Establishment of an in‐house real‐time RT‐PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource‐limited country

BackgroundThe COVID‐19 pandemic caused by SARS‐CoV‐2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real‐time RT‐PCR are critical for the detection of SARS‐CoV‐2 in clinical specimens from patients suspected of COVID‐19.ObjectiveWe aimed to establish and validate an in‐house real‐time RT‐PCR for the detection of SARS‐CoV‐2.MethodologyPrimers and probes sets in our in‐house real‐time RT‐PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real‐time RT‐PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance.ResultsThe limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of C _t values observed between two tests with r ² = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively.ConclusionIn the present study, we established and validated an in‐house real‐time RT‐PCR for molecular detection of SARS‐CoV‐2 in a resource‐limited country.     

Evaluation of conventional and point‐of‐care real‐time RT‐PCR tests for the detection of SARS‐CoV‐2 through a pooled testing strategy

BackgroundThe rapid identification and isolation of individuals infected with SARS‐CoV‐2 are fundamental countermeasures for the efficient control of the COVID‐19 pandemic, which has affected millions of people around the world. Real‐time RT‐PCR is one of the most commonly applied reference methods for virus detection, and the use of pooled testing has been proposed as an effective way to increase the throughput of routine diagnostic tests. However, the clinical applicability of different types of real‐time RT‐PCR tests in a given group size remains inconclusive due to inconsistent regional disease prevalence and test demands.MethodsIn this study, the performance of one dual‐target conventional and two point‐of‐care real‐time RT‐PCR tests in a 5‐specimen pooled testing strategy for the detection of SARS‐COV‐2 was evaluated.ResultsWe demonstrated the proof of concept that all of these real‐time RT‐PCR tests could feasibly detect SARS‐CoV‐2 from nasopharyngeal and oropharyngeal specimens that contain viral RNA loads in the range of 3.48 × 10⁵ to 3.42 × 10² copies/ml through pooled testing in a group size of 5 with overall positive percent agreement (pooling vs. individual testing) ranging from 100% to 93.75%. Furthermore, the two POC real‐time RT‐PCR tests exhibited comparable sensitivity to that of the dual‐target conventional one when clinical specimens were tested individually.ConclusionOur findings support the feasibility of using real‐time RT‐PCR tests developed as a variety of platforms in routine laboratory detection of suspected COVID‐19 cases through a pooled testing strategy that is beneficial to increasing the daily diagnostic capacity.     

Establishing an internal quality control method for the stable extraction of nucleic acids of severe acute respiratory syndrome coronavirus 2 and RT‐PCR‐based detection

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the causative agent of the coronavirus disease 2019 (COVID‐19), is detected using real‐time RT‐PCR. However, there are limitations pertaining to quality control, particularly with respect to establishing quality control measures for extraction of viral nucleic acids. Here, we investigated the quality control measures for the various processes using an extrinsic quality control substance and quality control charts.MethodsAn extrinsic quality control substance was added to the sample, and then, real‐time RT‐PCR was performed. Samples with negative test results and the corresponding data were analyzed; a quality control chart was created and examined.ResultsData analysis and the quality control charts indicated that SARS‐CoV‐2 could be reliably detected using real‐time RT‐PCR, even when different nucleic acid extraction methods were used or when different technicians were employed.ConclusionWith the use of quality control substances, it is possible to achieve quality control throughout the process—from nucleic acid extraction to nucleic acid detection—even upon using varying extraction methods. Further, generating quality control charts would guarantee the stable detection of SARS‐CoV‐2.     

Comparison and optimization of various moving patient‐based real‐time quality control procedures for serum sodium

BackgroundPatient‐based real‐time quality control (PBRTQC) is a valuable tool for monitoring the performance of testing processes. We aimed to compare and optimize various PBRTQC procedures for serum sodium.MethodsIn a computer simulation, artificial errors were added to 680,000 real patients’ results. The characteristics of error detection of various algorithms—moving average, moving median, moving SD and moving proportion of normal results including different control limits (CLs)—were assessed on their ability to detect critical errors early.ResultsThe moving average and moving median were sensitive to system error, and the moving SD tended to detect random error. P_3SD (moving proportion of normal results, CLs based on mean and SD of proportion of normal results) demonstrated excellent performance for both system error and random error. The increase of block sizes (N) leads to the delay of error detection and the decrease of false rejection, except for QC procedures with minimum and maximum as CLs. CLs calculation with “0.1% false alarm rate” had more effective performance than that set false alarm to zero (minimum and maximum as CLs). The impact of truncation on QC performance depended on truncation limits, algorithms and the types of error. The significant improvement in QC performance due to truncation was only found in moving SD.Conclusion“P_3SD,N = 50, without truncation” and “moving SD, N = 25, set 0.1% false alarm as CLs and set 1% outliers exclusion as truncation limits” were recommended as the optimized procedures for serum sodium to monitor system error and random error, respectively.     

Assessment of patient based real‐time quality control on comparative assays for common clinical analytes

BackgroundIt is critical for laboratories to conduct multianalyzer comparisons as a part of daily routine work to strengthen the quality management of test systems. Here, we explored the application of patient‐based real‐time quality controls (PBRTQCs) on comparative assays to monitor the consistency among clinical laboratories.MethodsThe present study included 11 commonly tested analytes that were detected using three analyzers. PBRTQC procedures were set up with exponentially weighted moving average (EWMA) algorithms and evaluated using the AI‐MA artificial intelligence platform. Comparative assays were carried out on serum samples, and patient data were collected. Patients were divided into total patient (TP), inpatient (IP), and outpatient (OP) groups.ResultsOptimal PBRTQC protocols were evaluated and selected with appropriate truncation limits and smoothing factors. Generally, similar comparative assay performance was achieved using both the EWMA and median methods. Good consistency between the results from patients'' data and serum samples was obtained, and unacceptable bias was detected for alkaline phosphatase (ALP) and gamma‐glutamyl transferase (GGT) when using analyzer C. Categorizing patients'' data and applying specific groups for comparative assays could significantly improve the performance of PBRTQCs. When monitoring the inter‐ and intraanalyzer stability on a daily basis, EWMA was superior in detecting very small quality‐related changes with lower false‐positive alarms.ConclusionsWe found that PBRTQCs have the potential to efficiently assess multianalyzer comparability. Laboratories should be aware of population variations concerning both analytes and analyzers to build more suitable PBRTQC protocols.     

A case report of prolonged COVID‐19‐positive RT‐PCR for five months

The COVID‐19 gold standard assessment tool remained the RT‐PCR of upper respiratory tract specimen extracted by the nasopharyngeal swab. A positive result would decrease through a three‐week course and eventually be undetectable. The maximum duration of viral shedding is 83 days. Besides, COVID‐19 RT‐PCR remained positive for 74 days in a patient suffering from lymphoma. In this study, we have presented a 56‐year‐old male patient, a known case of lymphoma since 2015, who experienced many episodes of chemotherapy with a five‐month positive RT‐PCR COVID‐19 laboratory test and finally was intubated and then died of opportunistic pulmonary infections. COVID‐19 patients with concurrent lymphoma failed to remove the virus thoroughly, despite providing appropriate treatment regimens.     

Smaller reaction volume of triplex taqman real‐time reverse transcription‐PCR assays for diagnosing coronavirus disease 2019

BackgroundCoronavirus disease 2019 (COVID‐19) has had a devastating impact on public health services worldwide. Currently, there are no standard remedies or therapies for COVID‐19. it is important to identify and diagnose COVID‐19 to control the spread. But clinical symptoms of COVID‐19 are very similar to those of other respiratory viruses.ResultsAs a result, the diagnosis of COVID‐19 relies heavily on detecting pathogens. We established a bunch of triplex new TaqMan real‐time PCR assays. Three sets of primers and probes (targeting the ORF1ab, N, and E genes, respectively) are poorly consistent with other human coronaviruses and the human influenza virus. The sensitivity of established PCR assays notices as few as 100 copies per PCR of the ORF1ab, N, and E genes. Meanwhile, standard curves concluded from constant PCR reaction all showed glorious linear correlations between Ct values and the polymer loading copy variety (correlation coefficient (R²) of ORF1ab, N, and E genes is 0.996, 0.991, and 0.998, respectively). Surveillance of RNA‐based pseudovirus demonstrated that they were identified to be positive with respect to SARS‐CoV‐2 and that established PCR assays are achievable.ConclusionThe assays established provide a smaller reaction volume for diagnosing COVID‐19.     

Allergen immunotherapy in MASK‐air users in real‐life: Results of a Bayesian mixed‐effects model

BackgroundEvidence regarding the effectiveness of allergen immunotherapy (AIT) on allergic rhinitis has been provided mostly by randomised controlled trials, with little data from real‐life studies.ObjectiveTo compare the reported control of allergic rhinitis symptoms in three groups of users of the MASK‐air^® app: those receiving sublingual AIT (SLIT), those receiving subcutaneous AIT (SCIT), and those receiving no AIT.MethodsWe assessed the MASK‐air^® data of European users with self‐reported grass pollen allergy, comparing the data reported by patients receiving SLIT, SCIT and no AIT. Outcome variables included the daily impact of allergy symptoms globally and on work (measured by visual analogue scales—VASs), and a combined symptom‐medication score (CSMS). We applied Bayesian mixed‐effects models, with clustering by patient, country and pollen season.ResultsWe analysed a total of 42,756 days from 1,093 grass allergy patients, including 18,479 days of users under AIT. Compared to no AIT, SCIT was associated with similar VAS levels and CSMS. Compared to no AIT, SLIT‐tablet was associated with lower values of VAS global allergy symptoms (average difference = 7.5 units out of 100; 95% credible interval [95%CrI] = −12.1;−2.8), lower VAS Work (average difference = 5.0; 95%CrI = −8.5;−1.5), and a lower CSMS (average difference = 3.7; 95%CrI = −9.3;2.2). When compared to SCIT, SLIT‐tablet was associated with lower VAS global allergy symptoms (average difference = 10.2; 95%CrI = −17.2;−2.8), lower VAS Work (average difference = 7.8; 95%CrI = −15.1;0.2), and a lower CSMS (average difference = 9.3; 95%CrI = −18.5;0.2).ConclusionIn patients with grass pollen allergy, SLIT‐tablet, when compared to no AIT and to SCIT, is associated with lower reported symptom severity. Future longitudinal studies following internationally‐harmonised standards for performing and reporting real‐world data in AIT are needed to better understand its ‘real‐world’ effectiveness.     

The sensitivity and specificity of COVID‐19 rapid anti‐gene test in comparison to RT‐PCR test as a gold standard test

BackgroundCoronavirus disease 2019 (COVID‐19) is a modern infectious disease, first identified in December 2019 in Wuhan, China. The etiology is via severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), in a pandemic manner. The study aimed to compare between RT‐PCR and rapid anti‐gene tests for COVID‐19 with regard to sensitivity and specificity.MethodsThis is a cohort hospital‐based study done during the period of July to September 2020. Both rapid anti‐gene test kit (SARS‐CoV‐2) and RT‐qPCR were used for the detection of COVID‐19 in suspected cases.ResultsA total of 148 cases were tested using both the RT‐qPCR and rapid test. Twenty‐nine (19.6%) of these cases had positive results for RT‐qPCR and 119 (80.4%) were negative, whereas 52 (35.1%) patients were positive to rapid anti‐gene test and 96 (64.9%) of them negative. The sensitivity of the rapid test was 37.9%, the specificity was 65.5% and the accuracy was 64.44%. Rapid IgG test was positive in 47 (31.8) of cases. Although, rapid IgM test was positive in 18 (12.2%). The rapid IgG test was more sensitive than rapid IgM (Sensitivity 34.48% vs. 3.45%), but it was less specific than rapid IgM test (Specificity 68.91% vs. 85.71%).ConclusionWe cannot consider rapid anti‐gene test alone as a diagnostic method for COVID‐19. We should also conduct RT‐PCR test and other investigations like imaging CT scan of chest to confirm the diagnosis. The rapid IgG test is more sensitive than rapid IgM, but it was less specific.     

Impact of cytotoxic T‐lymphocyte‐associated protein 4 codon 17 variant and expression on vitiligo risk

BackgroundCytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) is one of the essential brakes expressed on T cells that prevent T‐cell hyperactivation‐associated autoimmune disorders. Several CTLA4 polymorphisms were implicated in the regulation of gene expression. We aimed to explore the association of CTLA4 expression and rs231775 (c.49A>G) variant with vitiligo risk and severity of the disease in a sample of the Middle Eastern population.MethodsThe CTLA4 gene expression and genotyping for rs231775 (A/G) variant were assessed in 161 vitiligo patients and 165 controls using a real‐time polymerase chain reaction. Vitiligo Area Severity Index (VASI) and Vitiligo Disease Activity score (VIDA) were evaluated.ResultsA higher frequency of rs231775 G allele was observed in vitiligo cases than controls (45% vs. 33%, p = 0.002). After adjustment of age, sex, family history of vitiligo, and CTLA expression level, using multivariate analysis, G/G carriers were associated with a higher risk of vitiligo under recessive (OR = 2.94, 95% CI = 1.61–5.35, p < 0.001), dominant (OR = 1.87, 95% CI = 1.14–3.06, p = 0.013), and homozygote comparison (OR = 3.34, 95% CI = 1.73–6.42, p = 0.001) models. Although the CTLA4 relative expression levels were comparable to that of controls, G/G carriers exhibited a significantly lower expression profile (median = 0.63, IQR = 0.34–1.75) than A/A (median = 1.43, IQR = 0.39–4.25, p = 0.018) and A/G carriers (median = 1.68, IQR = 0.49–3.92, p = 0.007). No significant associations of CTLA4 variant/expression with disease severity and/or activity were observed.ConclusionThe CTLA4 rs231775 variant was associated with vitiligo susceptibility and gene expression; the risky genotype (GG) was associated with lower CTLA4 relative expression levels than the other genotypes. Further large‐scale studies in different populations are warranted.     

Development and evaluation of time‐resolved fluorescent immunochromatographic assay for quantitative detection of SARS‐CoV‐2 spike antigen

BackgroundThe spread of COVID‐19 worldwide caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has necessitated efficient, sensitive diagnostic methods to identify infected people. We report on the development of a rapid 15‐minute time‐resolved fluorescent (TRF) lateral flow immunochromatographic assay for the quantitative detection of the SARS‐CoV‐2 spike protein receptor‐binding domain (S1‐RBD).ObjectivesOur objective was to develop an efficient method of detecting SARS‐CoV‐2 within 15 min of sample collection.MethodsWe constructed and evaluated a portable, disposable lateral flow device, which detected the S1‐RBD protein directly in nasopharyngeal swab samples. The device emits a fluorescent signal in the presence of S1‐RBD, which can be captured by an automated TRF instrument.ResultsThe TRF lateral flow assay signal was linear from 0 to 20 ng/ml and demonstrated high accuracy and reproducibility. When evaluated with clinical nasopharyngeal swabs, the assay was performed at >80% sensitivity, >84% specificity, and > 82% accuracy for detection of the S1‐RBD antigen.ConclusionThe new S1‐RBD antigen test is a rapid (15 min), sensitive, and specific assay that requires minimal sample preparation. Critically, the assay correlated closely with PCR‐based methodology in nasopharyngeal swab samples, showing that the detected S1‐RBD antigen levels correlate with SARS‐CoV‐2 virus load. Therefore, the new TRF lateral flow test for S1‐RBD has potential application in point‐of‐care settings.     

检测EV71和EV-U的TaqMan-LNA探针多重荧光RT-PCR的建立

目的 建立一种针对手足口病病原体的快速准确检测方法。 方法 利用TaqMan-LNA探针建立多重荧光定量RT-PCR方法,同时检测肠道病毒71型(EV71)和肠道病毒通用型(EV-U),用含有目的序列RNA的标准品制备标准曲线,对病毒进行准确定量,同时对该方法的特异性、灵敏度、重复性进行评估。结果 用TaqMan-LNA探针多重荧光RT-PCR法检测CoxA16、CoxB2、EV71病毒和其他人类病毒RNA,证实其特异性为100%;对EV71和EV-U的检测灵敏度均达到10^{2拷贝/μl;将EV71和EV-U的RNA标准品进行重复性实验,其批内、批间变异系数分别为0.97%～2.02%和0.89%～2.13%。 结论 本方法灵敏度较高,特异性强,重复性好,适用于手足口病的临床检测。}     

Accompanying a semi‐nested PCR assay to support histopathology findings of fungal keratitis in formalin‐fixed paraffin‐embedded corneal samples

BackgroundFungal species are responsible for 40%–50% of all microbial keratitis cases. Due to the low amount of extracted DNA in ocular Formalin‐fixed Paraffin‐embedded (FFPE) samples, selecting a reliable molecular method is a substantial issue in this field.MethodsSixty‐six samples were collected via the penetrating keratoplasty (PK) technique. Histopathology assays were performed using hematoxylin–eosin (H&E) and periodic acid Schiff (PAS) staining methods. The ITS1/ITS4 and ITS1/ITS2 primer pairs were used in a semi‐nested polymerase chain reaction (PCR) to target the universal internal transcribed spacer (ITS) region. Some PCR results were validated through sequencing.ResultsFungal DNA was detected in 44 of 66 samples (66.7%), and histopathology was positive for 41 of 66 samples (62.1%). Of 41 histopathologically proven fungal‐positive cases, 39 were PCR‐positive (95%). Moreover, of 44 PCR‐positive samples, 39 (88.6%) were histopathology‐positive, and 5 (11.3%) were histopathology‐negative. Totally in 39 cases (59%), both histopathology and PCR yielded positive results. The Kappa agreement rate between the two diagnostic methods, including histopathology and PCR, was 0.77. Sensitivity, specificity, positive predictive value, and false predictive value were reported as 88.64%, 90.9%, 95.12%, and 80%, respectively.ConclusionAs we reached the acceptable Kappa agreement rate, we concluded that applying the semi‐nested PCR assay is a promising method for supporting the evidence by histopathology. Finally, we suggest targeting more specific gene regions using primer pairs that amplify smaller amplicon sizes and surveying novel molecular methods such as NGS to achieve higher sensitivity and Kappa agreement rates.     

Analysis of anti‐apoptotic PVT1 oncogene and apoptosis‐related proteins (p53, Bcl2, PD‐1, and PD‐L1) expression in thyroid carcinoma

BackgroundAn aberrant expression of long non‐coding RNA PVT1 has been associated with apoptosis in various cancer types. We aimed to explore the PVT1 and four apoptosis‐related proteins (p53, Bcl2, and PD‐1/PD‐L1) signature in thyroid cancer (TC).MethodsThe PVT1 expression level was measured in 64 FFPE TC paired samples by real‐time quantitative PCR. Overall and stratified analyses by different clinicopathological features were done. The apoptotic proteins were evaluated by immunohistochemistry staining.ResultsOverall analysis showed significant PVT1upregulation in TC tissues (p < 0.001). Similarly, subgroup analysis by BRAF ^V600E mutation showed consistent results. Lower expression of p53 was associated with mortality (p = 0.001). Bcl2 overexpression was associated with greater tumor size (p = 0.005). At the same time, HCV‐positive cases were associated with repressed Bcl2 expression levels (54.3% in HCV‐negative vs. 6.9% in HCV‐positive cases, p = 0.011). PD‐1 expression was associated with lymph node metastasis (p = 0.004). Enhanced PD‐L1 expression in the tumor was associated with a higher tumor stage, lymphovascular invasion, and mortality risk. Kaplan–Meier curves for overall survival showed that low p53 and high PD‐L1 expressions were associated with lower survival time. The p53‐positive staining is associated with a 90% decreased mortality risk (HR = 0.10, 95%CI = 0.02–0.47, p = 0.001), while patients with high PD‐L1 were five times more likely to die (HR = 4.74, 95%CI = 1.2–18.7, p = 0.027).ConclusionOur results confirm the upregulation of PVT1 in TC. The apoptosis‐related proteins (p53, Bcl2, and PD‐1/PD‐L1) showed different prognostic utility in TC patients; in particular, low p53 and high PD‐L1 expressions associated with low survival times. Further large‐scale and mechanistic studies are warranted.     

Recent patents in allergy and immunology: A quantitative real‐time polymerase chain reaction method for diagnosing asthma and asthma exacerbation

BackgroundAsthma is diagnosed based on a history of the characteristic symptoms and evidence of expiratory airflow limitation. However, asthma diagnosis using the existing tests is associated with a risk of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 spread. In this study, we developed a quantitative real‐time polymerase chain reaction (qRT‐PCR)‐based asthma diagnosis tool.MethodsWe detected nectin‐4 in the plasma of patients with asthma using qRT‐PCR, explored the relationship of clinical variables.Resultsquantitative real‐time polymerase chain reaction revealed that plasma nectin‐4 mRNA levels were higher in asthmatics than controls. These results correlated with lung function.ConclusionsThose results suggest that qRT‐PCR for nectin‐4 may aid asthma diagnosis and monitoring.     

The prognostic values of serum markers in hepatocellular carcinoma after invasive therapies based on real‐world data

Background and aimsHepatocellular carcinoma (HCC) is one of the most common malignancy with poor prognosis, and the mortality rate remains high. More than 70% of HCC patients have recurrence within 5 years after treatment. The purpose of this study is to evaluate the prognostic values of serum markers with retrospective data.MethodsWe applied real‐world data (RWD) to analyze the prognostic values of six serum markers for HCC patients after treatment, including α‐fetoprotein (AFP), α‐fetoprotein‐L3 (AFP‐L3), Golgi protein73 (GP73), alanine aminotransferase (ALT), albumin (ALB), and total bilirubin (TBil). A total of 268 cases were enrolled to analyze recurrence‐free survival (RFS), and 104 cases were used to analyze overall survival (OS).ResultsOur results demonstrated that patients with higher AFP and AFP‐L3 had shorter RFS (p = 0.016 and 0.004), while higher GP73, ALT, and TBil experienced longer RFS (p = 0.000, 0.020, and 0.019). Patients with high‐level GP73, ALT, TBil, and low‐level ALB had significantly higher mortality rate (p=0.035, 0.008, 0.010, and 0.005). Multivariate analysis revealed that GP73 (HR = 1.548, p = 0.001) and ALT (HR = 1.316, p = 0.046) were identified as independent prognostic factors for RFS, ALB (HR = 0.127, p = 0.007), and ALT (HR = 0.237, p = 0.01) were identified as independent prognostic factors for OS. Subgroups analysis showed that GP73 had better prognostic values than other serum markers in early‐stage HCC (p = 0.023).ConclusionsOur study demonstrates that AFP, AFP‐L3, and GP73 can be used as prognostic indicators for predicting the recurrence of HCC, while liver function tests have better survival prediction values. GP73 can act as a promising prognostic marker for early‐stage HCC.     

A study of the moving rate of positive results for use in a patient‐based real‐time quality control program on a procalcitonin point‐of‐care testing analyzer

ObjectiveTo establish an applicable and highly sensitive patient‐based real‐time quality control (PBRTQC) program based on a data model constructed with patients’ results of a procalcitonin point‐of‐care testing (POCT) analyzer.MethodsPatients’ results were retrospectively collected within one year. The Excel software was used to establish quality control (QC) programs of the moving average (MA) and the moving rate of positive results (MR). A Monte Carlo simulation was used to introduce positive and negative biases between 0.01 and 1 ng/ml at random points of the testing data set. Different parameters were used to detect the biases, and the detection efficiency was expressed using the median number of patient samples affected until error detection (MNPed). After comparing the MNPeds of different programs, MA and MR programs with appropriate parameters were selected, and validation plots were generated using MNPeds and maximum number of the patient samples affected (MAX). β curves were generated using the power function of the programs, the performances were compared with that of the conventional QC program.ResultsNeither the conventional QC nor MA program was sensitive to small bias, While MR program can detect the minimum positive bias of 0.06 ng/ml and negative of 0.4 ng/ml at an average daily run size of 10 specimens, with FRs < 1.0%, βs < 1%.ConclusionThe MR program, which is more sensitive to small biases than conventional QC and MA programs, with low FR and β. As such, it can be used as a PBRTQC program with high performance.