Effect of collection tube type and preanalytical handling on myeloperoxidase concentrations

BACKGROUND: Myeloperoxidase (MPO) has shown potential as a marker for cardiovascular disease. Limited studies have been published with a variety of sample types, resulting in a wide range of MPO values. Little is known or understood about the impact of collection tube type and preanalytical handling of specimens for MPO determination. METHOD: MPO concentration was determined by use of the ARCHITECT(R) MPO research use assay, which is currently under development. Samples were collected into multiple anticoagulant collection tubes from donors and patients presenting to the emergency department with symptoms of acute coronary syndromes. Whole blood was stored on ice or at room temperature for predetermined time periods. We also evaluated serum and plasma after centrifugation followed by storage at room temperature, 2-8 degrees C, and below -10 degrees C. RESULTS: Baseline sample concentrations were dependent on collection tube type as well as handling conditions. MPO concentrations were consistently higher in samples collected in serum and heparin plasma tubes than in samples in EDTA or citrate tubes. Spike recovery was acceptable in all sera and plasma tested, indicating that the increased MPO concentrations were not due directly to an anticoagulant interference. CONCLUSIONS: The collection tube type and preanalytical handling are critical for accurate and consistent MPO measurement. The preferred anticoagulant and tubes are the EDTA or EDTA plasma preparation tube. MPO concentrations in samples collected in these tubes are stable before centrifugation as whole blood as well as plasma after processing.     

Stability of YKL-40 concentration in blood samples

The stability of YKL-40, a mammalian member of the family of 18 glycosylhydrolases, in blood samples handled under different temperatures and different time intervals before centrifugation was studied in paired serum and plasma samples from 25 healthy premenopausal Danish women. Significant elevations of YKL-40 were found in 8 paired serum samples left on the clot for more than 3 h at room temperature compared to paired serum samples left on the clot for 3 h or less. Significant elevations of YKL-40 were found in 8 paired plasma (EDTA) samples left on the blood cells for more than 8 h at room temperature compared to paired plasma (EDTA) samples left on the blood cells for 8 h or less. No elevations were found in YKL-40 levels in serum samples left on the clot at 4 degrees C for 24 h or in plasma (EDTA) samples left on the blood cells for 72 h before centrifugation. Significantly lower concentrations of YKL-40 were measured in plasma (EDTA) compared with paired serum samples with a serum/plasma ratio of 1.4 in samples left on the clot or on blood cells at 4 degrees C for up to 24 h. Repetitive freezing and thawing had no significant effect on the measured YKL-40 concentrations. In conclusion, we have shown that YKL-40 is very dependent on the handling procedures. All the blood samples must be processed into plasma (EDTA) within 8 h at room temperature or into serum in less than 3 h at room temperature. If this is not possible, the blood samples must be stored at 4 degrees C until processed.     

Choline in whole blood and plasma: sample preparation and stability

BACKGROUND: Choline is critical for a variety of biological functions and has been investigated as a biomarker for various pathological conditions including acute coronary syndrome. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to quantify choline in whole blood and plasma in freshly collected samples prepared with ultrafiltration or protein precipitation. We investigated the effects of preanalytical variables including types of anticoagulants and storage temperature and time. RESULTS: We observed no significant differences in whole-blood choline concentration in EDTA-anticoagulated vs heparin-anticoagulated samples: mean (SD) difference 0.9% (3.2%), P = 0.80. For plasma, choline concentrations with heparin in 5 of 12 volunteers were >10% higher than with EDTA, P = 0.01. One freeze-thaw cycle led to significant mean (SD) increases in choline concentrations in heparin whole blood, 19.3% (11.4%), P <0.01, and the effect was not significant for other sample types studied (P >0.33). For freshly collected samples stored at ambient temperature, choline concentrations in all types of samples increased with storage time. For EDTA whole blood, EDTA plasma, and heparin plasma, the choline concentration increased for the first 60 min and then stabilized. For heparin whole blood, the choline concentration continued to increase linearly with storage time for >4 h, at which time the choline concentrations were increased by approximately 50%. CONCLUSIONS: Sample collection, storage, and sample preparation procedures are critical for clinical measurements of choline in whole blood and plasma.     

Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma

BACKGROUND: Plasma and serum samples have been used to detect cell-free genomic DNA in serum or plasma in certain pathologic conditions such as systemic lupus erythematosus, pulmonary embolism, and malignancies, as well as in fetal cell chimerisms in maternal serum and/or plasma. In this study, baseline concentrations of cell-free DNA in serum and plasma samples were evaluated for the study of posttransfusion chimerism. STUDY DESIGN AND METHODS: DNA was extracted from fresh or stored (4 degrees C for 1-6 days) normal donor serum or plasma samples (ACD; EDTA) by using reagents from an HIV assay kit. After incubation and washing of samples, purified DNA was amplified with HLA DQ-alpha primers (GH26 and 27) or human Y-chromosome primers (SA and SD) to quantitate the concentration of genomic DNA. RESULTS: Fresh serum samples had concentrations of cell-free DNA that were about 20-fold higher than the concentrations in fresh plasma samples. The concentration of cell-free genomic DNA in serum samples increased daily, to a level more than 100 times baseline after clotted blood tubes were stored at 4 degrees C for 4 to 5 days. There was a small increase in cell-free plasma DNA in stored ACD whole blood samples. Male WBCs, spiked into fresh nonanticoagulated female blood, were lysed during the process of clotting, with male DNA liberated into the serum samples. CONCLUSION: Most cell-free DNA in serum samples is generated during the process of clotting in the original collection tube. The concentration of cell-free genomic DNA in fresh plasma is probably the same as that in circulation. Consequently, while serum samples should not be used to monitor the concentration of cell-free DNA in a patient's circulation, serum collected from sample tubes containing clots (i.e., without anticoagulant), 3 to 5 days after the date of phlebotomy, could be useful as a source of DNA with which to screen for posttransfusion microchimerism.     

Influence of sample matrix and storage on BNP measurement on the Bayer Advia Centaur

The assessment and management of congestive heart failure relies increasingly on the measurement of B-type natriuretic peptide (BNP). However, the effective contribution of this biochemical test in the clinical decision making is influenced by reliability of the measure, which also depends on several preanalytical issues. Since there is controversy on the influence of the matrix and the storage conditions on BNP measurement, we compared results of BNP in serum, K2 ethylene diamine tetra-acetic acid (EDTA) plasma and lithium heparin plasma fresh samples and in matching samples stored at -20 and -80 degrees C for 1 week. BNP measured on the Bayer Advia Centaur was systematically underestimated in heparin plasma (-47%) and serum (-62%) when compared to K2 EDTA plasma. According to the established 100 ng/L cutoff value, 25% and 37% of the fresh samples collected in heparin plasma or serum were misclassified from the reference K2 EDTA fresh specimen, respectively. When compared to the fresh specimens, the mean and interindividual bias observed for samples stored at either -20 degrees C or -80 degrees C was, overall, modest for K2 EDTA plasma (-2%) and heparin plasma (+6% and -4%, respectively), though it appeared clinically meaningful in serum (+47% and +28%, respectively). Although we can not rule out that other BNP assays using different antibodies may be not affected from degradation during storage to the same extent, results of our investigation demonstrate that K2 EDTA plasma is the most suitable specimens for BNP testing on fresh and frozen samples stored at either -20 degrees C or -80 degrees C for up to 1 week.     

不同处理和保存条件下体外HCV RNA稳定性研究

对献血者或病人进行HCV核酸检测时，如果标本的采集、处理、保存不当，会造成病毒核酸降解，从而影响检测结果的真实性。本研究的目的是对不同抗凝剂、不同温度、不同保存时间下的HCV RNA病毒稳定性进行研究，以考察常规采供血过程中，标本采集及保存方式对NAT检测的影响。采集7例HCV RNA阳性献血者的血样，采用不同的抗凝剂、经不同的温度和不同的时间保存后，采用荧光定量PCR方法测定HCV RNA病毒含量，考察HCV RNA的稳定性。结果表明：①不同抗凝剂抗凝的全血于4℃保存48小时过程中，病毒含量下降至原滴度的42．7％；EDTA抗凝组各时间点的滴度均低于其它3组（分别相当于其它3组的67．6％-25．1％）。②ACD抗凝的全血在4、25和37℃保存48小时．病毒含量分剐下降到原滴度的53．8％、72．5％、29．8％。③ACD抗凝的全血离心分离出血浆，4℃或25℃继续保存7天，病毒含量分别下降至原滴度的70．9％和25．1％。④ACD抗凝的全血分离的血浆，反复冻融4次，病毒含量下降到原滴度的38．9％。结论：①用于核酸检测的标本应该用无菌采血管采样；②在无菌采血管采样的前提下，核酸检测标本用未抗凝血、EDTA、ACD、CPDA抗凝血均可；③采集的全血应避免放置37℃以上，ACD抗凝全血在4℃、25℃保存48小时内、37℃保存14小时内，HCV RNA病毒仍较为稳定；④分离后的血浆应避免放置25℃以上，ACD抗凝全血分离后的血浆在4℃保存7天，25℃保存3天，HCV RNA病毒仍比较稳定；⑤血浆标本应避免多次冻融，但冻融3次的血浆HCV RNA病毒含量仍然较为稳定；⑥无菌采样对维持病毒的稳定性非常重要；单纯的机械性溶血并不会明显导致病毒的降解；只要注意无菌的问题和有合适的核酸提取方法去除血红素，HCV RNA病毒实际上比以前认为的要稳定得多。     

Effect of blood collection and processing on radioimmunoassay results for apolipoprotein A-I in plasma

We studied the effects of different procedures of blood collection and processing on quantification of apolipoprotein A-I (apoA-I) by radioimmunoassay. ApoA-II and apolipoproteins of low- and very-low-density lipoproteins did not cross react in the assay. Analytical recovery of apoA-I at different doses was complete. ApoA-I concentration in pooled human plasma was stable for as long as a year stored at -70 degrees C. Inter- and intra-assay CVs averaged 7% and 5%, respectively. We collected blood from 20 subjects into tubes containing EDTA alone or EDTA with antiproteolytic agents, then separated the plasma either immediately or after 3 h at 4 degrees C. We tested various formulations of antibacterial, antiproteolytic, and anti-oxidant agents added to plasma, measuring apoA-I concentrations either within 24 h of blood collection or after storage of plasma for 6 weeks at -70 degrees C. No significant difference in the concentrations of apoA-I was found in these specimens, regardless of the conditions studied. We conclude that addition of protective agents other than EDTA is not necessary during blood collection or specimen processing for reliable quantification of apoA-I in fresh or frozen human plasma.     

Evaluation of hepatitis C virus RNA stability in room temperature and multiple freeze–thaw cycles by COBAS AmpliPrep/COBAS TaqMan HCV

To assess the stability of various sample types and storage conditions for quantitative detectability of hepatitis C virus (HCV) RNA viral loads, we studied serum and EDTA/citrate plasma samples obtained from 10 patients known to be positive for HCV RNA. Samples were subjected to the following conditions: 1) 10 freeze–thaw (FT) cycles, and 2) storage at room temperature for 24, 48, and 72 h. Detection of HCV RNA was performed by COBAS AmpliPrep/COBAS TaqMan HCV. The following conclusions were reached: 1) no significantly different viral loads were observed in different blood compartments; 2) no significantly different viral loads were observed after 24, 48, and 72 h at room temperature; 3) no significantly different viral loads were observed after 10 FT cycles in serum and plasma samples; and 4) HCV RNA is quite stable in serum and plasma (EDTA/citrate) samples.     

Stability of YKL-40 concentration in blood samples

The stability of YKL-40, a mammalian member of the family of 18 glycosyl-hydrolases, in blood samples handled under different temperatures and different time intervals before centrifugation was studied in paired serum and plasma samples from 25 healthy premenopausal Danish women. Significant elevations of YKL-40 were found in 8 paired serum samples left on the clot for more than 3 h at room temperature compared to paired serum samples left on the clot for 3 h or less. Significant elevations of YKL-40 were found in 8 paired plasma (EDTA) samples left on the blood cells for more than 8 h at room temperature compared to paired plasma (EDTA) samples left on the blood cells for 8 h or less. No elevations were found in YKL-40 levels in serum samples left on the clot at 4°C for 24 h or in plasma (EDTA) samples left on the blood cells for 72 h before centrifugation. Significantly lower concentrations of YKL-40 were measured in plasma (EDTA) compared with paired serum samples with a serum/plasma ratio of 1.4 in samples left on the clot or on blood cells at 4°C for up to 24 h. Repetitive freezing and thawing had no significant effect on the measured YKL-40 concentrations. In conclusion, we have shown that YKL-40 is very dependent on the handling procedures. All the blood samples must be processed into plasma (EDTA) within 8 h at room temperature or into serum in less than 3 h at room temperature. If this is not possible, the blood samples must be stored at 4°C until processed.     

Stability studies of twenty-four analytes in human plasma and serum

BACKGROUND: The stability and stoichiometric changes of analytes in plasma and serum after prolonged contact with blood cells in uncentrifuged Vacutainer tubes were studied. METHODS: We simultaneously investigated the stability of 24 analytes (a) after prolonged contact of plasma and serum with blood cells and (b) after immediate separation of plasma and serum (centrifuged twice at 2000g for 5 min). We verified biochemical mechanisms of observed analyte change by concomitant measurement of pH, PCO(2), and PO(2). Hemolysis was qualitatively and semiquantitatively assessed. All specimens were maintained at room temperature (25 degrees C) and analyzed in duplicate 0.5, 4, 8, 16, 24, 32, 40, 48, and 56 h after collection. Statistically significant changes from the 0.5 h mean were determined using repeated-measures ANOVA. The significant change limit was applied to determine clinically significant changes in measured analytes. RESULTS: Fifteen of 24 analytes in plasma and serum maintained in contact with cells showed clinically relevant changes, with the degree of change more pronounced in most plasma specimens. All analytes in plasma and serum immediately separated from cells after collection were stable. CONCLUSION: Storage of uncentrifuged specimens beyond 24 h caused significant changes in most analytes investigated because of (a) glucose depletion and Na(+),K(+)-ATPase pump failure; (b) the movement of water into cells, causing hemoconcentration; and (c) leakage of intracellular constituents and metabolites. Immediate separation of plasma or serum from cells provides optimal analyte stability at room temperature. When prolonged contact of plasma or serum with cells is unavoidable, use of serum is recommended because of the higher instability of plasma analytes.     

Effects of storage temperature and time before centrifugation on ionized calcium in blood collected in plain vacutainer tubes and silicone-separator (SST) tubes

We studied the stability of ionized calcium and pH in samples stored at either room temperature or 4 degrees C, in centrifuged and uncentrifuged blood-collection tubes and in centrifuged tubes containing a silicone-separator gel (SST tubes). At room temperature, in uncentrifuged blood from healthy individuals, mean ionized calcium usually increased no more than 10 mumol/L per hour; at 4 degrees C it did not change detectably for 70 h. This stability was fortuitous, however: the concentrations of both hydrogen and lactate ions in these samples increased, apparently with offsetting effects on the concentration of ionized calcium. Blood stored for 70 h at 4 degrees C in centrifuged SST tubes, although showing a slightly greater change in ionized calcium, had less change of pH and no change in the ionized calcium corrected to pH 7.4. In 11 heparinized whole-blood samples from eight patients in intensive care, the mean change per hour in ionized calcium and pH after storage at room temperature was +10 mumol/L and -0.04 units, respectively.     

Effect of anticoagulants and storage temperatures on stability of plasma and serum hormones

OBJECTIVES: To determine the effect of different anticoagulants and storage conditions on the stability of hormones in plasma and serum. DESIGN AND METHODS: Human blood samples were collected from volunteers into EDTA, lithium heparin, sodium fluoride/potassium oxalate, or tubes without anticoagulant, plasma and serum left at -20 degrees C, 4 degrees C or 30 degrees C for 24 and 120 hours then assayed for ACTH, aldosterone, alpha-subunit, AVP, CRH, C-peptide, estradiol, FSH, glucagon, GH, IGF-1, IGFBP-3, insulin, leptin, LH, PPP, PTH, prolactin and VIP, or at room temperature for 0 to 72 hours (BNP, NT-BNP)(n = 6 per condition). RESULTS: The anticoagulant altered the measured concentrations for 9 hormones when compared to EDTA. All hormones except ACTH were stable for > 120 hours in EDTA or fluoride at 4 degrees C, but only 13 hormones were stable in all anticoagulants. At 30 degrees C, 8 hormones were stable for > 120 hours in EDTA, and 3 hormones in all anticoagulants. BNP and NT-BNP were stable for < 24 hours when stored in EDTA or heparin at room temperature. DISCUSSION: Storage of samples in EDTA plasma at 4 degrees C is suitable for most hormones (except ACTH) for up to 120 hours.     

Effects of temperature on stability of blood homocysteine in collection tubes containing 3-deazaadenosine

BACKGROUND: The accuracy of homocysteine (Hcy) results is currently compromised by the requirement to separate the plasma within 1 h of sample collection. We studied the effect of temperature on the stability of plasma Hcy over a 72-h time course in blood collected into evacuated tubes containing either EDTA alone or both EDTA and 3-deazaadenosine (3DA). METHODS: We recruited 100 volunteers, including both diseased and healthy individuals with a range of baseline plasma Hcy values, from two centers. Blood samples were collected into tubes containing EDTA, and EDTA plus 3DA and stored at ambient temperature (20-25 degrees C) or refrigerated (2-8 degrees C). Aliquots of blood were centrifuged at various times up to 72 h, the plasma was removed, and Hcy was measured by HPLC. RESULTS: Plasma Hcy measurement covering the sample collection and storage conditions during the whole time course was possible on samples from 59 of those recruited. One-way ANOVA for repeated measures within subjects revealed that only samples that were collected into tubes containing EDTA plus 3DA and stored refrigerated were stable over 72 h (P = 0.2761). CONCLUSIONS: A combination of 3DA and storage at 2-8 degrees C will allow collection of samples for plasma Hcy measurement outside of the hospital setting and wider population screening.     

Effects of processing and storage conditions on amyloid beta (1-42) and tau concentrations in cerebrospinal fluid: implications for use in clinical practice

BACKGROUND: Reported concentrations of amyloid beta (1-42) (A beta 42) and tau in cerebrospinal fluid (CSF) differ among reports. We investigated the effects of storage temperature, repeated freeze/thaw cycles, and centrifugation on the concentrations of A beta 42 and tau in CSF. METHODS: Stability of samples stored at -80 degrees C was determined by use of an accelerated stability testing protocol according to the Arrhenius equation. A beta 42 and tau concentrations were measured in CSF samples stored at 4, 18, 37, and -80 degrees C. Relative CSF concentrations (%) of the biomarkers after one freeze/thaw cycle were compared with those after two, three, four, five, and six freeze/thaw cycles. In addition, relative A beta 42 and tau concentrations in samples not centrifuged were compared with samples centrifuged after 1, 4, 48, and 72 h. RESULTS: A beta 42 and tau concentrations were stable in CSF when stored for a long period at -80 degrees C. CSF A beta 42 decreased by 20% during the first 2 days at 4, 18, and 37 degrees C compared with -80 degrees C. CSF tau decreased after storage for 12 days at 37 degrees C. After three freeze/thaw cycles, CSF A beta 42 decreased 20%. CSF tau was stable during six freeze/thaw cycles. Centrifugation did not influence the biomarker concentrations. CONCLUSIONS: Repeated freeze/thaw cycles and storage at 4, 18, and 37 degrees C influence the quantitative result of the A beta 42 test. Preferably, samples should be stored at -80 degrees C immediately after collection.     

Serum and plasma fragments of C-telopeptides of type I collagen (CTX) are stable during storage at low temperatures for 3 years

BACKGROUND: Control of pre-analytical variables is essential for successful application of biological markers, including bone resorption markers, in clinical trials and routine use. The effect of storage temperature on stability of bone resorption markers have not been subject of systematically investigation, and therefore the present study was set out to determine the stability of C-telopeptides of type I collagen (CTX) in serum and plasma samples stored frozen for 3 years. METHODS: The serum and plasma levels of CTX were determined in samples aliquoted and stored frozen for up to 3 years. RESULTS: No significant decrease could be detected in neither serum nor plasma samples after 3 years of storage at -20, -80 or -150 degrees C. However, at elevated temperature, i.e. 4 and 37 degrees C, improved stability of CTX was observed in EDTA plasma samples compared to serum. CONCLUSIONS: CTX is stable in frozen serum and plasma samples for up to 3 years. EDTA plasma might be the preferred matrix due to improved stability at elevated temperatures.     

Effects of storage and handling conditions on concentrations of individual carotenoids, retinol, and tocopherol in plasma

We investigated the effects of storage and handling on measured values for carotenoids, retinol, and tocopherol in plasma. We found no significant differences in the concentrations of these analytes measured in plasma samples that were frozen immediately after separation as compared with replicate samples maintained at room temperature in the dark for 24 h. Analytes were stable in solvents for at least 18 h at 23 degrees C after extraction. Purging samples with nitrogen gas before freezing had no detectable beneficial effects. All analytes were stable in plasma stored at -70 degrees C for at least 28 months or at -20 degrees C for five months. By 15 months the concentrations of carotenoids were significantly less (P less than 0.05) in plasma stored at -20 degrees C than in plasma stored at -70 degrees C, while retinol and tocopherol concentrations were not significantly different. Concomitant with the decrease in carotenoids was the appearance of unidentified peaks in the ultraviolet. Adding ascorbic acid or butylated hydroxytoluene antioxidants to the precipitating solvent did not alter the losses of carotenoids or alter the appearance of unidentified peaks. Under appropriate conditions, plasma carotenoids, retinol, and tocopherol are stable for more than two years.     

Evaluation of three different specimen types (serum, plasma lithium heparin and serum gel separator) for analysis of certain analytes: clinical significance of differences in results and efficiency in use.

BACKGROUND: There is a lack of consensus regarding the most appropriate specimen type for analysis of many biochemistry analytes. The aim of this study was to compare renal and lipid analyte profiles and phenytoin values in plain serum (S), serum gel (G) and plasma (lithium heparin, P) tubes and to investigate the stability of these analytes after prolonged contact with cells or gel at room temperature (RT, 20 degrees C) and as aliquoted and stored at 4 degrees C. METHODS: Primary specimens were centrifuged once, maintained at RT and analysed within 2 h (T(0)) and after 24 h (T(24)) and 48 h (T(48)). For assessment of stability at 4 degrees C, two cell-free aliquots were separated from each of the primary tubes and stored at 4 degrees C and then analysed at T(24) and T(48). Differences in analyte concentrations between tubes at T(0) and following storage (at T(24) and T(48)) were evaluated for both statistical and clinical significance. RESULTS: At T(0) all analytes, except potassium, demonstrated equivalence between serum, gel and plasma tubes. Potassium and creatinine were more stable in gel tubes than in serum/plasma tubes. In contrast, phentytoin was stable in plain serum and plasma up to T(48) at RT, but showed a progressive and clinically significant decrease in concentration in gel tubes at T(24) and T(48) at RT. All analytes except CO(2) were stable up to T(48) when aliquoted and stored at 4 degrees C. CONCLUSIONS: We concluded that the serum gel tube has advantages over plain serum and plasma tubes for measurement of the analytes investigated in this study, with the exception of phenytoin. In practice, the gel tubes demonstrate enhanced analyte stability and reduce the need to aliquot specimens, with greater protection against possible contamination. Further investigation would be required to evaluate a broader range of analytes.     

Hormone stability in human whole blood

OBJECTIVE: To determine whether significant changes in the plasma concentrations of 17 hormones occur when human whole blood is held at 4 or 24 degrees C for up to 24 h before separation of the plasma fraction. DESIGN AND METHODS: Blood samples (EDTA) from healthy human volunteers were held at 4 degrees C or 24 degrees C for 0.5, 6 or 24 h before separation. Plasma concentrations of ACTH, aldosterone, gonadotrophin alpha-subunits, AVP, C-peptide, estradiol, FSH, GH, glucagon, IGF-1, IGFBP3, insulin, leptin, LH, prolactin, PTH and VIP were measured and the results compared to baseline values. Nonlinear regression was used to test for a significant mean rate of change. The time interval for median concentrations to change by 10% was determined. RESULTS: Significant changes were observed for ACTH (decrease at 18.6 hr, 4 degrees C; 17.5 hr, 24 degrees C); AVP (increase at 2.6 h, 24 degrees C); insulin (decrease at 16.8 hr, 4 degrees C; 16.9 hr, 24 degrees C) and VIP (increase at 18.6 h, 24 degrees C). No changes were detected for the remaining analytes.B CONCLUSIONS: The measurement of some hormones is compromised by a delay in plasma separation from normal human blood. While many hormones appear stable in normal whole blood, we recommend that processing occurs without delay.     

Homocysteine export from erythrocytes and its implication for plasma sampling.

The concentration of free and total homocysteine in plasma increases in time if blood is stored uncentrifuged after sampling. The increase is temperature dependent and the maximal increase in total plasma homocysteine at 37 degrees C was 3.0 mumol.L-1.h-1. Even at 4 degrees C there is a substantial increase, particularly of free plasma homocysteine. Plasma glutathione, cysteinylglycine, and gamma-glutamylcysteine also show an increase in time if whole blood is stored, whereas cysteine decreases. We show that the erythrocytes are responsible for most of the increase in plasma homocysteine and suggest that homocysteine is derived from adenosylmethionine-dependent protein carboxymethylations in the cells. We conclude that strict sampling conditions are necessary when plasma homocysteine and especially its free fraction are assayed.     

Preanalytical variables affecting the quantification of fatty acid ethyl esters in plasma and serum samples

BACKGROUND: Fatty acid ethyl esters (FAEEs) are cytotoxic nonoxidative ethanol metabolites produced by esterification of fatty acids and ethanol. FAEEs are detectable in blood up to 24 h after ethanol consumption. The objective of this study was to assess the impact of gender, serum or plasma triglyceride concentration, time and temperature of specimen storage, type of alcoholic beverage ingested, and the rate of ethanol consumption on FAEE concentrations in plasma or serum. METHODS: For some studies, subject were recruited volunteers; in others, residual blood samples after ethanol quantification were used. FAEEs were isolated by solid-phase extraction and quantified by gas chromatography-mass spectrometry. RESULTS: For weight-adjusted amounts of ethanol intake, FAEE concentrations were twofold greater for men than women (P /=24 h. The type of alcoholic beverage and rate of consumption did not affect FAEE concentrations. CONCLUSION: These studies advance plasma and serum FAEE measurements closer to implementation as a clinical test for ethanol intake.