Introduction

The fine-needle aspirate biopsy of the spleen can provide a good source of material for cytology, serving as alternative to splenectomy or to the sacrifice of animals for obtaining cell samples during necropsy, particularly in experimental conditions. Advances of immunological methods and reagents for analyses of dog cells have contributed to the understanding of canine immune system (Cobbold & Metcalfe 1994). The flow cytometry technique (FACS - Fluorescence Activated Cell Sorting) allows the analysis and separation of cells, as well as of other intracellular structures (nuclei, chromosomes, mitochondria, granules) in suspension, thus simultaneous evaluation of different characteristics of several particles (Bacal & Faulhaber 2003). Following these technological achievements, many reports have given emphasis to populations and subpopulations of leukocytes in canine blood (Byrne et al. 2000, Faldyna et al. 2001, HogenEsch et al. 2004) or other biological fluids (Tipold et al. 1998, Guglielmino et al. 2004) in the study of canine diseases. Some works have described diagnostic procedures by FACS using samples of solid organs and tissues obtained in necropsy or surgically removed organs (Sakai et al. 2003, Stain et al. 2004, Faldyna et al. 2005). In the present study, we describe the standardization of a method for preparing single cell suspensions from samples obtained by fine needle spleen aspiration of living dogs, in order to provide suitable material for accurate immunophenotyping of dog splenocytes by flow cytometry. We aimed to obtain a good sample for a feasible diagnostic tool for routine clinical and experimental conditions.

Materials and Methods

Seventeen healthy young adult dogs were utilized for the standardization of blood and spleen leukocyte processing for obtaining suitable cell suspensions for morphological identification and immunophenotyping with monoclonal antibodies (MoAb) by FACS. Ten dogs were mixed breed blood-donors kept in a kennel at the Gonçalo Moniz Research Center--FIOCRUZ and seven lived with their owners. All procedures carried out on the animals were in accordance with guidelines defined by the Committee of Ethics in Animal Experimentation of the FIOCRUZ, Bahia, Brazil. Spleen aspirations and blood collection were performed in accordance with a technique detailed previously (Barrouin-Melo et al. 2006a), under sedation with 0.5 mg/kg of acepromazine. Each sample was carefully flushed into a sterile tube containing 10 mL of RPMI 1640 medium (Sigma) with 50 UI/ml of heparin. Erythrocytes were lysed with a 10% (w/v) ammonium chloride solution in H₂O. Each cell pellet was re-suspended to a concentration of 1 x 10⁷cells/mL in RPMI and subjected to different treatments, for disruption of cell debris and preparation of single cell suspensions, as needed for the acquiring step of FACS, each treatment in groups of two to six sampled dogs: 1) collagenase/dispase (Sigma) 1 mg/mL; 2) collagenase/dispase 0.5 mg/mL; 3) collagenase/dispase 0.25 mg/mL; 4) collagenase/dispase (1mg/mL) followed by BSA (10mg/mL); 5) collagenase/dispase (1mg/mL) followed by 10% skimmed milk and (6) cells in medium (RPMI) alone as negative controls for enzyme activity against diagnostic target molecules. Blood cells were subjected to all treatments and medium alone as individual controls for leukocyte molecule expression. Each enzymatic treatment lasted for ten minutes, followed by centrifugation at 400 x g for five minutes and resuspension in cold EDTA/FACS buffer. Then, the cell suspensions were filtered in gauze. Cell viability and numbers were assessed by Trypan Blue dying and counting in Neubauer camera under optical microscopy. The monoclonal antibodies (MoAbs) used were the anti-canine CD4 (rat--clone YKIX 302.9) and CD8 (rat--clone YCATE 55.9) (Serotec UK) and the AB6 MoAb, developed in our laboratory -anti-canine CD45 (Aguiar et al. 2004). The cells (5-106) were dispensed into 96-well round bottomed plates. Primary antibodies (50 μL) were incubated with resuspended cells for 20 min at 4°C. Secondary antibodies (biotinylated and direct conjugates anti-rat or mouse Ig, Sigma) were then added, incubated and washed, followed by appropriate streptavidin conjugates. Then, cells were resuspended in 150 μl of EDTA/FACS buffer for acquisition. Labelled cells were analyzed using a FACScan flow cytometer and Cell Quest software (Becton Dickinson / Flow Jo, Tree Star Inc). Forward scatter was set at 1.00 A, side scatter was set at 391 V and ampere gain was set at 1.38. Fluorescence intensity was set on logarithmic gain with 589V for green (FL1) fluorescence. A minimum of 10,000 events, gated to exclude dead cells, were analyzed. Cell populations were displayed as scatter plots based on FSC (size) and SSC (complexity).

Results and Discussion

The volume of spleen samples, obtained by transcutaneous fine needle aspirations, varied from 100 to 400μL, approximately. After the processing steps for obtaining cell suspensions, as standardized previously (Barrouin-Melo et al. 2006b), cell yields varied from 2.0 to 8.0 x 10⁷ total viable purified spleen or peripheral blood leukocytes. Prior to enzymatic digestion or mechanical disruption, spleen cell suspensions contains, among single leukocytes, cell clusters that match with the splenic architecture, including white and red pulp, as seen by optical microscopy of cytospin slides (data not shown). A major obstacle in studying those splenic cells has been their reproducible isolation for comparative analysis and enrichment in high numbers for molecular study by FACS, since the method requires single particle suspensions. A number of techniques for this purpose have been described in the literature, showing that proteolytic enzymatic treatment should generally be avoided as they may disrupt surface markers (Abuzakouk et al. 1996). Nevertheless, in our present study, we could verify that mechanical filtration of those spleen suspensions has resulted in too low cell yields, unviable for FACS evaluation. Likewise, enrichment techniques, such as density gradients, select certain cell populations excluding others, as in the case of Ficoll treatment with granulocytes (Byrne et al. 2000) and still do not allow the assess to the cells inside clusters. Therefore, in order to assess the cell populations present in those clusters, different concentrations of collagenase/dispase enzyme preparations were tested in either, splenic and blood cells suspensions. Cell and molecular viability for FACS were assessed by dying with Trypan Blue followed by microscopy counting and identification by monoclonal antibodies against canine CD4, CD8 and CD45, known markers for T-cell and leukocytes, respectively. All concentrations of collagenase/dispase in RPMI medium tested leaded to reduction of CD4 and CD8 cell counts in a dose-dependent manner. There was no effect on the expression of CD45 among enzyme treated our non-treated cell suspensions. On the other hand, the highest concentration utilized for the enzyme preparation, of 1mg/mL, was the only capable of disrupting the cell clusters, as could be seen in cytospin slides (data not shown). The possibility of regeneration of possible damaged surface proteins by enzyme preparations was assessed by the addiction and incubation for 1 hour prior to diluting in EDTA/FACS buffer, with two kinds of proteins, bovine serum albumin and skimmed milk, after one washing step of enzyme treated cells. In fact, the protein treatment resulted in higher FACS counts of CD4 and CD8 cells, being the number of milk treated cells equivalent to the control blood cell suspensions in medium alone (Figure 1). Dispase (Abuzakouk et al. 1996) or collagenase (Van Damme et al. 2000) treatments have been shown to induce changes and even disappearance of CD4 and CD8 molecules in cell suspensions from different solid organs. However, to the best of our knowledge, no evidences of recovering of those molecules expression after enzymatic treatment followed by incubation with a protein with stabilizing function, such as skimmed milk, has been described so far. The repeatability and mechanisms relying on this finding will be further explored using higher numbers of animals and samples.

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Figure 1. Enrichment of dog splenocytes obtained by enzymatic isolation from transcutaneous fine needle sampling of spleen, and blood controls. Flow cytometric analysis of CD4 expression on total nucleated cells in blood w/ medium alone (A), blood treated w/ 1.0 mg/mL of collagenase/dispase (B), blood treated w/ 1.0 mg/mL of collagenase/dispase plus BSA (C), blood treated w/ 1.0 mg/mL of collagenase/dispase plus skimmed milk (D), spleen treated w/ 1.0 mg/mL of collagenase/dispase plus BSA (E); and spleen treated w/ 1.0 mg/mL of collagenase/dispase plus skimmed milk (F). Relevant populations are gated and numbers indicate the percentage of CD4+ cells within corresponding gates. Plots are representative of 2-3 experiments.

Conclusions

Considering the diagnostic potential of flow cytometry in a variety of canine diseases, by using fine needle aspirate samples of solid organs, the development of procedures to obtain samples which could reflect, as closest as possible, the natural condition, is fundamental for the accuracy of the method. This study shows that different concentrations of collagenase/dispase enzyme preparations promote changes on the readings of CD4 and CD8, but not of CD45, in canine splenocytes and blood by FACS; and that further incubation with skimmed milk seems to restore the expression of those molecules. Because the spleen is an important lymphoid organ, involved in different canine pathologies, including infectious diseases, its evaluation by FACS represents a valuable diagnostic tool, but should be interpreted with caution depending on the method used for processing cell samples.

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