Assessing immunophenotyping performance: proficiency-validation for adopting improved flow cytometry methods.

BACKGROUND: The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform "switch" studies to validate performance before methods for T-cell subset analysis can be changed. METHODS: Switch studies were conducted among the four flow cytometry laboratories of the Multicenter AIDS Cohort Study (MACS), comparing a 2-color, lyse-wash method and a newer, 3-color, lyse no-wash method. Two of the laboratories twice failed to satisfy the criteria for acceptable differences from the previous method. Rather than repeating more switch studies, these laboratories were allowed to adopt the 3-color, lyse no-wash method. To evaluate the impact of the switch to the new method at these two sites, their results with the new method were evaluated within the context of all laboratories participating in the NIH-NIAID-Division of AIDS Immunology Quality Assurance (IQA) proficiency-testing program. RESULTS: Laboratory performance at these two sites substantially improved relative to the IQA standard test results. Variation across the four MACS sites and across replicate samples was also reduced. CONCLUSIONS: Although switch studies are the conventional method for assessing comparability of laboratory methods, two alternatives to the requirement of repeating failed switch studies should be considered: (1) test the new method and assess performance on the proficiency testing reference panel, and (2) prior to adoption of the new methods, use both the old and the new method on the reference panel samples and demonstrate that performance with the new method is better according to standard statistical procedures. These alternatives may help some laboratories' transition to a new and superior methodology more quickly than if they are required to attempt multiple, serial switch studies.

Chikungunya virus RNA and antibody testing at a National Reference Laboratory since the emergence of Chikungunya virus in the Americas.

Since first reported in the Americas in December 2013, chikungunya virus (CHIKV) infections have been documented in travelers returning from the Caribbean, with many cases identified by CHIKV antibody and/or RNA testing at our laboratory. We used our large data set to characterize the relationship between antibody titers and RNA detection and to estimate IgM persistence. CHIKV RNA was measured by nucleic acid amplification and CHIKV IgG/IgM by indirect immunofluorescence. Of the 1,306 samples submitted for RNA testing in January through September 2014, 393 (30%) were positive; for 166 RNA-positive samples, CHIKV antibody testing was also ordered, and 84% were antibody negative. Of the 6,971 sera submitted for antibody testing in January through September 2014, 1,811 (26%) were IgM positive; 1,461 IgM positives (81%) were also IgG positive. The relationship between the CHIKV antibody titers and RNA detection was evaluated using 376 IgM-positive samples (138 with RNA testing ordered and 238 deidentified and tested for RNA). RNA detection showed no significant association with the IgM titer but was inversely related to the IgG titer; 63% of the IgG negative sera were RNA positive, compared to 36% of sera with low IgG titers (1:10 to 1:80) and 16% with IgG titers of ≥1:160. Using second-sample results from 62 seroconverters, we estimated that CHIKV IgM persists for 110 days (95% confidence interval, 78 to 150 days) after the initial antibody-negative sample. These findings indicate that (i) RNA detection is more sensitive than antibody detection early in CHIKV infection, (ii) in the absence of RNA results, the IgG titer of the IgM-positive samples may be a useful surrogate for viremia, and (iii) CHIKV IgM persists for approximately 4 months after symptom onset.

Persistent alterations in the T-cell repertoires of HIV-1-infected and at-risk uninfected men.

OBJECTIVE: We examined the association between immunogenic exposure and T-cell receptor (TCR) diversity to more clearly assess the impact of HIV-1 infection on the T-cell repertoire. METHODS: : To estimate the extent of T-cell clonality attributable to HIV-1 infection, we evaluated T-cell repertoires in low-risk and at-risk seronegative men and HIV-1 seropositive men by assessment of T-cell receptor beta-chain (TCR beta) complimentary determining region 3 (CDR3) lengths. RESULTS: The frequency of T-cell clonality in both HIV-1 infected and at-risk uninfected men was elevated in comparison to low-risk uninfected men. Among low-risk and at-risk seronegative, and HIV-1 seropositive men, clonal expansions were present in 3, 8, and 10% of CD4+ CDR3 lengths, and 18, 22, and 28% of CD8+ CDR3 lengths respectively. In addition, the longitudinal conservation of clonal expansions was observed in at-risk seronegative men. Based on comparisons to at-risk seronegative men, we estimate that at-risk seropositive men with chronic HIV-1 infection exhibit a 27% increase in the number of expanded CD8+ CDR3 lengths. CONCLUSION: These findings provide an approximation of the magnitude of the T-cell response in individuals undergoing chronic HIV-1 infection and demonstrate a significant association between the history of immunogenic challenge and the magnitude of clonality within the T-cell repertoire. In addition, these findings underscore the necessity of selecting controls with similar antigenic exposure histories when investigating T-cell dynamics in HIV-infected individuals.

Optimization of methods to assess human mucosal T-cell responses to HIV infection.

The majority of HIV-1 infections occur via sexual transmission at mucosal epithelia lining the vagina, cervix or rectum. Mucosal tissues also serve as viral reservoirs. However, our knowledge of human mucosal T-cell responses is limited. There is a need for reliable, sensitive, and reproducible methods for assessing mucosal immunity. Here we report on the collaborative efforts of two laboratories to optimize methods for processing, culturing, and analyzing mucosal lymphocytes. Rectal biopsy tissue was obtained by flexible sigmoidoscopy, which is rapid, minimally invasive, and well tolerated. Of the four methods compared for isolating mucosal mononuclear cells (MMC), collagenase digestion reproducibly yielded the most lymphocytes (4-7 x 10(6)). Furthermore, 0.5-1 x 10(6) MMC could be polyclonally expanded to yield 17 x 10(6) CD8+ T cells allowing mapping of responses to overlapping peptides spanning the HIV-1 genome using IFN-gamma enzyme-linked immunospot (ELISpot). Expansion also reduced the spontaneous IFN-gamma production normally detected in fresh MMC. Piperacillin-tazobactam and amphotericin B reduced contamination of MMC cultures to 4%. Taken together, these methods will be useful for studies of mucosal immunity to HIV-1 and other pathogens during natural infection and following vaccination.

Immunologic profile of highly exposed yet HIV type 1-seronegative men.

The host immune factors that determine susceptibility to HIV-1 infection are poorly understood. We compared multiple immunologic parameters in three groups of HIV-1-seronegative men: 14 highly exposed (HR10), 7 previously reported possibly to have sustained transient infection (PTI), and a control group of 14 low risk blood bank donors (BB). Virus-specific cellular immune assays were performed for CD4(+) T helper cell responses, CD8(+) cytotoxic T lymphocyte activity, CD8(+) cell chemokine release, and CD8(+) cell-derived antiviral soluble factor activity. General immune parameters evaluated included CCR5 genotype and phenotype, interferon alpha production by PBMCs, leukocyte subset analysis, and detailed T lymphocyte phenotyping. Comparisons revealed no detectable group-specific differences in measures of virus-specific immunity. However, the HR10 group differed from the BB group in several general immune parameters, having higher absolute monocyte counts, higher absolute CD8(+) T cell counts and percentages, lower naive and higher terminal effector CD8(+) cells, and lower levels of CD28(+)CD8(+) cells. These changes were not associated with seropositivity for other chronic viral infections. The PTI men appeared to have normal levels of monocytes and slightly elevated levels of CD8(+) T cells (also with increased effector and decreased naive cells). Although we cannot entirely exclude the contribution of other chronic viral infections, these findings suggest that long-lived systemic cellular antiviral immunity as detected by our assays is not a common mechanism for resistance to infection, and that resistance may be multifactorial. General immune parameters reflected by CD8(+) T cell levels and activation, and monocyte concentrations may affect the risk of infection with HIV-1, and/or serve as markers of exposure.

Estimation of dengue virus IgM persistence using regression analysis.

Dengue virus IgM persistence was estimated using follow-up sera from 98 patients (60 with primary infections and 38 with secondary infections) whose first-specimen IgM index was strongly positive, suggesting recent disease onset. Regression analysis of the follow-up IgM index versus days between samples yielded a trend line that reached the cut-point index (1.10) at 179 days for the primary infection group and 139 days for the secondary infection group. This difference reflected significantly higher first-sample IgM indices in primary infections than in secondary infections rather than differences in IgM decay rates.

Dengue virus immunoglobulin M detection in a reference laboratory setting during the 2010 dengue virus outbreak on Caribbean islands.

A large outbreak of dengue virus (DV) infections occurred on Caribbean islands during 2010, with cases peaking during the second half of the year. In conjunction with the outbreak, we observed an unprecedented spike in the number of sera submitted for DV antibody testing between June and December 2010, with a concomitant increase in the number of IgM-positive specimens, indicative of acute DV infection. Analysis of the place of residence of the IgM-positive patients identified from June to December of 2010 revealed that 58.1% were residents of Caribbean islands (Puerto Rico and the U.S. Virgin Islands), whereas 40.6% were residents of the U.S. mainland or Hawaii. The U.S. residents represented 42 states plus the District of Columbia, but most (53%) were from just 3 states (California, Florida, and New York). In comparison to the Caribbean IgM-positive patient group, the U.S. IgM-positive patient group contained proportionately more adults 21 to 60 years old and fewer individuals <21 years old. These findings indicate that the 2010 Caribbean DV outbreak affected many U.S. residents (mostly adults, presumably travelers) from diverse geographic areas and emphasize the potential for a viremic DV-infected returning traveler to spark a local DV outbreak by introducing DV into a community with competent mosquito vectors.

Evaluation of a multiplex bead-based screening assay for detection of binding antibodies to interferon-beta.

Multiple sclerosis patients treated with interferon-beta (IFNbeta) can develop neutralizing binding antibodies (BAbs) that reduce the agent's effectiveness. Screening for these antibodies can be performed by ELISA. We investigated a multianalyte immune detection (MAID) assay as an alternative to ELISA to detect anti-IFNbeta-1a and -1b. For 146 sera representing both 1a and 1b treated groups, MAID concordance with ELISA was 94% and 92%, respectively. For all discordant results, the corresponding ELISA and MAID values were within 4 units of each other, and all discordant values but one fell within 2 units of the BAbs cutoff value for reflexing to neutralization testing (4 units). Our data indicate that the MAID assay is an accurate and cost-effective alternative to ELISA for detecting BAbs to IFNbeta.

Virus and antibody dynamics in acute west nile virus infection.

BACKGROUND: The dynamics of the early stages of West Nile virus (WNV) infection can be assessed by follow-up studies of viremic blood donors. METHODS: A total of 245 donors with WNV viremia were followed up weekly for 4 weeks and then monthly for up to 6 additional months or until seroconversion. Plasma samples were tested for WNV RNA by transcription-mediated amplification (TMA) and for WNV-specific IgM and IgG antibodies. RNA persistence was investigated by 6 replicate TMA tests; samples that were viremic for >40 days were tested for WNV-neutralizing activity. Follow up of 35 additional viremic donors for up to 404 days was conducted to evaluate persistence of WNV-specific antibody. RESULTS: The median time from RNA detection to IgM seroconversion was 3.9 days; to IgG seroconversion, 7.7 days; to RNA negativity by single-replicate TMA, 13.2 days; and to RNA negativity by 6-replicate TMA, 6.1 additional days after results of single-replicate TMA are negative. For 4 donors in whom RNA persisted for >40 days after the index donation, all samples obtained after this threshold were also positive for WNV IgG and neutralizing activity. The mean times to IgM and IgA negativity were 156 and 220 days, respectively. CONCLUSIONS: IgM and IgG develop rapidly after viremia and before RNA levels become undetectable, which occurred a mean of 13.2 days after the index donation among donors in this study. WNV RNA detection by replicate TMA rarely persists for >40 days after the index donation and is accompanied by WNV-specific neutralizing antibody, consistent with an absence of WNV transmission via transfusion of seropositive blood components.

Evaluation of a tetraplex microsphere assay for Bordetella pertussis antibodies.

To increase testing efficiency, a microsphere-based multianalyte immune detection (MAID) system was developed to measure serum immunoglobulin G (IgG) and IgA recognizing two Bordetella pertussis antigens, pertussis toxin (PT) and filamentous hemagglutinin antigen (FHA). The assay was performed as two separate duplexes. One duplex measured IgG to PT and FHA, and the other measured IgA to PT and FHA. The two duplexes were then combined and analyzed as a tetraplex. The MAID system and an in-house enzyme-linked immunosorbent assay (ELISA) system were used to evaluate 100 sera from blood donors and 220 consecutive sera submitted for B. pertussis antibody testing. For both the MAID and ELISA systems, antibody levels were defined as increased if greater than the blood donor group 95th percentile value. The qualitative concordance rates between MAID and ELISA results for the 220 consecutively submitted sera were as follows: PT IgG, 99%; PT IgA, 94%; FHA IgG, 93%; FHA IgA, 94%. The overall concordance rate was 95% (836 of 880 result sets). For 29 of 44 (66%) discordant result sets, the discordant MAID result was supported by the MAID and ELISA results for other B. pertussis antibodies. The MAID and in-house ELISA systems were also used to evaluate 20 sera previously tested for pertussis antibodies at a pertussis vaccine research laboratory; MAID results for all four analytes did not significantly differ from results obtained by the research laboratory. These findings show that antibodies to B. pertussis antigens can be measured easily and accurately using a tetraplex microsphere system.

Parallel human immunodeficiency virus type 1-specific CD8+ T-lymphocyte responses in blood and mucosa during chronic infection.

Gut-associated lymphoid tissue is the major reservoir of lymphocytes and human immunodeficiency virus type 1 (HIV-1) replication in vivo, yet little is known about HIV-1-specific CD8+ T-lymphocyte (CTL) responses in this compartment. Here we assessed the breadth and magnitude of HIV-1-specific CTL in the peripheral blood and sigmoid colon mucosa of infected subjects not on antiretroviral therapy by enzyme-linked immunospot analysis with 53 peptide pools spanning all viral proteins. Comparisons of blood and mucosal CTL revealed that the magnitude of pool-specific responses is correlated within each individual (mean r2 = 0.82 +/- 0.04) and across all individuals (r2 = 0.75; P < 0.001). Overall, 85.1% of screened peptide pools yielded concordant negative or positive results between compartments. CTL targeting was also closely related between blood and mucosa, with Nef being the most highly targeted (mean of 2.4 spot-forming cells [SFC[/10(6) CD8+ T lymphocytes/amino acid [SFC/CD8/aa]), followed by Gag (1.5 SFC/CD8/aa). Finally, comparisons of peptide pool responses seen in both blood and mucosa (concordant positives) versus those seen only in one but not the other (discordant positives) showed that most discordant results were likely an artifact of responses being near the limit of detection. Overall, these results indicate that HIV-1-specific CTL responses in the blood mirror those seen in the mucosal compartment in natural chronic infection. For protective or immunotherapeutic vaccination, it will be important to determine whether immunity is elicited in the mucosa, which is a key site of initial infection and subsequent HIV-1 replication in vivo.

Effects of HIV-1 infection on lymphocyte phenotypes in blood versus lymph nodes.

Most immunopathogenesis studies of HIV-1 use peripheral blood. Most lymphocytes reside in lymphoid tissues, however, and the extent to which blood mirrors tissues is unclear. Here, we analyze lymphocytes in blood and lymph nodes of HIV-1-uninfected and -infected persons. Baseline comparison of node and blood lymphocytes in seronegative persons demonstrates a lower ratio of CD8+ versus CD4+ T lymphocytes, a lower number of effector cells (CD28-) within the CD8+ compartment, and greater activation (D-receptor [DR+]) within the CD4+ compartment. In infected versus uninfected persons, nodes exhibit elevated CD8+ T lymphocytes with an increased memory-effector phenotype (CD62L-/CD45RA-) and activation (CD38+ and DR+) but minimal differences in the CD4+ compartment. Changes attributable to HIV-1 infection are markedly greater in node lymphocytes than in blood. Comparisons of CD8+ T-lymphocyte parameters and viremia in infected persons reveal positive correlations of CD38+ expression on cells in blood and nodes and a negative correlation of terminal effector cells (CD62L-/CD45RA+) in the nodes to viremia. Multiple linear regression analysis indicates that CD38 expression on node (not blood) CD8+ T lymphocytes is the sole independent predictor for viremia. Thus, blood indirectly reflects processes in lymphoid tissues, and caution should be applied when interpreting immunopathogenesis studies of blood.

MaGiK method of T-Cell receptor repertoire analysis.

T-cell receptor diversity enables the cellular immune response to recognize a broad range of viral and other pathogenic agents. An increasingly common method of characterizing T-cell receptor diversity and usage in response to antigenic challenges involves the identification of clonal expansions by PCR amplification of the CDR3 region of distinct TCRVbeta families. Though clonal expansions often appear evident upon visual inspection of the results, a systematic method is needed for the valid enumeration of these expansions. Here, we describe a novel analysis method, termed the MaGiK method, for systematically identifying and enumerating clonal T-cell expansions and for applying the results to investigations of the T-cell receptor repertoire.

Simultaneous flow cytometric analysis of two cell surface markers, telomere length, and DNA content.

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.

Epitope escape mutation and decay of human immunodeficiency virus type 1-specific CTL responses.

To investigate possible mechanisms behind HIV-1 escape from CTL, we performed detailed longitudinal analysis of Gag (SLYNTVATL)- and RT (ILKEPVHGV)-specific CTL responses and plasma epitope sequences in five individuals. Among those with CTL against consensus epitope sequences, epitope mutations developed over several years, invariably followed by decay of the CTL targeting the consensus epitopes. The maturation state of the CTL varied among individuals and appeared to affect the rate of epitope mutation and CTL decay, despite similar IFN-gamma production. Escape mutations were oligoclonal, suggesting fitness constraints. The timing of escape indicated that the net selective advantage of escape mutants was slight, further underscoring the importance of understanding factors determining selective pressure and viral fitness in vivo. Our data show surprisingly consistent decay of CTL responses after epitope escape mutation and provide insight into potential mechanisms for both immune failure and shifting CTL specificities.

Predictive value of immunologic and virologic markers after long or short duration of HIV-1 infection.

Laboratory markers that predict HIV-1 disease progression include plasma viral burden, CD4+ T-cell count, and CD38 expression on CD8 T cells. To better understand whether the predictive value of these markers is dependent on how long an individual has been infected, we analyzed data from the Multicenter AIDS Cohort Study early (median = 2.8 years) and late (median = 8.7 years) in the course of infection. Overall, we found that HIV RNA and CD38 levels were similarly predictive of AIDS early on compared with a relatively weaker CD4 cell count signal. Later in the course of infection, CD38 level remained the strongest predictive marker and CD4 cell count registered a marked increase in prognostic power. Among untreated individuals, there was little difference in prognosis (median time to AIDS) associated with given marker values regardless of infection duration. The prognosis given a specific viral load level tended to deteriorate late in the course of infection among those undergoing treatment with monotherapy or combination therapy, however. These data provide a unique historical look at the predictive value and prognostic significance of HIV-1 disease markers at different stages of infection in a large cohort, with direct relevance to current patients who are untreated or for whom treatment is ineffective.