Coagulation Testing in Real-World Setting: Insights From a Comprehensive Survey.

The objective of this survey was to gain a real-world perspective on coagulation testing by evaluating the availability of various coagulation laboratory tests, assessing specific analytic and postanalytic steps in clinical laboratories in Korea.Participants were surveyed using a 65-question questionnaire specifically focused on their coagulation testing practices related to prothrombin time (PT), activated partial thromboplastin time (aPTT), plasma-mixing studies, lupus anticoagulant (LA) tests, platelet function tests, coagulation factor assays, and the composition of hemostasis and thrombosis test panels. The survey was performed between July and September 2022.The survey achieved a 77.9% (81 of 104) response rate. PT or aPTT tests were performed directly at all participating institutions, followed by D-dimer and fibrinogen tests, platelet function test, and plasma-mixing studies in order of frequency. Variations existed in the performance of mixing test and LA assessment. Patterns of coagulating testing differed depending on the size of the hospital. The survey revealed that most laboratories conducted coagulation tests following the international guidelines such as Clinical Laboratory Standards Institute guidelines and the Korean Laboratory Certification system. However, some coagulation tests, including mixing test and LA tests, are yet to be standardized in Korea.Continuous education on coagulation test methods and internal and external quality control are required to encourage laboratories to enhance the performance of coagulation testing.

Performance evaluation of the Sysmex CN-6000-automated coagulation analyzers for routine coagulation tests.

The Sysmex CN-6000 is a novel automated multiparameter coagulometer that performs clotting, chromogenic and immunological assays, and platelet aggregation tests in a single system. Here we evaluated its performance of routine coagulation assays. The precision, linearity, carryover and establishment of reference ranges of the CN-6000, as well as correlations between it and the currently used Diagnostica Stago STA-R Max were determined according to Clinical and Laboratory Standards Institute guidelines. The evaluated parameters included prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FBG), antithrombin (AT), d-dimers (DDi), and fibrin and FBG degradation products (FDP). The intra-run and inter-run precisions of the six tests were determined using normal and pathological control materials; all coefficients of variation were acceptable and within the allowable ranges. The CN-6000 showed excellent linearity for FBG, AT, DDi, and FDP (R = 0.999-1.00). Passing-Bablok regression (R2 > 0.95) demonstrated good agreement between the analyzers. In the carryover study, APTT, PT, FBG, AT, DDi, and FDP values were all acceptable. The establishing reference intervals revealed that each manufacturer's range was acceptable. Significant differences were observed in the APTT reference range because of using different detection systems and reagents. The CN-6000 analyzer showed reliable performance and good correlation with the currently used STA-R Max automated hemostatic analyzer. As CN-6000 uses an optical clot-detection method, its reference ranges for PT and APTT are lower than those of the STA-R Max; thus, the difference should be considered before its use.

Sample collection for pre-transfusion crossmatching: Benefits of using an electronic identification system.

BACKGROUND: Transfusion of ABO blood group-mismatched blood or administration to the wrong recipient may result in fatal adverse events. To prevent these types of errors, various strategies have been employed. Recently, we developed a novel sample collection workflow for the pre-transfusion crossmatching test and patient recognition. This study aimed to analyse the usage of the new workflow and improvements in outcomes. METHODS: We analysed the number of crossmatching and wrong-patient errors among the blood transfusion cases during 3 years of data collection (from August 2018 to July 2021). From May 2021 to July 2021, the new workflow was implemented. Outcomes were calculated according to the department type, patient age and processing time. The sample processing time was defined as the time from placing the order to lab arrival. RESULTS: The new workflow utilisation increased from 50.7% to 80.3% and wrong-patient errors decreased annually. The new workflow was used for more adults (3001/3680 samples, 81.5%) than paediatric cases (345/522 samples, 65.5%; p < 0.001) and in general wards than in the emergency room or intensive care unit. The sample processing time differed according to ward type and timing of the request (day: 28.80, 2.43-3889.43 min, night: 3.36, 2.72-1671.47 min; p < 0.001). CONCLUSION: Wrong-patient errors were reduced without increasing sample-processing time after introducing the new workflow which included using an electronic identification system. The time needed for the blood processing differed according to the ward type, patient age, and timing of the request. Patient safety can be promoted by managing these factors and using an electronic identification system.

Effects of Different Centrifugation Protocols on the Detection of EGFR Mutations in Plasma Cell-Free DNA.

OBJECTIVES: Various preanalytical factors, including the collection tube, storage conditions, and centrifugation, affect the detection results of plasma cell-free DNA (cfDNA). We compared the effect of different centrifugation protocols on the detection of EGFR mutations in cfDNA. METHODS: We analyzed 117 plasma specimens from 110 patients with non-small cell lung cancer using the cobas EGFR Mutation Test v2 (Roche Diagnostics). We compared the identified EGFR mutations and semiquantitative index values from the 1- and 2-step centrifugation groups and confirmed the clinical impact of differences in the results after further high-speed centrifugation. RESULTS: We detected EGFR mutations in 44 (37.6%) and 47 (40.2%) samples that were centrifuged once and twice, respectively; the 2 groups showed an 89.7% (105/117) concordance and a strong correlation in their semiquantitative index values (r = 0.929). Among the 12 inconsistent result pairs, 9 samples of 2-step centrifugation (75%) were consistent with the results of a recent tissue biopsy. CONCLUSIONS: Additional high-speed centrifugation has been shown to increase the sensitivity of EGFR mutation detection in a commercial in vitro diagnostic real-time polymerase chain reaction device and is an optimal preanalytical factor for detecting low-allele frequency gene mutations using low concentrations of cfDNA.

Comparison of Serologic Response of Hospitalized COVID-19 Patients Using 8 Immunoassays.

BACKGROUND: In Korea, there were issues regarding the use of immunoassays for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies to detect infection. So, we compared antibody results of eight kinds of commercial immunoassays using clinical remnant specimens. METHODS: We compared the results of several immunoassay kits tested on 40 serum samples from 15 confirmed patients and 86 remnant serum samples from clinical laboratory. Eight kinds of IVD kits-four enzyme-linked immunosorbent assay, two lateral flow rapid immunochromatographic assays, and two chemiluminescent immunoassays with one RUO kit were tested. RESULTS: Among 40 serum samples from 15 coronavirus disease 2019 (COVID-19) patients, 35 yielded at least one positive result for detecting antibodies in the combined assessment. There were inconsistent results in 12 (28%) samples by single immunoassay. Forty samples collected in 2019 before the first COVID-19 Korean case showed negative results except for one equivocal result. CONCLUSION: The discrepant results obtained with different immunoassay kits in this study show that serological assessment of SARS-CoV-2 by a single immunoassay requires caution not only in detecting infection but also in assessing immunologic status.

Chimerism Assay Using Single Nucleotide Polymorphisms Adjacent and in Linkage-Disequilibrium Enables Sensitive Disease Relapse Monitoring after Hematopoietic Stem-Cell Transplantation.

BACKGROUND: Short tandem repeat (STR)-based chimerism analysis has been widely used for chimerism monitoring after hematopoietic stem-cell transplantation (HSCT), but technical artifacts can be problematic. We designed a chimerism assay using single nucleotide polymorphisms (SNPs) adjacent and in linkage-disequilibrium (CASAL), which doubly checked for SNP pairs, and thus could reduce background errors and increase analytical sensitivity. METHODS: CASAL targeted 84 SNP pairs within 10 bp distance and in perfect linkage-disequilibrium. Using undiluted and serially diluted samples, baseline error rates, and linearity was calculated. Clinical performance of CASAL was evaluated in comparison with a conventional STR assay, using 191 posttransplant samples from 42 patients with HSCT. RESULTS: CASAL had ∼10 times lower baseline error rates compared to that of ordinary next-generation sequencing. Limit of detection and quantification of CASAL were estimated to be 0.09 and 0.39%, respectively, with a linear range of 0.1-100%. CASAL correlated well with STR assay (r2 = 0.99) and the higher sensitivity enabled detection of low-level recipient chimerism and earlier prediction of relapse. CONCLUSIONS: CASAL is a simple, analytically sensitive and accurate assay that can be used in clinical samples after HSCT with a higher performance compared to that of traditional assays. It should also be useful in other forensic and archeological testing.

Comparison of single nucleotide polymorphisms and short tandem repeats as markers for differentiating between donors and recipients in solid organ transplantation.

Background: To analyze transplant rejection and to distinguish between donor and recipient, it is necessary to select a marker from single nucleotide polymorphism (SNP), short tandem repeat (STR), and human leukocyte antigen (HLA) testing. SNPs are bi-allelic and the polymerase chain reaction method used for SNP testing has the advantage of lower cost than sequencing methods. In this study, we aimed to distinguish donors from recipients using a combination of existing commercialized STRs and the SNPs identified. Methods: All selected SNPs complied with the following criterion known and validated minor allele frequency (MAF) ≥43% in Korean and reported ethnicities from global populations (HapMap, 1000 Genomes, and the Korean Reference Genome project). The STR assays were performed for 16 tetranucleotide repeat loci. Results: DNA from the 52 donor/recipient pairs were tested for informative markers. The median age of the recipients was 47 years. MAF in the 52 pairs was 1.0%-76.0%. The probability of informative genotypes (I) was 0.001-0.124. The summation of I was 0.680. In the 52 donor recipient pairs, the selected SNPs showed a 0.031 average probability of being informative. The probability of identity in our study was 0.122-0.348. SNP panel configuration distinguished 100% of 52 donors/recipient pairs. Conclusions: Donors and recipients were distinguished by STR and 22 SNPs with MAF identified from SNP databases. Seventeen SNPs were able to distinguish between donors and recipients (I value=0.039).

Evaluation of conductivity-based osmolality measurement in urine using the Sysmex UF5000.

BACKGROUND: Automated flow cytometry-based urine analyzer is increasingly being used to identify and enumerate cells and particles in urine specimens. It measures electrical conductivity which could be transformed to osmolality. Using this machine, all urine specimens could be screened for osmolality without requiring a separate dedicated device. We evaluated the performance of the new instrument, the UF-5000 (Sysmex Corporation), in the measurement of urine osmolality. METHODS: The precision of urine osmolality measurement by the UF-5000 was evaluated for 20 days and 4 times a day for 2 concentrations. The linearity and detection capability were evaluated according to the Clinical and Laboratory Standards Institute guidelines. For comparison, 270 random urine specimens from patients were tested simultaneously using the UF5000 and the OsmoPro micro-osmometer (Advanced instruments). RESULTS: The laboratory-based coefficient variations were less than 5%. Urine osmolality using the UF-5000 has a verified linear range (y = 1.097x + 16.91, R2  = .997). Within the comparison analysis, the mean difference was not large (-7.72%) but each differences were largely dispersed with 95% limits of agreement (LoA) from -70.5 to 55.06%, and the mean absolute difference -28.3 mOsm/kg with 95% LoA from -295.13 to 238.45 mOsm/kg. Cohen's kappa value was 0.54 (95% CI, 0.45-0.63). CONCLUSIONS: The UF-5000 measured conductivity and generated an acceptable quantitative analysis of urine osmolality. When compared with the results of the freezing point depression method used by the OsmoPro, a percentage of the measured urine osmolality by the UF-5000 was outside the allowable limit.

Transfusion Requirements and Blood Bank Support in Heart and Lung Transplantation.

OBJECTIVE: Transplantations may require massive transfusion of blood products. Therefore, blood banks need to predict, prepare, and supply the required amount of blood products. METHODS: We measured the volume of transfused blood components as red blood cells, fresh frozen plasma, platelets, and cryoprecipitate in 54 and 89 patients who received heart and lung transplantation, respectively, in our hospital between January 2012 and December 2019. RESULTS: Platelets were the most frequently transfused blood component. Transfusion volumes during heart and lung transplantation surgeries differed: red blood cells, 7.83 units vs 14.84 units; fresh frozen plasma, 2.67 units vs 12.29 units; platelets, 13.13 units vs 23.63 units; and cryoprecipitate, 1.74 units vs 2.57 units; respectively. The average transfusion volume of transplants was different each year. CONCLUSION: Periodic evaluation of transfusion requirements will facilitate the efficient management of blood products at the time of transplantation and help blood banks predict changes in blood requirements.

Platelet Inventory Management Program: Development and Practical Experience.

BACKGROUND: Patients with ongoing or expected bleeding require platelet (PLT) transfusions; however, owing to the testing required after a blood donation, manufacturing PLT products may take 1.5-2.0 days after a request is made. This supply-demand mismatch leads clinicians to retain spare PLTs for transfusions, leading to increased PLT discard rates. We developed a PLT inventory management program to supply PLTs more efficiently to patients requiring PLT transfusions within the expiration date, while reducing PLT discard rates. METHODS: PLT concentrates (58,863 and 58,357 units) and apheresis products (7,905 and 8,441 units) were analyzed from May 2015 to November 2017 and from December 2017 to January 2020, respectively. We developed a program to manage total PLT inventories and prospective PLT transfusion patients based on blood type, blood product, and remaining period of efficacy; the program facilitates PLT preparation transfer to non-designated patients within the remaining period of efficacy. RESULTS: The overall PLT concentrate discard rate was 3,254 (2.78%): 1,811 (3.07%) units before and 1,443 units (2.41%) after program application (P<0.001). The discard rate owing to expiration was reduced from 69 units (3.81%) before to two units (0.14%) after program application (P<0.001). CONCLUSIONS: This program can guide the allocation of PLT preparations based on the remaining period of efficacy, enabling PLT products to be used before their expiration date and reducing PLT product discard rate.

Potential misdiagnosis of von Willebrand disease caused by insufficient mixing after thawing frozen plasma.

INTRODUCTION: von Willebrand disease (vWD) is a common inherited bleeding disorder caused by a deficiency in von Willebrand factor (vWF), but many laboratories and clinicians continue to struggle with diagnosing or excluding vWD. Its diagnosis requires laboratory testing, which may be compromised by preanalytical events, including poor specimen quality. This study assessed 17 different preanalytical conditions as potential causes of vWD misdiagnosis. METHODS: Specimens from healthy controls (N = 21) were obtained. vWF antigen and vWF activity were analyzed using a newly developed automatic coagulation analyzer according to various preanalytic conditions such as centrifugation conditions, storage room temperature before centrifugation, cold storage temperature after centrifugation, thawing conditions, and inadequate mixing of thawed citrated plasma following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) H21-A5 guidelines. RESULTS: The only condition that was significantly different from the reference condition was lack of mixing after thawing frozen citrated plasma (vWF activity and antigen were reduced by 58.7% and 49.6%, respectively). Our study showed that mixing after thawing was more important than the chosen method of mixing. CONCLUSION: Thawed plasma should be mixed because of the risk of misdiagnosing vWD. Further education regarding the importance of appropriate mixing is warranted to achieve results comparable to those of freshly centrifuged samples.

Evaluation of the Barricor Tube in 28 Routine Chemical Tests and Its Impact on Turnaround Time in an Outpatient Clinic.

BACKGROUND: We recently introduced the Barricor (BD, Franklin Lakes, NJ, USA) plasma separation tube, which uses a mechanical separator instead of a gel. We evaluated the effects of using the Barricor tube in a stat (statin) laboratory on the results and turnaround time (TAT) of routine chemical tests. We verified the impact of Barricor tube on reducing TAT and providing results similar to those obtained using serum separator tubes (SSTs). METHODS: We collected venous blood samples from 166 outpatients in Barricor tubes and SSTs and measured 28 routine analytes using an AU5800 instrument (Beckman Coulter, Brea, CA, USA). TAT indexes were compared before and after using Barricor tube. RESULTS: Mean percent differences were <5%, except for alanine aminotransferase , total CO2, high-density lipoprotein, phosphate, total protein, and direct bilirubin. The median TAT decreased from 45 to 38 minutes, and the rate of a TAT >60 minutes decreased from 7.84% to 2.66%, which was approximately one-third of that for SST. The reduction in TAT was attributable to a decrease in centrifugation time. Incomplete clotting and repeated centrifugation, which occurred frequently when using SST, also decreased after using the Barricor tubes. CONCLUSIONS: The Barricor tube is an alternative to SST for routine chemical tests in institutions aiming to reduce TAT, with clinically allowable differences in test results.

Minimal residual disease in acute lymphoblastic leukemia: technical aspects and implications for clinical interpretation.

Minimal residual disease (MRD) monitoring has proven to be one of the fundamental independent prognostic factors for patients with acute lymphoblastic leukemia (ALL). Sequential monitoring of MRD using sensitive and specific methods, such as real-time quantitative polymerase chain reaction (qPCR) or flow cytometry (FCM), has improved the assessment of treatment response and is currently used for therapeutic stratification and early detection. Although both FCM and qPCR yield highly consistent results with sensitivities of 10‒4, each method has several limitations. For example, qPCR is time-consuming and laborious: designing primers that correspond to the immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements at diagnosis can take 3‒4 weeks. In addition, the evolution of additional clones beyond the first or index clone during therapy cannot be detected, which might lead to false-negative results. FCM requires experienced technicians and sometimes does not achieve a sensitivity of 10‒4. Accordingly, a next generation sequencing (NGS)-based method has been developed in an attempt to overcome these limitations. With the advent of high-throughput NGS technologies, a more in-depth analysis of IG and/or TCR gene rearrangements is now within reach, which impacts all applications of IG/TR analysis. However, standardization, quality control, and validation of this new technology are warranted prior to its incorporation into routine practice.

Mutation analysis in Korean patients with T-cell acute lymphoblastic leukemia.

Genomic studies have illuminated the alterations in pathways underlying T-cell acute lymphoblastic leukemia (T-ALL) pathogenesis, but detailed mutation data by next-generation sequencing have not been reported in Korean patients. We aimed to investigate mutation frequency, spectrum, and pattern in the Korean patients with T-ALL. We designed a multigene panel targeting 101 genes and validated it using 10 reference materials. The mutation analysis was done in a total of 10 patients with T-ALL. Clinical data and laboratory tests including immunophenotyping, cytogenetics, and molecular genetic tests were also investigated. All of the 10 patients harbored at least one mutation (range 1-6 per patient). A total of 34 clinically significant mutations including 15 novel mutations were identified in 23 genes. The median of variant allelic frequencies (VAFs) and blasts were counted upto 33% (range 5-91%) and 79% (range 38-90%), respectively. Recurrent mutations were involved in epigenetic regulators (60%), NOTCH1 signaling (40%), PI3K-AKT (40%), JAK-STAT (30%), and transcription factors (30%). We found that both NOTCH signaling and JAK-STAT signaling were positively associated with epigenetic regulators, while showed mutually exclusive patterns with PI3K-AKT pathway. This study showed that the frequency of mutations in epigenetic regulators in Korean patients was significantly higher than expected. Distribution of VAF as well as mutation spectrum is considerably heterogeneous in Korean patients with T-ALL. Although from a limited number of patients, this study provides the first detailed mutational portrait of T-ALL of Korean patients, and gives additional insight into molecular pathogenesis of the disease.

Temporary Cessation of Immunosuppression for Infection May Contribute to the Development of Graft-vs-Host Disease After ABO-Incompatible Living Donor Liver Transplantation: A Case Report.

Graft-vs-host disease (GVHD) after liver transplantation is a rare complication with a high mortality rate. A complex interplay between donor and recipient immunity plays a role in the development of GVHD. Infection following liver transplantation is one of the most common complications in a recipient of an organ transplant who is immunosuppressed. On clinical signs of infection, the immune reaction of the recipient can be reconstituted by withdrawal of immunosuppression in order to help combat infection. However, the discontinuation of immunosuppression could restore the donor's immune activity rather than that of the recipient. There is little information available as to whether the discontinuation of immunosuppression for severe infection could contribute to the development of GVHD in a patient who underwent ABO-incompatible (ABO-I) living donor liver transplantation (LDLT). Herein, we present a unique case of GVHD following ABO-I LDLT, for which the cessation of immunosuppression could be responsible.

T-Cell Receptor Rearrangements Determined Using Fragment Analysis in Patients With T-Acute Lymphoblastic Leukemia.

BACKGROUND: Chromosomal abnormalities and common genetic rearrangements related to T-acute lymphoblastic leukemia (T-ALL) are not clear. We investigated T-cell receptor (TCR) rearrangement in Korean T-ALL patients by fragment analysis, examining frequency, association between clinicopathologic characteristics and TCR clonality, and feasibility for detecting minimal residual disease (MRD). METHODS: In 51 Korean patients diagnosed as having T-ALL, TCR rearrangement was analyzed using the IdentiClone TCR gene clonality assay (InVivoScribe Technologies, San Diego, CA, USA) from archived bone marrow specimens. Limit of detection (LOD) and clonal stability at relapse were evaluated. The association between clinical prognosis and TCR clonality was examind by age and immunophenotypic classification. RESULTS: Thirty-eight patients (74.5%) had 62 clonal products of TCRß, TCRγ, and/or TCRδ rearrangements at diagnosis. Children with T-ALL (<12 years) showed a higher frequency of clonality (93.8%) than adolescents/adults (65.7%; ≥12 years). Patients with a mature immunophenotype (84.4%) showed a relatively higher frequency of clonality than those with the immature immunophenotype (57.9%). Survival and event-free survival were not influenced by immunophenotype or TCR clonality. The LOD was 1%. Clonal evolution at the relapse period was noted. CONCLUSIONS: The overall detection rate of TCR clonality was 74.5%. Survival did not differ by TCR clonality or immunophenotype and age group. Fragment analysis of TCR rearrangement cannot be used to assess MRD due to low sensitivity. Further research on the relationship between prognosis and frequency of TCR rearrangements is needed, using more sensitive methods to detect clonality and monitor MRD.

Characteristics of Bilirubin According to the Results of the Direct Antiglobulin Test and Its Impact in Hemolytic Disease of the Newborn.

BACKGROUND: Hyperbilirubinemia, which is a sign of hemolytic disease of the newborn (HDN), can irreversibly damage the central nervous system. OBJECTIVES: To determine the etiology of HDN in affected patients and characterize the changing pattern of bilirubin using direct antiglobulin testing (DAT). METHODS: We collected clinical data from newborns who underwent perinatal DAT and from their mothers, between August 2008 and July 2017. RESULTS: Among 303 neonates, 37 (12.2%) showed positive DAT results. The positive predictive values (PPVs) and negative predictive values (NPVs) based on DAT results were 75.7% and 28.9%, respectively, for starting phototherapy. Bilirubin levels increased more rapidly in the DAT-positive group, compared with the DAT-negative group. The initial bilirubin level differed significantly according to the etiology of hyperbilirubinemia. Further, neonates with anti-D showed higher delta bilirubin per day than neonates with other antibodies. CONCLUSION: Our results may help to determine the measurement period for bilirubin according to DAT results and etiology.

Increased Anticolitic Effects in C57BL/6 Mice Based on Functional Ingredients of Ramyeon Noodles and Soup.

We investigated the preventive effects of Ramyeon made from brown rice-sorghum noodles and doenjang-bamboo salt soup (BS+DB) on colitis induced by dextran sulfate sodium (DSS) in C57BL/6 mice. Noodles were prepared with 10% brown rice and 10% sorghum powders added to wheat flour and potato powder, and soup was made using starter fermented (SF) doenjang powder (32%) and bamboo salt (13.5%). The experimental animals were divided into five groups: Normal, Control, BS (brown rice and sorghum noodles)+DB (doenjang and bamboo salt soup) (BS+DB Ramyeon), W (white flour noodles, commercial one)+DB, W+dC (W+doenjang commercial soup), and W + D (W+SF doenjang powder [100%]). The BS+DB and W + D groups showed significant reduction of DSS-induced colitis symptoms (P < .05). Doenjang soup (100%) (W+D) also showed a strong anticolitic effect even though the noodles were prepared with W. Histological observation of the colon revealed that BS+DB Ramyeon markedly alleviated colitis development in mice. Serum protein and mRNA levels of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) were significantly suppressed in colon tissue of the BS+DB group compared with those of the W+DB and W+dC groups. BS+DB Ramyeon also reduced colon mRNA levels of inducible nitric oxide synthase and cyclooxygenase-2 compared with those of other groups (P < .05). Our results show that modification of noodle ingredients using brown rice and sorghum as well as alteration of soup composition using doenjang and bamboo salt improved the health benefits of Ramyeon.