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Abstract Previous studies on the immunogenicity of blood group antigens have utilized a formula incorporating antigen frequencies and relative frequencies of unexpected antibodies to the corresponding antigens. This study was aimed at investigating other variables potentially affecting the estimation of immunogenicity using this formula. We examined the effect of multiple transfusions, as there are more chance for a recipient to receive repeated transfusions rather than only once; the effect of antigen density, which may vary depending on homozygote/heterozygote; and the effect of unreliability of the observed frequency of rare antibodies and antigens. For multiple transfusions, the expected antibody frequency increased as the number of transfusions increased. For antigen density, the immunogenicity was falsely low for the low-prevalence antigen, and this tendency intensified as the effect of antigen density increased. Expected antibody frequencies were significantly affected by the uncertainties caused by estimation of small numbers. This study showed that the effects of various factors on the immunogenicity of blood group antigens depended on the antigen frequency. Estimating the immunogenicity of blood group antigens requires acknowledging the diverse factors that can affect it and interpreting the findings with caution.

Abstract Background B‐ and T‐lymphocyte attenuator (BTLA) and programmed cell death‐1 (PD‐1) inhibit γδ T cell homeostasis and activation. This study aimed to determine whether BTLA and PD‐1 signaling pathways were convergent or independent in human peripheral blood γδ T cells. Herein we demonstrate that the signalings of BTLA and PD‐1 regulated proliferation and cytotoxicity of human γδ T cells, respectively. Methods Human peripheral blood γδ T cells were cultured with inactivated Jurkat cells in the presence of interleukin‐2 and zoledronate (Zol) for 14 days. Flow cytometry was performed to evaluate the phenotypes and functions of γδ T cells. Results The proliferation of the γδ T cells was increased when PBMCs were cocultured with inactivated herpes virus entry mediator (HVEM)low Jurkat cells. The cytotoxicity of the expanded γδ T cells was not affected by coculture with inactivated HVEMlow Jurkat cells and was further increased in the presence of anti‐PD‐L1 mAb. These results suggest that the inactivation of the BTLA signaling pathway during expansion could help produce more γδ T cells without compromising γδ T cell function. The inhibition of BTLA or PD‐1 signaling repressed phosphorylation of the src homology region 2‐containing protein tyrosine phosphatase 2 and increased the phosphorylation of protein kinase B in γδ T cells. However, there were no synergistic or additive effects by a combination of BTLA and PD‐1 blockade. Conclusion These results suggest that BTLA signaling is crucial in regulating γδ T cell proliferation and function and that the BTLA and PD‐1 signaling pathways act independently on the proliferation and cytotoxicity of human peripheral γδ T cells.

Background/Aims:Postal distribution of a fecal immunochemical test (FIT) kit has been recommended as an effective method of increasing participation in colorectal cancer (CRC) screening. The present study was performed to assess the impact of the round-mailed FIT kit on screening participation in underserved regions of Korea and to identify factors related to nonparticipation. Methods: Residents were recruited from three rural regions of Korea that lack screening units for the National Cancer Screening Program. A package containing a FIT kit for stool self-sampling and a return envelope addressed to the local health center was postally distributed to each subject. Thirty days after the kits were mailed, nonresponders were reminded via telephone as the second intervention. The participation rates and odds ratios with 95% confidence intervals (CIs) for each intervention response were calculated to evaluate the effect of the interventions and factors related to screening participation in response to the interventions. Results: CRC screening participation rates increased from 24.5% (95% CI, 21.6% to 27.4%) to 42.6% (95% CI, 39.3% to 46.0%) as a result of postal screening and increased further to 51.4% (95% CI, 48.0% to 54.9%) after the telephone reminder. After controlling for the sex, age, and household type of each subject, factors associated with poor response to postal screening were identified as low educational attainment and poor previous participation in the National Cancer Screening Program. Conclusions: Round-mailed FIT kits with phone call reminders were an effective intervention, nearly doubling the screening rate in underserved regions of Korea.

Rapid, simple, and inexpensive diagnostic point-of-care tests (POCTs) are essential for controlling infectious diseases in resource-limited settings. In this study, we developed a new detection system based on nanoparticle–DNA aggregation (STat aggregation of tagged DNA, STAT-DNA) to yield a visual change that can be easily detected by the naked eye. This simplified optical detection system was applied to detect hepatitis C virus (HCV). Reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers labeled with biotin and digoxigenin. Streptavidin-coated magnetic particles (1 μm) and anti-digoxigenin antibody-coated polystyrene particles (250–350 nm) were added to form aggregates. The limit of detection (LoD) and analytical specificity were analyzed. The STAT-DNA results were compared with those of the standard real-time PCR assay using serum samples from 54 patients with hepatitis C. We achieved visualization of amplified DNA with the naked eye by adding nanoparticles to the PCR mixture without employing centrifugal force, probe addition, incubation, or dilution. The LoD of STAT-DNA was at least 101 IU/mL. STAT-DNA did not show cross-reactivity with eight viral pathogens. The detection using STAT-DNA was consistent with that using standard real-time PCR.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the simple, quick, and low-cost detection of various viral genes. LAMP assays are susceptible to generating non-specific amplicons, as high concentrations of DNA primers can give rise to primer dimerization and mismatched hybridizations, resulting in false-positive signals. Herein, we reported that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly enhance the specificity of LAMP, owing to its ability to adsorb single-stranded DNA (ssDNA). By adsorbing surplus ssDNA primers, PEG-nGO minimizes the non-specific annealing of ssDNAs, including erroneous priming and primer dimerization, leading to the enhanced specificity of LAMP. The detection of complementary DNAs transcribed from the hepatitis C virus (HCV) RNA was performed by the PEG-nGO-based LAMP. We observed that the inclusion of PEG-nGO significantly enhances the specificity and sensitivity of the LAMP assay through the augmented difference in fluorescence signals between the target and non-target samples. The PEG-nGO-based LAMP assay greatly facilitates the detection of HCV-positive clinical samples, with superior precision to the conventional quantitative real-time PCR (RT-qPCR). Among the 20 clinical samples tested, all 10 HCV-positive samples are detected as positive in the PEG-nGO-based LAMP, while only 7 samples are detected as HCV-positive in the RT-qPCR. In addition, the PEG-nGO-based LAMP method significantly improves the detection precision for the false-positive decision by 1.75-fold as compared to the LAMP without PEG-nGO. Thus, PEG-nGO can significantly improve the performance of LAMP assays by facilitating the specific amplification of target DNA with a decrease in background signal.

DDX41 mutations are associated with hematologic malignancies including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but the incidence in idiopathic cytopenia of undetermined significance (ICUS) is unknown. We investigated the incidence, genetic characteristics, and clinical features of DDX41 mutations in Korean patients with ICUS, MDS, or AML. We performed targeted deep sequencing of 61 genes including DDX41 in 457 patients with ICUS (n=75), MDS (n=210), or AML (n=172). Germline DDX41 mutations with causality were identified in 28 (6.1%) patients, of whom 27 (96.4%) had somatic mutations in the other position of DDX41. Germline origins of the DDX41 mutations were confirmed in all of the 11 patients in whom germline-based testing was performed. Of the germline DDX41 mutations, p.V152G (n=10) was most common, followed by p.Y259C (n=8), p.A500fs (n=6), and p.E7* (n=3). Compared with non-mutated patients, patients with a DDX41 mutation were more frequently male, older, had a normal karyotype, low leukocyte count, and hypocellular marrow at diagnosis. Three of the four ICUS patients with germline DDX41 mutations progressed to MDS. The incidence of DDX41 mutations in Korean patients was high and there was a distinct mutation pattern, in that p.V152G was a unique germline variant. ICUS harboring germline DDX41 mutations may be regarded as a hereditary myeloid neoplasm. Germline DDX41 mutations are not uncommon and should be explored when treating patients with myeloid malignancies.

Background and objectives: The ABO antibody (Ab) titration tests are used in monitoring in ABO-incompatible (ABOi) solid organ transplantation (SOT). However, currently developed ABO Ab tests show Ab binding reactions. This study attempted to measure ABO Ab level using complement-dependent cytotoxicity (CDC). Materials and methods: We studied 93 blood group O serum samples from patients who underwent ABOi SOT from January 2019 to May 2021. Patients’ sera were incubated with A1 or B cells and added to a human complement solution. Supernatants were collected after centrifugation, and free hemoglobin (Hb) was measured by spectrophotometry. We converted plasma Hb value to hemolysis (%), which were compared with ABO Ab titer. Results: We found a mild correlation between hemolysis and ABO Ab titers. In simple regression analysis, the correlation coefficients were within 0.3660–0.4968 (p < 0.0001) before transplantation. In multiple linear regression analysis, anti-A hemolysis (%) was higher in immunoglobulin M (IgM) (β = 12.9) than in immunoglobulin G (IgG) (β = −3.4) (R2 = 0.5216). Anti-B hemolysis was higher in IgM (β = 8.7) than in IgG (β = 0.0) (R2 = 0.5114). There was a large variation in hemolysis within the same Ab titer. Conclusions: CDC can be used in a new trial for ABO Ab measurement. Furthermore, IgM rather than IgG seems to play a significant role in vivo activity, consistent with previous knowledge. Thus, this study may help in the development of the ABO Ab titration supplement test for post-transplant treatment policy establishment and pre-transplant desensitization.

Abstract Background Identifying preferences for stool collection devices may help increase uptake rates for colorectal cancer screening via fecal immunochemical test (FIT). This study surveyed satisfaction with different devices utilized to collect stool samples for FIT: a conventional container and a sampling bottle (Eiken OC-Sensor). Methods This cross-sectional study was conducted at the National Cancer Center, Korea. Participants aged 50–74 years who used either a conventional container or a sampling bottle to collect a stool sample for FIT were asked to complete a questionnaire designed to survey their satisfaction with the stool collection process and their intentions to undergo FIT in subsequent screening rounds. In total, 1657 participants (1224 conventional container, 433 sampling bottle) were included for analysis. Results Satisfaction with the sampling bottle was higher than that with the conventional container (79.9% vs.73.0%, p = 0.005, respectively; aOR = 1.52, 95% CI: 1.16–2.00). Participants satisfied with the sampling bottle were more likely to be female, be of younger age (50–64 years old), have higher household income, and have prior experience with FIT. Intentions to undergo subsequent screening were stronger among those given the sampling bottle than those given the conventional container (aOR = 1.78, 95% CI: 1.28–2 .48). Conclusions Satisfaction with the stool collection process was higher with the sampling bottle. However, additional studies are needed to validate whether the increased satisfaction and stronger intentions to undergo subsequent screening with the sampling bottle could actually lead to increased uptake in subsequent rounds, along with analysis of the device’s cost effectiveness.

One of the most effective strategies for eliminating new and emerging infectious diseases is effective immunization. The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) warrants the need for a maximum coverage vaccine. Moreover, mutations that arise within the virus have a significant impact on the vaccination strategy. Here, we built a comprehensive in silico workflow pipeline to identify B-cell- and T-cell-stimulating antigens of SARS-CoV-2 viral proteins. Our in silico reverse vaccinology (RV) approach consisted of two parts: (1) analysis of the selected viral proteins based on annotated cellular location, antigenicity, allele coverage, epitope density, and mutation density and (2) analysis of the various aspects of the epitopes, including antigenicity, allele coverage, IFN-γ induction, toxicity, host homology, and site mutational density. After performing a mutation analysis based on the contemporary mutational amino acid substitutions observed in the viral variants, 13 potential epitopes were selected as subunit vaccine candidates. Despite mutational amino acid substitutions, most epitope sequences were predicted to retain immunogenicity without toxicity and host homology. Our RV approach using an in silico pipeline may potentially reduce the time required for effective vaccine development and can be applicable for vaccine development for other pathogenic diseases as well.

Background/AimsWe are in the process of conducting a randomized trial to determine whether compliance with the fecal immunochemical test (FIT) for colorectal cancer screening differs according to the stool-collection method. This study was an interim analysis of the performance of two stool-collection devices (sampling bottle vs conventional container).Methods : In total, 1,701 individuals (age range, 50 to 74 years) were randomized into the sampling bottle group (intervention arm) or the conventional container group (control arm). In both groups, we evaluated the FIT positivity rate, the positive predictive value for advanced neoplasia, and the detection rate for advanced neoplasia.Results : The FIT positivity rates were 4.1% for the sampling bottles and 2.0% for the conventional containers; these values were significantly different. The positive predictive values for advanced neoplasia in the sampling bottles and conventional containers were 11.1% (95% confidence interval [CI], −3.4 to 25.6) and 12.0% (95% CI, −0.7 to 24.7), respectively. The detection rates for advanced neoplasia in the sampling bottles and conventional containers were 4.5 per 1,000 persons (95% CI, 2.0 to 11.0) and 2.4 per 1,000 persons (95% CI, 0.0 to 5.0), respectively.Conclusion : sThe impact of these findings on FIT screening performance was unclear in this interim analysis. This impact should therefore be evaluated in the final analysis following the final enrollment period.

Ginsenosides are the principal active components of ginseng and are considered attractive candidates for combination cancer therapy because they can kill tumors and have favorable safety profiles. However, the overall benefit of ginsenosides remains unclear, particularly in cancer immunosurveillance, considering the controversial results showing repression or promotion of immune responses. Here we identify a potentiating role of ginsenoside F1 (G-F1) in cancer surveillance by natural killer (NK) cells. Among 15 different ginsenosides, G-F1 most potently enhanced NK cell cytotoxicity in response to diverse activating receptors and cancer cells. G-F1 also improved cancer surveillance in mouse models of lymphoma clearance and metastatic melanoma that rely on NK cell activity. G-F1-treated NK cells exhibited elevated cytotoxic potential such as upregulation of cytotoxic mediators and of activation signals upon stimulation. NK cell potentiation by G-F1 was antagonized by insulin-like growth factor (IGF)-1 blockade and recapitulated by IGF-1 treatment, suggesting the involvement of IGF-1. Thus, our results suggest that G-F1 enhances NK cell function and may have chemotherapeutic potential in NK cell-based immunotherapy. We anticipate our results to be a starting point for further comprehensive studies of ginsenosides in the immune cells mediating cancer surveillance and the development of putative therapeutics.

Recently, because the quality of laboratory analyses has increased along with the need for quality improvement, several external quality control bodies have adapted performance specifications using the Desirable Biological Variation Database, termed "Ricos goals"; these criteria are more stringent than those presented in CLIA 88. In this study, we aimed to validate newly introduced serum separator tubes, Improvacutor, for routine clinical chemistry testing in accordance with Ricos goals and CLIA 88. Blood samples were collected from 100 volunteers into three types of serum vacuum tubes: Greiner Vacuette, Becton Dickinson (BD) Vacutainer, and Improve Improvacutor. The samples were subjected to 16 routine chemistry tests using a TBA-200fr NEO chemistry autoanalyzer. In the comparison analysis, all 16 test results were acceptable according to CLIA 88. However, in the comparison of Improve and BD tubes, creatinine showed 4.31% (+0.08 μmol/L) bias. This slightly exceeded the Desirable Specification for Inaccuracy Ricos limit of ±3.96%, but still satisfied the CLIS88 limit of ±26.52 μmol/L. The remaining 15 analytes performed acceptably according to the Desirable Specifications of Ricos. The correlation coefficient of 12 analytes was greater than 0.95 in Passing-Bablok regression analysis among the three tubes, but was lower for four analytes: calcium, sodium, potassium, and chloride. In the stability assay, only potassium tested in the Greiner tube revealed a larger positive bias (2.18%) than the Ricos Desirable Specification for Inaccuracy based on biologic variation (1.8%). The BD tube also showed a positive bias of 1.74%, whereas the new Improve tube showed the smallest positive bias of 1.17% in potassium level after 72 h storage. Thus, the results of this study demonstrate that recently introduced analytical performance specifications based on components of biological variation (Rico's goal) could be extended to criterion for performance evaluation and applied.

There is a growing body of evidence demonstrating an association between smoking and DNA methylation. Accordingly, DNA methylation is now considered a promising biomarker of smoking exposure. We evaluated the relationship between methylation markers (AHRR and F2RL3) and urine cotinine as well as self-reported smoking status. DNA methylation levels of AHRR and F2RL3 in blood as well as urine cotinine were measured in 330 adults (46 to 87 years of age). Pyrosequencing was performed to measure DNA methylation of AHRR and F2RL3 associated with smoking exposure. The lung cancer risk associated with DNA methylation and urine cotinine was analyzed using logistic regression analysis. The AHRR and F2RL3 genes were significantly hypomethylated in current smokers compared to in individuals who have never smoked. An inverse relationship was observed between urine cotinine and methylation levels. Methylation of AHRR and F2RL3 distinguished current smokers from never-smokers with high accuracy. Logistic multivariate analysis showed that AHRR methylation is significantly associated with the risk of lung cancer (OR = 0.96, P = 0.011). Our study validated the smoking-associated DNA methylation markers reported in a Korean population-based cohort. In conclusion, DNA methylation of AHRR and F2RL3 provided accurate measures for smoking exposure. Methylation markers reflecting the long-term effect of smoking on the risk of lung cancer showed better performance in distinguishing former smokers from never-smokers.

Passive exposure to tobacco smoke significantly contributes to morbidity and mortality in children. Children, in particular, seem to be the most susceptible population to the harmful effects of environmental tobacco smoke (ETS). Paternal smoking inside the home leads to significant maternal and fetal exposure to ETS and may subsequently affect fetal health. ETS has been associated with adverse effects on pediatric health, including preterm birth, intrauterine growth retardation, perinatal mortality, respiratory illness, neurobehavioral problems, and decreased performance in school. A valid estimation of the risks associated with tobacco exposure depends on accurate measurement. Nicotine and its major metabolite, cotinine, are commonly used as smoking biomarkers, and their levels can be determined in various biological specimens such as blood, saliva, and urine. Recently, hair analysis was found to be a convenient, noninvasive technique for detecting the presence of nicotine exposure. Because nicotine/cotinine accumulates in hair during hair growth, it is a unique measure of long-term, cumulative exposure to tobacco smoke. Although smoking ban policies result in considerable reductions in ETS exposure, children are still exposed significantly to tobacco smoke not only in their homes but also in schools, restaurants, child-care settings, cars, buses, and other public places. Therefore, more effective strategies and public policies to protect preschool children from ETS should be consolidated.

Graphene oxide (GO) has proven to be a satisfactory DNA-sensor platform for applications in enzyme-free signal amplification, fluorescence-based amplification, and nanoparticle-based platforms because of its excellent electrical, thermal, and optical properties. In this study, we designed a novel platform for the fluorescence detection of biomolecules, using a fluorescent dye-labeled primer and GO. We applied this system for the detection of the IS6110 insertion sequence of the Mycobacterium tuberculosis complex (MTB) and evaluated its feasibility for use in molecular diagnostics. Fifty-four sputum specimens were collected at our institution from October 2010 to March 2012. To detect MTB in the samples, we performed PCR amplification of the IS6110 DNA sequence using FAM-labeled primers, after which the PCR amplicon was incubated with GO and the fluorescence was measured. The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR). The fluorescence intensity observed increased in a concentration-dependent manner with the FAM-labeled IS6110 amplicon. The results of the PCR-GO system for detecting IS6110 DNA were in good agreement with those obtained with conventional RQ-PCR (kappa statistic = 0.925). The PCR-GO system detected MTB DNA in 23 of 25 RQ-PCR-positive sputum samples (92.0%; 95% CI, 75.0-98.0%), but not in 29 of 29 RQ-PCR-negative sputum samples (100%; 95% CI, 88.1-100.0%). These results indicate the utility of the PCR-GO system in molecular diagnostics.

Nanoparticles (NPs) are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. Photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. Nucleic acids detection based on upconversion nanoparticles (UCNPs), which display a high signal-to-noise ratio and no photobleaching, has been widely applied. We evaluated whether UCNPs can improve polymerase chain reaction (PCR) specificity and affect PCR amplification. The effects of UCNPs with a diameter size of 40, 70, and 250 nm were evaluated using 3 PCR kits (AccuPower PCR PreMix, AmpliTaq Gold 360 Master Mix, and HotStarTaq Plus Master Mix) and 3 real-time PCR kits (AccuPower GreenStar qPCR PreMix, SYBR Green PCR Master Mix, and QuantiTect SYBR Green PCR Kit). Quantum dots were used for comparison with the UCNPs. In the presence of an appropriate concentration of UCNPs, PCR specificity was optimized. UCNPs of 40-nm size improved PCR specificity more effectively than did UCNPs sized 70 or 250 nm. As the size and concentrations of the UCNPs were increased, PCR amplification was more severely inhibited. At lower annealing temperatures (25°C-45°C), addition of the 40 nm UCNP (1 µg/µL) to the PCR reagent produced specific PCR products without nonspecific sequence amplification. Therefore, UCNPs of different sizes, with different DNA polymerases used in the commercial kits, showed different inhibitory effects on PCR amplification. These results demonstrate that optimization of UCNPs, added to reaction mixtures at appropriate concentrations, can improve PCR specificity. However, the mechanism underlining UCNPs effect on PCR remains unclear and will require further investigation.

Background: Flow cytometric immunophenotyping of hematolymphoid neoplasms (FCI-HLN) is essential for diagnosis, classification, and minimal residual disease (MRD) monitoring. FCI-HLN is typically performed using in-house protocols, raising the need for standardization. Therefore, we surveyed the current status of FCI-HLN in Korea to obtain fundamental data for quality improvement and standardization. Methods: Eight university hospitals actively conducting FCI-HLN participated in our survey. We analyzed responses to a questionnaire that included inquiries regarding test items, reagent antibodies (RAs), fluorophores, sample amounts (SAs), reagent antibody amounts (RAAs), acquisition cell number (ACN), isotype control (IC) usage, positive/negative criteria, and reporting. Results: Most hospitals used acute HLN, chronic HLN, plasma cell neoplasm (PCN), and MRD panels. The numbers of RAs were heterogeneous, with a maximum of 32, 26, 12, 14, and 10 antibodies used for acute HLN, chronic HLN, PCN, ALL-MRD, and multiple myeloma-MRD, respectively. The number of fluorophores ranged from 4 to 10. RAs, SAs, RAAs, and ACN were diverse. Most hospitals used a positive criterion of 20%, whereas one used 10% for acute and chronic HLN panels. Five hospitals used ICs for the negative criterion. Positive/negative assignments, percentages, and general opinions were commonly reported. In MRD reporting, the limit of detection and lower limit of quantification were included. Conclusions: This is the first comprehensive study on the current status of FCI-HLN in Korea, confirming the high heterogeneity and complexity of FCI-HLN practices. Standardization of FCI-HLN is urgently needed. The findings provide a reference for establishing standard FCI-HLN guidelines. [ABSTRACT FROM AUTHOR]

Background: Human immunodeficiency virus (HIV) infection presents a major global health challenge. According to the World Health Organization, approximately 39.0 million people have HIV, as of 2022, with 1.3 million new infections annually. In Korea, the first HIV cases were reported in 1985, and the number of cases has increased continuously since 1988. In 2022, 1,066 cases were newly diagnosed, increasing the total number of cases to approximately 15,880. Current Concepts: Diagnostics play a crucial role in the management of HIV, for early diagnosis and initiation of antiretroviral therapy. Approval of the first HIV antibody test in 1985 by the US Food and Drug Administration marked a notable advance in the ability to diagnose HIV infection. The screening test for HIV infection typically consists of a two-step approach, in which a fourth-generation combination HIV-1/2 immunoassay is followed by a confirmatory Western blot. Despite its high accuracy for chronic infections, the window period and the early-stage of HIV infections are problematic, and populations with low HIV prevalence show a low positive predictive value. A false positive result can occur from blood transfusions, vaccinations, and autoantibodies. Novel diagnostic tools, such as nucleic acid testing for viral RNA/DNA and antigen testing for p24 antigen have reduced the window period, allowing for earlier detection and differentiation between HIV-1 and HIV-2. Point- of-care tests are beneficial in resource-limited settings although their lower sensitivity and specificity require additional confirmatory follow-up testing. Discussion and Conclusion: In this review, we provide a historical perspective on the evolution of HIV diagnostics (serological and molecular) and describe the roles of conventional and point-of-care testing within the laboratory diagnostic network. [ABSTRACT FROM AUTHOR]

The adoption of high-sensitivity flow cytometry (HSFC) in routine laboratory settings has been slow owing to concerns regarding the reliability and reproducibility of results. Validation is an essential prerequisite for conducting assays, and implementing the CLSI guidelines has been confusing, primarily because many aspects are not yet established. We aimed to validate an HSFC protocol for detecting follicular helper T (Tfh) cells in a realworld laboratory environment. The analytical validity of the Tfh cell panel was ensured through rigorous testing, including evaluations of precision, stability, carryover, and sensitivity, following the CLSI H62 guidelines. We found that Tfh cells, present in very small numbers in the blood, could be sufficiently detected through HSFC, and concerns about the reliability and reproducibility of the results in real-world laboratories could be solved through systematic validation. Establishing the lower limit of quantification (LLOQ) is a critical step in HSFC evaluations. By selecting an appropriate sample, for example, collecting residual cells from CD4 isolation in our experiment and using them as low-level samples, the LLOQ could be accurately established. The strategic validation of flow cytometry panels can facilitate the adoption of HSFC in clinical laboratories, even with limited resources. [ABSTRACT FROM AUTHOR]