High Circulating Levels of Neutrophil Extracellular Traps Parameters Predicting Poor Outcome in COVID-19.

OBJECTIVE: Exploration of biomarkers to predict the severity of COVID-19 is important to reduce mortality. Upon COVID-19 infection, neutrophil extracellular traps (NET) are formed, which leads to a cytokine storm and host damage. Hence, the extent of NET formation may reflect disease progression and predict mortality in COVID-19. METHODS: We measured 4 NET parameters - cell-free double stranded DNA (cell-free dsDNA), neutrophil elastase, citrullinated histone H3 (Cit-H3), and histone - DNA complex - in 188 COVID-19 patients and 20 healthy controls. Survivors (n=166) were hospitalized with or without oxygen supplementation, while non-survivors (n=22) expired during in-hospital treatment. RESULTS: Cell-free dsDNA was significantly elevated in non-survivors in comparison with survivors and controls. The survival rate of patients with high levels of cell-free dsDNA, neutrophil elastase, and Cit-H3 was significantly lower than that of patients with low levels. These three markers significantly correlated with inflammatory markers (absolute neutrophil count and C-reactive protein). CONCLUSION: Since the increase in NET parameters indicates the unfavourable course of COVID-19 infection, patients predisposed to poor outcome can be rapidly managed through risk stratification by using these NET parameters.

Association of HLA-DRB1 and -DQB1 Alleles with Susceptibility to IgA Nephropathy in Korean Patients.

BACKGROUND: Associations between IgA nephropathy (IgAN) and HLA-DRB1 and -DQB1 alleles have been reported in several ethnic groups. We investigated the association of HLA-DRB1 and -DQB1 alleles with the predisposition for IgAN and disease progression to end-stage kidney disease (ESKD) in Korean patients. METHODS: We analyzed HLA-DRB1 and -DQB1 genotypes in 399 IgAN patients between January 2000 and January 2019 using a LIFECODES sequence-specific oligonucleotide (SSO) typing kit (Immucor, Stamford, CT, USA) or a LABType SSO Typing Test (One Lambda, Canoga Park, CA, USA). Alleles with a significant difference in two-digit resolution were further analyzed using in-house sequence-based typing and sequence-specific primer PCR. As controls, 613 healthy hematopoietic stem cell donors were included. Kidney survival was analyzed in 281 IgAN patients with available clinical and laboratory data using Cox regression analysis. Where needed, P-values were adjusted using Bonferroni correction. RESULTS: The allele frequencies of HLA-DRB1*04:05 (corrected P [Pc]<0.001), -DQB1 *04:01 (Pc=0.048), and -DQB1*03:02 (Pc=0.021) were significantly higher in IgAN patients than in controls, whereas those of HLA-DRB1*07:01, -DRB1*15:01, -DQB1*02:02, and -DQB1*06:02 (Pc<0.001 for all) were significantly lower in IgAN patients than in controls. The allele frequency of HLA-DQB1*05:03 (Pc=0.016) was significantly lower in the ESKD group than in the non-ESKD group; however, there was no significant difference for ESKD progression between these groups. CONCLUSIONS: We report novel associations of HLA-DRB1*15:01, DQB1*02:02, -DQB1*03:02, and -DQB1*04:01 with IgAN. Further studies of HLA alleles associated with IgAN progression in a larger cohort and in various ethnic groups are needed.

Prevalence of a Single-Nucleotide Variant of SARS-CoV-2 in Korea and Its Impact on the Diagnostic Sensitivity of the Xpert Xpress SARS-CoV-2 Assay.

The sensitivity of molecular diagnostics could be affected by nucleotide variants in pathogen genes, and the sites affected by such variants should be monitored. We report a single-nucleotide variant (SNV) in the nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., G29179T, which impairs the diagnostic sensitivity of the Xpert Xpress SARS-CoV-2 assay (Cepheid, Sunnyvale, CA, USA). We observed significant differences between the threshold cycle (Ct) values for envelope (E) and N genes and confirmed the SNV as the cause of the differences using Sanger sequencing. This SNV, G29179T, is the most prevalent in Korea and is associated with the B.1.497 virus lineage, which is dominant in Korea. Clinical laboratories should be aware of the various SNVs in the SARS-CoV-2 genome and consider their potential effects on the diagnosis of coronavirus disease 2019.

Acceptable Donor-Specific Antibody Levels Before and After Desensitization Therapy in Living Donor Kidney Transplantation.

BACKGROUND: Plasmapheresis (PP) is commonly used for desensitization in highly sensitized patients with donor-specific antibodies (DSA) in living donor kidney transplantation. We analyzed the impact of DSA levels before and after desensitization on renal allograft outcome. METHODS: Twenty-three patients who underwent desensitization with PP, intravenous immunoglobulin (IVIG), and rituximab before kidney transplantation in Seoul National University Hospital from August 2006 to August 2016 were enrolled. The association of median fluorescent intensity (MFI) value of DSA with graft outcome was analyzed. RESULTS: The frequency of positive HLA class II DSA after desensitization was lower in patients without antibody-mediated rejection (AMR) compared to those with AMR (p = 0.006). The cutoff value of MFI sum of HLA class II DSA after desensitization for predicting AMR was 2,122 with 63% sensitivity and 94% specificity. The frequency of moderate HLA class II DSA (MFI 5,000 - 10,000) after desensitization was significantly higher in patients with graft loss compared to those without graft loss (p = 0.02). CONCLUSIONS: Weak HLA class II DSA after desensitization including PP, IVIG, and rituximab was related to AMR and moderate levels of HLA class II DSA after desensitization was related to graft loss in living donor kidney transplantation.

Association of Foxp3 Polymorphism With Allograft Outcome in Kidney Transplantation.

BACKGROUND: Forkhead box P3 (Foxp3) is the most reliable marker for regulatory T cells, which play an important role in maintaining renal allograft tolerance. Recently, Foxp3 polymorphisms have been reported to be associated with graft outcome in kidney transplantation. We analyzed the association of Foxp3 polymorphisms with renal allograft outcome. METHODS: Foxp3 polymorphisms (rs3761548 A/C, rs2280883 C/T, rs5902434 del/ATT, and rs2232365 A/G) were tested by PCR with sequence-specific primers (PCR-SSP) in 231 adult kidney transplantation recipients from 1996-2004 at Seoul National University Hospital. RESULTS: Patients with the rs3761548 CC genotype showed better graft survival than those with the AC or AA genotype (log rank test, P=0.03). Patients with the rs3761548 CC genotype also showed a lower rate of recurrence of the original glomerular disease than those with the AC or AA genotype (P=0.01). The frequency of acute rejection (AR) in patients with the rs2280883 TT genotype was lower than that in patients with the rs2280883 CT or CC genotype (26.9% vs 53.3%, P=0.038). Patients with the rs2280883 TT genotype also showed better graft survival than those with the CT or CC genotype (P=0.03). CONCLUSIONS: Foxp3 rs3761548 CC and rs2280883 TT genotypes were associated with superior graft outcome of kidney transplantation. Further studies involving a larger number of patients are needed.

Association of HLA Types with Non-Specific Binding of Negative Control Beads in Luminex Panel Reactive Antibody (PRA) Screening Assay.

BACKGROUND: Luminex panel reactive antibody (PRA) screening assays using microbeads are widely used for organ transplantation. Anti-HLA serum reactivity is calculated by correcting for non-specific binding to the negative control (NC) beads. High mean fluorescence intensity (MFI) value of NC beads are observed in some patients and can result in false negative results in the PRA screening assay. We analyzed the clinical characteristics and HLA types of those patients with high MFI values of NC beads. METHODS: Sixty-six patients with high MFI values of NC beads (> 300) in the PRA LABScreen Mixed assay (One Lambda) tested were included as the high NC group. Age and gender matched controls with low MFI values of NC beads (< 100) (n = 132), tested with PRA, were selected as the low NC group and 207 healthy Koreans were used as normal controls. Association of clinical characteristics and HLA types with the high NC group were analyzed using Chi-square test or Fischer's exact test, as appropriate. RESULTS: The proportion of patients with underlying liver disease was higher in the high NC group compared to the low NC group (18.1% vs. 1.5%, p < 0.001, OR = 14.2). The seropositivity of anti-nuclear antibody and rheumatoid factor, the frequency of use of intravenous immunoglobulin G, anti-thymocyte globulin, and rituximab showed no difference between two groups. The phenotype frequency (PF) of HLA-B46 was higher in the high NC group than in the low NC group (8.0% vs. 3.2%, p = 0.036, OR = 2.8). The PF of HLA-B7 was lower in the high NC group than in the healthy controls (0.0% vs. 6.5%, p = 0.008, OR = 0.1). The PF of HLA-DR1 was lower in the high NC group than in the low NC group (0.8% vs. 6.6%, p = 0.015, OR = 0.1) or healthy controls (0.8% vs. 7.4%, p = 0.003, Pc = 0.042, OR = 0.1). CONCLUSIONS: Increased non-specific binding to NC beads was associated with underlying liver disease and HLAB46. HLA-B7 and HLA-DR1 were related to a lower chance of non-specific binding to NC beads. The mechanism of those associations, such as differences in non-specific antibody response according to HLA phenotype or underlying disease, needs to be elucidated in further studies.

Association of aplastic anemia and FoxP3 gene polymorphisms in Koreans.

OBJECTIVES: Aplastic anemia (AA) is characterized by pancytopenia and bone marrow failure, and most acquired AA is an immune-mediated disorder. Regulatory T cells (Tregs) suppressing autoreactive T cells were decreased in AA patients. FoxP3 is a major regulator for the development and function of Tregs. Polymorphism in FoxP3 was shown to be associated with various autoimmune diseases, however, has not yet been studied in AA. In this study, we examined the association between FoxP3 polymorphisms and AA in Korean patients. METHODS: The study population consisted of 94 patients diagnosed by bone marrow examination in Seoul National University Hospital (SNUH) during 1997-2012 and 195 healthy controls. FoxP3 polymorphisms (rs5902434 del/ATT, rs3761548 C/A, rs3761549 C/T, rs2232365 A/G) were analyzed by PCR-sequencing method. We analyzed differences of genotype and allele frequencies between patients and controls. We also compared differences of genotype and allele frequencies between responder and non-responder in patients treated with immunosuppressive therapy (IST). For the statistical analysis, the chi-square test and Fisher's exact test were used and P < 0.05 was regarded as statistically significant. RESULTS: There was no significant difference in the genotype frequencies of FoxP3 polymorphisms between patients and controls. With regards to the allele frequencies, rs3761548 C allele was significantly higher in AA patients than in controls (87.4% vs. 79.7%, P = 0.047). In patients treated with IST, rs3761549 C allele was significantly higher in non-responder patients than in responders (89.6% vs. 66.7%, P = 0.036) and female rs3761549 C/C genotype carriers were associated with greater risk for non-response to IST (84.2% vs. 16.7%, P = 0.006). CONCLUSION: Polymorphisms in rs3761548 and rs3761549 of FoxP3 in our population were associated with disease susceptibility and response for IST, respectively. This study suggests an association between FoxP3 polymorphisms and AA in Korean patients and will be useful in further understanding the genetic basis of disease susceptibility and response to IST in AA patients.

Association of HLA-DP variants with the responsiveness to Hepatitis B virus vaccination in Korean Infants.

Recently, HLA-DP single nucleotide polymorphisms (SNPs) have been reported to be related to responsiveness to hepatitis B virus (HBV) vaccination. The aim of this study was to investigate associations between HLA-DP SNPs and responsiveness to HBV vaccine in Korean infants. A total of 290 healthy Korean infants who were registered to Seoul Metropolitan Public Cord Blood Bank during the period of February 2007 to December 2011 were enrolled. Anti-HBs antibody level was analyzed after three doses of HBV vaccination. Genotyping of HLA-DPA1 SNPs (rs3077 and rs3830066) and HLA-DPB1 SNPs (rs7770370, rs7770501, rs3128961, and rs9277535) were performed by PCR-sequencing. HLA-A, -B, and -DRB1 genotyping was also performed by PCR-sequence-specific oligonucleotide probe kits. HLA-DPB1 SNPs (rs7770370, rs7770501, rs3128961, and rs9277535) were associated with HBV vaccine response. Allele frequencies of rs7770370 A, rs7770501 C, rs3128961 G, and rs9277535 A were significantly higher in responders than in non-responders (all p<0.01). Anti-HBs antibody levels were different according to genotypes of DPB1 rs7770370, rs7770501, rs3128961, and rs9277535 (all p<0.01). In multivariate analysis, HLA-DPB1 rs7770370 AA genotype was significantly associated with HBV vaccine response (relative risk, RR=2.5, p=0.033) and high-titer vaccine response (RR=2.7, p<0.001). In conclusion, HLA-DPB1 SNPs were significantly associated with responses to HBV vaccination in Korean infants.

Desensitization Using Bortezomib and High-dose Immunoglobulin Increases Rate of Deceased Donor Kidney Transplantation.

Combination therapy of intravenous immunoglobulin (IVIG) and rituximab showed a good transplant rate in highly sensitized wait-listed patients for deceased donor kidney transplantation (DDKT), but carried the risk of antibody-mediated rejection. The authors investigated the impact of a new combination therapy of bortezomib, IVIG, and rituximab on transplantation rate.This study was a prospective, open-labeled clinical trial. The desensitization regimen consisted of 2 doses of IVIG (2  g/kg), a single dose of rituximab (375  mg/m), and 4 doses of bortezomib (1.3  mg/m). The transplant rate was analyzed. Anti-Human leukocyte antigen (HLA) DRB antibodies were determined by a Luminex solid-phase bead assay at baseline and after 2, 3, and 6 months in the desensitized patients.There were 19 highly sensitized patients who received desensitization and 17 patients in the control group. Baseline values of class I and II panel reactive antibody (%, peak mean fluorescence intensity) were 83  ±â€Š 16.0 (14952  ±  5820) and 63  ±  36.0 (10321  ±  7421), respectively. Deceased donor kidney transplantation was successfully performed in 8 patients (42.1%) in the desensitization group versus 4 (23.5%) in the control group. Multivariate time-varying covariate Cox regression analysis showed that desensitization increased the probability of DDKT (hazard ratio, 46.895; 95% confidence interval, 3.468-634.132; P = 0.004). Desensitization decreased mean fluorescence intensity values of class I panel reactive antibody by 15.5% (20.8%) at 2 months. In addition, a liberal mismatch strategy in post hoc analysis increased the benefit of desensitization in donor-specific antibody reduction. Desensitization was well tolerated, and acute rejection occurred only in the control group.In conclusion, a desensitization protocol using bortezomib, high-dose IVIG, and rituximab increased the DDKT rate in highly sensitized, wait-listed patients.

Performance of LIFECODES HLA-A, -B, and -DRB1 Typing Kits Using Luminex Platform in Koreans.

BACKGROUND: Recently, HLA typing using PCR sequence-specific oligonucleotide (SSO) and the Luminex platform has been introduced and widely used in clinical laboratories. METHODS: We evaluated the performance of LIFECODES HLA-A, -B, and -DRB1 typing kits (Immucor, USA) on 108 samples that had been tested with WAKFlow kits (Wakunaga, Japan) and 54 samples (18 for each A, B, and DRB1 kits) including rare alleles in Koreans (gene frequency < 1.0%) that had been assigned by sequence-based typing (SBT). RESULTS: LIFECODES 2-digit typing results were 100% concordant with WAKFlow and SBT results. For allelic group assignment, ambiguous results were frequently observed for A*02:01g/*02:07g (n = 47), *02:06g/*02:10g (n = 15), *24:02g/*24:20g (n = 40) and DRB1*04 allelic groups (n = 11). False assignment was observed in 5 cases (2 cases of A*11:02g to A*11:01g; 3 cases of A*26:02g to A*26:01g) due to false reactions of probe 206 and 368, respectively. Additional false reaction did not affect allelic group assignment. CONCLUSIONS: LIFECODES HLA-A, -B, and -DRB1 typing kits showed good performance in Koreans with correct designation of 2-digit assignment and possible assumption of HLA allelic groups in most cases. They can be properly used for organ transplantation and donor screening of hematopoietic stem cell transplantation in Korea.

Allele and Haplotype Frequencies of Human Leukocyte Antigen-A, -B, -C, -DRB1, and -DQB1 From Sequence-Based DNA Typing Data in Koreans.

BACKGROUND: Data on allele frequencies (AFs) and haplotype frequencies (HFs) of HLA-C and -DQB1 are limited in Koreans. We investigated AFs and HFs of HLA-A, -B, -C, -DRB1, and -DQB1 in Koreans by high-resolution sequence-based typing (SBT). METHODS: Hematopoietic stem cells were obtained from 613 healthy, unrelated donors to analyze HLA-A, -B, -C, -DRB1, and -DQB1 genotypes by using AlleleSEQR HLA-A, -B, -C, -DRB1, and -DQB1 SBT kits (Abbott Molecular, USA), respectively. Alleles belonging to HLA-C*07:01/07:06 group were further discriminated by using PCR-sequence specific primer analysis. AFs and HFs were calculated by direct counting and maximum likelihood method, respectively. RESULTS: In all, 24 HLA-A, 46 HLA-B, 24 HLA-C, 29 HLA-DRB1, and 15 HLA-DQB1 alleles were identified. AFs and HFs of HLA-A, -B, and -DRB1 were similar to those reported previously. For the HLA-C locus, C*01:02 was the most common allele, followed by C*03:03, C*03:04, C*14:02, C*03:02, and C*07:02 (AF ≥7%). AFs of C*07:01 and C*07:06 were 0.16% and 3.18%, respectively. For the HLA-DQB1 locus, DQB1*03:01 was the most common allele, followed by DQB1*03:03, *03:02, *06:01, *05:01, *04:01, and *06:02 (AF ≥7%). AFs of DQB1*02:01 and DQB1*02:02 were 2.12% and 6.69%, respectively. HFs of A*33:03-C*07:06 and C*07:06-B*44:03 were 3.09% and 3.10%, respectively, while those of DRB1*07:01-DQB1*02:02 and DRB1*03:01-DQB1*02:01 were 6.61% and 2.04%, respectively. CONCLUSIONS: This study reported AFs and HFs of HLA, including HLA-C and -DQB1, in Koreans by using high-resolution SBT. These data can be used to resolve ambiguous results of HLA typing for organ and hematopoietic stem cell transplantations.

The impact of HLA and KIR ligand mismatching on unrelated allogeneic hematopoietic stem cell transplantation in Korean adult patients.

BACKGROUND: The impact of HLA and KIR ligand mismatching on the outcome of hematopoietic stem cell transplantation (HSCT) remains unclear. Previous reports have identified considerable ethnic differences in the impact of HLA and KIR ligand mismatches, as well as KIR ligand status, on HSCT; however, to date, no data has been acquired in Korean adult patients. METHODS: We investigated the association of high-resolution HLA matching on five loci (HLA-A, -B, -C, -DRB1, and -DQB1), KIR ligand mismatching, and KIR ligand status on the outcome of allogeneic HSCT from unrelated donors in 154 Korean adult patients treated at Seoul National University Hospital. RESULTS: In a multivariate analysis, less than 9/10 allelic matches in five HLA loci was an independent risk factor for acute graft-versus-host disease (GVHD) (grade II to IV) (P=0.019, odds ratio [OR]=2.7). In addition, HLA-A allele mismatching was increasingly prevalent in patients with acute GVHD compared to patients without (61.9% vs. 34.5%, P=0.06). For KIR ligand status, the patient and donor combination of both C1/C1 ligands showed better event-free and overall survival than combinations with C2 ligand patients or donors (P=0.048, P=0.034, respectively) by log-rank test. CONCLUSIONS: Korean adult transplant patients with less than 9 of 10 HLA allele matches in the HLA-A, -B, -C, -DRB1, and DQB1 loci have a higher likelihood of developing acute GVHD (grade II to IV). Impact of KIR ligand status on clinical outcome should be further studied in a larger patient population.

Performance of LIFECODES HLA-DQB1 typing kit using Luminex platform in Koreans.

Intermediate-resolution HLA-DQ typing has gained importance in organ transplantation recently. We evaluated the performance of the LIFECODES HLA-DQB1 typing kit (Immucor, USA) using sequence-specific oligonucleotide (SSO) probe and Luminex platform (Luminex Corp., USA) on 100 samples tested by sequence-based typing (SBT) using the AlleleSEQR HLA-DQB1 kit (Abbott Molecular, USA) in Korean individuals. No sample showed ambiguity in the assignment of 4-digit HLA-DQB1 allele with the LIFECODES HLA-DQB1 SSO typing kit, and the results were fully concordant with those of high-resolution typing of AlleleSEQR HLA-DQB1 SBT up to 4-digit level. Three samples required adjustment of false reactions (3/100, 3.0%): two samples with DQB1(*)03:03/(*)06:01 showed false-positive result in probe 253, and 1 sample with DQB1(*)04:02/(*)05:02 showed false-negative result in probe 217. We tested an additional sample with DQB1(*)03:03/(*)06:01, which showed same false-positivity in probe 253 and 2 samples with DQB1(*)04:02/(*)05:02, which showed no false reaction. The false reactions did not result in ambiguity or change in the HLA allele assignment. We could assign HLA-DQB1 alleles to 4 digit-level without ambiguity, with 100% concordance with the SBT results. Thus, LIFECODES HLA-DQB1 SSO typing kit showed good performance for intermediate-resolution HLA-DQB1 typing in clinical laboratory for organ transplantation in Koreans.

Association of HLA alleles with the responsiveness to hepatitis B virus vaccination in Korean infants.

Hepatitis B virus (HBV) vaccination is the most effective means of countering HBV-related morbidity and mortality, and individuals who do not respond to vaccination (non-responders) are problematic. The aim of the present study was to investigate associations between HLA and responsiveness to HBV vaccine in Korean infants. A total of 944 healthy Korean infants 9-12 months old were enrolled, and HLA distribution was compared among subgroups in accordance with the response to HBV vaccination. The HLA distribution of the subjects was similar to known Korean population data and did not deviate from the HWE proportions. The alleles that showed positive associations with non-responsiveness (<10mIU/mL) or low antibody titer (<100mIU/mL) were HLA-A*33, B62, DRB1*04, and DRB1*07, while the alleles A*02 and DRB1*08 showed negative associations. Among these alleles, B62, DRB1*07 and DRB1*08(-) showed significant associations with a poor or decreased response to vaccination even after correction (OR=1.83, 1.99, 5.63; pc<0.05) and also showed dose effects. After stratification by other associated alleles at different loci, B62 and DRB1*07 were independently associated with non-responsiveness, but A*02(-) and DRB1*08(-) lost their individual associations. The combined association of A*02(-)-DRB1*08(-) and B62-DRB1*08(-) was significant (OR=25.2 and 24.5; pc<0.05). Although the hierarchy is not clear, we can assume the following: (i) B62 and DRB1*07 have independent effects, (ii) DRB1*08(-) has a very strong and synergic effect, and (iii) there is probability of a third factor controlling A*02(-) and DRB1*08(-) with an effect on non-responsiveness to HBV vaccination in Korean infants.

Diagnostic and prognostic significance of neutrophil gelatinase-associated lipocalin in disseminated intravascular coagulation.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) exists as monomers, homodimers, and NGAL/matrix metalloproteinase-9 (MMP-9) complexes. Circulating neutrophils are activated in ongoing disseminated intravascular coagulation (DIC); therefore, plasma NGAL levels are likely to be increased in DIC. We investigated the diagnostic performance of the plasma NGAL level in predicting acute kidney injury (AKI) in patients with DIC and determined the prognostic value of NGAL. METHODS: A total of 126 patients with suspected DIC were enrolled. The plasma free NGAL was analyzed with a point-of-care immunoassay. Plasma total NGAL and NGAL/MMP-9 complex levels were measured using commercial kits. RESULTS: Median free and total NGAL levels were markedly higher in patients with AKI than those without AKI. However, no significant difference in the NGAL/MMP-9 complex level was found between the groups. In patients without AKI, the plasma free and total NGAL levels were significantly higher in those with overt-DIC than in those without overt-DIC. Of note, the free NGAL level showed a significant prognostic value in DIC. CONCLUSION: Plasma free and total NGAL proved to be powerful markers for AKI in DIC patients and plasma free NGAL also has a prognostic relevance in DIC patients without AKI.