Verification of a method using magnetic bead enrichment and nucleic acid extraction to improve the molecular detection of bacterial contamination in blood components.

Bacterial contamination of blood products poses a significant risk in transfusion medicine. Platelets are particularly vulnerable to bacterial growth because they must be stored at room temperature with constant agitation for >5 days. The limitations of bacterial detection using conventional methods, such as blood cultures and lateral flow assays, include the long detection times, low sensitivity, and the requirement for substantial volumes of blood components. To address these limitations, we assessed the performance of a bacterial enrichment technique using antibiotic-conjugated magnetic nanobeads (AcMNBs) and real-time PCR for the detection of bacterial contamination in plasma. AcMNBs successfully captured >80% of four bacterial strains, including Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae, in both plasma and phosphate-buffered saline. After 24-h incubation with bacterial enrichment, S. aureus and B. cereus were each detected at 101 CFU/mL in all trials (5/5), E. coli at 101 CFU/mL in 1/5 trials, and K. pneumoniae at 10² CFU/mL in 4/5 trials. Additionally, without incubation, the improvement was also achieved in samples with bacterial enrichment, S. aureus at 10² CFU/mL and B. cereus at 101 CFU/mL in 1/5 trials each, E. coli at 10³ CFU/mL in 3/5 trials, and K. pneumoniae at 10¹ CFU/mL in 2/5 trials. Overall, the findings from this study strongly support the superiority of bacterial enrichment in detecting low-level bacterial contamination in plasma when employing AcMNBs and PCR.IMPORTANCEThe study presents a breakthrough approach to detect bacterial contamination in plasma, a critical concern in transfusion medicine. Traditional methods, such as blood cultures and lateral flow assays, are hampered by slow detection times, low sensitivity, and the need for large blood sample volumes. Our research introduces a novel technique using antibiotic-conjugated magnetic nanobeads combined with real-time PCR, enhancing the detection of bacteria in blood products, especially platelets. This method has shown exceptional efficiency in identifying even low levels of four different species of bacteria in plasma. The ability to detect bacterial contamination rapidly and accurately is vital for ensuring the safety of blood transfusions and can significantly reduce the risk of infections transmitted through blood products. This advancement is a pivotal step in improving patient outcomes and elevating the standards of care in transfusion medicine.

False-positive results of galactomannan assays in patients administered glucose-containing solutions.

Galactomannan (GM) is a polysaccharide cell wall component released by Aspergillus spp., and an immunoenzymatic GM assay is used for the diagnosis of invasive pulmonary aspergillosis. We evaluated the cause of strong positivity for GM in patients with no typical signs of aspergillosis. Repeat assays were performed using different instruments and reagent lots, but there were no differences in results among the assays. Patients with strongly positive GM results were investigated. Medication histories revealed that 14 of 23 patients had been administered total parenteral nutrition solution from one manufacturer and 4 patients had been administered dextrose solution from a different manufacturer before being tested. The results of GM assays conducted on samples of dextrose solution and the glucose fraction of the total parenteral nutrition solution were strongly positive, confirming the causes of the false-positive reactions. We hypothesize that a trace amount of GM was introduced into the glucose-containing solutions because glucoamylase, which is necessary for the saccharification step of glucose synthesis, was derived from Aspergillus niger. To enhance patient care and prevent unnecessary antifungal prescriptions, healthcare providers and manufacturers of healthcare products need to be aware of the possibility of false-positive reactions for GM.

A Korean family with RHD*DNT only detectable after anti-D alloimmunization.

OBJECTIVES: Identification of DNT, a rare partial D, can be challenging, as it is difficult to distinguish from D+. This study aimed to identify DNT individuals by analyzing the DNT proband's family members, characterize DNT, and propose management strategies. METHODS: Family members of the first Korean DNT proband were recruited. RHD genotyping was conducted, and weak D tests were carried out using several anti-D reagents. RESULTS: Three DNT individuals were identified among 6 family members, including 1 with an anti-D alloantibody. As DNT red cells exhibited strong reactivity with all anti-D clones, DNT was serologically indistinguishable from D+. Moreover, unusual serologic findings in DNT individuals only became apparent after anti-D alloimmunization. CONCLUSIONS: We recommend DNT individuals as candidates for Rh immune globulin prophylaxis during the perinatal period and transfusions with D- blood components. An anticipatory RHD genotyping is suggested for partial D family members to prevent potential partial D individuals from becoming alloimmunized.

A voluntary transfusion recipient registry in Korea as a database for blood group antibodies.

BACKGROUND: Several types of transfusion-related registries have been developed to improve patient outcomes and blood banks. In Korea, a transfusion program functioning as a blood group antibody database and a reference laboratory has been in operation since July 2013. This study was conducted to determine the current status of blood group antigens and antibodies in Korea and propose a model for registries in the field of transfusion medicine. MATERIALS AND METHODS: Cases with unexpected red cell antibodies were registered online in the voluntary transfusion registry. Specific antigen-negative frequencies were calculated based on the recorded data. To determine the frequencies of RhCE antigens, data added via the Blood Information Sharing System were also analyzed. Data added to the registries between July 2013 and June 2022 were included in the analysis. RESULTS: Among 9,048 antibody cases registered from 29 hospitals, anti-E alone was identified most commonly, followed by anti-E and c, anti-C and e, anti-Lea, and anti-M (2,202, 1,792, 757, 618, and 383 cases, respectively). The frequencies of E-, E-c-, C-e-, Le(a-), and M- were 49.1%, 41.6%, 9.1%, 69.4%, and 21.8%, respectively. DISCUSSION: The distributions of antibodies and antigen frequencies were estimated through the transfusion registry. Antigen frequencies were calculated based on the results of antigen typing of red blood cell components performed at the time of issuing. The online transfusion registry serving as a blood group antibody database is useful for determining the frequencies of blood group antigens and antibodies.

Allele and haplotype frequencies of 11 HLA loci in Koreans by next-generation sequencing.

Data on HLA genotype distribution, including DQA1 and DPA1, in the Korean population are limited. We aimed to investigate the allele and haplotype frequencies of 11 HLA loci in 339 Korean subjects using next-generation sequencing (NGS)-based HLA typing. A total of 339 samples from unrelated healthy subjects were genotyped for HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQB1, -DQA1, -DPB1, and -DPA1 using two different NGS-based HLA typing kits (166 tested using the NGSgo-MX11-3 kit [GenDx, Netherlands] and 173 by the AllType NGS 11 Loci Amplification kit [One Lambda, USA]). PyPop software was used to estimate allele and haplotype frequencies and linkage disequilibrium between the loci. Additionally, a principal component analysis was performed to compare the allele distribution of Koreans with that of other populations. A total of 214 HLA alleles (97 class I and 117 class II alleles) were assigned. The most frequent alleles for each locus were A*24:02:01 (24.78%), B*15:01:01 (10.18%), C*01:02:01 (18.44%), DRB1*04:05:01 (9.59%), DRB3*02:02:01 (13.72%), DRB4*01:03:01 (25.81%), DRB5*01:01:01 (9.0%), DQA1*01:02:01 (16.96%), DQB1*03:01:01 (14.31%), DPA1*01:03:01 (44.4%), and DPB1*05:01:01 (35.1%), respectively. The most frequent haplotypes were A*33:03:01-C*03:02:02-B*58:01:01 for HLA class I (5.01%) and DRB1*04:05:01-DQA1*03:03:01-DQB1*04:01:01-DPA1*02:02:02-DPB1*05:01:01 for HLA class II (6.23%). The total allelic ambiguities by NGS were estimated to be minimal and considerably decreased compared with those by Sanger sequencing. The Japanese population had the most similar allele distribution to Koreans, followed by the Chinese population. Frequency data of 11 HLA loci in Koreans can provide essential data for population genetics and disease association studies.

Molecular detection of bacterial contamination in plasma using magnetic-based enrichment.

Bacterial contamination of blood products is a major problem in transfusion medicine, in terms of both morbidity and mortality. Platelets (PLTs) are stored at room temperature (under constant agitation) for more than 5 days, and bacteria can thus grow significantly from a low level to high titers. However, conventional methods like blood culture and lateral flow assay have disadvantages such as long detection time, low sensitivity, and the need for a large volume of blood components. We used real-time polymerase chain reaction (PCR) assays with antibiotic-conjugated magnetic nanobeads (MNBs) to detect enriched Gram-positive and -negative bacteria. The MNBs were coated with polyethylene glycol (PEG) to prevent aggregation by blood components. Over 80% of all bacteria were captured by the MNBs, and the levels of detection were 101 colony forming unit [CFU]/mL and 102 CFU/mL for Gram-positive and -negative bacteria, respectively. The detection time is < 3 h using only small volumes of blood components. Thus, compared to conventional methods, real-time PCR using MNBs allows for rapid detection with high sensitivity using only a small volume of blood components.

Prediction of various blood group systems using Korean whole-genome sequencing data.

AIMS: This study established blood group analysis methods using whole-genome sequencing (WGS) data and conducted blood group analyses to determine the domestic allele frequency using public data from the Korean whole sequence analysis of the Korean Reference Genome Project conducted by the Korea Disease Control and Prevention Agency (KDCA). MATERIALS AND METHODS: We analyzed the differences between the human reference sequences (hg19) and the conventional reference cDNA sequences of blood group genes using the Clustal Omega website, and established blood group analysis methods using WGS data for 41 genes, including 39 blood group genes involved in 36 blood group antigens, as well as the GATA1 and KLF1 genes, which are erythrocyte-specific transcription factor genes. Using CLC genomics Workbench 11.0 (Qiagen, Aarhus, Denmark), variant analysis was performed on these 41 genes in 250 Korean WGS data sets, and each blood group's genotype was predicted. The frequencies for major alleles were also investigated and compared with data from the Korean rare blood program (KRBP) and the Erythrogene database (East Asian and all races). RESULTS: Among the 41 blood group-related genes, hg19 showed variants in the following genes compared to the conventional reference cDNA: GYPA, RHD, RHCE, FUT3, ACKR1, SLC14A1, ART4, CR1, and GCNT2. Among 250 WGS data sets from the Korean Reference Genome Project, 70.6 variants were analyzed in 205 samples; 45 data samples were excluded due to having no variants. In particular, the FUT3, GNCT2, B3GALNT1, CR1, and ACHE genes contained numerous variants, with averages of 21.1, 13.9, 13.4, 9.6, and 7.0, respectively. Except for some blood groups, such as ABO and Lewis, for which it was difficult to predict the alleles using only WGS data, most alleles were successfully predicted in most blood groups. A comparison of allele frequencies showed no significant differences compared to the KRBP data, but there were differences compared to the Erythrogene data for the Lutheran, Kell, Duffy, Yt, Scianna, Landsteiner-Wiener, and Cromer blood group systems. Numerous minor blood group systems that were not available in the KRBP data were also included in this study. CONCLUSIONS: We successfully established and performed blood group analysis using Korean public WGS data. It is expected that blood group analysis using WGS data will be performed more frequently in the future and will contribute to domestic data on blood group allele frequency and eventually the supply of safe blood products.

Performance evaluation of three automated quantitative immunoassays and their correlation with a surrogate virus neutralization test in coronavirus disease 19 patients and pre-pandemic controls.

BACKGROUND: SARS-CoV-2 pandemic is currently ongoing, meanwhile vaccinations are rapidly underway in some countries. The quantitative immunoassays detecting antibodies against spike antigen of SARS-CoV-2 have been developed based on the findings that they have a better correlation with the neutralizing antibody. METHODS: The performances of the Abbott Architect SARS-CoV-2 IgG II Quant, DiaSorin LIAISON SARS-CoV-2 TrimericS IgG, and Roche Elecsys anti-SARS-CoV-2 S were evaluated on 173 sera from 126 SARS-CoV-2 patients and 151 pre-pandemic sera. Their correlations with GenScript cPass SARS-CoV-2 Neutralization Antibody Detection Kit were also analyzed on 173 sera from 126 SARS-CoV-2 patients. RESULTS: Architect SARS-CoV-2 IgG II Quant and Elecsys anti-SARS-CoV-2 S showed the highest overall sensitivity (96.0%), followed by LIAISON SARS-CoV-2 TrimericS IgG (93.6%). The specificities of Elecsys anti-SARS-CoV-2 S and LIAISON SARS-CoV-2 TrimericS IgG were 100.0%, followed by Architect SARS-CoV-2 IgG II Quant (99.3%). Regarding the correlation with cPass neutralization antibody assay, LIAISON SARS-CoV-2 TrimericS IgG showed the best correlation (Spearman rho = 0.88), followed by Architect SARS-CoV-2 IgG II Quant and Elecsys anti-SARS-CoV-2 S (all rho = 0.87). CONCLUSIONS: The three automated quantitative immunoassays showed good diagnostic performance and strong correlations with neutralization antibodies. These assays will be useful in diagnostic assistance, evaluating the response to vaccination, and the assessment of herd immunity in the future.

Molecular Characteristics of the Serological Weak D Phenotype in Koreans.

Serological weak D is a reaction of 2+ or less to anti-D reagent and includes weak D and partial D phenotypes. Although identifying the RhD subtype is important for transfusion safety, serological tests are insufficient for defining the RhD subtype, and molecular tests are needed. To analyze the molecular characteristics of D variants in Koreans to facilitate the formulation of individualized transfusion strategies, molecular tests such as RhD genotyping using real-time polymerase chain reaction (PCR) and partial-D and/or weak-D sequence-specific amplification (SSP) were performed on 105 Korean Rare Blood Program (KRBP) patients exhibiting serological weak D. In total, 58 out of 68 serologically determined weak D KRBP patients were typed as having weak D or partial D phenotypes via RhD genotyping. In detail, eight (13.8%) were typed as partial DVa or DBS, nine (15.5%) as weak D type 15, and four others (6.8%) as partial DVI, partial DVII, weak D type 2, or weak D type 41 or 45, whereas the rest (n = 37, 63.8%) was typed as having either weak D or partial D. This suggests that serological weak D Koreans who require transfusion should be treated as D-negative.

Is elevated microsatellite alterations at selected tetranucleotide repeats (EMAST)-negative/MSI-high colorectal cancer a distinct subtype of the disease?

BACKGROUND AND OBJECTIVES: Microsatellite instability (MSI) plays a prognostic and predictive role in colorectal cancer (CRC). Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a novel type of MSI, was recently identified. METHODS: A retrospective analysis of a prospective cohort database was performed. Patients who attempted curative surgery for MSI-high (MSI-H) CRC and had available testing results of EMAST were included for analysis. The difference in clinical characteristics, immunohistochemistry profile, and 3-year recurrence-free and overall survival between EMAST-negative and EMAST-positive tumors was measured. RESULTS: EMAST status was successfully evaluated in 86 cases among patients who received EMAST testing, and only 16.3% (14/86) of these patients were EMAST-negative/MSI-H. Patients with EMAST-negative tumors were younger; their tumors exhibited well differentiation, less venous invasion, and greater mutS homolog 3 expression. There was no distant metastasis or cancer-specific death among EMAST-negative patients. Yet no statistically significant difference was found between the two groups in 3-year overall or recurrence-free survival. CONCLUSIONS: Patients with EMAST-negative/MSI-H CRC seem to have different clinicopathological characteristics. Future large-scale studies could clarify the role of EMAST genotype as a sub-classifier of MSI-H CRC.

Differential prognostic impact of CD8+ T cells based on human leucocyte antigen I and PD-L1 expression in microsatellite-unstable gastric cancer.

BACKGROUND: The aim of the study was to determine the human leucocyte antigen class-I (HLA-I), programmed death-ligand 1 (PD-L1) expression and tumour-infiltrating lymphocytes (TILs) of microsatellite instability-high gastric cancer. METHODS: The HLA-I expression type was determined by immunohistochemistry of HLA-A, HLA-B, HLA-C and ß2-microglobulin in the centre of the tumour (CT) and in the invasive margin (IM) of samples from 293 patients (total loss vs. preserved type). PD-L1 expression and TIL density was examined immunohistochemically. HLA-I genotyping was also performed. RESULTS: The expression loss of the HLA-I molecules was significantly associated with low TIL density. According to survival analyses, the HLA-I expression type and PD-L1 positivity were not independent prognostic factors. The TIL density had no prognostic implication when survival analysis was performed for the whole patient group; however, high CD8+ TIL infiltration was significantly associated with good prognosis in only HLA-I-preserved-type/PD-L1-positive group (p = 0.034). The homozygosity of the HLA-I allele was more frequently observed in the total loss type group. CONCLUSIONS: We confirmed differential prognostic implication of CD8+ TILs according to the HLA-I and PD-L1 expression. Determination of the HLA-I expression could be helpful to select patients who would benefit from anti-PD-1/PD-L1 therapy.

Recommendations Regarding Practical DEL Typing Strategies for Serologically D-Negative Asian Donors.

BACKGROUND: DEL, the weakest D variant, is mistyped as D-negative by routine serological assays. Transfusion of red blood cells expressing the DEL phenotype has the potential to elicit anti-D alloimmunization in D-negative recipients. The goal of this study was to recommend DEL typing strategies for serologically D-negative Asian donors. METHODS: RhCE phenotyping and the adsorption-elution test were performed on 674 serologically D-negative samples. RHD genotyping using real-time polymerase chain reaction and melting curve analysis were also undertaken to identify DEL alleles. Costs and turnaround time of RhCE phenotyping, the adsorption-elution test, and RHD genotyping were estimated. RESULTS: Sensitivity and specificity of the adsorption-elution test for serologically D-negative samples were 94.9% (93/98) and 91.5% (527/576), respectively. C+ phenotypes were detected in all 98 samples with DEL alleles. Despite comparable costs, RHD genotyping was more accurate and rapid than the adsorption-elution test. CONCLUSIONS: Two practical DEL typing strategies using RhCE phenotyping as an initial screening method were recommended for serologically D-negative Asian donors. Compared with DEL typing using RHD genotyping, serological DEL typing using adsorption-elution test is predicted to increase the incidence of anti-D alloimmunization and decrease the D-negative donor pool without having any cost-competitiveness but can be used in laboratories where molecular methods are not applicable.

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.

Removal of Non-specific Reactions of LABScreen Single Antigen Class II Assay by Fetal Bovine Serum Treatment.

Recently, the removal of false reaction from beads 10 (DRB1 *04:04), 30 (DRB1 *16:01), and 31 (DRB1 *16:02) by fetal bovine serum (FBS) treatment in LABScreen Single Antigen Class II Assay was reported. We aimed to confirm the reaction in many cases. Fifty-nine sera showed positivity on at least two among beads 10, 30 and 31 from Nov 2017 to Oct 2018 in Seoul National University Hospital were included. FBS treatment was performed on 59 sera, and Single Antigen Class II Assay was repeated. Among 59 cases, the negative conversion rates of DR16 (57/59, 96.6%), DR4 (37/39, 94.9%), and accompanied other antibodies, e.g.) DP19 (39/45, 86.7%) were very high. The prior use of intravenous immunoglobulin in a non-specific binding group was significantly higher than the sex, age-matched control group (P=0.005).

A novel strategy for interpreting the T-SPOT.TB test results read by an ELISPOT plate imager.

BACKGROUND: The T-SPOT.TB can be read by an ELISPOT plate imager as an alternative to a labor-intensive and time-consuming manual reading, but its accuracy has not been sufficiently discussed to date. METHODS: 1,423 test results obtained from manual reading using a microscope and an ELISPOT plate imager were compared. The agreement of qualitative test results was assessed using Cohen's kappa coefficient. The relationship of spot counts was studied using Bland-Altman analysis. RESULTS: The overall percent agreement of the qualitative test results was 95.43% with a kappa coefficient of 0.91. Positive test results with the maximum net spot count of 8 and borderline test results showed relatively high discordance. The agreement of spot counts in panel A, panel B, and nil control was good, and variability did not increase with higher spot counts. On the basis of study findings, a novel strategy for interpreting the test results by an ELISPOT plate imager was proposed. CONCLUSIONS: To increase diagnostic accuracy, positive test results with the maximum net spot count of 8 and borderline test results should be manually confirmed. Our strategy could be a practical guide for laboratories to build their own strategies for interpreting the test results by an ELISPOT plate imager.

Microarray-Based Nucleic Acid Assay and MALDI-TOF MS Analysis for the Detection of Gram-Negative Bacteria in Direct Blood Cultures.

Objectives: To compare a microarray-based identification and resistance determination system (blood culture gram-negative [BC-GN]; Nanosphere, Northbrook, IL) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct blood cultures (BCs). Methods: BC-GN and MALDI-TOF MS assay results from direct BCs were compared with conventional test results after pure culture. Results: Among 124 BCs, 130 gram-negative rods (GNRs), including six cultures with mixed GNRs (117 bacteria were covered by the BC-GN panel), were detected. The BC-GN test presented 116/117 (99.1%) concordance for the identification of targeted GNRs. Among the six polymicrobial BCs, 10 targeted GNRs were correctly identified. Among the 100 BCs tested by MALDI-TOF MS, 88/106 (86.7%) GNRs were correctly identified, and 18 GNRs were not identified. Among the six polymicrobial samples, seven of 12 GNRs (58.3%) were correctly identified. Conclusions: The BC-GN assay exhibited superior performance compared with MALDI-TOF MS for the identification of targeted GNRs in direct BCs, particularly in polymicrobial samples.

Frequency of Red Blood Cell Antigens According to Parent Ethnicity in Korea Using Molecular Typing.

Frequencies of red blood cell (RBC) blood group antigens differ by ethnicity. Since the number of immigrants is increasing in Korea, RBC antigens should be assessed in children/youths with parents of different ethnicities to ensure safe transfusions. We investigated the frequency of RBC antigens, except for ABO and RhD, in 382 children and youths with parents having Korean and non-Korean ethnicities. Subjects were divided into those with ethnically Korean parents (Korean group; N=252) and those with at least one parent of non-Korean ethnicity (non-Korean group; N=130). The 37 RBC antigens were genotyped using the ID CORE XT system (Progenika Biopharma-Grifols, Bizkaia, Spain). The frequencies of the Rh (E, C, e, hr(S), and hr(B)), Duffy (Fy(a)), MNS (Mi(a)), and Cartwright (Yt(b)) antigens differed significantly between the two groups. Eight and 11 subjects in the Korean and non-Korean groups, respectively, exhibited negative expression of high-frequency antigens, whereas 14 subjects in the non-Korean group showed positive expression of low-frequency antigens. The frequency of RBC antigens has altered alongside demographic changes in Korea and might lead to changes in distribution of RBC antibodies that cause acute or delayed hemolytic transfusion reaction.