CC BY
02022, Scienceline Publication
Worlds Veterinary Journal
World Vet J, 12(1): 74-80, March 25, 2022
DOI: https://dx.doi.org/10.54203/scil.2022.wvj10
The Impact of Camel Leukocytes Fixation on Cell Count and Monoclonal Antibodies Reactivity in Flow Cytometry
Hams Almohammed, Gader Abdulaziz Alhafiz, Fatema Hassan Alghatam, and Jamal Hussen*
Department of Microbiology, College of Veterinary Medicine, King Faisal University, Al-Ahsa, Saudi Arabia »Corresponding author's Email: jhussen@kfa.edu.sa; : 0000-0001-8942-005X
ABSTRACT
Immunophenotyping of separated leukocytes is a common technique used to evaluate the changes in cellular immunity during clinical studies. For fixed cells or blood specimens infected with hazardous pathogens, cell fixation is performed before immunofluorescence. The impact of camel leukocytes fixation before staining on the reactivity of cell surface markers with monoclonal antibodies has not been investigated so far. The aim of the present study was, therefore, to compare cell staining of fixed and unfixed camel leukocytes with monoclonal antibodies to several cell surface antigens. Leukocytes were separated from camel blood and were fixed with paraformaldehyde (PFA) or left without fixation. Cells were labeled with monoclonal antibodies to several leukocyte antigens and the expression pattern of the antigens was compared between fixed and non-fixed cells using flow cytometry. The mean fluorescence intensity of each cell marker was calculated and compared between fixed and unfixed cells. Leukocyte fixation with PFA changed the binding activity of the monoclonal antibodies to CD163 and WC1 markedly, making it unable to stain any cell population. Although the cell staining efficacy of other molecules (such as CD14, CD172a, MHCII, CD11a, CD18, CD44, and CD45) was reduced, they were still able to define the target cells. The fixation-induced changes in the expression density of the analyzed monocytic markers may, however, lead to the misinterpretation of immunophenotyping studies of fixed monocytes or macrophages. Collectively, the obtained results indicated significant changes in the staining efficacy of monoclonal antibodies against several cell surface antigens of camel leukocytes, which should be considered when PFA-fixed cellular targets on camel leukocytes are to be analyzed.
Keywords: Antibodies, Cell fixation, Dromedary camel, Flow cytometry, Leukocytes INTRODUCTION
The analysis of immune cell composition and phenotype has become an indispensable part of veterinary diagnostics and the evaluation of the animal immune status (Hussen and Schuberth, 2020; Hussen et al., 2020b). For such analysis, immune cells are identified based on their cell markers using monoclonal antibody labeling (Gaashan et al., 2020). In the dromedary camel, several cell marker antigens have been recently identified for the characterization of immune cell phenotypes. This mainly includes CD14, CD172a, MHCII, CD11a, CD18, CD44, and CD45 (Hussen and Schuberth, 2020). Immune cell testing usually includes separation of blood leukocytes for ex vivo phenotypic and functional analysis. One of the major challenges when analyzing the immunophenotype of cells is the time between sample collection and analysis, which should be abbreviated to avoid protein denaturation and loss of epitope antigenicity (Laurin et al., 2015). Cell fixation represents an alternative to extend sample age and preserve their morphological integrity (Ng et al., 2012; Qin et al., 2021). For intra-cytoplasmic antigens, fixation and permeabilization of the studied cells are required to access the target antigen by antibodies and to prevent cellular activity during the staining procedures (Paavilainen et al., 2010; Cheng et al., 2019) .
Paraformaldehyde (PFA), which works by cross-linking cellular proteins, is one of the most commonly used cellular fixatives (Cheng et al., 2019). It is mainly used, when stained cells are to be preserved for later analysis, or before cellular permeabilization to detect antigens inside the cells. For fixed histological specimens or blood specimens infected with hazardous pathogens, paraformaldehyde fixation is performed before immunological detection of epitopes (Schuberth et al., 1998). In this case, it is important that the antigenicity of the epitopes and the binding specificity of monoclonal antibodies are not affected by the fixation process. Studies in other species demonstrated different effects of fixative agents on the binding of antibodies to cell surface antigens. For porcine mononuclear cells, fixation with PFA affected the reactivity of 38 out of 134 positive reacting mAbs. The changes included reduced as well as elevated expression densities of the analyzed cell markers (Schuberth et al., 1998). Similarly, fixation of human leukocytes resulted in a marked decrease in forward and side scatter values with significant changes in the expression density of several cell surface antigens (Stewart et al., 2007). In a previous study on the fixation of human blood leukocytes, paraformaldehyde fixation after removal of red blood cells resulted in poor results in term of leukocyte cell count and
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staining with several cell markers (Ng et al., 2012). Fixation of human mononuclear cells before staining especially reduced the cell staining density with antibodies to the cell antigens CD8, CD19, CD16, and CD56 (Pinto et al., 2005).
As no previous studies investigated the effect of cell fixation on the reactivity of camel leukocytes to monoclonal antibody staining, the present work employed flow cytometry to report on the binding activity of selected monoclonal antibodies on camel blood leukocytes fixed with paraformaldehyde.
MATERIALS AND METHODS
Ethical Approval
The study was approved by the Ethics Committee of King Faisal University, Saudi Arabia (Approval no. KFU-REC/2020-09-25).
Animals and blood sampling
Six apparently healthy dromedary camels (housed at the farm of the Camel Research Center, King Faisal University, Al-Ahsa, Saudi Arabia) were used for blood sample collection. The animals were non-lactating female camels from Almajaheem breed aged between 8 and 10 years. Blood sampling was performed by venipuncture of the jugular vein into EDTA tubes. The ambient temperatures during blood collection from the animals and during cell separation and staining in the lab were 34°C and 24°C, respectively.
Cell separation
Leukocytes isolation from blood was done within one hour after blood collection by inducing erythrolysis. For this 1 ml of blood was incubated (at room temperature) in 5 ml of distilled water for 20 seconds and 5 ml of double concentrated phosphate-buffered saline (PBS) were added. In case of no complete lysis, the procedure was repeated. Before fixation, the cells were washed two times in PBS (500 xg, 250 x g, 10 minutes, 10°C). Separated leukocytes were finally suspended in staining buffer (PBS containing 5 g/l BSA, 100 mg /l NaN3) at 5 x 106 cells/ml (Gaashan et al., 2020).
Cell fixation
For the preparation of a 4% PFA solution, 4 g PFA (Roth, Karlsruhe, Germany) were dissolved in 50 ml of distilled water at 60°C for 30 min. After mixing, 50 ml double concentrated PBS was added to the solution. Cell fixation was performed as described previously (Schuberth et al., 1998). Briefly, separated cells were incubated with PFA solution for 15 minutes at room temperature (RT) and with constant rotation. After that, the fixed cell sample was washed two times in PBS (250 x g, 3 minutes, RT) and finally, resuspended in staining buffer for antibody labeling.
Immunolabeling and flow cytometry
For cell staining the wells of a round-bottomed 96-well microtiter plate were filled with 5 x 105 leukocytes (100 ^l). In the first labeling step, primary monoclonal antibodies (mAbs) to the cell surface molecules, CD4, WC-1, CD14, CD163, CD172a, MHC-II, CD11a, CD18, CD44, and CD45 (Hussen and Schuberth, 2020) were added to the wells followed by incubation for 15 minutes. After two washings with staining buffer, the second staining step was done by adding fluorochrome-labeled antibodies to mouse IgM, IgG1, and IgG2a (Invitrogen), and the plate was incubated for 15 minutes in the dark. Staining with isotype controls was also included. After two washings, labeled cells were analyzed by flow cytometry (Accurie C6 flow cytometer from BD Biosciences). Data analysis was performed using the CFlow Software (V 1.0.264.21; BD Biosciences). The Neubauer counting chamber was used to estimate the total number of leukocytes in blood as previously described (Hussen, 2021b). For this, a blood sample was stained with Türk solution (1 to 10 dilution) and a stained sample was counted under the microscope. All incubation and centrifugation steps were performed at 4°C. Cell washing was done using staining buffer and centrifugation of the plates at 250 xg for 3 minutes.
Statistical analysis
The statistical software program GraphPad Prism was used for calculating column statistics including minimal (min) and maximal (max) values, mean, and SEM. The paired student's t-test was used to compare the means for each parameter. A p-value less than 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
The current work evaluated the extent to which PFA fixation of camel blood leukocytes before staining affects the cell count and staining of leukocyte subsets with monoclonal antibodies to cell surface molecules by flow cytometry (Figure 1).
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A
SSC-H FL-1 FSC-A
B
Unfixed
PFA-fixed
Unfixed
PFA-fixed
CD4-APC
CD14-FITC
WC1-FITC
MHCII-PE
CD11a-PE
CD163-FITC
CD44-PE
CD45-PE
Figure 1. Flow cytometric analysis of cell staining with cell surface markers. A: Gating scheme used for the analysis of neutrophils (PMN), eosinophils (Eos), mononuclear cells (MNC), and their subpopulations, monocytes (Mon) and lymphocytes (Ly). After the exclusion of cell duplicates (using SSC-H against SSC-A), PMN, Eos, and MNC were gated based on SSC-A and autofluorescence (Eos). After selecting MNC, lymphocytes, and monocytes were identified in a separate dot plot. B: Representative dot plots for staining patterns of selected monoclonal antibodies on unfixed and PFA-fixed cells.
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Fixation-induced changes in forward and side scatter characteristics of camel leukocytes
PFA fixation induced a marked (p < 0.05) shift (increase) in the SSC and FSC of neutrophils, eosinophils, and monocytes (Table 1). The FSC signal was increased for neutrophils and eosinophils, while decreased for monocytes and lymphocytes (p < 0.05).
Impact of PFA fixation on the composition of camel leukocytes
Percentages of gated neutrophils, eosinophils, lymphocytes, and monocytes of unfixed and PFA-fixed cells are shown in Table 1. The analysis of relative leukocyte composition revealed significantly (p < 0.05) higher percentage and numbers of monocytes in samples fixed with PFA compared to unfixed cells (Table 1). For neutrophils, eosinophils, and lymphocytes both relative and absolute counting of cells revealed comparable (p > 0.05) values between fixed and unfixed samples (Table 1 and Figure 2). The comparative analysis of lymphocyte composition between PFA-fixed and unfixed samples revealed lower percentages and numbers of B cells (p < 0.05) and yS T cells (p < 0.05), while the number of helper T cells (Th) did not change significantly after fixation (Figure 2). However, the effect of fixation on the staining of yS T cells was significantly stronger altering the WC1 epitope; therefore, it could no longer detected within fixed lymphocytes by anti-WC1 antibodies.
Table 1. Impact of camel blood leukocyte fixation on cell composition and phenotype
Control leukocytes Fixed leukocytes P value
Min Max Mean SEM Min Max Mean SEM
PMN 63.0 73.9 68.5 1.8 59.8 70.4 66.3 1.9 0.07
WBC sub (% of Eos 4.6 13.5 6.8 1.7 5.5 11.4 8.0 1.1 0.36
WBC) Ly 8.1 16.7 13.9 1.6 7.9 18.3 14.6 1.8 0.19
Mon 4.2 11.2 6.4 1.2 6.2 14.3 9.3 1.3 0.001
PMN 5128 10053 6959 849 4913 9445 6718 782.5 0.10
WBC sub Eos 420.4 1295.0 670.8 161.7 414.2 1295.0 825.0 160.7 0.34
(cell/^l) Ly 777.6 1791.0 1390.0 176.9 759.4 1852.0 1447.0 189.3 0.15
Mon 402.8 1030.0 626.8 106.5 717.4 1311.0 908.2 103.6 0.001
Ly sub Th cell 19.1 28.4 22.6 1.9 16.7 32.0 24.2 1.8 0.43
Percentage of y8 T cell 3.1 8.6 6.6 1.0 0.1 0.3 0.2 0.04 0.001
lymphocytes) B cell 10.1 29.3 22.6.2 3.0 7.7 22.0 17.6 2.7 0.01
Th cell 157.9 435.3 357.4 52.3 232.0 447.0 325.4 48.3 0.21
Ly sub (cell/^l) y8 T cell 24.1 131.8.8 97.6 21.4 1.5 4.2 3.0 0.5 0.01
B cell 153.6 524.1 315.4 52.9 117.2 407.5 266.2 55.6 0.04
PMN 405627 479906 450361 12917 512356 596505 551713 14011 0.001
SSC (MFI) Eos 441870 502519 474379 10388 557446 584669 571546 5265 0.001
Ly 68855 71479 70335 502 68798 73900 70379 907.1 0.95
Mon 131068 134548 132770 606 136538 148587 144267 2623.0 0.02
PMN 1704000 1838000 1764000 22942 1921000 1986000 1946000 13183 0.001
FSC (MFI) Eos 1486000 1620000 1548000 24319 1732000 1902000 1808000 29057 0.001
Ly 1362000 1419000 1393000 9180 1308000 1324000 1318000 3704 0.001
Mon 1947000 2029000 1976000 13997 1370000 1578000 1460000 33983 0.001
PMN 1297.0 1397.0 1353.0 16.7 530.0 685.0 590.0 26.7 0.001
CD11a Ly 580.0 1353.0 963.6 156.3 367.0 511.0 442.6 31.5 0.01
Mon 5146 6755 6094 311 1995 2370 2197 61.5 0.001
PMN 15082 18880 17134 727 9545 11143 10494 291 0.001
CD18 Ly 5773 11191 7866 1141 1609 2849 2247 198 0.01
Mon 35606 45635 41786 1877 16429 22752 19290 1048 0.001
PMN 22188 33041 26855 1786 6322 8267 7361 310 0.001
CD44 (MFI) Eos 361 12121 5922 2509 6510 8543 7343 367 0.60
Ly 13139 21689 17567 1391 4696 7268 5946.4 408 0.001
Mon 44664 53220 49494 1422 10371 12828 11334 438 0.001
PMN 9959 17506 14259 1412 4173 7502 6023 601 0.001
CD45 (MFI) Eos 11064 14640 13166 735 4340 6105 5274 344 0.001
Ly 20343 36882 27098 2735 12781 19524 15356 1160 0.001
Mon 27611 51514 40851 4490 11153 19902 15942 1538 0.001
PMN 40025 48093 43316 1322 4340 6105 5274 344 0.001
CD172a (MFI) Eos 16246 19698 17778 575 12781 19524 15356 1160 0.01
Mon 54180 80112 63118 4431 11153 19902 15942 1538 0.001
CD163 (MFI) Mon 16202 26508 19349 1984 23558 26322 25040 474 0.001
B cell 4692 6778 5349 391 14080 16153 15691 403.1 0.001
MHC-II (MFI) PMN 93.0 115.0 106.0 5.1 31062 38029 33981 1187 0.68
Mon 3302 6585 4958 568 841 1140 927 56 0.001
CD14 (MFI) PMN 11348 13824 12735 549 556 1380 1102 164 0.001
Mon 46887 55130 49436 1465 106.0 110.0 108.2 0.9 0.001
WBC: White blood cells, PMN: Neutrophils, Eos: eosinophils, Mon: Monocytes, Ly: Lymphocytes, Sub: Subsets, MFI: Median fluorescence intensity, Th: T helper cell, y8: Gamma delta T cell, MHC II: Major histocompatibility class II molecules
77
Neutrophils
Eosinophils
o o
<u o
Ö
o o
13 o
10000-1 80006000400020000
Monocytes
1500-1 10005000
500-1 4003002001000
Helper T cell
Unfixed Fixed
1500 n
1000-
500-
2000-1 15001000500-
150-,
100-
50-
Lymphocytes
gd T cell
Unfixed Fixed
500-1 400300200 100 0
B cell
Unfixed Fixed
Figure 2. The impact of cell fixation on camel leukocyte composition. The absolute cell numbers of major leukocyte populations and lymphocyte subsets in blood from dromedary camels were calculated by using standard clinical hematology laboratory procedures. Data for unfixed and fixed cells were presented as mean and standard error of the mean. Differences between the means were calculated using the t-test and were considered significant (*) if p < 0.05.
0
0
0
PFA fixation affected the staining activity of adhesion and myeloid marker molecules
The abundance of the cell markers (as measured by the mean fluorescence intensity of stained cells) CD11a, CD18, CD44, and CD45 was significantly reduced after PFA fixation of leukocytes (p < 0.05). Although the staining efficiency of the monoclonal antibodies to all monocytic markers was reduced on PFA-fixed cells, the strongest reduction in fluorescence intensity was seen for CD163 molecules expression on monocytes (twenty times signal reduction) (Table 1).
During recent years, several camel myeloid and lymphoid immune cell populations and subpopulations have been characterized using membrane antibody staining of fresh-separated blood leukocytes and flow cytometric analysis (Hussen and Schuberth, 2020; Hussen, 2021a; Hussen, 2021b). For fixed histological specimens or for blood specimens infected with hazardous pathogens, however, cell fixation is performed before immunological detection of epitopes (Schuberth et al., 1998; Jamur and Oliver, 2010). In this case, it is important that the antigenicity of the epitopes and the binding specificity of monoclonal antibodies are not affected by the fixation process (Suthipintawong et al., 1996). In the current work, the impact of PFA fixation of camel blood leukocytes before staining on the cell count and staining of leukocyte subsets with monoclonal antibodies to cell surface molecules was evaluated by flow cytometry.
The observed changes in side scatter (SSC) and forward scatter (FSC) properties of fixed leukocytes indicate a significant fixation-induced shape-change effect for all leukocyte populations. As the major leukocyte populations are sometimes identified according to their shape, this effect should be considered when gating PFA-fixed camel leukocytes
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based on FSC and SSC properties. In addition, forward scatter and side scatter correlate with the cell size and granulation, respectively (MacDonald and Zaech, 1982; Stern et al., 2017). Therefore, the fixation-induced change in FSC and SSC values may be mistakenly interpreted as cell activation or degranulation of camel leukocytes. Fixation-induced shape-changes have been reported for human PBMC or whole blood samples fixed with PFA before or after staining with monoclonal antibodies (Pinto et al., 2005).
The comparable cell count of camel neutrophils, eosinophils, and total lymphocytes argues for a stable leukogram after PFA fixation. However, the higher percentage and numbers of monocytes in samples fixed with PFA compared to unfixed cells should be considered during the analysis of fixed samples. In addition, further studies are required to investigate the impact of PFA fixation on the composition of monocytes to see whether distinct monocyte subsets are affected by this increase (Hussen et al., 2020a). Although the number of lymphocytes was not affected by PFA fixation, the fixed lymphocyte population contained lower percentages and numbers of B cells and y8 T cells indicating an epitope-specific effect of PFA fixation. Studies on human lymphocytes reported no effect of PFA-fixation on total lymphocyte count or the number of their subsets (Pinto et al., 2005). The differences between camel and human lymphocytes may be due to different susceptibility to paraformaldehyde treatment, which should be evaluated in future comparative studies.
Testing of the monoclonal antibodies on unfixed and PFA-fixed cells showed different effects of cell fixation on the cell surface antigens with the CD163 staining being markedly reduced. It is well recognized that PFA fixation may differently affect cell staining with certain molecules being changed stronger than others (Tanaka et al., 2010). PFA fixation of human whole blood induced a higher decrease in the labeling efficiency of integrins than other cell surface antigens (Bateman et al., 1993; Celie et al., 2005).
CD14, CD172a, CD163, and MHCII are well-established markers of monocyte and macrophage polarization (Hussen and Schuberth, 2017). CD14 is a cell surface marker with an expression on monocytes, macrophages and to a lower extent on neutrophils (Ibeagha-Awemu et al., 2012). CD14 acts as a co-receptor for the cell-wall component of gram-negative bacteria, LPS. The CD172a, which is also called the signal regulatory protein alpha with inhibitory potential on cell activity, is a myeloid marker expressed on blood granulocytes and monocytes (Oronsky et al., 2020). MHCII is an antigen presentation receptor with an essential role in presenting peptide antigens to helper T cells (Busch et al., 2000). In the present study, the fixation-induced change in the expression density of the analyzed monocytic markers may lead to misinterpretation of immunophenotyping studies of fixed monocytes or macrophages. In addition, the significant change in the abundance of the cell adhesion molecules CD18, CD11a, and CD44 could result in the biased interpretation of the adhesion and migration capability of blood leukocytes (Harjunpaa et al., 2019).
CONCLUSION
Collectively, the results of the present study indicate significant changes in staining efficacy of monoclonal antibodies against several cell surface antigens after fixation of camel leukocytes. The results of the present study indicate that leukocyte fixation with PFA may change the antigenic structures of some cell surface molecules like CD163 and WC1, while only reducing the antigen-antibody binding efficacy for other molecules like CD14, CD172a, MHCII, CD11a, CD18, CD44, and CD45. Further studies are required to investigate the impact of sample storage time and temperature on the observed effects of PFA fixation on camel leukocyte composition and phenotype.
DECLARATIONS
Authors' contribution
HA collected the samples and wrote the original manuscript. GAA performed the flow cytometric analysis and wrote the manuscript. FHA prepared the samples for cell staining and did the data analysis. JH acquired the funding, analyzed the flow cytometric data, and wrote the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors have not declared any conflict of interest.
Ethical consideration
The authors declare that the manuscript has not been published before and is not currently being considered for publication elsewhere.
Acknowledgments
The study was supported through the Annual Funding track by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Project No. AN00031).
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