Journal of Immunotoxicology

ISSN: 1547-691X (Print) 1547-6901 (Online) Journal homepage: http://www.tandfonline.com/loi/iimt20

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: 10.3109/1547691X.2014.1001098 To link to this article: http://dx.doi.org/10.3109/1547691X.2014.1001098

Published online: 20 Jan 2015.

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Date: 13 November 2015, At: 21:23

http://www.tandfonline.com/iimt ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, 2016; 13(1): 64–76 ! 2015 Covance DOI: 10.3109/1547691X.2014.1001098

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

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Covance Laboratories, Madison, WI, USA

Abstract

Keywords

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.

Immunophenotyping, flow cytometry, non-human primate, peripheral blood, validation, reference range, lymphocytes, cynomolgus

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, E-mail: michael. [email protected]

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

DOI: 10.3109/1547691X.2014.1001098

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

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Reagents BD Biosciences (San Diego, CA) manufactured three customized antibody cocktails for Covance Laboratories. Each cocktail contained an antibody against CD45 (IgG1, Clone D058-1283, peridinin chlorophyll protein [PerCP]). Cocktail 1 also contained three additional isotype control antibodies (IgG1, Clone MOPC21; fluorescein isothiocyanate [FITC], phycoerythrin [PE], and allophycocyanin [APC]). Cocktail 2 also contained antibodies against CD3 (IgG1, Clone SP34-2, FITC), CD4 (IgG1, Clone L200, PE), and CD8 (IgG1, Clone SK1, APC). Cocktail 3 also contained antibodies against CD3 (IgG1, Clone SP34-2, FITC), CD16 (IgG1, Clone 3G8, PE), and CD20 (IgG1, 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 18–20 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.

Immunophenotyping validation for non-human primates

65

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 (K3EDTA). 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 10–20 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, &3 ml 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 (CD45highSSClow) (Schnizlein-Bick et al., 2002). At least 10 000 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

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J Immunotoxicol, 2016; 13(1): 64–76

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.

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.

Linearity

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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.

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).

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.

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, Blymphocytes, 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.

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

Table 1. Inter-sample precision.

Animal number I00074 I00097 I00116 I00124 I00738

Total T-cells

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Parameter

#/ml

%

#/ml

%

#/ml

%

#/ml

%

#/ml

%

Mean SD %CV Mean SD %CV Mean SD %CV Mean SD %CV Mean SD %CV

4559.1 15.8 0.35 4786.8 26.4 0.55 4662.3 37.0 0.79 5698.4 77.4 1.36 5201.7 51.2 0.98

80.30 0.28 0.35 71.40 0.39 0.55 71.80 0.57 0.79 62.50 0.85 1.36 72.30 0.71 0.98

2482.8 24.3 0.98 2331.1 24.3 1.04 2351.5 38.4 1.63 3541.2 65.5 1.85 2641.7 34.6 1.31

43.70 0.43 0.98 34.80 0.36 1.04 36.20 0.59 1.63 38.80 0.72 1.85 36.70 0.48 1.31

1833.7 30.3 1.65 2219.2 26.8 1.21 2032.6 31.1 1.53 1817.8 37.2 2.05 2178.4 34.0 1.56

32.30 0.53 1.65 33.10 0.40 1.21 31.30 0.48 1.53 19.90 0.41 2.05 30.30 0.47 1.56

731.1 18.0 2.46 1612.7 12.3 0.76 1080.3 28.9 2.68 1832.1 30.7 1.68 1401.1 31.2 2.23

12.90 0.32 2.46 24.10 0.18 0.76 16.60 0.45 2.68 20.10 0.34 1.68 19.50 0.43 2.23

220.4 27.4 12.44 178.2 36.4 20.41 622.8 24.1 3.87 1371.0 72.9 5.32 461.3 28.6 6.19

3.90 0.48 12.44 2.70 0.54 20.41 9.60 0.37 3.87 15.00 0.80 5.32 6.40 0.40 6.19

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).

Immunophenotyping validation for non-human primates

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Linearity Lymphocyte subset values were determined using nominal (50 ml) as well as serially diluted blood volumes (50–6.25 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 50–6.25 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

67

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 10–20 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

Table 2. Blood volume linearity. Total T-cells Animal number I01337

I01346

I01554

I01353

I01356

a

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Blood (mL)

Actual

Expected

Actual

Expected

Actual

Expected

Actual

Expected

Actual

Expected

50 25 12.5 6.25 50 25 12.5 6.25 50 25 12.5 6.25 50 25a 12.5 6.25 50 25 12.5 6.25

7466 3940 2206 610 10 819 5468 2904 1489 7161 3756 2004 1022 6643 – 1887 870 8214 4369 2327 1132

7466 3733 1867 933 10 819 5410 2705 1352 7161 3581 1790 895 6643 – 1661 830 8214 4107 2054 1027

3961 2101 1171 320 5157 2625 1408 713 3235 1724 922 447 3442 – 965 441 3730 2013 1053 520

3961 1981 990 495 5157 2579 1289 645 3235 1618 809 404 3442 – 860 430 3730 1865 932 466

3069 1611 901 248 4592 2285 1197 614 3297 1707 911 476 2668 – 754 350 4025 2099 1140 549

3069 1535 767 384 4592 2296 1148 574 3297 1649 824 412 2668 – 667 333 4025 2013 1006 503

3159 1642 887 238 3169 1661 820 409 1622 853 436 228 2052 – 601 283 1086 565 301 149

3159 1580 790 395 3169 1585 792 396 1622 811 405 203 2052 – 513 257 1086 543 271 136

508 243 129 36 479 299 139 64 408 190 110 51 364 – 104 51 268 129 63 34

508 254 127 63 479 240 120 60 408 204 102 51 364 – 91 46 268 134 67 33

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 50–6.25 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 50–6.25 ml could be used to identify all lymphocyte subsets.

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stability experiments using a comparable assay (lyse/no-wash) indicated that K3EDTA, 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 24–48 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. Total T-cells Animal number

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I01337

I01346

I01554

I01353

I01356

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Antibody (ml)

#/ml

% Change

#/ml

% Change

#/ml

% Change

#/ml

% Change

#/ml

% Change

20 15 10 5 20 15 10 5 20 15 10 5 20 15 10 5 20 15 10 5

7395 7397 7309 7453 10 845 10 944 10 824 10 827 7072 7199 7087 7073 6578 6608 6504 6576 8229 8308 8215 8281

– 0 1 1 – 1 0 0 – 2 0 0 – 0 1 0 – 1 0 1

3951 3963 3794 3980 5256 5195 5171 5163 3176 3253 3262 3270 3295 3356 3378 3324 3775 3815 3844 3718

– 0 4 1 – 1 2 2 – 2 3 3 – 2 3 1 – 1 2 2

2999 2991 3101 3043 4492 4590 4556 4582 3285 3329 3176 3227 2737 2693 2557 2681 3950 4018 3912 4040

– 0 3 1 – 2 1 2 – 1 3 2 – 2 7 2 – 2 1 2

3254 3205 3175 3241 3201 3296 3265 3271 1620 1593 1659 1622 2192 2127 2096 2172 1167 1163 1082 1133

– 2 2 0 – 3 2 2 – 2 2 0 – 3 4 1 – 0 7 3

543 500 522 576 567 552 588 494 413 397 454 379 400 391 381 335 294 301 289 232

– 8 4 6 – 3 4 13 – 4 10 8 – 2 5 16 – 2 2 21

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. Total T-cells Animal number I01337

I01346

I01554

I01353

I01356

Helper T-cells

Cytotoxic T-cells

Antibody (ml)

%

% Change

%

% Change

%

% Change

%

B-cells % Change

%

NK cells % Change

20 15 10 5 20 15 10 5 20 15 10 5 20 15 10 5 20 15 10 5

64.98 65.00 64.23 65.49 72.88 73.55 72.74 72.76 75.47 76.83 75.63 75.49 70.96 71.28 70.16 70.94 84.57 85.39 84.43 85.11

– 0 1 1 – 1 0 0 – 2 0 0 – 0 1 0 – 1 0 1

34.72 34.82 33.34 34.97 35.32 34.91 34.75 34.70 33.90 34.72 34.81 34.90 35.54 36.20 36.44 35.86 38.80 39.21 39.51 38.21

– 0 4 1 – 1 2 2 – 2 3 3 – 2 3 1 – 1 2 2

26.35 26.28 27.25 26.74 30.19 30.85 30.62 30.79 35.06 35.53 33.90 34.44 29.53 29.05 27.58 28.92 40.60 41.30 40.21 41.52

– 0 3 1 – 2 1 2 – 1 3 2 – 2 7 2 – 2 1 2

28.59 28.16 27.90 28.48 21.51 22.15 21.94 21.98 17.29 17.00 17.71 17.31 23.65 22.95 22.61 23.43 11.99 11.95 11.12 11.64

– 2 2 0 – 3 2 2 – 2 2 0 0 3 4 1 – 0 7 3

4.77 4.39 4.59 5.06 3.81 3.71 3.95 3.32 4.41 4.24 4.84 4.04 4.32 4.22 4.11 3.61 3.02 3.09 2.97 2.38

– 8 4 6 – 3 4 13 – 4 10 8 – 2 5 16 – 2 2 21

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.

Immunophenotyping validation for non-human primates

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Table 5. Blood sample stability. Absolute lymphocyte subset concentrations. Total T-cells Animal number I01598

I09982

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I09997

I01564

I01573

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Timepoint

#/ml

% Change

#/ml

% Change

#/ml

% Change

#/ml

% Change

#/ml

0 24 48 72 INTER 0 24 48 72 INTER 0 24 48 72 INTER 0 24 48 72 INTER 0 24 48 72 INTER

6300 6186 6333 5860 5877 4870 4830 4493 4650 4535 6481 6665 6537 5659 6141 5195 5119 5282 5255 5103 3708 3735 3718 3567 3364

–

3166 3062 3136 2974 2844 2236 2266 2037 2141 2074 3132 3243 3181 2690 2995 3061 3040 3159 3090 3045 2287 2274 2214 2176 2058

–

2695 2680 2660 2397 2557 2277 2124 2071 2028 2094 2495 2501 2434 2060 2321 1822 1776 1790 1738 1727 1134 1169 1168 1037 1023

–

2434 2243 2121 1654a 2008 2253 2060 1865 1742a 1936 1889 1831 1652 1455a 1699 1866 1793 1646 1341a 1752 1255 1191 1069 802a 1032

–

201 178 171 138 206 887 882 821 747 804 589 572 479 411 511 751 792 746 610 778 743 692 652 419 722

2 1 7 7 – 1 8 5 7 – 3 1 13 5 – 1 2 1 2 – 1 0 4 9

3 1 6 10 – 1 9 4 7 – 4 2 14 4 – 1 3 1 1 – 1 3 5 10

1 1 11 5 – 7 9 11 8 – 0 2 17 7 – 3 2 5 5 – 3 3 9 10

8 13 32 17 – 9 17 23 14 – 3 13 23 10 – 4 12 28 6 – 5 15 36 18

% Change – 11 15 31 3 – 1 7 16 9 – 3 19 30 13 – 5 1 19 4 – 7 12 44 3

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 24–48 h and shipped between laboratories at ambient conditions.

Table 6. Blood sample stability. Relative lymphocyte subset values. Total T-cells Animal number I01598

I09982

I09997

I01564

I01573

a

Timepoint 0 24 48 72 INTER 0 24 48 72 INTER 0 24 48 72 INTER 0 24 48 72 INTER 0 24 48 72 INTER

a

Helper T-cells

Cytotoxic T-cells

B-cells

%

% Change

%

% Change

%

% Change

%

70.31 70.86 73.81 77.92 72.74 60.12 62.56 61.55 61.75 61.29 70.83 72.68 73.70 69.27 72.59 65.93 66.66 69.14 70.54 66.97 64.48 65.64 67.72 69.80 65.70

– 1 5 11 3 – 4 2 3 2 – 3 4 2 2 – 1 5 7 2 – 2 5 8 2

35.33 35.07 36.55 39.55 35.20 27.61 29.35 27.90 28.43 28.03 34.23 35.36 35.86 32.93 35.40 38.85 39.58 41.35 41.48 39.96 39.77 39.96 40.32 42.58 40.20

0 1 3 12 0 – 6 1 3 2 – 3 5 4 3 – 2 6 7 3 – 0 1 7 1

30.08 30.70 31.00 31.87 31.64 28.11 27.51 28.37 26.93 28.30 27.27 27.27 27.44 25.21 27.44 23.12 23.12 23.43 23.33 22.66 19.73 20.54 21.27 20.29 19.99

0 2 3 6 5 – 2 1 4 1 – 0 1 8 1 – 0 1 1 2 – 4 8 3 1

27.16 25.69 24.72 21.99a 24.85 27.81 26.68 25.55 23.14a 26.16 20.65 19.97 18.63 17.81a 20.08 23.68 23.35 21.54 18.00a 22.99 21.83 20.93 19.48 15.69a 20.16

NK cells

% Change 0 5 9 19 9 – 4 8 17 6 – 3 10 14 3 – 1 9 24 3 – 4 11 28 8

% 2.24 2.04 1.99 1.83 2.55 10.95 11.43 11.24 9.92 10.86 6.44 6.24 5.40 5.03 6.04 9.53 10.31 9.77 8.19 10.21 12.92 12.17 11.88 8.20 14.11

% Change 0 9 11 18 14 – 4 3 9 1 – 3 16 22 6 – 8 3 14 7 – 6 8 37 9

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 24–48 h and shipped between laboratories at ambient conditions.

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Table 7. Fixed sample stability. Absolute lymphocyte subset concentrations. Total T-cells Animal number I01598

I09982

I09997

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I01564

I01573

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Timepoint

#/ml

% Change

#/ml

% Change

#/ml

% Change

#/ml

% Change

#/ml

0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72

5581 5386 5602 5425 4441 4229 4142 4076 6281 6260 6203 6028 6153 5745 5856 5748 4091 4070 3980 4017

– 3 0 3 – 5 7 8 – 0 1 4 – 7 5 7 – 1 3 2

2562 2566 2618 2557 2150 2052 2118 2098 2853 2871 2804 2776 3456 3334 3446 3410 2550 2506 2461 2469

– 0 2 0 – 5 2 2 – 1 2 3 – 4 0 1 – 2 3 3

2495 2357 2512 2412 1941 1829 1709 1669 2512 2377 2412 2370 2291 2007 2003 1955 1219 1245 1226 1210

–

2180 2096 2089 2034 1846 1830 1809 1815 1743 1739 1684 1656 2384 2282 2299 2300 1283 1189 1249 1205

– 4 4 7 – 1 2 2 – 0 3 5 – 4 4 4 – 7 3 6

262 290 287 267 651 637 523 528 573 579 599 585 1105 1138 1121 1062 960 957 883 874

6 1 3 – 6 12 14 – 5 4 6 – 12 13 15 – 2 1 1

% Change – 11 10 2 – 2 20 19 – 1 5 2 – 3 1 4 – 0 8 9

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).

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Table 8. Fixed sample stability. Relative lymphocyte subset values. Total T-cells Animal number I01598

I09982

I09997

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I01564

I01573

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Timepoint

%

% Change

%

% Change

%

% Change

%

% Change

%

0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72

69.76 67.32 70.02 67.81 62.91 59.90 58.67 57.74 71.78 71.54 70.89 68.89 63.24 59.04 60.18 59.08 64.53 64.19 62.77 63.36

– 3 0 3 – 5 7 8 – 0 1 4 – 7 5 7 – 1 3 2

32.02 32.07 32.72 31.96 30.46 29.07 30.00 29.71 32.60 32.81 32.04 31.73 35.52 34.27 35.42 35.05 40.22 39.53 38.82 38.95

– 0 2 0 – 5 2 2 – 1 2 3 – 4 0 1 – 2 3 3

31.19 29.46 31.40 30.15 27.49 25.90 24.21 23.64 28.71 27.17 27.56 27.08 23.55 20.63 20.59 20.09 19.23 19.64 19.34 19.08

–

27.25 26.20 26.11 25.42 26.15 25.92 25.62 25.71 19.92 19.87 19.24 18.92 24.50 23.45 23.63 23.64 20.24 18.75 19.70 19.01

– 4 4 7 – 1 2 2 – 0 3 5 – 4 4 4 – 7 3 6

3.27 3.62 3.59 3.34 9.22 9.02 7.41 7.48 6.55 6.62 6.85 6.68 11.36 11.70 11.52 10.91 15.14 15.10 13.93 13.79

6 1 3 – 6 12 14 – 5 4 6 – 12 13 15 – 2 1 1

% Change – 11 10 2 – 2 20 19 – 1 5 2 – 3 1 4 – 0 8 9

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 CD45highSSClow 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 24–48 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,

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Table 9. Species reference ranges. Absolute lymphocyte subset concentrations. Total T-cells Animal (Origin) Cynomolgus (Vietnam)

Cynomolgus (China)

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Cynomolgus (Mauritius)

Cynomolgus (Philipines)

Rhesus (China)

Baboon (United States)

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Parameter

Male

Female

Male

Female

Male

Female

Male

Female

Male

Female

Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number

6.55 2.740 1.07 12.03 2.49 12.90 18 4.27 1.96 0.34 8.19 1.10 11.78 210 3.24 1.06 1.12 5.35 1 7.28 80 4.48 2.04 0.39 8.56 1.42 11 93 6.36 1.832 2.70 10.03 3.47 9.25 20 2.00 0.787 0.43 3.58 0.80 3.64 20

6.00 4.077 0.00 14.15 1.08 19.96 20 3.51 1.65 0.21 6.81 0.80 11.41 197 2.88 0.93 1.03 4.74 1.1 6.04 80 3.65 1.80 0.05 7.24 1.18 8.54 88 4.83 2.026 0.78 8.88 2.06 9.29 20 1.63 0.989 0.00 3.60 0.65 3.96 20

3.54 1.387 0.77 6.32 1.30 6.70 18 2.12 0.92 0.28 3.97 0.52 5.70 210 1.56 0.60 0.36 2.77 0.5 3.95 80 2.24 0.94 0.36 4.11 0.66 5.4 93 2.96 1.022 0.92 5.01 1.72 4.78 20 1.03 0.408 0.21 1.84 0.46 1.88 20

3.05 1.611 0.00 6.27 0.58 6.96 20 1.86 0.86 0.14 3.58 0.50 5.10 197 1.45 0.50 0.46 2.44 0.4 3.18 80 1.87 0.84 0.20 3.54 0.65 4.33 88 2.29 0.914 0.46 4.12 1.11 4.33 20 0.82 0.445 0.00 1.71 0.33 1.84 20

2.53 1.275 0.00 5.08 0.72 5.10 18 1.81 0.99 0.18 3.80 0.40 5.90 210 1.42 0.55 0.31 2.53 0.4 3.29 80 1.82 1.00 0.19 3.82 0.29 5 93 2.84 0.906 1.02 4.65 1.35 4.40 20 0.78 0.354 0.07 1.49 0.26 1.58 20

2.46 2.251 0.00 6.96 0.41 10.99 20 1.36 0.77 0.17 2.89 0.20 5.32 197 1.19 0.48 0.24 2.15 0.3 2.51 80 1.46 0.88 0.30 3.22 0.39 3.91 88 2.11 1.066 0.00 4.24 0.60 4.76 20 0.62 0.439 0.00 1.50 0.24 1.91 20

3.27 1.786 0.00 6.84 0.75 7.05 18 1.77 1.12 0.46 4.01 0.35 9.10 210 1.03 0.49 0.05 2.01 0.25 2.76 80 2.18 1.94 1.70 6.05 0.24 8.62 93 2.94 1.143 0.66 5.23 1.07 5.08 20 1.04 0.355 0.33 1.75 0.58 1.78 20

3.12 1.552 0.02 6.22 0.76 6.56 20 1.75 1.16 0.57 4.07 0.20 7.55 197 0.84 0.45 0.06 1.73 0.2 2.87 80 1.71 0.93 0.14 3.56 0.2 4.17 88 2.48 1.182 0.12 4.85 0.91 5.02 20 1.15 0.822 0.00 2.79 0.38 3.41 20

0.88 0.434 0.01 1.74 0.15 1.56 18 0.73 0.45 0.18 1.64 0.10 2.61 210 1.16 0.62 0.09 2.40 0.1 2.87 80 0.78 0.44 0.10 1.67 0.03 2.31 93 0.90 0.396 0.11 1.69 0.32 1.70 20 0.28 0.231 0.00 0.75 0.08 0.94 20

0.54 0.386 0.00 1.32 0.10 1.40 20 0.51 0.32 0.13 1.14 0.06 1.90 197 1.00 0.69 0.39 2.39 0 3.18 80 0.65 0.38 0.11 1.42 0.1 2 88 0.63 0.377 0.00 1.39 0.14 1.18 20 0.13 0.101 0.00 0.34 0.02 0.51 20

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 (FcRgIII) 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

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Table 10. Species reference ranges. Relative lymphocyte subset values. Total T-cells Animal (origin) Cynomolgus (Vietnam)

Cynomolgus (China)

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Cynomolgus (Mauritius)

Cynomolgus (Philipines)

Rhesus (China)

Baboon (United States)

Helper T-cells

Cytotoxic T-cells

B-cells

NK cells

Parameter

Male

Female

Male

Female

Male

Female

Male

Female

Male

Female

Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number Mean SD Mean 2SD Mean +2SD Range (min) Range (max) Number

60.42 6.234 47.95 72.99 50.26 72.13 18 61.16 9.44 42.27 80.04 36.68 80.30 210 57.20 7.94 41.32 73.09 39.72 74.4 80 59.31 11.18 36.96 81.66 26.76 79.58 93 61.18 7.149 46.88 75.48 47.28 71.58 20 57.81 8.853 40.11 75.52 40.23 69.65 20

58.51 9.838 38.84 78.19 39.07 75.93 20 59.43 9.11 41.21 77.65 31.05 79.24 197 58.07 9.84 38.39 77.74 35.8 86.25 80 57.81 10.39 37.02 78.60 37.11 81.64 88 59.01 8.496 42.02 76.01 42.15 75.91 20 54.82 7.838 39.15 70.50 39.45 65.27 20

33.51 5.573 22.36 44.65 22.58 42.37 18 31.02 6.58 17.85 44.18 13.62 50.39 210 27.55 5.80 15.95 39.15 13.97 42.11 80 30.69 7.99 14.71 46.67 13.36 47.94 93 28.26 4.770 18.72 37.80 20.12 35.97 20 29.78 5.085 19.61 39.95 19.64 38.86 20

31.06 6.383 18.29 43.82 18.71 39.46 20 32.18 7.27 17.63 46.72 12.31 48.47 197 29.35 6.70 15.96 42.74 13.42 42.41 80 30.55 5.93 18.69 42.41 19.58 45.1 88 28.52 5.785 16.95 40.09 20.37 41.23 20 28.52 5.807 16.90 40.13 17.03 40.59 20

22.61 4.352 13.91 31.32 13.90 28.48 18 25.49 6.23 13.03 37.95 12.70 42.68 210 25.08 6.28 12.53 37.64 12.04 41 80 25.96 12.10 1.77 50.15 5.88 59.03 93 27.41 6.002 15.41 39.41 18.48 37.87 20 22.26 5.157 11.95 32.58 15.07 30.74 20

22.71 6.349 10.01 35.40 13.90 38.85 20 22.41 5.47 11.47 33.35 7.65 40.73 197 23.77 6.71 10.36 37.19 10.21 43.09 80 22.29 6.62 9.05 35.53 11.87 40.47 88 25.25 5.850 13.55 36.95 16.79 35.53 20 20.45 4.252 11.95 28.96 12.34 30.57 20

29.12 7.102 14.92 43.33 20.06 43.14 18 24.95 8.36 8.22 41.67 9.27 55.55 210 18.17 6.81 4.55 31.79 5.16 41.95 80 25.96 12.10 1.77 50.15 5.88 59.03 93 28.00 7.989 12.03 43.98 12.88 48.93 20 31.16 7.050 17.06 45.26 23.18 46.16 20

33.87 9.213 15.45 52.30 20.06 54.87 20 28.80 10.12 8.56 49.03 6.33 67.27 197 17.21 8.43 0.36 34.07 7.19 45.31 80 27.81 10.50 6.81 48.82 7.05 48.06 88 30.63 9.292 12.05 49.22 14.96 51.92 20 37.51 7.533 22.45 52.58 25.98 53.29 20

8.43 3.896 0.64 16.22 1.79 16.99 18 11.10 6.20 1.31 23.51 0.74 38.63 210 20.01 8.99 2.04 37.99 2.76 40.33 80 11.55 7.30 3.05 26.15 0.8 43.58 93 9.05 4.146 0.75 17.34 2.29 19.54 20 8.89 7.907 0.00 24.71 2.67 36.84 20

5.81 3.429 0.00 12.66 1.72 13.21 20 9.22 5.58 1.93 20.37 0.89 41.38 197 18.76 9.53 0.30 37.82 1.94 41.89 80 11.05 5.14 0.76 21.34 2.51 26.61 88 7.84 4.068 0.00 15.97 2.42 18.84 20 5.58 5.391 0.00 16.36 1.54 25.80 20

Relative values presented as a percentage of total lymphocytes. The mean, standard deviations, mean ± 2 SD, and number (per gender) of relative (%) immunophenotyping values are presented for cynomolgus monkeys obtained from referenced geographies as well as rhesus monkeys and baboons.

CD56 for NK cell enumeration would have precluded the use of the validated method on rhesus and baboon toxicology studies as this epitope is not expressed on rhesus or baboon NK cells (Carter et al., 1999; Choi et al., 2008; Jayashankar et al., 2003; Locher et al., 2003; Malyguine et al., 1996; Poaty-Mavoungou et al., 2001; Webster and Johnson, 2005). The data presented herein indicate that the validated assay robustly identifies NK cells in non-human primate species. As with any assay applied to small group size cohorts, it is beneficial to obtain at least two baseline samples in order to quantify individual animal normal ranges for NK (and other lymphocyte subset) populations prior to interrogating the impact of experimental procedures on individual animals. Historic publications evaluating cynomolgus monkeys lymphocyte subset populations have utilized differing types of flow cytometry methodologies; including alternate antibody combinations (CD2 staining of T-cells, CD56 staining of NK cells), single-color immuno-phenotyping (e.g. identifying T-cell subsets as only CD4+ or CD8+ without CD3 co-staining), often

only calculating relative lymphocyte subset values (not absolute values), and generally use homogeneous (forward-scatter vs sidescatter) lymphocyte gating strategies (Baker et al., 2008; Bleavins et al., 1993; Nam et al., 1998; Tryphonas et al., 1996; Verdier et al., 1995). Among the published references using three-color T-cell phenotyping, CD20+ B-cell phenotyping, and/or CD16+ NK cell phenotyping (Baker et al., 2008; Nam et al., 1998); the lymphocyte subset reference ranges are comparable to the data obtained during the validation experiments presented in the current research. It has been suggested that Chinese-origin (continental) cynomolgus monkeys exhibit appreciably different lymphocyte subset values as compared to Indonesian and Mauritius (extra-continental) cynomolgus monkeys (Krejsa et al., 2013). While subtle changes can be identified from continental, island, and hemispheric geographic ranges presented in the current research; the geographic data-sets presented herein are too small to discern substantive differences. It is also important to note that, while the data in the current research is all concurrent amongst analytical timepoints,

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Figure 1. Absolute (a and b) and relative (c and d) immunophenotyping values are presented for two cynomolgus monkeys during a 1-year period. Longitudinal immunophenotyping data. Absolute values (a) Male I02121; (b) Female I01279. Relative values (c) Male I02121; (d) Female I01279.

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Figure 1. Continued.

techniques, instrumentation, and reagents, other research comparing age/gender differences have compiled temporally-disparate data-sets generated utilizing different analytical techniques, instruments, and reagents (Krejsa et al., 2013). There are benefits to both types of analyses and the data amongst the cumulative research do not preclude each other. All of these data suggest that, when possible, animals from the same geographic source should be used within a particular research program in order to reduce any confounding differences introduced by differing hematologic (e.g., lymphocyte) parameters, immunologic capacity, endogenous enzootic differences, or genetic drift. The rhesus and baboon immunophenotyping ranges generated with the validated cynomolgus monkey immunophenotyping assay are also comparable to published species-specific data-sets (Autissier et al., 2010; Jayashankar et al., 2003; Sopper et al., 1997). The non-human primate immunophenotyping methods described in this current research can potentially be further adapted to utilize higher order multi-color staining reagents and increase processing efficiently though high throughput/smaller volume automated systems (Autissier et al., 2010). However, care should be taken to confirm that matrix-dependent interferences do not confound analytical accuracy as described for NK cells during the validation methodology. The reference values presented in this research can serve as baseline comparator information for other methodologies for a particular laboratory. However, reference range values should be generated for each laboratory and method, which are updated on a periodic basis.

Acknowledgments The authors would like to acknowledge the contributions of the Covance Laboratories vivarium staff and the statistics department who were essential for sample collection and data collection; as well as the custom antibody technology group at BD Biosciences for preparation of the antibody cocktails.

Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

References Autissier, P., Soulas, C., Burdo, T. H., and Williams, K. C. 2010. Immunophenotyping of lymphocyte, monocyte and dendritic cell subsets in normal rhesus macaques by 12-color flow cytometry: Clarification on DC heterogeneity. J. Immunol. Meth. 360:119–128. Baker, D. L., Finco-kent, D. L., Reagan, W. J., et al. 2008. Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis). Vet. Clin. Pathol. 37:42–48. Bergeron, M., Nicholson, J. K., Phaneuf, S., et al. 2002. Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens. Cytometry 50:53–61. Bleavins, M. R., Brott, D. A., Alvey, J. D., and de la Iglesia, F. A. 1993. Flow cytometric characterization of lymphocyte subpopulations in the cynomolgus monkey (Macaca fascicularis). Vet. Immunol. Immunopathol. 37:1–13. Calvelli, T., Denny, T. N., Paxton, H., et al. 1993. Guideline for flow cytometric immunophenotyping: A report from the National Institute of Allergy and Infectious Diseases, Division of AIDS. Cytometry 14: 702–715. Carter, D. L., Shieh, T. M., Blosser, R. L., et al. 1999. CD56 identifies monocytes and not natural killer cells in rhesus macaques. Cytometry 37:41–50. Choi, E. I., Wang, R., Peterson, L., et al. 2008. Use of an anti-CD16 antibody for in vivo depletion of natural killer cells in rhesus macaques. Immunology 124:215–222. Cunliffe, J., Derbyshire, N., Keeler, S., and Coldwell, R. 2009. An approach to the validation of flow cytometry methods. Pharm. Res. 26: 2551–2557. Davis, C., Wu, X., Li, W., et al. 2011. Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry. J. Immunol. Meth. 363:158–165. Dixon, W. J. 1993. Program 1R, Biomedical Data Processing (BMDP) Statistical Software Manual: To Accompany 1990 Software Release. Berkeley, CA: University of California Press. Draper, N. R., and Smith, H., (Eds.). 1966. Applied Regression Analysis. New York: John Wiley & Sons. Fleit, H. B., Kobasiuk, C. D., Daly, C., et al. 1992. A soluble form of FcgRIII is present in human serum and other body fluids and is elevated at sites of inflammation. Blood 79:2721–2728. Giorgi, J. V., and Landay A. 1994. HIV Infection: Diagnosis an disease progression evaluation. In: Methods in Cell Biology, Volume 42, Flow Cytometry Part B. (Darzynkiewicz, Z., Robinson, J. P., and Chrissman, H.A, eds., San Diego, CA: Academic Press, pp. 437–455.

Downloaded by [University of Wisconsin Oshkosh] at 21:23 13 November 2015

76

R. G. Caldwell et al.

Gratama, J. W., Kraan, J., Kenney, M., et al. 2007. Enumeration of immlunolocially defined cell populations by flow cytometry. Approved Guideline H42-A2, 2nd edn. Clinical and Laboratory Standards Institute. Harmonization, I.C.O. 2009. Addendum to ICH S6: Pre-clinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. ICH. 1997. S6: Pre-clinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. Federal Register. 62:61515. ICH. 2006. S8: Immunotoxicity Studies for Human Pharmaceuticals. Federal Register. 71:19193–19194. Jayashankar, L., Brasky, K. M., Ward, J. A., and Attanasio, R. 2003. Lymphocyte modulation in a baboon model of immunosenescence. Clin. Diagn. Lab. Immunol. 10:870–875. ICH. 2009. Addendum to ICH S6: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. Federal Register. 77:29665– 29666. Krejsa, C. M., Neradilek, M. B., Polissar, N. L., et al. 2013. An interlaboratory retrospective analysis of immunotoxicological endpoints in non-human primates: Flow cytometry immunophenotyping. J. Immunotoxicol. 10:361–272. Locher, C. P., Fujimura, S., Murthy, K. K., et al. 2003. Expression patterns of phenotypic markers on lymphocytes from human immunodeficiency virus type 2-infected baboons. AIDS Res. Human Retroviruses 19:31–40. Luster, M. I., and Rosenthal, G. J. 1993. Chemical agents and the immune response. Environ. Health Perspect. 100:219–226. Luster, M. I., Munson, A. E., Thomas, P. T., et al. 1988. Development of a testing battery to assess chemical-induced immunotoxicity: National Toxicology Program’s guidelines for immunotoxicity evaluation in mice. Fundam. Appl. Toxicol. 10:2–19. Luster, M. I., Pait, D. G., Portier, C., et al. 1992a. Qualitative and quantitative experimental models to aid in risk assessment for immunotoxicology. Toxicol. Lett. 64–65:Spec No. 71–78. Luster, M. I., Portier, C., Pait, D. G., et al. 1993. Risk assessment in immunotoxicology. II. Relationships between immune and host resistance tests. Fundam. Appl. Toxicol. 21:71–82. Luster, M. I., Portier, C., Pait, D. G., et al. 1992b. Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam. Appl. Toxicol. 18:200–210. Malyguine, A. M., Saadi, S., Platt, J. L., and Dawson, J. R. 1996. Differential expression of natural killer cell markers: Human versus baboon. Transplantation 62:1319–1324.

J Immunotoxicol, 2016; 13(1): 64–76

Mandy, F. F., Nicholson, J. K., and McDougal, J. S. 2003. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Centers for Disease Control and Prevention. MMWR Recomm. Rep. 52: 1–13. Nam, K. H., Akari, H., Terao, K., et al. 1998. Age-related changes in major lymphocyte subsets in cynomolgus monkeys. Exp. Anim. 47: 159–166. Nicholson, J. K. A., Hearn, T. L., Cross, G. D., and White, M. D. 1997. 1997 revised guidelines for performing CD4+ T-cell determinations in persons infected with human immuno-deficiency virus (HIV). Centers for Disease Control and Prevention. Morbidity and Mortality Weekly Report, Vol 46, No. RR-2, pp 1–30. Poaty-Mavoungou, V., Onanga, R., Yaba, P., et al. 2001. Comparative analysis of natural killer cell activity, lymphoproliferation and lymphocyte surface antigen expression in nonhuman primates housed at the CIRMF Primate Center, Gabon. J. Med. Primatol. 30: 26–35. Rogers, K. A., Scinicariello, F., and Attanasio, R. 2006. IgG Fc receptor III homologues in non-human primate species: Genetic characterization and ligand interactions. J. Immunol. 177:3848–3856. Schnizlein-Bick, C. T., Mandy, F. F., O’Gorman, M. R., et al. 2002. Use of CD45 gating in three and four-color flow cytometric immunophenotyping: Guideline from the National Institute of Allergy and Infectious Diseases, Division of AIDS. Cytometry 50:46–52. Schumacher, M. J., and Burkhead, T. 2000. Stability of fresh and preserved fetal and adult lymphocyte cell surface markers. J. Clin. Lab. Anal. 14:320–326. Sopper, S., Stahl-Hennig, C., Demuth, M., et al. 1997. Lymphocyte subsets and expression of differentiation markers in blood and lymphoid organs of rhesus monkeys. Cytometry 29:351–362. Tryphonas, H., Lacroix, F., Hayward, S., et al. 1996. Cell surface marker evaluation of infant Macaca monkey leukocytes in peripheral whole blood using simultaneous dual-color immunophenotypic analysis. J. Med. Primatol. 25:89–105. Verdier, F., Aujoulat, M., Condevaux, F., and Descotes, J. 1995. Determination of lymphocyte subsets and cytokine levels in cynomolgus monkeys. Toxicology 105:81–90. Webster, R. L., and Johnson, R. P. 2005. Delineation of multiple subpopulations of natural killer cells in rhesus macaques. Immunology 115:206–214.