Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethical Statement
2.2. Animals and Management
2.3. Experiment 1: Assessment of PMN Viability in Blood and Endometrial Samples
2.3.1. Blood Sample Collection and PMN Isolation
2.3.2. Endometrial Sample Collection and PMN Isolation
2.3.3. Blood and Endometrial PMN Immunolabeling
2.3.4. Sorting CH138A Positive Cells
2.3.5. Flow Cytometric Approach to Evaluate PMN Viability
2.4. Experiment 2: Blood and Endometrial PMN Functionality Tests
2.4.1. Oxidative Burst
2.4.2. Phagocytosis
2.4.3. Intracellular Proteolytic Degradation by DQ Ovalbumin
2.4.4. Flow Cytometric Approach to Evaluate PMN Functionality
2.4.5. Intra-Assay Validation Approach
2.4.6. Inter-Assay Validation Approach
2.5. Statistics
3. Results
3.1. PMN Isolation and Immunolabeling
3.2. PMN Viability
3.3. Blood and Endometrial PMN Functionality Tests
3.3.1. Intra-Assay Validation Approach
3.3.2. Inter-Assay Validation Approach
3.4. Blood Versus Endometrial PMN Functionality
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Acknowledgments
Conflicts of Interest
References
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Variable | Sample | Mean ± SD (in %) | Range (in %) | CCC (95% CI) | p-Value |
---|---|---|---|---|---|
PMN in endometrial cytology | A | 29.3 ± 30.7 | 0–80 | 0.97 (0.90–0.99) | <0.001 |
B | 28.7 ± 30.5 | 1–90 | |||
CH138A+ cells in endometrium | A | 31.7 ± 22.0 | 1.4–62.0 | 0.85 (0.62–0.95) | <0.001 |
B | 30.6 ± 21.5 | 5.7–68.1 | |||
Viable PMN in blood | A | 91.6 ± 5.82 | 80.3–99.1 | 0.86 (0.66–0.95) | <0.001 |
B | 92.1 ± 4.76 | 82.5–99.0 | |||
Apoptotic PMN in blood | A | 7.4 ± 5.7 | 0.3–18.9 | 0.86 (0.67–0.95) | <0.001 |
B | 6.9 ± 4.7 | 0.3–15.9 | |||
Necrotic PMN in blood | A | 0.7 ± 0.29 | 0.2–1.1 | 0.67 (0.26–0.87) | 0.006 |
B | 0.7 ± 0.3 | 0.3–1.2 | |||
Viable PMN in endometrium | A | 40.0 ± 15.7 | 14.2–75.7 | 0.68 (0.25–0.88) | 0.008 |
B | 37.6 ± 19.4 | 10.9–75.1 | |||
Apoptotic PMN in endometrium | A | 24.3 ± 17.6 | 4.6–59.9 | 0.95 (0.25–0.88) | <0.001 |
B | 23.0 ± 16.8 | 5.5–63.1 | |||
Necrotic PMN in endometrium | A | 32.2 ± 19.1 | 6.1–58.3 | 0.77 (0.44–0.92) | 0.001 |
B | 34.7 ± 23.2 | 8.0–70.6 |
Variable | Gating Strategy | Outcome | Range | CCC (95% CI) | p-Value |
---|---|---|---|---|---|
PPC in blood (in %) | Morphometrics | 83.82 | 69.36–92.03 | 0.70 (0.38–0.87) | 0.021 |
CH138A+ | 88.21 | 71.96–96.56 | |||
MFIPC in blood | Morphometrics | 1,780,888 | 483,827–4,043,406 | 0.89 (0.72–0.96) | <0.001 |
CH138A+ | 2,156,790 | 505,748–4,659,214 | |||
MFIOB in blood | Morphometrics | 242,081 | 58,924–573,332 | 0.97 (0.93–0.99) | <0.001 |
CH138A+ | 273,634 | 114,311–595,864 | |||
MFIDQ in blood | Morphometrics | 2519 | 1092–4631 | 0.94 (0.86–0.98) | <0.001 |
CH138A+ | 2845 | 1223–5447 | |||
PPC in endometrium (in %) | Morphometrics | 40.35 | 15.44–70.15 | 0.65 (0.24–0.87) | <0.001 |
CH138A+ | 49.92 | 20.02–91.76 | |||
MFIPC in endometrium | Morphometrics | 90,579 | 445–421,532 | 0.86 (0.56–0.96) | <0.001 |
CH138A+ | 140,235 | 384–424,773 | |||
MFIOB in endometrium | Morphometrics | −3073 | −172,034–116,458 | 0.87 (0.71–0.95) | 0.07 |
CH138A+ | 14,643 | −198,069–234,887 | |||
MFIDQ in endometrium | Morphometrics | 11,498 | 2392–19,693 | 0.41 (−0.25–0.81) | 0.15 |
CH138A+ | 8410 | 1982–18,191 |
Variable | CH138A | Outcome | Range | CCC (95% CI) | p-Value |
---|---|---|---|---|---|
PPC in blood (in %) | No | 78.20 | 61.38–87.82 | 0.57 (0.20–0.80) | 0.031 |
Yes | 83.82 | 69.36–92.03 | |||
MFIPC in blood | No | 1,116,458 | 450,894–1,841,761 | 0.20 (−0.19–0.53) | 0.06 |
Yes | 1,780,888 | 483,827–4,043,406 | |||
MFIOB in blood | No | 373,274 | 85,403–1,013,163 | 0.69 (0.39–0.85) | 0.007 |
Yes | 242,081 | 58,924–573,332 | |||
MFIDQ in blood | No | 974 | 404–1998 | 0.19 (0.01–0.35) | 0.001 |
Yes | 2519 | 1092–4631 | |||
PPC in endometrium (in %) | No | 37.67 | 15.05–57.66 | 0.80 (0.44–0.94) | 0.017 |
Yes | 40.35 | 15.44–70.15 | |||
MFIPC in endometrium | No | 38,637 | 375–244,415 | 0.61 (0.23–0.83) | 0.021 |
Yes | 169,632 | 445–421,532 | |||
MFIOB in endometrium | No | 56,358 | −13,006–477,606 | 0.43 (0–0.73) | 0.067 |
Yes | −3073 | −172,034–116,458 | |||
MFIDQ in endometrium | No | 16,513 | 763–66,290 | 0.74 (0.11–0.95) | 0.17 |
Yes | 11,498 | 2392–19,693 |
Variable | Outcome | Range | CCC (95% CI) | p-Value | |
---|---|---|---|---|---|
PPC (in %) | Blood | 78.20 | 61.38–87.82 | 0.04 (−0.04–0.12) | 0.34 |
Endometrium | 37.67 | 15.05–57.66 | |||
MFIPC | Blood | 1,116,458 | 450,894–1,841,761 | 0.01 (−0.03–0.04) | 0.61 |
Endometrium | 38,637 | 375–244,415 | |||
MFIOB | Blood | 373,274 | 85,403–1,013,163 | 0.08 (−0.21–0.36) | 0.18 |
Endometrium | 56,358 | −13,006–477,606 | |||
MFIDQ | Blood | 974 | 404–1998 | −0.01 (−0.03–0.00) | 0.005 |
Endometrium | 16,513 | 763–66,290 |
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Lietaer, L.; Demeyere, K.; Heirbaut, S.; Meyer, E.; Opsomer, G.; Bogado Pascottini, O. Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows. Animals 2021, 11, 1081. https://doi.org/10.3390/ani11041081
Lietaer L, Demeyere K, Heirbaut S, Meyer E, Opsomer G, Bogado Pascottini O. Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows. Animals. 2021; 11(4):1081. https://doi.org/10.3390/ani11041081
Chicago/Turabian StyleLietaer, Leen, Kristel Demeyere, Stijn Heirbaut, Evelyne Meyer, Geert Opsomer, and Osvaldo Bogado Pascottini. 2021. "Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows" Animals 11, no. 4: 1081. https://doi.org/10.3390/ani11041081