The influence of different anticoagulants and time-delayed sample processing and measurements on human monocyte subset and monocyte-platelet aggregate analyses.
Author(s): Ji WJ, Lu RY, Liu JX, Ma YQ, Zeng S, Shi R, Zhao JH, Chen SB, Zhou X, Li YM
Publication: Cytometry B Clin Cytom, 2017, Vol. 92, Page 371-379
PubMed ID: 26861109 PubMed Review Paper? No
Purpose of Paper
This paper compared the effects of a 2 h blood processing delay and post-staining storage on monocyte subsets and monocyte-platelet aggregation in EDTA and sodium citrate anticoagulated blood.
Conclusion of Paper
Citrated blood had more CD14++CD16-, CD14++CD16+, and CD14+CD16++ monocyte platelet aggregates than EDTA blood when processed immediately and after 2 h. Further, the percentage CD14++CD16+ and CD14++CD16- monocytes and CD14++CD16-, CD14++CD16+, and CD14+CD16++ monocyte-platelet aggregates in citrated blood were significantly affected by pre-processing storage and post-staining storage, but there was no effect of preprocessing storage in EDTA blood and post-staining storage only affected the percentage CD14++CD16+ monocyte platelet aggregates. Although citrated plasma had more monocyte platelet aggregates than EDTA specimens at all time points, the difference increased with post-staining storage and citrated specimens had fewer CD14++CD16- monocytes when stored post-staining for 1 h or more and more CD14++CD16+ monocytes when stored post-staining for 3 h or more compared to EDTA specimens.
Studies
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Study Purpose
This study compared the effect of a 2 h blood processing delay and post-staining storage on monocyte subsets and monocyte-platelet aggregation in EDTA and sodium citrate anticoagulated blood. Blood was collected from 12 healthy donors using a 21-gauge (G) needle and a light tourniquet (discarding the first 3 mL) into tubes containing 3.8% sodium citrate or 2.0 mg/mL EDTA. Specimens were either immediately subjected to red blood cell lysis and staining or stored for 2 h at 4˚C with 10 sec agitation every 30 min. Monocytes were incubated with fluorescently-labeled antibodies against CD14, CD16, CD86, and CD41 and red blood cells were lysed with red blood cell lysis buffer. After lysis and staining, specimens were stored at 4˚C and analyzed 0, 1, 3, and 5 h later by four-color flow cytometry. Viability was determined by 7-aminoactinomycin D staining.
Summary of Findings:
Citrated blood had more CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocyte platelet aggregates than EDTA blood when processed immediately (10.4%, P<0.01; 11.9%, P<0.01; and 8.9%, P<0.05; respectively) and even more so after 2 h (27.8%, P<0.01; 26.9%, P<0.01; and 19.4%, P<0.01; respectively). Storage of citrated blood at 4˚C for 2 before processing resulted in an increase in the percentage of CD14++CD16+ (P<0.01) and a decrease in CD14++CD16- (P<0.01) monocytes as well as an increase in CD14++CD16-, CD14++CD16+, and CD14+CD16++ monocyte-platelet aggregates (P<0.01, all), but had no effect on cell viability or the subset of CD14+CD16++ monocytes. In contrast, storage of EDTA anticoagulated blood had no effect on monocyte subsets, monocyte platelet aggregates, or cell viability. Post-staining storage resulted in a significant trend toward decreasing percentage of CD14++CD16- monocytes (P<0.01), and increasing percentages of CD14++CD16+ monocytes (P<0.01), CD14++CD16 monocyte platelet aggregates (P<0.01), CD14++CD16+ monocyte platelet aggregates (P<0.01), and CD14+CD16++ monocyte platelet aggregates (P<0.01) in citrated specimens and a trend toward increasing CD14++CD16+ monocyte platelet aggregates in EDTA specimens (P<0.05). Although citrated plasma had more monocyte platelet aggregates than EDTA specimens at all time points (P<0.01, all), the difference increased with post-staining storage. Additionally, citrated specimens had fewer CD14++CD16- monocytes when stored post-staining for 1 h or more and more CD14++CD16+ monocytes when stored post-staining for 3 h or more compared to EDTA specimens.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 h
2 h
1 h
3 h
5 h
Biospecimen Acquisition Anticoagulant EDTA
Sodium citrate