Sources of canine specimens

The Oregon State University Veterinary Diagnostic Laboratory provided 8 formalin-fixed, paraffin-embedded blocks of canine lymph node specimens (5 neoplastic and 3 nonneoplastic specimens from 6 dogs) that had been submitted between 2012 and 2017. Histologic evaluation of all lymph node specimens had been performed by board-certified veterinary pathologists at the veterinary diagnostic laboratory; a diagnosis of lymphoma was made for the 5 dogs with neoplastic lymph nodes, although information regarding the immunophenotype (T-cell or B-cell) was not available. The age of the dogs ranged from 6 to 12 years. Neoplastic specimens were obtained from 3 spayed females (1 Cocker Spaniel, 1 mixed-breed dog, and 1 dog of unknown breed), 1 sexually intact female (Bernese Mountain Dog), and 1 neutered male (mixed-breed dog); 3 nonneoplastic specimens were obtained from 1 neutered male German Shepherd Dog. Testicular tissue was obtained from a separate adult dog that had undergone routine castration for use as a positive control during immunohistochemical analysis of lymph node specimens.

Twelve healthy dogs were recruited from a local kennel club to provide blood samples from which circulating nonneoplastic lymphocytes were isolated. Informed consent was provided by all dog owners before samples were collected, and all procedures were approved by the Oregon State University Institutional Animal Care and Use Committee (protocol No. 4792). Among the 12 healthy dogs, there were 3 spayed females, 3 sexually intact females, 3 neutered males, and 3 sexually intact males. The mean age of the dogs was 6.7 years (range, 1 to 12 years); breeds included Brittany (n = 9) and Beagle, Chesapeake Bay Retriever, and German Shorthaired Pointer (1 each).

Three cryopreserved immortalized T-cell lymphoma cell lines (CLC, CLK, and EMA) derived from 3 adult female (reproductive status unknown) dogs (1 French Bulldog, 1 English Springer Spaniel, and 1 Great Pyrenees; age range, 6 to 8 years) with primary lymphoma were provided by the Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan, for use in establishing cell cultures. The cells were shipped to our laboratory on dry ice.

Immunohistochemical analysis of lymph node specimens

Serial 6-μm-thick sections were cut from each tissue block and placed onto positively charged slides; 3 slides were prepared for each specimen, including 1 for use as a negative control and 1 that was stained with H&E stain only (for histologic examination). All sections were deparaffinized with xylene, rehydrated with graded ethanol solutions (100%, 75%, and 50%), and subjected to heat-induced epitope retrieval.^d

The following procedures were performed for all slides, except those that were prepared for H&E staining only. Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide, and nonspecific binding was blocked with 1% horse serum^e before application of a 1:50 dilution of goat polyclonal anti-human LH receptor (primary) antibody.^f Negative controls for each tissue specimen were treated in a similar manner except that the primary antibody was not applied. Biotinylated horse anti-goat IgG (secondary) antibody was applied at a 1:10 dilution; slides were then incubated with preformed avidin-biotin-peroxidase complex^e followed by horseradish peroxidase-based substrate.^g Slides were counterstained with hematoxylin, dehydrated, and then mounted. The percentage of cells positive for LH receptors in each of 4 fields/slide was used to calculate the mean percentage of LH receptor-positive cells per slide; all slides were examined by 1 investigator (AME) at 400X magnification.

Isolation of circulating nonneoplastic lymphocytes

For each of the 12 healthy dogs, 6 mL of blood was collected into blood collection tubes containing EDTA by use of standard aseptic jugular venipuncture technique. Within 90 minutes after collection, each blood sample was sterilely transferred into a 15-mL conical centrifuge tube; an equal volume of PBSS-BSA-SA was added to each tube, and the tube contents were mixed by inversion. Six milliters of each blood sample mixture was then slowly layered on top of 4 mL of centrifugation medium^h (density, 1.077 g/mL) in a 15-mL centrifuge tube (ie, 12 separate tubes); the tubes were centrifuged at 400 × g for 30 minutes at room temperature (approx 23°C). Cells from the plasma-separation medium interface from each sample were collected by use of a plastic transfer pipette and placed into new 15-mL centrifuge tubes with up to 10 mL of PBSS-BSA-SA; the tubes were then centrifuged at 400 × g for 10 minutes at room temperature. After discarding the supernatant, the cell pellet from each sample was transferred to an Eppendorf tube containing up to 1 mL of PBSS-BSA-SA at 4°C, then centrifuged at 400 × g for 10 minutes at room temperature. The resulting pellets (circulating lymphocytes) were each resuspended in 700 μL of PBSS-BSA-SA at 4°C (resulting concentration, 130,000 to 200,000 lymphocytes/mL).

Flow cytometry

Circulating nonneoplastic lymphocytes—Six Eppendorf tubes containing isolated circulating lymphocytes were prepared for each dog; nonspecific binding by the cells was blocked by adding blocking serumⁱ (1:10 dilution) to each tube and then incubating the samples on ice for 10 minutes. Ten microliters of goat polyclonal anti-human LH receptor (primary) antibody^f (1:10 dilution) was added to tubes 1 to 3 to identify LH receptors on lymphocytes; 10 μL of fluorescein isothiocyanate-conjugated mouse anti-dog CD3 antibody^j (1:10 dilution) and 10 μL of fluorochrome-conjugated mouse anti-dog CD21 antibody^k (1:10 dilution) were also added to tubes 2 and 3, respectively. To identify the percentage of T and B lymphocytes, 10 μL of fluorescein isothiocyanate-conjugated mouse anti-dog CD3 antibody^j (1:10 dilution) and 10 μL of fluorochrome-conjugated mouse anti-dog CD21 antibody^k (1:10 dilution) were added to tubes 4 and 5, respectively. Tube 6 was prepared with no primary antibody (negative control). After incubation at room temperature for 20 minutes, the lymphocytes were washed twice with PBSS-BSA-SA at 4°C to remove unbound primary antibody; 100 μL of PBSS-BSA-SA at 4°C was then added to each tube, and the contents were mixed by pipetting action. Ten microliters of a phycoerythrin-conjugated secondary antibody (donkey anti-goat IgG^l; 1:10 dilution) was added to tubes 1, 2, 3, and 6 for each of the 12 healthy dogs. All tubes (n = 72) were then held on ice for 20 minutes. Each sample was washed twice with PBSS-BSA-SA to remove unbound antibodies, 200 μL of PBSS-BSA-SA at 4°C was added to each sample, and the tube contents were mixed by pipetting action. Pipette tip strainers^m were used to transfer 200 μL of sample from each tube into a separate well of a 96-well plate (samples run in singlicate). The samples were analyzed at various wavelengths (ie, 375, 405, 488, 561, 638, and 808 nm) by use of a flow cytometer.ⁿ The data were analyzed by use of flow cytometry software.° Single cell staining was used to perform compensation, and fluorescence-minus-one controls were used to determine where gates should be set for LH receptor-positive cells.¹⁷

Cultured T-cell lymphoma cell lines—The thawed cell lines were each cultured separately in R10 complete medium^p supplemented with 10% fetal bovine serum substitute^q and penicillin (100 U/mL) with streptomycin (100 μg/mL)^r in a humidified 5% CO₂ incubator at 37°C. Fresh R10 complete medium was added to cell cultures every 2 to 3 days at a ratio of 1:4 or 1:9. On reaching 75% to 85% confluence, the cells were washed to remove culture medium, counted with a hemocytometer, and transferred to Eppendorf tubes. Nonspecific binding by cells was blocked by the described procedures for the circulating nonneoplastic lymphocytes; a 1:10 dilution of the primary antibody^f was then added, and the cells were incubated at room temperature for 20 minutes. A negative control was prepared in a similar manner for each cell line except that the primary antibody was omitted. Cell washing and addition of secondary antibody^l were performed as described for the preparation of circulating nonneoplastic lymphocytes for flow cytometry. Each cell line and its negative control were analyzed in triplicate (analysis of 40,000 to 50,000 lymphocytes/well) on a flow cytometer,ⁿ and the data were analyzed.° Single cell staining was used to perform compensation, and fluorescence-minus-one controls were used to determine where gates should be set for LH receptor-positive cells.¹⁷

Statistical analysis

Statistical analyses were performed with statistical software.^s The Shapiro-Wilk test was used to evaluate the normality of the data on the mean percentage of LH receptor-positive cells per sample as obtained by immunohistochemical analysis and flow cytometry. The Student t test was used to compare the mean percentage of lymphocytes that expressed LH receptors in nonneoplastic versus neoplastic lymph node specimens. The Student t test was also used to compare the mean percentage of circulating nonneoplastic lymphocytes that expressed LH receptors in sexually intact versus gonadectomized dogs and in male versus female dogs. Significance was defined as a value of P < 0.05.