Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method.
Author(s): Loeffen R, Kleinegris MC, Loubele ST, Pluijmen PH, Fens D, van Oerle R, Ten Cate H, Spronk HM
Publication: J Thromb Haemost, 2012, Vol. 10, Page 2544-54
PubMed ID: 23020632 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
-
Study Purpose
The purpose of this study was to determine the effects of blood collection method and tube type on hemolysis, ETP and peak height in plasma triggered with different amounts of TF. 21-gauge conventional and butterfly needles were used along with a 20-gauge intravenous catheter. All collection containers contained 3.2% citrate. After collection, plasma was separated by a two-step centrifugation protocol, frozen in liquid nitrogen, and stored at -80 degrees C until analysis.
Summary of Findings:
Significantly more hemolysis was observed after centrifugation when specimens were collected through butterfly needles or intravenous catheters than when collected through a conventional straight needle, but there were only insignificant difference in ETP and thrombin peak height, regardless of TF trigger. Blood collection tube type affected thrombin generation triggered without TF or with 1 pM TF, but not 5 pM TF. The highest mean ETPs were observed in plasma from blood collected in glass Vacutainer tubes followed by plastic SCAT tubes, but the addition of CTI to SCAT tubes completely inhibited clot formation.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Morphology Hematology/ auto analyzer Morphology Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Glass vacutainer
Plastic vacutainer
Plastic Venoject
Plastic Monovette
Plastic SCAT
Plastic SCAT with corn trypsin inhibitor
Biospecimen Acquisition Method of fluid acquisition Butterfly device
IV catheter
Venipuncture
Needle
Hematology/ auto analyzer Specific Reaction solution 0 pM TF
1 pM TF
5 pM TF
-
Study Purpose
The purpose of this study was to determine the effects of discarding the first collection tube, delayed centrifugation, and using a one-step rather than two-step centrifugation on ETP and peak height in plasma collected in glass Vacutainer or plastic Monovette tubes and triggered with different concentrations TF. Blood was collected by standard venipuncture on alternating arms into the two types of collection tubes, each of which contained 3.2% citrate. After centrifugation, plasma was frozen in liquid nitrogen and stored at -80 degrees C until analysis.
Summary of Findings:
When specimens were collected in glass Vacutainers, ETP and thrombin peak height triggered without TF or with 1 pM TF were significantly higher in the first compared to the second collection tube (p<0.0001, both), but this was not observed when blood was collected into plastic Monovette tubes or when triggered with 5 pM TF. When whole blood was stored in glass Vacutainer tubes, ETP and peak height were not affected by a 6 h delay before centrifugation, but when whole blood was stored in a plastic Monovette tube during such a delay, ETPs triggered without TF or with 1 pM TF were increased compared to those observed when specimens were centrifuged immediately (p=0.008), and the largest increases were observed when storage was at room temperature rather than 4 or 37 degrees C. Peak height was increased in specimens stored for 6 h, in plastic Monovette tubes, at 4 degrees C or room temperature, prior to centrifugation, and triggered without TF compared to peak heights observed for specimens that were centrifuged immediately (p=0.0092 and p=0.0005, respectively). ETP was higher when specimens in plastic Monovette tubes triggered without TF 0 or with 1 pM TF were centrifuged at 2,000 x g for 15 min instead of being centrifuged at 2,000 x g for 5 min and then 10,000 x g for 10 min (p=0.0005 and p=0.0256, respectively) but not triggered with 5 pM TF. A similar increase ETP after one-step rather than two-step centrifugation was observed in specimens collected in glass Vacutainer tubes and triggered with 1 pM TF (p=0.0466), but no significant effect of a one-step rather than two-step centrifugation was observed when glass Vacutainer specimens were triggered with 0 or 5 pM TF.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Morphology Hematology/ auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Glass Vacutainer
Plastic Monovette,
Storage Storage duration 0 h
6 h
Storage Storage temperature 4 degrees C
Room temperature
37 degrees C
Biospecimen Aliquots and Components Aliquot sequential collection 1st collection
2nd collection
Hematology/ auto analyzer Specific Reaction solution 0 pm TF
1 pM TF
5 pM TF
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Centrifugation delays investigated
-
Study Purpose
The purpose of this study was to determine the effects of post-thaw storage temperature and duration on thrombin peak height and ETP in plasma triggered without TF or with 1 pM TF. Blood was collected by standard venipuncture into glass Vacutainers, and plasma was obtained with a two-step centrifugation protocol and immediately frozen in liquid nitrogen and stored at -80 degrees C. All specimens were thawed for 10 min at 37 degrees C and analyzed immediately or exposed to experimental storage.
Summary of Findings:
When plasma triggered without TF was stored post-thaw at room temperature for up to 60 min the thrombin peak height and ETP were within 10% of that observed in plasma analyzed immediately after thawing, but when triggered with 1 pM TF, peak height and ETP were higher in plasma stored for 60 min at room temperature compared to specimens analyzed immediately. Post-thaw storage at 37 degrees C led to lower ETP and peak height in response to 0 or 1 pM TF compared to immediately analyzed plasma. Post-thaw storage at 4 degrees C led to increased peak height and ETP in response to 1 pM TF compared to immediately analyzed plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Morphology Hematology/ auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 4 degrees C
Room temperature
37 degrees C
Storage Storage duration 10 min
20 min
30 min
40 min
50 min
60 min
Hematology/ auto analyzer Specific Reaction solution 0 pM TF
1 pM TF