Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates

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1 Journal of Immunotoxicology ISSN: X (Print) (Online) Journal homepage: Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates Robert G. Caldwell, Peggy Marshall & Jared Fishel To cite this article: Robert G. Caldwell, Peggy Marshall & Jared Fishel (2016) Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates, Journal of Immunotoxicology, 13:1, 64-76, DOI: / X To link to this article: Published online: 20 Jan Submit your article to this journal Article views: 617 View related articles View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 23 November 2017, At: 09:49

2 ISSN: X (print), (electronic) J Immunotoxicol, 2016; 13(1): 64 76! 2015 Covance DOI: / X RESEARCH ARTICLE Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates Robert G. Caldwell*, Peggy Marshall, and Jared Fishel Covance Laboratories, Madison, WI, USA Abstract A peripheral blood immunophenotyping assay was developed and validated for determination of total T-lymphocytes, helper T-lymphocytes, cytotoxic T-lymphocytes, B-lymphocytes, and natural killer cells in cynomolgus monkeys. Validation parameters included assessment of precision, linearity, antibody optimization, stability of peripheral blood samples, and stability of fixed immunophenotyping samples. Total lymphocyte populations were determined using a heterogeneous lymphocyte gating strategy consisting of CD45 fluorescent staining and sidescatter demarcation. Relative lymphocyte subset values were determined using antigen-specific gating strategies. Absolute subset concentrations for each lymphocyte subset were subsequently determined using a dual-platform methodology wherein relative lymphocyte subset values (via flow cytometry analyses) were multiplied by the absolute total lymphocyte (via hematology analyses) values. Reference ranges are presented for cynomolgus monkey, rhesus monkey, and baboon. Additional 1-year longitudinal immunophenotyping values are presented for the cynomolgus monkey. The method validation and reference ranges presented in this research provide a robust analytical methodology for determination of peripheral blood lymphocyte subsets in various non-human primate species. Introduction Pharmaceutical products can exert intended pharmacologic or unintended toxicologic effects on the immune system. Monitoring these immunomodulatory effects in pre-clinical toxicology studies can aid in risk assessment for human clinical trial study design and therapeutic application of new molecular/biologic entities. Potential immunomodulation of investigational therapeutic compounds can be detected during pre-clinical toxicology studies using nominal study design features including evaluation of opportunistic infection, routine hematology parameters, and histology of immune tissues. More specialized immunoassays used in pre-clinical toxicology studies include immunophenotyping of peripheral blood and/or lymphoid tissues, T-cell-dependent antibody responses to parenterally-administered antigens, natural killer (NK) cell cytotoxicity assays, evaluation of soluble factors including cytokines and complement, as well as evaluation of host resistance to sub-lethal doses of prototypic pathogens. Multi-center studies sponsored by the National Toxicology Program during the 1980s and 1990s evaluated a tiered system of these types of immunoassays for predicting the immunosuppressive potential of various environmental chemicals and therapeutic drugs (Luster et al., 1988, 1992a,b, 1993; Luster and Rosenthal, 1993). Among the various assays evaluated, lymphocyte immunophenotyping and T-cell-dependent antibody responses were among the most predictive assays. The results of these pivotal *Current affiliation: AbbVie Inc., North Chicago, IL, USA. Address for correspondence: Michael Holsapple, michael. holsapple@covance.com Keywords Immunophenotyping, flow cytometry, non-human primate, peripheral blood, validation, reference range, lymphocytes, cynomolgus History Received 14 October 2014 Accepted 11 December 2014 Published online 20 January 2015 studies and other immunoassay evaluations supported the inclusion of lymphocyte immunophenotyping as one of several recommended immunoassays during non-clinical toxicology studies of new chemical pharmaceuticals (ICH S8, 2006). While not specifically discussed in the respective biopharmaceutical guidance documents (ICH S6, 1997 and ICH S6 R1, 2009), lymphocyte immunophenotyping is also utilized during preclinical toxicology studies evaluating new biopharmaceuticals. While ostensibly used for monitoring toxicology end-points, immunophenotyping assays can also be used to monitor intended pharmacologic effects not limited to numeric modulation of specific cell types, alteration of cell surface markers, and receptor occupancy assessments. The pre-clinical utility of this assay benefits from a wide array of reagents and analytical equipment, the ability to conduct serial peripheral blood analyses in conscious/live animals, and the potential to apply comparable translational assays to a human clinical trial design. As with any analytical methodology, standard technical validation parameters should be evaluated prior to research application of peripheral blood immunophenotyping assays. Extensive literature exists regarding validation procedures and parameters for peripheral blood immunophenotyping assays with human samples (Calvelli et al., 1993; Cunliffe et al., 2009; Davis et al., 2011; Gratama et al., 2007; Nicholson et al., 1997; Schnizlein-Bick et al., 2002). While cynomolgus monkeys are often utilized for toxicologic testing of small molecule and biologic pharmaceutical agents, there are rare published examples of peripheral blood immuno-phenotyping assay validations specific for this species (Baker et al., 2008; Bleavins et al., 1993; Krejsa et al., 2013). The purpose of this article is to present

3 DOI: / X Immunophenotyping validation for non-human primates 65 validation methodologies and corresponding analytical results for a readily applicable cynomolgus monkey peripheral blood immunophenotyping assay. The assay utilizes CD45 staining of peripheral blood lymphocytes. In addition to method validation results, immunophenotyping reference ranges are presented for cynomolgus and rhesus monkeys as well as baboons. Additional immunophenotyping data are presented for 1 year of longitudinal peripheral blood immunophenotyping data from cynomolgus monkeys. Materials and methods Reagents BD Biosciences (San Diego, CA) manufactured three customized antibody cocktails for Covance Laboratories. Each cocktail contained an antibody against CD45 (IgG 1, Clone D , peridinin chlorophyll protein [PerCP]). Cocktail 1 also contained three additional isotype control antibodies (IgG 1, Clone MOPC- 21; fluorescein isothiocyanate [FITC], phycoerythrin [PE], and allophycocyanin [APC]). Cocktail 2 also contained antibodies against CD3 (IgG 1, Clone SP34-2, FITC), CD4 (IgG 1, Clone L200, PE), and CD8 (IgG 1, Clone SK1, APC). Cocktail 3 also contained antibodies against CD3 (IgG 1, Clone SP34-2, FITC), CD16 (IgG 1, Clone 3G8, PE), and CD20 (IgG 1, Clone L27, APC). At the time of this research, 4-color immunophenotyping cocktails for use with non-human primates were not commercially available. FACS Lysing Solution (BD Biosciences) was utilized to lyse red blood cells. As this reagent contains formaldehyde, it also served as the fixative for stained cells. Staining buffer was purchased from BD Biosciences as 2% bovine serum albumin and 0.1% sodium azide in phosphate buffered saline. Equipment and software All flow cytometry experiments were conducted on a FACSCalibur (BD Biosciences) flow cytometer. Data acquisition and analysis was conducted using CellQuest Pro (BD Biosciences) software. Total leukocyte and differential blood cell counts were measured from each peripheral blood sample using an Advia 120 Hematology System (Siemens Healthcare Diagnostics). Animals During the assay validation phase, male and female cynomolgus monkeys (Macaca fascicularis) were utilized from the Covance Laboratories facility stock colony. The animals were &2 3-yearsold, with body weight ranges of 2 4 kg. Three different cohorts of five animals each were utilized for inter-sample precision, blood volume linearity and antibody cocktail volume optimization, and stability parameters, respectively. Following completion of sample collections, all animals were re-assigned to the testing facility stock colony. During the species reference range determination experiments, peripheral blood samples (&3 ml) were obtained from animals at vendor facilities and shipped at insulated ambient conditions by overnight express courier to the testing facility. A total of animals/sex/origin were evaluated. Blood samples from cynomolgus and rhesus (Macaca mulatta) monkeys were purchased from Covance Research Products (Alice, TX). The animals were 2 3-years-old, with body weight ranges of 2 4 kg. The cynomolgus monkeys originated from Vietnam, China, Mauritius, and the Philippines. Rhesus monkeys originated from China. Baboon (Papio hamadryas) samples were purchased from Mannheimer Foundation (Homestead, FL). The animals were 1 2-years-old, with body weight ranges of 3 8 kg. During the year-long longitudinal reference range experiments, peripheral blood samples were obtained from five animals/sex maintained in the Covance Laboratories facility stock colony. The males were of Philippine origin, were 2 3-years-old, and had body weight ranges of 2 3 kg. The females were of mixed origin (Mauritius, Indonesia, Vietnam, China), were 3 5-years-old, and had body weight ranges of 2 4 kg. These monkeys were maintained for the entire sampling period without any experimental test compound administration; instead, these animals were utilized for general husbandry and/or routine procedural training purposes. All animals were maintained in compliance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. All animal research was approved by the Covance Institutional Animal Care and Use Committee. Immunophenotyping analyses Animals were not fasted prior to blood collection. Blood samples (&1 ml, unless otherwise indicated) were collected via a femoral vein. The anti-coagulant was potassium EDTA (K 3 EDTA). Each blood sample was held at ambient conditions (unless otherwise noted) prior to immunophenotyping sample processing. Blood (50 ml, unless otherwise noted) was placed into each of the necessary number of sample tubes, after which 2 3 ml cold staining buffer was placed into each tube; the tube was briefly vortexed and then centrifuged for 5 min at 200 x g. Staining buffer was aspirated from each tube, sufficient to leave a small volume of staining buffer (estimated ml) with the pelleted cells. Appropriate antibody cocktail (20 ml, unless other-wise noted) was added to the appropriate sample tube. The tubes were briefly vortexed, then incubated at ambient conditions for &15 30 min. The incubated cells were washed with staining buffer as indicated above, &3ml FACS Lysing Solution was added to each tube; the tube was briefly vortexed and then utilized for flow cytometry analysis experiments. Unless otherwise noted for timed experiments; immunophenotyping samples were processed from blood collection through fixation within 3 6 h and then analyzed within 24 h of fixation. Lymphocyte populations were delineated on the flow cyto meter using a heterogeneous lymphocyte gating strategy consisting of high CD45 fluorescent staining and low side scatter (SSC) gating (CD45 high SSC low ) (Schnizlein-Bick et al., 2002). At least gated lymphocytes were acquired from each tube for each analysis. Lymphocyte subsets were delineated from the total CD45 + lymphocyte population as total T-lymphocytes (CD3 + ), helper (CD3 + CD4 + CD8 ) T-cells, cytotoxic (CD3 + CD4 CD8 + ) T-cells, B-cells (CD3 CD20 + CD16 ), and natural killer (NK) (CD3 CD20 CD16 + ) cells. Concurrent samples stained with control reagents were used to verify flow cytometry voltage and compensation settings, as well as subset gating strategies. Relative lymphocyte values were calculated from the flow cytometer. A dual-platform methodology was utilized to calculate absolute lymphocyte values wherein relative values for each lymphocyte subset (via immunophenotyping analysis) were multiplied by the absolute lymphocyte values (via hematology analysis) to enumerate absolute cell values (cells/ml) for each lymphocyte subset. The lymphosum (summation of relative values for total T-lymphocytes, B-lymphocytes, and NK cells) was calculated from each immunophenotyping sample to confirm 4 95% gating efficiency (data not presented). Validation parameters Precision The precision of the analytical procedure was defined as the closeness of agreement (degree of scatter) between a series of

4 66 R. G. Caldwell et al. J Immunotoxicol, 2016; 13(1): measurements obtained from multiple individual samplings of the same homogeneous sample under prescribed conditions. Intersample precision was determined by preparing and analyzing 10 replicate immunophenotyping samples from five animals. Linearity The linearity of the analytical procedure was defined as the ability to obtain test results that are directly proportional to the concentration of analyte in the sample. One immunophenotyping set was made from each of five animals using peripheral blood volumes of 50, 25, 12.5, and 6.25 ml. The blood samples were diluted in staining buffer to maintain a constant total blood volume of 50 ml, then processed as an immunophenotyping sample as described above. Total lymphocyte concentrations were measured from the diluted blood samples. Absolute values reported in the linearity data tables reflect the absolute number (cells/ml) in the diluted linearity sample. Antibody-cocktail optimization Antibody cocktails were evaluated to investigate the effect of varying volumes of antibody cocktail per tube. One immunophenotyping set was made from each of five animals using antibody cocktail volumes of 20, 15, 10, and 5 ml. The antibody cocktails were diluted in staining buffer to maintain a constant total antibody volume of 20 ml, then processed as an immunophenotyping sample as described above. Percentage change values reported in the antibody cocktail optimization table reflect relative change from assay using a 20-ml antibody volume. Stability The stability of the peripheral blood samples was determined for both fresh (intra- and inter-laboratory) and fixed preparations from each of five animals. Intra-laboratory blood sample stability was determined by preparing immunophenotyping samples following storage of the blood for within 6 h (Time 0) and 24, 48, and 72 h at ambient conditions. After shipment of the collected blood by overnight carrier (at insulated ambient temperature), inter-laboratory blood sample stability was evaluated. The blood was shipped from the testing facility via overnight carrier, with confirmed documented receipt in destination locations (i.e. FedEx airport offices, Nashville, TN) and ultimate return to testing facility (&24 h total transit time). Immunophenotyping analysis results of the shipped samples were compared to concurrent immunophenotyping analysis results conducted at the time of sample collection. Intra-laboratory fixed samples stability was conducted following analysis of one fixed immunophenotyping set within 6 h of blood sample collection (Time 0) and again following storage of the same fixed sample for &24, 48, and 72 h at insulated ambient conditions within the testing facility. Statistical analyses Statistical analysis of immunophenotyping data included calculation of means, standard deviations, and coefficient of variance as appropriate. The blood dilution linearity data was evaluated using individual and simultaneous regression analyses (Dixon, 1993). The stability of the fresh and fixed samples during the 72-h storage period was evaluated using linear regression over time analyses (Draper and Smith, 1966). Results Precision The inter-sample precision of the assay was assessed from 10 replicate immunophenotyping preparations from one blood sample from each animal (five animals, Table 1). The intersample precision for each absolute and relative lymphocyte subset was 5 3% coefficient of variance (CV) for all parameters, except NK cells. The inter-sample precision of absolute and relative NK cells ranged from 4 20% CV. By comparison, intra-sample (not inter-sample) precision assesses the reproducibility of the flow cytometer (rather than the analyst or reagents). Intra-sample precision (10 measurements of one immunophenotyping sample) was assessed in earlier immunophenotyping validation studies with a comparable (lyse/no-wash) assay. Intra-sample precision was 5 13% CV for NK cells and 10% CV for remaining phenotypes (data not shown). Based on these results, the current immunophenotyping assay provides precise identify-cation of total T-lymphocytes, helper and cytotoxic T-lymphocytes, B- lymphocytes, and NK cells. The relatively higher imprecision of the NK cell populations is likely a function of the small absolute and relative numbers of NK cells in any given sample. Table 1. Inter-sample precision. Animal number Parameter #/ml % #/ml % #/ml % #/ml % #/ml % I00074 Mean SD %CV I00097 Mean SD %CV I00116 Mean SD %CV I00124 Mean SD %CV I00738 Mean SD %CV Inter-sample precision was determined by preparing and analyzing 10 replicate immunophenotyping samples from five cynomolgus monkeys. Absolute (#/ml) and relative (%) values for each set of replicates are expressed as the mean values with standard deviation (SD) and coefficient of variance (%CV).

5 DOI: / X Immunophenotyping validation for non-human primates 67 Linearity Lymphocyte subset values were determined using nominal (50 ml) as well as serially diluted blood volumes ( ml, five animals, Table 2). The slopes and intercepts of the expected and actual dilutions were not statistically different over the tested dilution range for all lymphocyte subsets (p 0.01). For all lymphocyte populations, the mean marker fluorescence of positive staining cells was well-delineated relative to the unstained cells at all blood volume dilutions (data not shown). These data indicate that blood volumes from ml could be used to identify all lymphocyte subsets. Blood volumes of 50 ml were routinely used for the immunophenotyping staining in order to reduce the impact of potential pipetting errors. The entire experiment requires sufficient blood volume to conduct routine hematology analyses (assuming absolute subset values are required) and a nominal 150 ml of blood to prepare the immunophenotyping samples. Should analytical errors occur, individual immunophenotyping experiments could potentially be repeated using relatively small volumes (6.25 ml) obtained from residual hematology blood samples. Antibody cocktail optimization Lymphocyte subset values were determined using nominal (20 ml) as well as serially diluted antibody cocktail volumes (20 5 ml, five animals, Tables 3 and 4). For all lymphocyte populations, the mean marker fluorescence of positive staining cells was welldelineated relative to the unstained cells at all antibody cocktail dilutions (data not shown). For all lymphocyte subsets excluding NK cells, absolute and relative lymphocyte values from samples prepared with 5 15 ml of antibody cocktail were typically within 3% of values obtained using 20 ml of antibody cocktail. For NK cells, the absolute and relative values using 5 15 ml of antibody cocktail were 6 21% of values using a 20 ml antibody cocktail. The variances for NK cells were not consistently proportional to reagent dilution, did not always trend in the same direction with increasing dilution per animal, and were within the inter-sample Table 2. Blood volume linearity. precision (4 20% CV, see above) for this analyte. The NK cell value variances were always highest at the 5-ml antibody cocktail volume. These data indicated that antibody cocktail volumes from ml could readily be used to identify all lymphocyte subsets. This allows for analysis of immunophenotyping samples even during unexpected conditions of reagent shortage. Antibody cocktail volumes of 20 ml were routinely used with this methodology in order to reduce the impact of potential antibody cocktail pipetting errors. These data also suggest that 20 ml of antibody reagent is perhaps excess reagent for normal lymphocyte subsets; and, therefore, would be sufficient to detect numeric increases in particular lymphocyte populations induced by experimental conditions (as compared to untreated animals). Stability Intra-laboratory stability was assessed for blood samples stored at ambient conditions (five animals, Tables 5 and 6). For all T-lymphocyte populations, absolute and relative lymphocyte subsets were within 10% of initial values over 48 h of storage at ambient conditions. For B- and NK cells, absolute and relative lymphocyte subsets were typically within 10% of starting values at 24 h, and differed by as much as 20% of starting values by 48 h. All of the changes within 48 h were deemed acceptable given the inter-sample precision of the assay. However, the consistently decreased values following 72 h of ambient storage (often 4 20% of starting values for B- and NK cells) were deemed unacceptable for experimental purposes. However, using the heterogeneous gating strategy, analysts were easily able to delineate the total lymphocyte populations among the cellular debris at all timepoints (data not shown). The mean marker fluorescence of subsetdelineating surface markers was well-delineated relative to the unstained cells at all timepoints (data not shown). Linear regression analysis of intra-laboratory blood sample stability data confirm that the slope of the line is significantly decreased from Time 0 only for relative and absolute B-lymphocytes, and only at 72 h (p 0.05). Additional intra-laboratory blood sample Animal number Blood (ml) Actual Expected Actual Expected Actual Expected Actual Expected Actual Expected I I I I a I a Due to technical errors, the absolute lymphocyte counts (and therefore absolute immunophenotyping values) were not collected for this dilution. Assay linearity was assessed from serial dilutions of cynomolgus monkey blood ranging from ml. Actual measured and calculated expected absolute values (#/ml) are presented for each animal at each dilution. Slopes and intercepts of the expected and actual dilutions were not statistically different over the tested dilution range for all lymphocyte subsets (p 0.01). These data indicate that blood volumes from ml could be used to identify all lymphocyte subsets.

6 68 R. G. Caldwell et al. J Immunotoxicol, 2016; 13(1): stability experiments using a comparable assay (lyse/no-wash) indicated that K 3 EDTA, sodium heparin, or sodium citrate anticoagulants exhibited comparable stability profiles when retained at either insulated ambient or insulated refrigerated conditions (data not shown). Samples are routinely analyzed in this laboratory within a few hours of blood collection. There are instances where the uniqueness of a particular sample, laboratory resource constraints, and potential experimental errors would necessitate re-analysis of retained blood samples within h of sample collection. Inter-laboratory stability was assessed for blood samples shipped at insulated ambient conditions (five animals, Tables 5 and 6). For all lymphocyte populations, the identity of total lymphocyte populations and mean marker fluorescence of subset- Table 3. Antibody cocktail volume optimization. Absolute lymphocyte subset concentrations. Animal number Antibody (ml) #/ml % Change #/ml % Change #/ml % Change #/ml % Change #/ml % Change I I I I I Percentage change values are relative to the values obtained using 20 ml antibody cocktail. Antibody cocktail volume optimization was assessed using serial dilutions of antibody cocktail reagents ranging from 20 5 ml. Measured absolute (#/ml) lymphocyte subset values are presented for each cynomolgus monkey at each antibody cocktail dilution. Changes for measured values at each antibody cocktail dilution are presented relative (%) to the nominal 20 ml antibody cocktail volume. These data indicated that antibody cocktail volumes from 20 5 ml could be used to identify all lymphocyte subsets, and are sufficient to detect numeric increases in particular lymphocyte populations induced by experimental conditions. Table 4. Antibody cocktail volume optimization. Relative lymphocyte subset values. Animal number Antibody (ml) % % Change % % Change % % Change % % Change % % Change I I I I I Percentage change values are relative to the values obtained using 20 ml antibody cocktail. Antibody cocktail volume optimization was assessed using serial dilutions of antibody cocktail reagents ranging from 20 5 ml. Measured relative) lymphocyte subset values are presented for each cynomolgus monkey at each antibody cocktail dilution. Changes for measured values at each antibody cocktail dilution are presented relative (%) to the nominal 20 ml antibody cocktail volume. These data indicated that antibody cocktail volumes from 20 5 ml could be used to identify all lymphocyte subsets, and are sufficient to detect numeric increases in particular lymphocyte populations induced by experimental conditions.

7 DOI: / X Immunophenotyping validation for non-human primates 69 Table 5. Blood sample stability. Absolute lymphocyte subset concentrations. Animal number Timepoint #/ml % Change #/ml % Change #/ml % Change #/ml % Change #/ml % Change I a INTER I a INTER I a INTER I a INTER I a INTER a The parameter is statistically significant (slope is significantly different from 0) using linear regression over time (p 0.05). Blood sample stability was assessed using cynomolgus monkey blood samples retained at ambient conditions for up to 72 h, as well as blood sampled shipped/returned from an external site as a surrogate for inter-laboratory (INTER) shipments. Measured absolute (#/ml) lymphocyte subset values are presented for each animal at each timepoint. Linear regression analysis of intra-laboratory blood sample stability data confirm that the slope of the line is significantly decreased from Time 0 only for relative and absolute B-lymphocytes, and only at 72 h (p 0.05). These data indicate that blood samples can be retained at ambient conditions for h and shipped between laboratories at ambient conditions. Table 6. Blood sample stability. Relative lymphocyte subset values. Animal number Timepoint a % % Change % % Change % % Change % % Change % % Change I a INTER I a INTER I a INTER I a INTER I a INTER a The parameter is statistically significant (slope is significantly different from 0) using linear regression over time (p 0.05). Blood sample stability was assessed using cynomolgus monkey blood samples retained at ambient conditions for up to 72 h, as well as blood sampled shipped/returned from an external site as a surrogate for inter-laboratory (INTER) shipments. Measured relative (%) lymphocyte subset values are presented for each animal at each timepoint. Linear regression analysis of intra-laboratory blood sample stability data confirm that the slope of the line is significantly decreased from Time 0 only for relative and absolute B-lymphocytes, and only at 72 h (p 0.05). These data indicate that blood samples can be retained at ambient conditions for h and shipped between laboratories at ambient conditions.

8 70 R. G. Caldwell et al. J Immunotoxicol, 2016; 13(1): Table 7. Fixed sample stability. Absolute lymphocyte subset concentrations. Animal number Timepoint #/ml % Change #/ml % Change #/ml % Change #/ml % Change #/ml % Change I I I I I Fixed sample stability was assessed using stained immunophenotyping samples retained at ambient conditions for up to 72 h. Measured absolute (#/ml) lymphocyte subset values are presented for each cynomolgus monkey at each timepoint. Linear regression analysis of fixed sample stability data indicates that the slope of the line was not significantly different from Time 0 during the 72 h period for all phenotypes (p 0.05). These data indicate that fixed immunophenotyping samples can be retained at ambient conditions for up to 72 h at ambient conditions. delineating surface markers were well-delineated relative to the unstained cells at all timepoints (data not shown). Values for absolute and relative lymphocyte subsets were typically within 10% of initial values following shipment and, therefore, deemed acceptable given the analytical precision of the assay. These data indicate that blood samples can be shipped at ambient conditions. Intra-laboratory stability experiments using a comparable assay (lyse/no-wash) indicated that blood samples are also stable when stored at refrigerated conditions (data not shown). Therefore, inter-laboratory shipments are routinely conducted in this laboratory using insulated refrigerated conditions in order to reduce the potential for sample instability resulting from ambient temperature fluctuations related to airplane/airport transport and/or seasonal (summer/winter) temperature fluctuations. Stability data was assessed for fixed immunophenotyping samples prepared at the time of blood collection (five animals, Tables 7 and 8). For all lymphocyte populations, the identity of total lymphocyte populations and mean marker fluorescence of subset-delineating surface markers were well-delineated relative to the unstained cells at all timepoints (data not shown). For all lymphocyte populations, absolute and relative lymphocyte subsets were typically within 10% of starting values through 72 h of storage at ambient conditions. Linear regression analysis of fixed sample stability data indicates that the slope of the line was not significantly different from Time 0 during the 72 h period for all phenotypes (p 0.05). Blood samples are routinely processed through fixation and analyzed within a few hours of sample collection. However, these data indicate that fixed immunophenotyping samples can be re-analyzed for up to 72 h at ambient conditions. Additional fixed sample stability experiments using a comparable assay (lyse/no-wash) indicated that fixed samples are also stable when stored at refrigerated conditions (data not shown). Geographic immunophenotyping ranges Immunophenotyping were obtained from cynomolgus monkeys originating from four different geographic locations, as well as rhesus monkeys and baboons (Tables 9 and 10). All scatter plots for each animal from each species were reviewed and found to provide appropriate demarcation of respective immunophenotyping parameters. A detailed analysis of precision, sample volume linearity, antibody concentration optimization, whole blood stability, or fixed immunophenotyping stability was not conducted for rhesus monkey or baboon. No marked differences were evident among the various geographies and species examined, nor were there gender differences within any cohort. The mean values, standard deviations, and absolute ranges generally overlapped amongst all cohorts. Some minor trends were evident. Mean absolute lymphocyte subset values were generally similar amongst the continental (Vietnamese and Chinese) cynomolgus monkeys, as opposed to extra-continental cynomolgus (Mauritius, the Phillipines) cohorts. The Mauritius cohort generally exhibited the smallest mean absolute lymphocyte values amongst all cynomolgus geographies, and baboons generally exhibited the lowest mean absolute lymphocyte subset values amongst all the species. Mean relative lymphocyte subset values were generally similar among all geographies and species examined. Longitudinal variation One year of longitudinal immunophenotyping data was collected from a cohort of cynomolgus monkeys (five/sex). All animals were healthy during the course of the evaluations. Representative absolute and relative immunophenotyping values (and total absolute lymphocyte values) from one male and one female monkey are presented in Figures 1a d. Absolute lymphocyte subset values exhibit periods of static or active fluctuation over the course of the 1-year sampling interval, always associated with fluctuation patterns for the absolute total lymphocyte count. Absolute total lymphocyte values and lymphocyte subset values fluctuated by as much as 2-fold for any particular animal during the course of the year, with parallel trends for each parameter. Given these trends, it is not surprising that relative lymphocyte subset values exhibited relatively little fluctuation throughout the 1-year sampling interval. Trends for the remaining eight examined animals were comparable to the presented data (data not shown).

9 DOI: / X Immunophenotyping validation for non-human primates 71 Table 8. Fixed sample stability. Relative lymphocyte subset values. Animal number Timepoint % % Change % % Change % % Change % % Change % % Change I I I I I Fixed sample stability was assessed using stained immunophenotyping samples retained at ambient conditions for up to 72 h. Measured relative (%) lymphocyte subset values are presented for each cynomolgus monkey at each timepoint. Linear regression analysis of fixed sample stability data indicates that the slope of the line was not significantly different from Time 0 during the 72 h period for all phenotypes (p 0.05). These data indicate that fixed immunophenotyping samples can be retained at ambient conditions for up to 72 h at ambient conditions. Discussion An immunophenotyping assay has been validated for use with cynomolgus monkey peripheral blood samples. The lyse/no-wash assay generates precise, reproducible, and sensitive data for enumeration of total T-lymphocytes, helper and cytotoxic T-lymphocytes, B-lymphocytes, and NK cells. The assay utilizes a unique yet robust heterogeneous lymphocyte gating strategy consisting of CD45 high SSC low demarcation to identify total lymphocytes, with relative lymphocyte subset analyses conducted via antigen-specific antibody cocktails. Lymphocyte subsets were gated from the total CD45 + lymphocyte populations. Absolute lymphocyte subsets were then enumerated using a dual-platform methodology. While samples should typically be processed through fixation as soon as possible, the whole blood sample could be processed and analyzed within h after collection. Fixed immunophenotyping samples are stable for up to 3 days. Samples can also be collected from remote sites and shipped to the analytical facility, with sample processing/fixation occurring within &1 day of sample collection. This methodology represents the only evident published example of extensive validation data for a cynomolgus monkey immunophenotyping methodology using a heterogeneous lymphocyte gating strategy. During pre-validation feasibility testing, analytical results were compared using both the homogeneous (side-scatter vs forward-scatter) and heterogeneous (sidescatter vs CD45) gating methodologies. The analytical results were not markedly different between the two assays. However, there were a few aspects of the assay that ultimately led to the decision to use the heterogeneous gating strategy. Foremost, feasibility experiments suggested that the homogeneous lymphocyte gating strategy was more susceptible to signal deterioration during extended stability testing, which is consistent with published literature (Bergeron et al., 2002; Schnizlein-Bick et al., 2002; Schumacher and Burkhead, 2000). Secondly, the additional resource required to use the heterogeneous vs homogeneous gating methodologies is limited to one extra antibody per reaction tube (CD45). All other resource aspects of the homogeneous and heterogenous gating strategies are comparable, including the total number of tubes required to conduct the assay (assuming a maximum 4-color staining method). Finally, the validated heterogeneous gating and dual-platform methodology provides an interim methodology to a potentially more efficient single-platform methodology. The primary benefit of a potential single-platform methodology would be the removal of concomitant hematology analyses, thereby utilizing smaller blood collection volumes and the ability to collect multiple serial samples from one animal (particularly useful for rodent toxicology studies). The single-platform method requires the use of integrated sample calibration beads which necessitate use of CD45 gating strategies and the elimination of any sample aspiration steps (i.e. a lyse/no-wash assay). As described in the subsequent paragraph, the current assay (a lyse/ wash dual-platform assay) is hampered by CD16 matrix interference (see following discussion) that precluded transition to the lyse/no wash single-platform method. Taken together, the analytical and technological advantages afforded by the heterogeneous gating strategy outweigh the relatively minor resource advantages of the homogeneous gating strategy. These and other considerations have led to the utility of heterogenous gating strategies for various human clinical applications (Bergeron et al., 2002; Davis et al., 2011; Gratama et al., 2007; Mandy et al., 2003; Nicholson et al., 1997; Schnizlein-Bick et al., 2002). By using heterogenous gating methodologies as the platform method for pre-clinical studies, there is potential to develop translational flow cytometry methodologies for use in both pre-clinical and clinical biomarker assays. Pre-validation feasibility experiments suggested a plasma component interfered with CD16 staining of NK cells when using a lyse/no-wash assay (in preparation for a single-platform methodology as described above). When using the validated assay antibody clones in a single-platform methodology, the CD3 CD16 + NK cell populations were not well discriminated from the CD3 CD16 cell types. It was determined that none of the various reagent components (CD16 antibody clones, antibody cocktail interference, quantification beads, fixation solutions,

10 72 R. G. Caldwell et al. J Immunotoxicol, 2016; 13(1): Table 9. Species reference ranges. Absolute lymphocyte subset concentrations. Animal (Origin) Parameter Male Female Male Female Male Female Male Female Male Female Cynomolgus (Vietnam) Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Cynomolgus (China) Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Cynomolgus (Mauritius) Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Cynomolgus (Philipines) Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Rhesus (China) Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Baboon (United States) Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Absolute values presented as number of thousand cells per microliter (E3/mL). Relative values presented as percentage of total lymphocytes. The mean, standard deviations, mean ± 2 SD, and number (per gender) of absolute (#/ml) immunophenotyping values are presented for cynomolgus monkeys obtained from referenced geographies as well as rhesus monkeys and baboons. blood anticoagulants) or flow cytometer settings (voltage, compensation, gating strategies) contributed to the unexpected NK cell identification. However; introducing a lyse/wash step into the sample preparation procedure allowed for well-delineated and easily distinguished NK cell populations. For example, mean NK cell values were 1 4% (five animals; unpublished data) with the single-platform lyse/no-wash method as compared to 3 15% (five animals, Table 1) with the validated dual-platform lyse/wash method. These matrix interference results are consistent with soluble CD16 (F c R g III) interference, particularly inasmuch as soluble CD16 is expressed in normal human serum at levels ranging from 7 76 nm (Fleit et al., 1992). Serum levels of soluble CD16 in non-human primates were not evident in the literature. However, it is interesting to note that multiple amino acid sequence differences within the CD16 transmembrane domains between human and non-human primate species suggest that there may be differences in serum concentrations of soluble CD16 amongst human and non-human primate species (Rogers et al., 2006). In addition, other authors have indicated that soluble CD16 concentrations vary amongst humans and can interfere with CD16-dependent flow cytometry techniques (Giorgi and Landay, 1994). Nonetheless, the results from these experiments indicate that the serum must be removed prior to performing CD16 antibody staining of cynomolgus monkey peripheral blood. The necessity of the lyse/wash procedure excluded the use of quantification beads and the single-platform methodology, as counting beads that could be added after washing were not available at the time of these studies. Current methodologies can also use CD159a (NKG2A) in place of or alongside CD16 to help identify NK cells (Yokoyama et al., 1998), particularly when the pharmacologic mechanism involves interaction with the CD16 molecule. CD56 was also considered as a potential NK cell marker given its use in human NK cell activity and immunophenotyping experiments; however, CD56 antibodies did not react with expected cynomolgus NK cell populations during prevalidation feasibility testing (data not shown). In addition, use of