Comparison and evaluation of seven different bench-top flow cytometers with a modified six-plexed mycotoxin kit.

Many bench-top flow cytometers (b-FCs) are compatible with microsphere-based multiplexed assays. Disciplines implementing b-FCs-based assays are expanding; they include monitoring and validating food quality. A multiplexed platform protocol was evaluated for poly-mycotoxin assays, which is compatible with a variety of b-FC models. The seven instruments included: BD FACSCalibur(™) , BD FACSArray(™) Bioanalyzer, Accuri C6, Partec CyFlow(®) Space, Beckman Coulter FC 500, Guava EasyCyte Mini, and Luminex 100 (™) . Current reports related to the food industry describe fungal co-infections leading to poly-mycotoxin contamination in grain (Sulyok M, Berthiller F, Krska R, Schuhmacher R, Rapid Commun Mass Spectrom 2006;20:2649-2659). It is imperative to determine whether b-FC-based assays can replace traditional single-mycotoxin enzyme-linked immunosorbent assay (ELISA). A six-plexed poly-mycotoxin kit was tested on seven different b-FCs. The modified kit was initially developed for the BD FACSArray(™) Bioanalyzer (BD Biosciences) (Czeh A, Mandy F, Feher-Toth S, Torok L, Mike Z, Koszegi B, Lustyik G, J Immunol Methods 2012;384:71-80). With the multiplexed platform, it is possible to identify up to six mycotoxin contaminants simultaneously at regional grain collection/transfer/inspection facilities. In the future, elimination of contaminated food threat may be better achieved with the inclusion of b-FCs in the food protection arsenal. A universal protocol, matched with postacquisition software, offers an effective alternative platform compared to using a series of ELISA kits. To support side-by-side evaluation of seven flow cytometers, an instrument-independent fluorescence emission calibration was added to the protocol. All instrument performances were evaluated for strength of agreement based on paired sets of evaluation to predicate method. The results suggest that all b-FCs were acceptable of performing with the multiplexed kit for five of six mycotoxins. For OTA, the detection sensitivity was consistent only for five of the seven instruments.

A flow cytometry based competitive fluorescent microsphere immunoassay (CFIA) system for detecting up to six mycotoxins.

BACKGROUND: Exposure to multiple mycotoxins through the food chain represents a major potential health hazard to both humans and livestock. They can cause a variety of severe acute as well as chronic diseases. Eliminating mycotoxins from various grain crops is a global health priority. According to the Food and Agriculture Organization (FAO), world food production needs to double by 2050. Innovative solutions will be required to sustain toxin free grain supplies worldwide. METHODS: A competitive flow cytometry based multiplexed assay with fluorescent microspheres has been developed. The new multiplexed method can analyze simultaneously any one or all six major mycotoxins. They include: Ochratoxin A (OTA), Aflatoxin B1 (AFB1), Fumonisin B1 (FB1), T-2 toxin (T-2), Deoxynivalenol (DON) and Zearalenone (ZEA), which are all potential human health hazards. The CFIA described here includes a simplified single extraction step for mycotoxins from specimens and a comprehensive post acquisition software module. The new assay system was developed with a FACSArray™ BD Bioanalyzer flow cytometer (BD Biosciences, Belgium). RESULTS: The CFIA performs favourably when compared to commercial ELISA. Sensitivity range with CFIA increased between 13% and 100% with an average improvement of 50% for the six mycotoxins. CONCLUSIONS: The multiplexed assay presented here has the unique capacity to quantify up to six mycotoxins simultaneously from a single specimen extraction. CFIA's poly-mycotoxin detection sensitivity exceeds standard ELISA. CFIA may be part of a comprehensive assay system that will provide reliable and effective safeguard for agricultural commodities to be free of mycotoxins.

African regional external quality assessment for CD4 T-cell enumeration: development, outcomes, and performance of laboratories.

BACKGROUND: An independent African Regional External Quality Assessment Scheme (AFREQAS) was implemented from Johannesburg. The aim was to establish a network of CD4 laboratories supporting HIV/AIDS anti-retroviral therapy programs and improve the quality of regional CD4 testing with EQA assessment, feedback, remedial action, and technical training. The overall performance from 2002 to 2006 (Trials 1-20) is reported, together with cumulative longitudinal performance of the different CD4 methods used. METHODS: Stabilized blood samples with "normal" and/or "low" CD4 values were shipped over 20 Trials. Data was analyzed for each trial including trimmed mean, standard deviation, and percentage coefficient of variation (%CV); "Residual" and SDI values were also calculated for each participating laboratory for both absolute CD4 counts (CD4abs) and CD4 percentage of lymphocytes values (CD4%/Ly). Standardized individual laboratory SDI values across 20 trials were analyzed according to CD4 method. RESULTS: Average participation was 91.5%. Overall AFREQAS between-laboratory reproducibility (trimmed %CV) was 10.5% and 9.1% for absolute CD4 and CD4%/Ly, respectively. For the respective CD4abs and CD4%/Ly values in the trials where "normal" material was shipped trimmed %CV of 10.9 and 7.3% were noted, and in "low" value shipments %CV of 13.8% and 12.4% were noted. Cumulative absolute CD4 SDI analysis revealed the best between-laboratory precision amongst FACSCount and PanLeucogating (PLG-CD4) users (both SD of SDI = <1.2 and %CV of <<8%). Dual Platform or Single Platform algorithm-based systems and certain volumetric methods (laboratories who used Partec CyFlow instruments) had higher numbers of outlying laboratories (>12-25%CV and SD(SDI) > 2.2 noted), indicating that additional technical training and/or manufacturer support was required. CONCLUSIONS: Participation in an AFREQAS with feedback and remedial action improves the quality of CD4 testing. African laboratory professionals can easily master CD4 counting technologies. However, the introduction of the simplest and most cost-effective methodologies is required to take ownership, and enable the delivery of quality CD4 counts in vast numbers necessary to support expansion of African ART programs.

Diagnostics in the shadow of HIV epidemics.

As part of the effort to promote elimination of global health care disparities, this special supplement compiled 19 articles about practical diagnostic cytometry to recognize the recent achievements of laboratory scientists working in the shadow of the HIV/AIDS epidemics in resource limited settings. First, the clinical significance, diagnostic utility (as governed by international guidelines), and the historical perspectives of CD4+ T cell enumeration are reviewed. Then successful large-scale implementations of cost-effective CD4 counting are described for parts of Africa, USA, and the Caribbean. These activities are linked with both the training of personnel in fledgling laboratories as well as with external quality assessment implementations. Some of the more recent solutions related to pediatric CD4 testing using CD4% values are covered. Nevertheless, the need for further simplification and parsimony is still immense, and the potential solutions are catalogued in the articles written by experts operating in truly challenging rural environments. Cytometry is considered to be an expandable flexible technology for other assays beyond CD4 assessment, particularly within organized laboratory services in the Third World. These include haematological measurements, CD38/CD8 lymphocyte activation for viral load-related assessments, diagnosis of active tuberculosis and malaria, and bead-based serological assays for a variety of infectious diseases. The development and support of these emerging technologies by affluent countries is not entirely altruistic but is likely to be beneficial for both the Third and the First Worlds.

A North American multilaboratory study of CD4 counts using flow cytometric panLeukogating (PLG): a NIAID-DAIDS Immunology Quality Assessment Program Study.

BACKGROUND: The global HIV/AIDS pandemic and guidelines for initiating anti-retroviral therapy (ART) and opportunistic infection prophylaxis demand affordable, reliable, and accurate CD4 testing. A simple innovative approach applicable to existing technology that has been successfully applied in resource-challenged settings, PanLeukogated CD4 (PLG), could offer solutions for cost saving and improved precision. METHODS: Day-old whole blood from 99 HIV+ donors was simultaneously studied in five North-American laboratories to compare the performance of their predicate methods with the dual-platform PLG method. The predicate technology included varying 4-color CD45/CD3/CD4/CD8 protocols on different flow cytometers. Each laboratory also assayed eight replicate specimens of day-old blood from 10 to 14 local donors. Bias and precision of predicate and PLG methods was studied between- and within-participating laboratories. RESULTS: Significantly (P < 0.0001) improved between-laboratory precision/coefficient of variation (CV%) was noted using the PLG method (overall median 9.3% vs. predicate median CV 13.1%). Within-laboratory precision was also significantly (P < 0.0001) better overall using PLG (median 4.6% vs. predicate median CV 6.2%) and in 3 of the 5 laboratories. PLG counts tended to be 11% smaller than predicate methods (P < 0.0001) for shipped (median of predicate-PLG = 31) and local specimens (median of predicate-PLG = 23), both overall and in 4 of 5 laboratories (median decreases of 4, 16, 20, and 21% in shipped specimens); the other laboratory had a median increase of 5%. CONCLUSION: Laboratories using predicate CD4 methods similar to those in this study could improve their between-laboratory and their within-laboratory precision, and reduce costs, by switching to the PLG method after adequate training, if a change (usually, a decrease) in CD4 counts is acceptable to their health systems.

Early changes in T-cell activation predict antiretroviral success in salvage therapy of HIV infection.

OBJECTIVE: Because effective antiretroviral therapy (ART) reduces immune activation, we hypothesize that early changes in immune activation are associated with subsequent virologic response to therapy. DESIGN: Observational cohort study. SETTING: Institutional HIV clinic. SUBJECTS: Thirty-four adult HIV patients with virologic failure on their current antiretroviral regimen. INTERVENTION: Change to salvage regimen selected by patient's physician. MAIN OUTCOME MEASURES: Measures of immune activation at baseline and at 2, 4, 8, and 24 weeks after enrollment. Data were analyzed by proportional hazards (PH) models. RESULTS: PH models showed that reductions between baseline and week 2 in expression of CD38 (P = 0.02) or CD95 (P = 0.02) on CD4 T cells were associated with increased likelihood of achieving virologic suppression. Kaplan-Meier analysis demonstrated that patients who had reductions within the first 2 weeks of therapy in CD4 T-cell expression of CD38 (P = 0.003) or CD95 (P = 0.08) were more likely to achieve viral suppression than those who did not. CONCLUSIONS: Reduced CD4 T-cell expression of CD38 and CD95 occurring within 2 weeks of salvage therapy is associated with subsequent viral suppression. Monitoring CD38 and CD95 may allow earlier assessment of the response to ART.

Precise CD4 T-cell counting using red diode laser excitation: for richer, for poorer.

BACKGROUND: Measuring CD4 T-cell counts at low cost is relevant in dealing with the human immunodeficiency virus (HIV) epidemic throughout the developing world. The recently introduced novel concepts in gating strategies and sample stabilization facilitate affordable immunophenotyping by flow cytometry. However, the impact of these developments is still limited by the high cost of currently available flow cytometers. METHODS: Diode lasers emitting 10-15 mW at 635 nm are one-tenth the size and cost and require one thousandth the power of an equivalent 488-nm argon ion laser. We used the available 635-nm diode-based flow cytometers, including PA-II, Luminex 100, SuperMot, and FACSCalibur, to investigate whether these instruments can generate reliable CD4 counts when used with allophycocyanin (APC) and cyanin-5 (Cy5)-labeled CD4 antibodies. RESULTS: We document the feasibility of obtaining leucocyte differential counts using orthogonal side scatter (SSC) without the need for forward scatter (FSC). Accurate CD4% values among lymphocytes and leucocytes can be obtained by primary CD4 gating using a single CD4 monoclonal antibody conjugated to APC or Cy5. Double immunofluorescence (IF) staining with CD4-APC (FL1) and CD45-APC-Cy7 (FL2) introduces pan-leucogating for a convenient assessment of absolute CD4 counts on double platforms. We demonstrate that small flow cytometers with laser diodes are capable of delivering absolute CD4 T-cell counts with a precision similar to the performance of the current state-of-the-art single-platform instruments (e.g., the CytoronAbsolute; R(2) = 0.961). In this respect, they appear to be superior to the nonflow CD4 counting techniques. CONCLUSIONS: Accurate CD4 counts can be generated at minimal cost on red diode laser-operated flow cytometers, retaining the potential for high throughput capacity without compromising precision. With further improvements in volumetric technology and clinical software, these cytometers may develop into a new generation of inexpensive battery-operated laboratory hardware that combines cellular phenotyping with bead-based multiplexing immunoassays for (HIV) serology.