Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Agreement between blood draw techniques for assessing platelet activation by flow cytometry.

Author(s): Welch EL, Crooks MG, Hart SP

Publication: Platelets, 2019, Vol. 30, Page 530-534

PubMed ID: 30365337 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of blood draw method and discarding the first drawn specimen on platelet P-selectin expression and platelet-monocyte complexes in response to ADP stimulation. Two blood specimens were drawn sequentially from the median cubital vein of the right arm of 10 healthy individuals using a 21-gauge butterfly needle into Vacutainer tubes containing sodium citrate (Vacutainer 1 and Vacutainer 2). Blood was then collected from the left arm by manual aspiration into two 5 mL syringes containing sodium citrate (syringe 1 and syringe 2). Blood was processed within 20 min of collection and two-color flow cytometry using anti-CD14-APC and anti-CD42b-AF488, anti-CD62P-PE and ADP stimulation (0.1, 1, or 10 µM), or anti-CD42b-PerCP-Cy5 was performed within 3 h. Monocytes were identified by forward and side scatter properties and CD14 expression. Platelets were identified by forward and side scatter properties and CD42b expression. The percentage of platelet monocyte complexes was calculated as the percentage of CD14+ monocytes that were also CD42+.  

   Summary of Findings:

   The percentage platelet-monocyte complexes (the percentage of CD14+ monocytes that were also CD42+) and percentage of P-selectin positive platelets increased with increasing concentration of ADP (approximately 10 and 18-fold, respectively). The platelet-monocyte complexes were very strongly correlated between the specimen types. The regression lines for percentage platelet-monocyte complexes and P-selectin between tubes were close to unity for Vacutainer 1-Vacutainer 2 (1.02 and 0.979, respectively) Syringe 1-Syringe 2 (1.00 and 0.989, respectively), Vacutainer 1-Syringe 1 (0.996 and 0.953, respectively), and Vacutainer 2-Syringe 2 (1.01 and 0.963, respectively) indicating no effect of discarding blood prior to analysis. Further, tube type did not significantly affect platelet-monocyte complexes or percentage positive for P-selectin. Bland-Altman plots showed good agreement for platelet-monocyte complexes and P-selectin for all comparisons but variability in both directions increased with increasing ADP concentration.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot sequential collection	1st collection 2nd collection
   Biospecimen Acquisition	Method of fluid acquisition	Syringe draw Needle

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