EpCAM expression in squamous cell carcinoma of the uterine cervix detected by monoclonal antibody to the membrane-proximal part of EpCAM.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is a promising biomarker for squamous cell carcinoma (SCC) of the uterine cervix, because it is over-expressed in various cancers of epithelial origin. However, EpCAM expression reported in previous immunohistochemistry (IHC) studies was inconsistent. We hypothesize that the membrane-distal part of EpCAM may be lost during tissue preparation, leaving only the membrane-proximal part of EpCAM available for antibody binding and IHC staining. METHODS: Two new anti-EpCAM MAbs to the membrane-proximal part (WC-2) and the membrane-distal part (WC-1) of EpCAM were generated and characterized. WC-2 was selected for its ability to detect EpCAM in cervical tissues by IHC. One hundred thirty-five archival paraffin-embedded tissues previously diagnosed as cervical SCC (n=44), high-grade (HSIL) (n=43), or low-grade (LSIL) (n=48) squamous intraepithelial lesions were examined. IHC score was collected, recorded, and analyzed for distribution, intensity, and percentage of cancer cells stained for EpCAM. RESULTS: EpCAM expression was consistently detected on cervical tissues by WC-2, but not by WC-1. EpCAM was expressed with high IHC score in the majority of cervical SCC (37/44), but not in normal epithelial area adjacent to SCC. EpCAM was also highly expressed on precancerous lesion of the cervix, particularly in HSIL. More importantly, EpCAM expression could be used to distinguish between HSIL and LSIL, according to staining distribution. HSIL tissues displayed EpCAM expression in two-thirds to full thickness of the epithelium, while in LSIL the staining was limited to the lower one-third of the thickness. The IHC score of EpCAM expression was strongly correlated with cervical cancer and grades of precancerous lesions (r=0.875, p<0.001). CONCLUSION: Only the anti-EpCAM MAb to the membrane-proximal part is able to detect EpCAM on paraffin-embedded cervical cancer tissues. A strong positive correlation between EpCAM expression level and the grades of SILs provides the possibility that EpCAM can be used to predict prognosis and severity in these patients.

Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7.

Development of new cancer therapies based on specific recognition of molecules in cancer cells is a significant challenge, as this requires identification of such molecules (molecular targets) and subsequent development of high-affinity, selective binders (targeting molecules). While several molecular targets for cancer therapies are currently under evaluation in clinical trials, greater selectivity for cancer cells over normal cells is required to enhance efficacy. Migration-inducing gene 7 (Mig-7), a membrane protein found in various types of carcinoma cells, is a cancer-specific biomarker and a promising molecular target for targeted cancer therapies. The purpose of this study was to produce and characterize a novel monoclonal antibody (mAb) raised against an N-terminal peptide of human Mig-7 (Mig-7(1-30)). The Mig-7(1-30) peptide was conjugated with a KLH carrier protein for immunization, and the mAb specific to Mig-7 (STmAb-1) was produced using hybridoma technology. Western blot analysis showed that STmAb-1 specifically reacted with a 23-kDa Mig-7 protein expressed in cancer cell lines, and, crucially, not with primary human fibroblasts. The affinity constant (Kaff) of STmAb-1, as measured by non-competitive enzyme immunoassay, was 1.31 × 10(9) M(-1), indicating high mAb affinity against Mig-7. Immunofluorescence assays demonstrated that STmAb-1 could specifically recognize Mig-7 expressed in cancer cell lines, but not in primary human fibroblasts and keratinocytes. Moreover, STmAb-1 inhibited the growth of MCF7 and HeLa cell lines in contrast to primary human fibroblasts, highlighting its potential usefulness in the development of new cancer therapeutics.

Adult-onset immunodeficiency in Thailand and Taiwan.

BACKGROUND: Autoantibodies against interferon-γ are associated with severe disseminated opportunistic infection, but their importance and prevalence are unknown. METHODS: We enrolled 203 persons from sites in Thailand and Taiwan in five groups: 52 patients with disseminated, rapidly or slowly growing, nontuberculous mycobacterial infection (group 1); 45 patients with another opportunistic infection, with or without nontuberculous mycobacterial infection (group 2); 9 patients with disseminated tuberculosis (group 3); 49 patients with pulmonary tuberculosis (group 4); and 48 healthy controls (group 5). Clinical histories were recorded, and blood specimens were obtained. RESULTS: Patients in groups 1 and 2 had CD4+ T-lymphocyte counts that were similar to those in patients in groups 4 and 5, and they were not infected with the human immunodeficiency virus (HIV). Washed cells obtained from patients in groups 1 and 2 had intact cytokine production and a response to cytokine stimulation. In contrast, plasma obtained from these patients inhibited the activity of interferon-γ in normal cells. High-titer anti-interferon-γ autoantibodies were detected in 81% of patients in group 1, 96% of patients in group 2, 11% of patients in group 3, 2% of patients in group 4, and 2% of controls (group 5). Forty other anticytokine autoantibodies were assayed. One patient with cryptococcal meningitis had autoantibodies only against granulocyte-macrophage colony-stimulating factor. No other anticytokine autoantibodies or genetic defects correlated with infections. There was no familial clustering. CONCLUSIONS: Neutralizing anti-interferon-γ autoantibodies were detected in 88% of Asian adults with multiple opportunistic infections and were associated with an adult-onset immunodeficiency akin to that of advanced HIV infection. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research; ClinicalTrials.gov number, NCT00814827.).

Simultaneous discrimination and detection of influenza A(H1N1)pdm09 and seasonal influenza A viruses using a rapid immunogold biosensor.

A rapid immunogold biosensor for the simultaneous discrimination of influenza A(H1N1)pdm09 and seasonal influenza A viruses was developed successfully. Monoclonal antibodies (mAbs) that were specific for the hemagglutinin protein of the A(H1N1)pdm09 virus were produced, and the best mAb pairs were selected. Using an mAb that was specific for the influenza A nucleoprotein, a rapid immunogold biosensor for the discrimination and detection of A(H1N1)pdm09/seasonal influenza viruses was developed. When tested with 72 virus isolates, the system achieved 100 % detection of the A(H1N1)pdm09 virus without cross-reactivity against seasonal influenza A (H1, H3 subtypes) and B viruses, parainfluenza viruses, respiratory syncytial viruses, and adenoviruses. The detection limits for A(H1N1)pdm09 and seasonal strains were 5 × 10(2)-7.5 × 10(3) and 1 × 10(3)-7.5 × 10(5) TCID50/mL, respectively. When tested with 49 clinical specimens, the specificity was high (100 %). The sensitivity for the detection of A(H1N1)pdm09 and seasonal strains was 90 % and 100 %, respectively, which correlated with the results of real-time reverse transcription polymerase chain reaction as a reference method. The ability of the system to detect and discriminate the A(H1N1)pdm09 strain from the seasonal strains suggests that this method may be beneficial for investigation of outbreaks and diagnostic applications. Furthermore, this method might be a useful platform for developing a rapid diagnostic system for the simultaneous discrimination of other influenza virus subtypes during future outbreaks.

Cryptococcal osteomyelitis in a child with a novel compound mutation of the IL12RB1 gene.

The IL-12p40/IL-12Rbeta1 and IFN-gammaR1/IFN-gammaR2/STAT1 signaling pathways are important for clearing intracellular bacteria. Genetic defects within these pathways are associated with increased susceptibility to intracellular pathogens. Among these, IL-12Rbeta1 deficiency is the most common defect and leads to infections with Salmonella and Mycobacterium spp. We report a child who presented with Cryptococcal osteomyelitis and history of disseminated Mycobacterial infection and recurrent Salmonella septicemia. Flow cytometry showed defective expression of IL-12Rbeta1. Mutation analysis revealed a novel compound heterozygous mutation of IL12RB1, c.625C>T, p.Q209X was found in exon 7 on the paternal allele and c.710delC, p.P237HfsX5 was found in exon 8 on the maternal allele. As these mutations each result in a stop codon before the last spliceable exon, the transcripts likely underwent nonsense mediated decay, leading to a lack of IL12Rbeta1 expression on the cell surface and eradicating signaling via the IL12 signaling pathway.

Performance Evaluation of BD FACSPrestoTM Near-Patient CD4 Counter for Monitoring Antiretroviral Therapy in HIV-Infected Individuals in Primary Healthcare Clinics in Thailand.

HIV viral load is more reliable tool for monitoring treatment throughout the course of HIV/AIDS, but the test may be expensive in resource-limited settings. Therefore, enumeration of CD4 T-lymphocyte count remains important in these settings. This study evaluated the performance of BDFACSPresto, a near-patient CD4 counter planned to be used in primary healthcare clinics in Thailand. Results of percent, absolute CD4 count and hemoglobin (Hb) on the FACSPresto were compared with the TriTEST/TruCOUNT/BDFACSCalibur method and a Sysmex hematology analyzer. Phase I of the study was performed in an ISO15189 laboratory. Both percentage and absolute values showed Passing-Bablok slopes within 0.98-1.06 and 0.97-1.13, mean Bland-Altman biases of +1.2% and +20.5 cells/µL, respectively. In phase II, venous and some capillary blood samples were analyzed in four primary healthcare clinics. The results showed good correlation between capillary and venous blood. For venous blood samples, regression lines showed slopes of 1.01-1.05 and 1.01-1.07 for all percentage and absolute values. The overall mean biases were +0.9% and +17.0 cells/µL. For Hb, Passing-Bablok regression result gave slope within 1.01-1.07 and mean bias of -0.06 g/dL. Thus, CD4 enumeration in blood by the FACSPresto is reliable and can be performed to an identical standard at primary healthcare clinics.

Immunogenicity of a Two-Dose Human Papillomavirus Vaccine Schedule in HIV-Infected Adolescents with Immune Reconstitution.

HIV-infected patients are at increased risk of human papillomavirus (HPV) acquisition and HPV-associated diseases. This study set out to determine whether a two-dose (2D) HPV vaccination schedule was sufficient in HIV-infected adolescents with immune reconstitution (IR) following antiretroviral treatment. Participants aged 9-15 years who had CD4 cell counts > 500 cells/mm3 and HIV-1 RNA < 40 copies/mL for at least one year were assigned to the 2D schedule, while older participants or those without IR received a three-dose (3D) schedule. Antibodies to HPV-16 and -18 were measured using a pseudovirion-based neutralization assay. A total of 96 subjects were enrolled; 31.3% and 68.7% received the 2D and 3D schedule, respectively. Of these, 66.7% and 57.6% of the 2D and 3D participants, respectively, were male. The seroconversion rates for HPV-16 and HPV-18 were 100% in all cases, except for HPV-18 in males who received the 3D schedule (97.4%). In males, the anti-HPV-16 geometric mean titers (GMTs) were 6859.3 (95% confidence interval, 4394.3-10,707.1) and 7011.1 (4648.8-10,573.9) in the 2D and 3D groups (p = 0.946), respectively, and the anti-HPV-18 GMTs were 2039.3 (1432.2-2903.8) and 2859.8 (1810.0-4518.4) in the 2D and 3D (p = 0.313) groups, respectively. In females, the anti-HPV-16 GMTs were 15,758.7 (8868.0-28,003.4) and 26,241.6 (16,972.7-40,572.3) in the 2D and 3D groups (p = 0.197), respectively, and the anti-HPV-18 GMTs were 5971.4 (3026.8-11,780.6) and 9993.1 (5950.8-16,781.1) in the 2D and 3D groups (p = 0.271), respectively. In summary, a 2D schedule is as immunogenic in young adolescents with IR as a 3D schedule in older subjects and those without IR.

Flow cytometric CD4 enumeration of four different HIV-infected blood samples at the cost of one monoclonal antibody reagent.

BACKGROUND: The frequency and absolute number of CD4+ T-lymphocytes continue to be one of the major clinical markers for management of HIV/AIDS. The present standard dual-platform (DP) three-color and two-color PanLeucogating flow cytometric (FCM) methods for most developing countries are either expensive if manufacturers' monoclonal antibody reagents are used or limited due to an insufficient supply of generic reagents. Clearly, more affordable FCM methods are needed. OBJECTIVE: To develop a novel DP FCM method using biotin-streptavidin-fluorochrome labeling in combination with the two standard DP methods for 4 different white blood cells (WBC) using only one monoclonal antibody reagent. METHODS: The percentage of CD4+ T-lymphocytes in 116 HIV-infected blood samples was determined using our new method. Results were compared with the two standard methods. Correlation and agreement of the pair method were determined using linear regression, Bland-Altman and percent similarity analysis. RESULTS: Our study showed that percentage of CD4+ T-lymphocyte values obtained from the new method correlated highly with the standard three-color and the two-color methods (r2 = 0.95 {n=52} and 0.97 {n=64}). The mean bias and percent similarity for the new method compared with the two standard methods were -0.53% (limit of agreement {LOA}:-5.22% to +4.16% with percent similarity of 99.28; and -0.22% with LOA of -3.42% to +2.98%, the percent similarity of 98.15, respectively. CONCLUSIONS: Our FCM method using biotin to label 4 different WBC samples followed by streptavidin staining is reliable for determination of CD4+ T-lymphocytes. Such an approach will significantly reduce the cost for monitoring HIV-infected patients in resource-limited settings.

A novel mutation of the IL12RB1 gene in a child with nocardiosis, recurrent salmonellosis and neurofibromatosis type I: first case report from Thailand.

Genetic defects of interleukin (IL)-12/23-and interferon (IFN)-gamma-mediated immunity can cause increased susceptibility to intracellular microbes. Among these defects, a mutation of the gene encoding the IL-12 receptor beta1 (IL-12Rbeta1) is the most common worldwide. A 12-year old Thai boy with pre-existing neurofibromatosis type 1 (NF1) was evaluated for primary immunodeficiency after a history of tuberculous lymphadenitis, recurrent Salmonella infections and nocardiosis. Flow cytometry of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) revealed a defect in the IL-12Rbeta1 surface expression. A genetic study showed a novel nonsense homozygous mutation of the IL12RB1 gene in exon 4 (402C > A), confirming the diagnosis of IL-12Rbeta1 deficiency. This is the first case report of a primary IL-12Rbeta1 deficiency in Thailand with the interesting finding of a coexisting NF1.

Evaluation of a single-platform microcapillary flow cytometer for enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai patients.

BACKGROUND: Various assays are used to enumerate peripheral blood absolute CD4+ T-lymphocytes. Flow cytometry is considered the gold standard for this purpose. However, the high cost of available flow cytometers and monoclonal antibody reagents make it difficult to implement such methods in the resource-poor settings. In this study, we evaluated a cheaper, recently developed single-platform microcapillary cytometer for CD4+ T-lymphocyte enumeration, the personal cell analyzer (PCA), from Guava Technologies. METHODS: CD4+ and CD8+ T-lymphocyte counts in whole blood samples from 250 HIV-1 infected Thais were determined, using a two-color reagent kit and the Guava PCA, and compared with the results obtained with two reference microbead-based methods from Becton Dickinson Biosciences: the three-color TruCOUNT tube method and the two-color FACSCount method. Statistical correlations and agreements were determined using linear correlation and Bland-Altman analysis. RESULTS: Absolute CD4+ T-lymphocyte counts obtained using the Guava PCA method highly correlated with those obtained using TruCOUNT method (R(2) = 0.95, mean bias +13.1 cells/microl, limit of agreement [LOA]-117.9 to +144.1 cells/microl) and the FACSCount method (R2 = 0.94, mean bias = +33.2 cells/microl, LOA-101.8 to +168.3 cells/microl). Absolute CD8+ T-lymphocyte counts obtained using the Guava PCA method also highly correlated with those obtained with the two reference methods (R(2) = 0.92 and 0.88, respectively). CONCLUSION: This study shows that the enumeration of CD4+ T-lymphocytes using the Guava microcapillary cytometer PCA method performed well when compared with the two reference bead-based methods. However, like the two reference methods, this new method needs substantial technical expertise.

A novel mutation of the CYBB gene resulting in severe form of X-linked chronic granulomatous disease.

We evaluated a boy who had multiple Salmonella septicemia, Aspergillus pneumonia and brain abscesses. His nitroblue tetrazolium (NBT) test was reportedly abnormal. The dihydrorhodamine (DHR) flow cytometry assay was compatible with typical X-linked chronic granulomatous disease (X-CGD). CYBB analysis revealed a novel complex mutation atggacg --> ttca in exon 12 (base pairs 1532-1538). As a result, 3 amino acids Tyr 511, Gly 512 and Arg 513 were deleted and replaced by 2 amino acids, Phe and Gln. The DHR and mutation analysis of his mother showed normal DHR pattern and no mutations in exon 12 of CYBB gene. In conclusion, any children with multiple Salmonella and Aspergillus infection should be suspected of CGD. NBT test, DHR assay and gene analysis are helpful toolsto confirm the diagnosis e v en i n the case of de novo mutation.

A multicenter evaluation of the PanLeucogating method and the use of generic monoclonal antibody reagents for CD4 enumeration in HIV-infected patients in Thailand.

BACKGROUND: The current method of CD4 enumeration in Thailand, based on the three-tube, three-color method recommended by the Centers for Disease Control and Prevention, is expensive and thus unavailable to most patients who have the human immunodeficiency virus (HIV). Less expensive, simpler protocols (i.e., PanLeucogating and primary CD4 gating) have been described but require more published validation data to gain widespread acceptance. We describe a multicenter evaluation of the PanLeucogating method. METHODS: The PanLeucogating method using generic reagents was evaluated in comparison with the standard three-tube, three-color method using commercial reagents. Percentage of CD4+ T cells among lymphocytes and absolute CD4+ T-cell counts were determined in 611 HIV-infected individuals recruited from four sites. Linear regression and Bland-Altman tests were used for statistical analysis. RESULTS: The correlation of percentage of CD4+ T cells and absolute CD4+ T-cell counts obtained with the PanLeucogating strategy and the standard predicate method was high (r2 = 0.96 and 0.95, respectively, for the entire study population and r2 > 0.95 and 0.93, respectively, for each study group). Absolute CD4+ T-cell counts of the overall study pool and of the two subdivisions of absolute CD4+ T-cell counts (i.e., 0-250 cells/microl and > 250 cells/microl) derived from the two methods demonstrated excellent agreement, with mean biases of +18 cells/microl, +11 cells/microl, and +24 cells/microl, respectively. CONCLUSIONS: These observations demonstrate that CD4 enumeration by PanLeucogating is reliable and can be performed to an identical standard in a quality-assured network of collaborating laboratories as a new cost-effective approach to HIV monitoring.

Serodiagnosis of melioidosis by a competitive enzyme-linked immunosorbent assay using a lipopolysaccharide-specific monoclonal antibody.

Burkholderia pseudomallei is the causative agent of melioidosis, a severe and potentially fatal infectious disease in humans known to be endemic in Southeast Asia and northern Australia. The infection is also increasingly recognized in various animal species with a potential to spread to humans. With the potential as a biological warfare agent, specific serodiagnosis of melioidosis for surveillance in large populations at risk, humans or animals, would be highly valuable. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) using a lipopolysaccharide-specific monoclonal antibody was developed. The assay provides high specificity, based on a previously described monoclonal antibody to a specific epitope on the lipopolysaccharide (LPS) of B. pseudomallei. The assay sensitivity of 96.0% and specificity of 100% were achieved at a cutoff value of 50% inhibition in human culture-proven melioidosis cases. An optimal cutoff value of 65% inhibition for sera from a melioidosis endemic area was obtained by ROC analysis and resulted in an assay specificity of 86.2%, while maintaining assay sensitivity of 92.0%. A potential application of the assay in the serodiagnosis of melioidosis in animal species was also evaluated usina dolphin sera with satisfactory results.

Low-cost CD4 enumeration in HIV-infected patients in Thailand.

In Thailand, over one million people have been infected with HIV since the beginning of the epidemic. This has created a great burden on the country's limited health care budget. Monitoring CD4+ T-lymphocytes is important to determine the success of any antiretroviral therapy as well as HIV vaccine trials. However, the high cost of CD4 counts makes monitoring of every HIV-infected patient impossible in Thailand. Therefore, the development of affordable strategies is necessary in order to allow more HIV infected persons to access CD4 testing to control the disease. The current standard methods for enumeration of CD4+ T-lymphocytes are performed on whole blood by flow cytometric immunophenotyping using the 6-tube 2-color and 3-tube 3-color panels recommended by the Centers for Diseases Control (CDC). In this study, percentage CD4+ T-lymphocyte values (from 142 HIV-seropositive patients and 26 anti-HIV negative adult blood donors) generated by the use of just 2 reagents (CD45/CD4) in a 1-tube 2-color panel employing side scatter/CD45 morphospectral gating were compared to those obtained by state of the art methods. We also compared the use of generic monoclonal antibody reagents with commercial reagents and found the results to be comparable with an overall correlation coefficient (r) of more than 0.95 for both CD4+ and CD8+ T-lymphocytes. Bland-Altman analysis of the mean CD4 values plotted against the difference in values between the generic reagents and the commercial reagents showed no bias. The 1-tube 2-color method using generic monoclonal antibody reagents potentially permits more affordable but reliable CD4 testing and therefore could increase access for more HIV-infected patients in resource-poor countries.

Improved sensitivity of influenza A antigen detection using a combined NP, M, and NS1 sandwich ELISA.

A new modified triple-antigen detection test was developed for the direct detection of the influenza A virus. The nucleoprotein (NP), matrix (M), and non-structural (NS1) proteins were used as target antigens because they are abundant in infected cells. Monoclonal antibodies specific to the NP, M, and NS1 proteins were generated. The antibody pairs were selected and evaluated for their reactivity individually and in combination in the triple-antigen detection using sandwich ELISA. Triple-antigen detection demonstrated a higher sensitivity than individual antigen detection when tested with both the H1N1 and H3N2 influenza A viruses. This was illustrated by the 4-fold lower limit of detection of the triple-antigen test than the individual antigen detection test. The findings demonstrated that the sensitivity of influenza A antigen detection was improved with the triple-antigen detection system as compared to individual antigen detection. Therefore, this technique could be a useful tool for the direct detection of cell-associated influenza A antigen. Furthermore, it could provide a basis for the development of a rapid triple-antigen test for influenza A diagnosis.

Performance evaluation of the Alere PIMA CD4 test for monitoring HIV-infected individuals in resource-constrained settings.

BACKGROUND: Enumeration of CD4+ T-lymphocytes is important in the management of HIV. However, standard laboratory systems based on flow cytometry are expensive, complicated, and thus unavailable to most resource-limited settings where a low-cost and fully automated point-of-care CD4 testing system is required. In attempts to address this issue, a study was conducted to validate the Alere PIMA point-of-care CD4 test. METHOD: Duplicate values of the absolute number of CD4+ T-lymphocytes in 203 HIV-infected blood samples obtained using the PIMA system were compared with the two predicate single-platform FACSCount and the dual-platform FACSCan (Becton Dickinson Biosciences). RESULTS: The overall absolute CD4+ T-lymphocyte count obtained using the PIMA system correlated highly with the FACSCount (r = 0.957; mean bias, -54.2 cells/µL; limit of agreement, -190.9 to +82.5 cells/µL) and the FACSCan (r = 0.957; mean bias -44.0 cells/µL; limit of agreement, -179.7 to +91.6 cells/µL). Good correlation and low biases were also observed for samples with CD4+ T-lymphocyte count ranges of 0 to 200 and 0 to 350 cells/µL. Additionally, there was no significant difference in absolute CD4+ T-lymphocyte counts noted between the duplicate samples using the PIMA system. CONCLUSIONS: This new point-of-care product is a simple and reliable system and should contribute significantly to the simplification of performing CD4 testing and thus increase access for patients in resource-limited settings. The inability to obtain values for the frequency (%) of CD4+ T-lymphocyte count is one limitation of the PIMA system, the addition of which would be of value for clinical staging or monitoring in HIV-infected pediatric patients.

New BD FACSCount CD4 reagent system for simultaneous enumeration of percent and absolute CD4 T-lymphocytes in HIV-1-infected pediatric patients.

BACKGROUND: Absolute CD4+ T-lymphocyte counts are used in the initiation and monitoring of antiretroviral therapy in HIV-infected patients. Becton Dickinson's (BD) FACSCount system was introduced 12 years ago as a dedicated instrument for enumeration of absolute CD4+ T-lymphocytes. However, this system does not provide percent CD4+ T-lymphocyte that is the required monitoring parameter in pediatric patients. We evaluated a new BD FACSCount CD4 software and reagents for simultaneous percent and absolute CD4+ T-lymphocytes in HIV-infected blood. METHODS: Percent and absolute CD4+ T-lymphocytes in 149 HIV-infected blood samples were determined using a new FACSCount system. Results of percent and absolute CD4+ T-lymphocytes were compared between the dual-platform (DP) method, using BD FACScan flow cytometer plus hematology analyzer and the standard FACSCount system. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis. RESULTS: Percent CD4+ T-lymphocyte values obtained from the new FACSCount system correlated well with DP FACScan method (r2 = 0.977, P < 0.0001). Mean bias was only -0.36% [limit of agreement (LOA): -2.52% to +1.80%] and percent similarity was 101.36%. For absolute CD4+ T-lymphocyte, the new system correlated highly with standard FACSCount system (r2 = 0.986, P < 0.0001), with a percent similarity of 98.2. Mean bias was +3.39 cells/microl with LOA of -52.53 cells/mul to +59.31 cells/microl. CONCLUSION: This new FACSCount system is a simple and reliable system for enumeration of absolute and percent CD4+ T-lymphocytes. Having one system giving both results should reduce the cost and thus increase access to CD4 testing for pediatric and adult patients.

Comparison of 5 flow cytometric immunophenotyping systems for absolute CD4+ T-lymphocyte counts in HIV-1-infected patients living in resource-limited settings.

Enumeration of CD4+ T lymphocytes is important in management of HIV-infected patients. However, CD4 testing by current gold standard bead-based flow cytometer (FCM) system is expensive for developing countries. This study compared 2 affordable volumetric FCMs with the 3 predicate FCM systems. CD4+ T-lymphocyte counts on blood samples from 150 HIV-1-infected Thai patients were determined in parallel by 5 FCM systems: the 2 single-platform volumetric FCM systems, Guava and CyFlow(green); the 2 standard single-platform bead-based systems (2-color FACSCount and the TriTEST/TruCOUNT tube using a FACSCalibur FCM); and the dual-platform TriTEST system. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis. Results from these 2 volumetric systems gave similar results and excellent correlation: R2 > 0.93; mean biases ranged from +6.3 to +24.1 cells per microliter more for the Guava. In contrast, the CyFlow(green) showed the lowest values with R2 > 0.97; mean biases ranged from -9.8 to -27.6 cells per microliter. This indicates that the absolute CD4+ T-lymphocyte counts determined by CyFlow(green) are < FACSCount < DP TriTEST < TriTEST/TruCOUNT < Guava. Although the use of these 2 volumetric FCMs could make CD4+ T-lymphocyte enumeration more affordable in resource-poor settings, variations among these systems should be considered if these are to be interchanged.

Flow cytometric CD4 enumeration of four different HIV-Infected blood samples at the cost of one monoclonal antibody reagent.

Background: Thefrequency and absolute number of CD4+ T-lymphocytes continue to be one of the major clinical markers for management of HIV/AIDS. The present standard dual-platform (DP) three-color and two-color PanLeucogating flow cytometric (FCM) methods for most developing countries are either expensive if manufacturers’ monoclonal antibody reagents are used or limited due to an insufficient supply of generic reagents. Clearly, more affordable FCM methods are needed. Objective: To develop a novel DP FCM method using biotin-streptavidin-fluorochrome labeling in combination with the two standard DP methods for 4 different white blood cells (WBC) using only one monoclonal antibody reagent. Methods: The percentage of CD4+ T-lymphocytes in 116 HIV-infected blood samples were determined using our new method. Results were compared with the two standard methods. Correlation and agreement of the pair method were determined using linear regression, Bland-Altman and percent similarity analysis. Results: Our study showed that percentage of CD4+ T-lymphocyte values obtained from the new method correlated highly with the standard three-color and the two-color methods (r2= 0.95 {n=52} and 0.97 {n=64}). The mean bias and percent similarity for the new method compared with the two standard methods were -0.53% (limit of agreement {LOA}:-5.22% to +4.16% with percent similarity of 99.28; and -0.22% with LOA of -3.42% to +2.98%, the percent similarity of 98.15, respectively. Conclusions: Our FCM method using biotin to label 4 different WBC samples followed by streptavidin staining is reliable for determination of CD4+ T-lymphocytes. Such an approach will significantly reduce the cost for monitoring HIV-infected patients in resource-limited settings.

A novel mutation of the IL12RB1 gene in a child with nocardiosis, recurrent salmonellosis and neurofibromatosis type I: First case report from Thailand.

Genetic defects of interleukin (IL)-12/23-and interferon (IFN)-γ-mediated immunity can cause in-creased susceptibility to intracellular microbes. Among these defects, a mutation of the gene encoding the IL-12 receptor β1 (IL-12Rβ1) is the most common worldwide. A 12-year old Thai boy with pre-existing neurofibromatosis type 1 (NF1) was evaluated for primary immunodeficiency after a history of tuberculous lymphadenitis, recurrent Salmonella infections and nocardiosis. Flow cytometry of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) revealed a defect in the IL-12Rβ1 surface expression. A genetic study showed a novel nonsense homozygous mutation of the IL12RB1 gene in exon 4 (402C>A), confirming the diagnosis of IL-12Rβ1 deficiency. This is the first case report of a primary IL-12Rβ1 deficiency in Thailand with the interesting finding of a coexisting NF1.