Eun Jee Oh

Validation of the Elecsys® HIV combi PT assay for screening and reliable early detection of HIV-1 infection in Asia

BACKGROUND The Elecsys® HIV combi PT assay was developed to allow earlier detection of HIV infection with increased sensitivity and specificity. OBJECTIVES To validate the assay for screening and reliable early detection of HIV-1 infection in Asia. STUDY DESIGN Samples tested reflected those routinely screened in Asia and comprised: HIV-1 antigen lysate (25 samples) and antibody (20 samples) dilutions; seven HIV-1 seroconversion panels (46 samples); 39 patient samples from early infection; 183 known-positive sera; HIV-1 p24 antigen sensitivity panel (seven samples); >500 routine clinical samples per center. The Elecsys® HIV combi PT assay was compared with fourth- (ADVIA Centaur® HIV combo, ARCHITECT® HIV combo, Elecsys® HIV combi) and third-generation (VIRONOSTIKA® HIV Uni-Form II Plus O, Zhuhai Livzon Anti-HIV EIA, Serodia® Particle Agglutination) assays commonly used in the region. RESULTS Overall, the Elecsys® HIV combi PT showed superior or similar sensitivity to the comparators for detecting all subtypes. The assay correctly identified all positive samples, including those taken soon after infection, and detected seroconversion at a similar or shorter time interval than the comparators. The analytical sensitivity of Elecsys® HIV combi PT for HIV-1 p24 antigen was 0.90 IU/mL, which was lower than reported previously. The assay showed good specificity (99.86%) that was superior or equivalent to the other fourth-generation assays tested. CONCLUSIONS These robust data demonstrate the good subtype inclusivity of the Elecsys® HIV combi PT assay and its suitability for screening and reliable early detection of HIV infection in Asia.

Application of Calculated Panel Reactive Antibody Using HLA Frequencies in Koreans

Background Introduction of the Luminex panel reactive antibody (PRA)-single antigen (SA) assay has increased the detection rates of unacceptable antigens in sensitized patients; the calculated PRA (CPRA) level represents the percentage of actual organ donors that express 1 or more of these unacceptable antigens. We developed a CPRA calculator based on the HLA frequencies in Koreans to measure sensitization levels in Korean patients. Methods To develop the calculator, we obtained the HLA-A, HLA-B, and HLA-DR phenotypes of 1,622 Koreans, and compared these with previously reported frequencies in Koreans. Sera from patients awaiting kidney transplantation were tested for HLA antibodies by Luminex PRA-screen, PRA-identification (ID), and PRA-SA assays. The measured %PRA from the PRA-screen (N=55) and PRA-ID (N=71) were compared to the %CPRA for the unacceptable antigens obtained from PRA-SA. Results Phenotype frequencies used for the CPRA calculator agreed with previously reported data. The concordance rates among the 3 PRA methods for the detection of class I and class II antibodies were 76.1-81.8% (kappa, 0.519-0.636) and 72.7-83.6% (0.463-0.650), respectively. For the detection of broadly sensitized sera (>50% or >80%), the concordance rates were over 80%. In sera with 80-100% CPRA, 91.7% and 94.4% of the samples had concordant results (80-100% PRA) in the PRA-screen and PRA-ID assay, respectively. Conclusions Although further clinical studies are required to confirm the benefits of CPRA values, adoption of CPRA analysis based on HLA frequencies in Koreans may be useful for sensitization measurements and organ-allocation algorithms.

JAK2 V617F mutation in myelodysplastic syndrome, myelodysplastic syndrome/myeloproliferative neoplasm, unclassifiable, refractory anemia with ring sideroblasts with thrombocytosis, and acute myeloid leukemia

Background The JAK2 V617F mutation has been noted in the cases of polycythemia vera, essential thrombocythemia, and primary myelofibrosis patients. This mutation occurs less frequently in acute myeloid leukemia (AML) and other hematologic diseases, such as myelodysplastic syndrome (MDS); myelodysplatic syndrome/myeloproliferative neoplasm, unclassifiable (MDS/MPN-U); and refractory anemia with ring sideroblasts with thrombocytosis (RARS-T). Methods Patients diagnosed with hematologic diseases other than MPN who visited Seoul St Marys Hospital from January 2007 to February 2010 were selected. A total of 43 patients were enrolled in this study: 12 MDS, 9 MDS/MPN-U, 7 RARS-T, and 15 AML patients. The diseases were diagnosed according to the 2008 WHO classification criteria. Data obtained from JAK2 V617F mutation analysis and cytogenetic study as well as complete blood count and clinical data were analyzed. Results Of the 43 patients, 6 (13.9%) harbored the JAK2 V617F mutation. The incidence of the JAK2 V617F mutation in each patient group was as follows: 8.3% (1/12), MDS; 22.2% (2/9), MDS/MPN-U; 14.3% (1/7), RARS-T; and 13.3%, (2/15) AML. The platelet count was higher than 450×109/L in 3 of the 6 patients (50%) harboring the JAK2 V617F mutation, and it was in the normal range in the remaining 3 patients. Among the 6 patients, 1 MDS and 1 MDS/MPN-U patients had the 46,XX,del(20)(q11.2) karyotype. Conclusion The JAK2 V617F mutation is associated with an increased platelet count in MDS, MDS/MPN-U, RARS-T, and AML patients. Cytogenetic abnormalities of del(20)(q11.2) occurred in 1/3 of patients with the JAK2 V617F mutation but further studies are required to confirm this association.

Effects of ECM protein mimetics on adhesion and proliferation of chorion derived mesenchymal stem cells.

Background: We evaluated the effects of fibronectin, collagen, cadherin, and laminin based extracellular matrix (ECM) protein mimetics coated with mussel derived adhesive protein (MAP) on adhesion and proliferation of chorionic mesenchymal stem cells (cMSCs). Methods: Human placental chorionic tissues from term third-trimester pregnancies (n=3) were used. The cMSCs were cultured on rationally designed ECM protein mimetics coated with MAP on plastic surfaces with the addition of reduced fetal bovine serum (0.5%, 1% FBS). Adhesion capabilities were monitored by a real time cell analysis system (RTCA) utilizing an impedance method. Proliferation capabilities were monitored by RTCA and MTS assay. Results: Of the ECM protein mimetics tested, GRGDSP(FN) coated surfaces exhibited the highest adhesion and proliferation capabilities on RTCA at FBS concentration of 0.5% and 1%. When 0.5% FBS was added to ECM protein mimetics during the MTS assay, GRGDSP(FN), REDV(FN), and collagen mimetics, GPKGAAGEPGKP(ColI) showed higher cMSCs proliferation compared with the control. When 1% FBS was added, GRGDSP(FN) and TAIPSCPEGTVPLYS(ColIV) showed significant cMSCs proliferation capacity. Conclusions: Fibronectin mimetics, GRGDSP(FN) amino acid sequence showed the highest adhesion and proliferation capabilities. In addition, results from RTCA assessment of cell viability correlated well with the tetrazolium-based MTS assay.

Evaluation of NK cell function by flowcytometric measurement and impedance based assay using real-time cell electronic sensing system.

Although real-time cell electronic sensing (RT-CES) system-based natural killer (NK) cytotoxicity has been introduced, it has not been evaluated using human blood samples. In present study, we measured flowcytometry based assay (FCA) and RT-CES based NK cytotoxicity and analyzed degranulation activity (CD107a) and cytokine production. In 98 healthy individuals, FCA with peripheral blood mononuclear cells (PBMCs) at effector to target (E/T) ratio of 32 revealed 46.5 ± 2.6% cytolysis of K562 cells, and 23.5 ± 1.1% of NK cells showed increased degranulation. In RT-CES system, adherent NIH3T3 target cells were resistant to basal killing by PBMC or NK cells. NK cell activation by adding IL-2 demonstrated real-time dynamic killing activity, and lymphokine-activated PBMC (E/T ratio of 32) from 15 individuals showed 59.1 ± 6.2% cytotoxicity results after 4 hours incubation in RT-CES system. However, there was no significant correlation between FCA and RT-CES cytotoxicity. After K562 target cell stimulation, PBMC produced profound proinflammatory and immunoregulatory cytokines/chemokines including IL-2, IL-8, IL-10, MIP-1α β, IFN-γ, and TNF-α, and cytokine/chemokine secretion was related to flowcytometry-based NK cytotoxicity. These data suggest that RT-CES and FCA differ in sensitivity, applicability and providing information, and further investigations are necessary in variable clinical conditions.

B-cell Complement Dependent Cytotoxic Crossmatch Positivity is an Independent Risk Factor for Long-term Renal Allograft Survival

The clinical significance of positive B-cell complement-dependent cytotoxicity crossmatching (B-CDC) in renal transplant recipients remains unclear. We reviewed 20 recipients with isolated B-CDC positivity at the time of transplantation. We compared the clinical characteristics, acute rejection and long-term graft survival between positive and negative B-CDC patients (n = 602). The number of retransplant recipients and positivity for T- and B-flowcytometric crossmatch was greater in positive B-CDC patients than in negative B-CDC patients. The overall acute rejection rate of positive B-CDC patients was significantly higher (P < 0.001), and Banff grade II or III cellular rejection was more frequently observed in positive B-CDC patients (P = 0.037). Compared with negative B-CDC patients, acute cellular rejection as a cause of graft loss was more prevalent (P = 0.020) and rescue rejection therapy was more frequently needed in positive B-CDC patients (P = 0.007). The allograft survival rate of positive B-CDC patients was significantly lower than that of negative B-CDC patients (P < 0.001), and B-CDC positivity independently increased the risk of allograft failure 2.31-fold (95% CI 1.15-4.67; P = 0.019) according to multivariate analysis. In conclusion, isolated B-CDC positivity is an independent long-term prognostic factor for allograft survival.

Antibody monitoring system to support the single-antigen Luminex assay in donor-specific antibody detection

The antibody monitoring system (AMS) is a crossmatch test using enzyme-linked immunoassay to detect donor-specific anti-HLA antibodies (DSA). The aim of this study was to determine whether the AMS assay is useful for supporting DSA results measured with single-antigen Luminex assay (Luminex-DSA) in renal transplant recipients. Thirty sera from 12 sensitized recipients and 71 sera from 60 nonsensitized recipients were screened by complement-dependent cytotoxic crossmatch (CDC-XM), flow cytometric crossmatch (FCXM), AMS, and the Luminex assays. Twenty-two sera (73.3%) from sensitized patients were positive for Luminex-DSA, and the AMS assay revealed the great specificity and positive predictive value for detecting Luminex-DSA in sera from sensitized patients. Positivity in the AMS assay was observed most frequently in Luminex-DSA-positive sera with ≥10,000 mean fluorescence intensity (MFI) levels (66.7%). Of the 101 sera, the results of the AMS assay were compatible with CDC-XM results in 73 sera (91.3%) and with FCXM results in 72 sera (90.0%); these rates were significantly higher for the AMS assay than for the Luminex assay (p = 0.008 for CDC-XM and p = 0.001 for FCXM). In conclusion, the AMS assay is useful as a supportive solid-phase method for predicting actual crossmatch and high level of Luminex-DSA in sensitized patients.

Comparison of antibody monitoring system with flow cytometric crossmatch test in renal transplant recipients with high panel-reactive antibody.

Background/Aims: The antibody monitoring system (AMS) is a recently developed enzyme-linked immunosorbent assay (ELISA) crossmatch assay to detect donor-specific anti-HLA immunoglobulin G antibodies (DS-HLA Abs). This study was conducted to compare the AMS with the flow cytometric crossmatch (FCXM) test in renal transplant recipients with high panel-reactive antibody (PRA). Methods: Thirty-two sera were obtained from 10 patients with panel reactivity above 50%. When anti-HLA Ab was detected by ELISA PRA and the matched donor had the corresponding HLA antigen, it was considered to indicate DS-HLA Ab. The results of the AMS assay and FCXM were compared with the DS-HLA Abs. Results: Twenty-three (71.9%) sera were positive for DS-HLA Abs by ELISA PRA. The AMS assay showed that the number of compatible sera with DS-HLA Abs was 27 (84.4%), and it was significantly concordant (κ = 0.649, p < 0.0001). For FCXM, the number of compatible sera with DS-HLA Abs was 26 (81.3%), and it was also significantly concordant (κ = 0.614, p < 0.0001). There was a significant degree of concordance between the AMS assay and FCXM in detection of DS-HLA Abs (κ = 0.452, p = 0.010). Conclusion: The AMS assay is comparable to FCXM in detecting DS-HLA Abs in high PRA recipients.