The effect of specimen hemolysis on coagulation test results.

Author(s): Laga AC, Cheves TA, Sweeney JD

Publication: Am J Clin Pathol, 2006, Vol. 126, Page 748-55

PubMed ID: 17050072 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of hemolysis on PT, APTT, factor V, factor VII, factor X, FVIIa, FXIIa, and F1+2 in blood specimens from patients and to evaluate the effects of ultracentrifugation (at 35000 x g rather than centrifugation at 2500 x g) on the concentration of hemoglobin in plasma. After collection of a hemolyzed specimen, a second non-hemolyzed specimen was obtained from the same patient within 180 minutes of the first collection (average interval between specimen pairs was 72 min). PT and APTT were measured within 2 hours while plasma was stored at -80 degrees C prior to the measurement of coagulation factors and hemoglobin.

   Summary of Findings:

   Statistically significant but clinically insignificant decreases in PT and APTT were observed in hemolyzed specimens compared to nonhemolyzed specimens (each P<0.01). The authors conclude that rejection of specimens based on the degree of hemolysis may not be appropriate for PT and APTT measurement. Factor V, factor VIIa, factor X, and F1+2 were also significantly increased in hemolyzed specimens compared to nonhemolyzed specimens. Factor VIII and factor XIIa were not affected by hemolysis. High correlations were seen for all factors between the paired specimens (r=0.85-0.99) except for F1+2 (r=0.33). None of the changes observed in coagulation test results between hemolyzed and nonhemolyzed specimens were associated with supernatant hemoglobin levels (degree of hemolysis). Ultracentrifugation of supernatant did not significantly affect the hemoglobin concentration.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Morphology	Hematology/ auto analyzer
   Protein	Hematology/ auto analyzer
   Cell count/volume	Macroscopic observation
   Cell count/volume	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot sequential collection	1st collection 2nd collection
   Biospecimen Aliquots and Components	Hemolysis	Not induced Tissue homogenizer-induced
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared

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2. Study Purpose

   The purpose of this study was to determine the effects of mechanically induced hemolysis, duration of tissue homogenizer use, specimen collection order, and storage duration on PT, APTT, and the concentration of hemoglobin in plasma. After mechanical induction of hemolysis and centrifugation, plasma was either stored at room temperature for 3 h or stored at room temperature for 8 h plus overnight refrigeration for a total storage duration of 24 h.

   Summary of Findings:

   Despite increases in the percentage of hemolysis and supernatant hemoglobin after tissue homogenizer use, PT remained unchanged, regardless of the duration of tissue homogenizer use. On the other hand, APTT did progressively lengthen when specimens were subjected to longer durations of tissue homogenizer use (trend, P<0.01), but the differences were minor (<1 s), and only the specimens with the highest degree of mechanically induced hemolysis (uncommonly encountered in practice) showed a statistically significant change in APTT from specimens with no mechanically induced hemolysis. Specimen collection order did not affect PT or APTT values. Delayed analysis (24 h at room temperature and then refrigerated) had no effects on PT measurements in hemolyzed or nonhemolyzed specimens, however, APTT measurements were higher in both hemolyzed and nonhemolyzed specimens after 24 h than after 3 h at room temperature.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Morphology	Hematology/ auto analyzer
   Cell count/volume	Spectrophotometry
   Cell count/volume	Hematology/ auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Refrigeration
   Storage	Storage duration	3 h 24 h
   Biospecimen Aliquots and Components	Aliquot sequential collection	1st collection 2nd collection 3rd collection 4th collection
   Biospecimen Aliquots and Components	Biospecimen mixing	Tissue homogenizer for 0 s Tissue homogenizer for 30 s Tissue homogenizer for 60 s Tissue homogenizer for 75 s
   Biospecimen Aliquots and Components	Hemolysis	Not induced Tissue homogenizer-induced

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