For HLA-B51 typing, DNA was extracted from ethylenediaminetetraacetic acid blood samples and HLA-B5 allele genotyping was performed by the polymerase chain reaction (PCR)-sequence specific primer method. RESULTS: Thirty-nine of 75 patients with BD (52%) and 15 of 50 controls (30%) were found HLA-B51-positive. The SCE frequencies in HLA-B51-positive patients were higher than in HLA-B51-negative ones (P < 0.001), whereas no difference was detected in the control group. CONCLUSION: This study revealed that there was a significant association between elevated SCE frequencies and existence of HLA-B51 patients with BD.

HLA class I and class II genotyping in patients with Behcet's disease: a regional study of eastern part of Turkey.

Class I human leucocyte antigen (HLA)-B51 is well known to be associated with Behcet's disease in many ethnic groups. However, there has been no published paper with respect to its association with HLA class I and class II among the Turkish people who live in the eastern region of Turkey. Moreover, as it is known that B51 antigen is encoded by 21 alleles, B*5101-5121, HLA-B51 allele typing was performed, as well as HLA class I and class II genotyping of 75 patients with the disease and the 54 individuals in the matched control group. The result shows that the HLA-B51 frequency was significantly higher (58.66%) in the patient group, compared to that in the control group (18.51%) (OR = 6.245). In the subtyping of B51 alleles, 44 B51-positive patients possessed B*5101 (45.5%), B*5108 (25%), B*5105 (9.1%) and B*5104 (4.5%). There was no significant difference in the HLA-B51 allelic distribution between the patient group and the control group. However, homozygous carriers of HLA-B51 showed considerably high risk (OR = 2.647) in the patient group, compared to that in the control group. In the genotyping of class II HLA alleles, while HLA-DRB1*04 (45.3%) and HLA-DRB1*07 (24%) were the predominant alleles in the patient group, DRB1*11 (50%) appeared to be more common in the control group.

Association of sister chromatid exchange frequencies in patients with ankylosing spondylitis with and without HLA-B27.

BACKGROUND: The analysis of sister chromatid exchange (SCE) is a cytogenetic technique used to show DNA damage due to an exchange of DNA fragments between sister chromatids. OBJECTIVE: To determine whether HLA-B27 positive patients with ankylosing spondylitis (AS) were associated with higher SCE frequencies than patients without B27. METHODS: Lymphocytes from 38 patients with AS (15 women, 23 men) and 34 control subjects were examined. Peripheral lymphocytes were cultured in darkness for 72 hours in BrdU added culture. Metaphase chromosomes were stained with a fluorescence and a Giemsa stain after a standard harvest procedure. RESULTS: The frequency of SCE was significantly increased in patients with AS compared with controls (p<0.001). Furthermore, the SCE frequencies in patients with positive HLA-B27 was much higher than in patients with negative HLA-B27 (p<0.001). The difference between SCE frequencies in the control groups with and without HLA-B27 was not significant. CONCLUSION: There is a strong association between HLA-B27 and the frequencies of SCE in patients with AS.

Relationship between electroneurographic changes and serum ubiquitin levels in patients with type 2 diabetes.

OBJECTIVE: The aim of the present study was to investigate any relationship between serum ubiquitin levels and electroneurographic changes in peripheral nerves for patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: The study involved 34 patients (19 men, 15 women; mean age 46 +/- 13 years) with type 2 diabetes. Serum ubiquitin values were measured by sandwich enzyme-linked immunosorbent assay Measurement of nerve conduction velocity (NCV) was performed on three motor (median, tibial, and peroneal) and three sensory (median, ulnar, and sural) nerves. The value of motor compound muscle action potential (CMAP) was obtained from the sum of median, tibial, and peroneal motor nerve amplitudes, and sensory compound nerve action potential (CNAP) was computed as the sum of median and ulnar sensory nerve amplitudes. RESULTS: Patients with diabetes were divided into three groups: group 1 (n = 8) had normal electroneurography results, group 2 (n = 8) had slowed NCV, and group 3 (n = 18) had low values of motor CMAP and/or sensory CNAP as well as slowed NCV. Mean ubiquitin level in group 3 (20.4 +/- 2.9 ng/dl) was significantly higher than that in group 1 (11.2 +/- 1.1 ng/dl, t = 11.5, P < 0.0001) and group 2 (13.2 +/- 2.7 ng/dl, t = 5.9, P < 0.0001). Serum ubiquitin levels were inversely correlated with motor CMAP (r = -0.68) and sensory CNAP (r = -0.61) values. CONCLUSIONS: The results of this study indicate that there could be a relationship between the diminished amplitudes of axons of the peripheral nerve and the increase in serum ubiquitin levels in patients with type 2 diabetes. Further studies are required to confirm this relationship.

Altered cell-matrix associated ADAM proteins in Alzheimer disease.

Alterations in cell-matrix 'contact' are often related to a disruption of cell cycle regulation and, as such, occur variously in neoplasia. Given the recent findings showing cell cycle alterations in Alzheimer disease, we undertook a study of ADAM-1 and 2 (A Disintegrin And Metalloprotease), developmentally-regulated, integrin-binding, membrane-bound metalloproteases. Our results show that whereas ADAM-1 and 2 are found in susceptible hippocampal neurons in Alzheimer disease, these proteins were not generally increased in similar neuronal populations in younger or age-matched controls except in association with age-related neurofibrillary alterations. This increase in both ADAM-1 and 2 in cases of Alzheimer disease was verified by immunoblot analysis (P < 0.05). An ADAM-induced loss of matrix integration would effectively "reset" the mitotic clock and thereby stimulate re-entry into the cell cycle in neurons in Alzheimer disease. Furthermore, given the importance of integrins in maintaining short-term memory, alterations in ADAM proteins or their proteolytic activity could also play a proximal role in the clinico-pathological manifestations of Alzheimer disease.

Production of anti-polyubiquitin and anti-ubiquitin carboxyl terminal hydrolase antibodies and immunohistochemically assessment of them on brain sections of Alzheimer's disease and Lewy body disease.

Several ubiquitin (ub) moieties are lined up head to tail by function of class III genes which code for polyubiquitin proteins. Ubiquitin carboxyl terminal hydrolyses (UCTH) disassemble the polyubiquitin chains. In our study we synthetically produced polyubiquitin (last six amino acids of ub is linked with first five amino acids of ub, UBI(71-76 + 1-5)) and purified anti-UCTH from human brain to produce antibodies against them. These antibodies were used on Alzheimer's and Lewy body disease brains. Anti-UBI(71-76 + 1-5) antibody stained plaque neurites of both disease brains. Anti-UCTH antibody showed reactivity with cortical Lewy bodies within the neurons and bulbous neurites of Alzheimer's disease and Lewy body disease brains.

Ischemic rat brains contain immunoreactivity of 14-3-3 proteins.

14-3-3 proteins and tissue damage association were determined. The localization of 14-3-3 proteins in section of cortex from ischemia produced rats was examined by immunohistochemistry. Ischemia was produced in rats by unilaterally clamping the carotid artery for 6 hrs at the distal side. Following recovery for 6 hrs, the animals were sacrificed. The intensity of neuronal necrosis stained with antibody raised against 14-3-3 protein was markedly more in the cortex than the other parts of the brain, since there was occlusion of the artery which feeds this region. 14-3-3 antibody was confined to neuronal cell body. Since 14-3-3 proteins are central to mitogen-activated protein (MAP) kinase signaling, this pathway is in part responsible for the hyperphosphorylation of neurofilaments or cytoskeletal proteins of neuron, which may possibly lead to neuronal inclusions.