Interference and blood sample preparation for a pyruvate enzymatic assay.
BACKGROUND
To assess the severity of circulatory failure, a pyruvate enzymatic assay was performed on whole blood using lactate dehydrogenase to catalyze the conversion of pyruvate to lactate. We investigated factors related to blood sample collection and preparation that might influence the results, including the timing of blood deproteinization, temperature of sample storage, and hemolysis.


METHODS
A total of 25 whole blood specimens were collected for this study. Each sample was divided into 2 parts: one stored at room temperature (RT) and another kept on ice. The samples were deproteinizied by using 8% perchloric acid (PCA) at varying times after collection; the first deproteinization was immediately after the blood was drawn (0 h), then at 1 h intervals for 6 h and also in samples kept overnight. The supernatant samples were analyzed soon after deproteinization using a COBAS Centrifugal Analyzer. In another set of samples, the blood was immediately deproteinized, and the supernatants were stored at RT and 4 degrees C and assayed for pyruvate at varying times, as above. Finally, the effect of hemolysis on the blood pyruvate enzymatic assay was also evaluated.


RESULTS
When samples were stored at RT, pyruvate levels remained constant until the third h after deproteinization, when there was an approximately 13.3% increase in pyruvate concentration. When whole blood samples were kept at 4 degrees C before deproteinization, pyruvate levels were significantly reduced over time, ranging from 37.8% to 62.2% (paired t test showed a significant mean difference, P < 0.001). No significant differences in pyruvate concentration were observed in supernatant stored at either RT or 4 degrees C. Hemolysis caused a 33.7% increase in the pyruvate concentration, equivalent to 0.18 mg pyruvate per gram per deciliter of hemoglobin.


CONCLUSIONS
For a pyruvate enzymatic assay, keeping a whole blood sample at RT will not cause a significant difference in the pyruvate level as long as the sample is immediately deproteinized. Whole blood samples should not be stored in an ice bath for transport, nor should hemolyzed samples be used for a blood pyruvate enzymatic assay.
Chuang
Chih-Kuang
CK
Division of Genetics and Metabolism, Department of Medical Research, Taipei 10449, Taiwan.
Wang
Tuen-Jen
TJ
Yeung
Chun-Yan
CY
Hsieh
Wen-Shyang
WS
Lin
Dar-Shong
DS
Ho
Shinn-Chamg
SC
Lin
Shuan-Pei
SP
D000328:Adult; D001774:Blood Chemical Analysis; D005260:Female; D006801:Humans; D007770:L-Lactate Dehydrogenase; D019344:Lactic Acid; D008297:Male; D008875:Middle Aged; D009243:NAD; D010472:Perchlorates; D019289:Pyruvic Acid / Q000032:analysis* / Q000097:blood*
D010472:Perchlorates; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase