Influence of sample type and storage conditions on soluble CD40 ligand assessment

BACKGROUND: Several studies have consistently shown that soluble CD40 ligand (sCD40L) concentrations are increased in patients with acute coronary syndromes and can serve as a biomarker for risk stratification. However, few data are available on preanalytic conditions that impact sCD40L values. Thus, the aim of our prospective study was to evaluate the impact of sampling techniques and storage conditions on sCD40L concentrations. METHODS: We included a total of 30 patients with no, stable, or unstable coronary heart disease. Blood samples were collected in gel-filled tubes without additives, in EDTA-filled tubes, and in citrate-filled tubes and were kept at various storage conditions. RESULTS: Median (interquartile range) sCD40L values at baseline were higher in serum samples [5.29 (3.89-6.33) microg/L] than in either EDTA plasma [0.78 (0.39-1.12) microg/L; P <0.001] or citrate plasma [0.37 (0.22-0.51) microg/L; P <0.001]. Serum values increased with delayed processing [7.94 (5.97-9.62) microg/L after 1.5 h (P <0.001) vs baseline; 10.55 (7.58-11.55) microg/L after 3 h (P <0.001) vs baseline]. However, after centrifugation, sCD40L values remained stable for all 3 sample types. CONCLUSION: Plasma, but not serum, samples are appropriate for sCD40L measurements. In general, preanalytic conditions are critical in the assessment of sCD40L concentrations and thus should be carefully considered for future studies.     

Increased plasma concentrations of soluble CD40 ligand in acute coronary syndrome depend on in vitro platelet activation

BACKGROUND: Soluble CD40 ligand (sCD40L) was suggested as a novel biomarker of cardiovascular risk. We examined the effect of preanalytical variation on the measurement of sCD40L concentration. METHODS: From healthy control individuals (n = 20) and patients with acute coronary syndrome (ACS) (n = 20) or sepsis (n = 20), we obtained blood drawn into 5 tubes containing citrate or a mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD). The tubes were incubated for 30 min at room temperature or 0 degrees C before a single or double centrifugation (15 min, 2500 g) at room temperature or 4 degrees C, respectively. sCD40L, beta-thromboglobulin (betaTG), and platelet factor 4 (PF4) concentrations were measured using immunoassays. RESULTS: Concentrations of sCD40L were very low in all CTAD and citrated samples maintained at 0 degrees C (median < or = 0.076 microg/L). Although increased betaTG and PF4 confirmed disease-related in vivo platelet activation, sCD40L was not higher in patients than in controls. In contrast, if the samples were processed at room temperature, sCD40L was significantly higher in ACS patients than in controls (P <0.02 in CTAD and citrated plasma at room temperature). Moreover, the betaTG:PF4 ratio decreased in patient but not control CTAD samples, suggesting a greater susceptibility of patient platelets to in vitro activation. CONCLUSIONS: Increased sCD40L concentrations resulted from in vitro platelet activation during sample preparation. Disease-related in vivo activation did not contribute to sCD40L concentrations in plasma. Therefore, published studies of sCD40L demand cautious interpretation, because their preanalytical conditions were not standardized.     

Validation of a new automated renin assay

BACKGROUND: Measurement of plasma renin is important for the treatment of patients with congenital adrenal hyperplasia (CAH) and in the evaluation of patients with suspected hyperaldosteronism. Immunologic assays for plasma renin offer easier implementation and standardization than enzyme-kinetic assays for plasma renin activity, but their sensitivity and specificity have been questioned. We studied a renin immunochemiluminescence assay on an automated platform. METHODS: Renin was measured by an enzymatic assay, by IRMA, and by the new Nichols Advantage Specialty System immunochemiluminometric assay (ICMA), in plasmas from unselected individuals from our outpatient departments and in samples from patients with selected diagnoses. RESULTS: The detection limit in the ICMA was 0.1 mU/L. The recovery was >90%, and the imprecision (CV) was generally <9%. Mean (SD) concentrations measured by ICMA were 32 (21)% lower than those measured by IRMA. Renin concentrations as measured by ICMA were identical in serum and EDTA-, heparin-, and citrate-anticoagulated plasmas. Prolonged incubation of whole blood at room temperature before centrifugation did not affect renin concentrations. The central 95% interval for 80 healthy adults was 6-85.5 mU/L. Plasma renin as assessed by ICMA in patients with primary hyperaldosteronism was <0.2 mU/L. CONCLUSIONS: The performance characteristics of the new renin ICMA allow its use for patients with CAH and for the diagnosis of mineralocorticoid hypertension. In view of the variability of renin concentrations, use for other forms of hypertension or physiologic research calls for the development of uniform sampling protocols.     

血浆同型半胱氨酸及其相关硫醇物在全血中的稳定性研究

目的 观察含EDTA的全血样本放置时间、保存温度以及不同稳定剂对血浆同型半胱氨酸(homocysteine,Hcy)及其相关硫醇物水平的影响.方法 用含EDTA、EDTA-氟化钠(EDTA-NaF)、EDTA-3-Deazaadenosine(EDTA-3DA)的试管收集17名健康成人静脉血,置于碎冰上(0～4 ℃)或室温中(25 ℃)保存,放置0、3、6、24、48 h后分离血浆.用HPLC法测定血浆总同型半胱氨酸(tHcy)、总半胱氨酸(total cysteine,tCys)、总半胱氨酰甘氨酸(total cysteinylglycine,tCysGly)、总谷胱甘肽(total glutathione,tGSH)浓度.设定全血样本0 h分离血浆所测硫醇物浓度为基础值.结果 EDTA管在室温中放置3、6、24、48 h,tHcy分别增加38.5%、64.2%、141.9%、225.4%;tCysGly、tGSH在3 h分别增加20.0%、37.9%,tCys则降低3.5%.EDTA管在碎冰上保存,各硫醇物浓度6 h内增加不超过5%.EDTA-3DA和EDTA-NaF管在室温放置3 h,与各自基础值相比,血浆tHcy、tCys、tCysGly、tGSH浓度差异无统计学意义(EDTA-3DA管:F值分别为0.01、0.94、0.09、0.01,P值均>0.05;EDTA-NaF管:F值分别为0.85、0.04、0.03、0.02,P值均>0.05).结论 EDTA抗凝血浆,所测tHcy及其相关硫醇物浓度呈时间、温度依赖性增加.血浆tHcy等硫醇物测定的分析前处理条件必须标准化.EDTA-3DA和EDTA-NaF管可使血浆tHcy、tCys、tCysGly、tGSH在室温中至少稳定3 h.

Abstract：

Objective To investigate the effects of different stabilizers, time and temperature before centrifugation on the stability of homocysteine (Hcy) and other related thiols levels involving EDTA-containing whole blood.Methods Blood was drawn from 17 healthy adults and collected into tubes containing EDTA, EDTA plus NaF and EDTA plus 3-deazaadenosine(3DA),then stored on crush ice(0-4 ℃) immediately or at room temperature(25 ℃).Plasma was separated at 0, 3, 6, 24 and 48 hours, respectively.The levels of plasma total Hcy (tHcy), total cysteine (tCys), tatal cysteinylglycine (tCysGly) and tatal glutathione (tGSH) were measured by HPLC.The plasma immediately separated was used as baseline sample. Results In EDTA tubes stored at room temperature, tHcy levels increased by 38.5%, 64.2%, 141.9%, 225.4% for 3, 6, 24, and 48 h, respectively.The levels of tCysGly and tGSH increased by 20.0% and 37.9% within 3 h, however, tCys decreased by 3.5%.The levels of the thiols increase by less than 5% up to 6 h in EDTA tubes stored on crush ice.In EDTA-3DA and EDTA-NaF tubes, no statistical differences were observed in the plasma levels of tHcy, tCys,tCysGly and tGSH compared with their respective baseline values at room temperature for 3 h(EDTA-3DA tubes:F=0.01,0.94,0.09,0.01,all P>0.05;EDTA-NaF tubes:F=0.85,0.04,0.03,0.02,all P>0.05).Conclusions The EDTA-plasma levels of tHcy and other related thiols are time and temperature-dependent. There is a strong need for standardization of blood sample collection and processing in tHcy and other thiols assays. The plasma concentrations of tHcy, tCys, tCysGly and tGSH were stable for 3 h at least in the EDTA-3DA and EDTA-NaF tubes kept at room temperature.     

Reproducibility of plasma angiotensin-converting enzyme activity in human subjects determined by fluorimetry with Z-phenylalanine-histidyl-leucine as substrate

Despite the major physiologic role of angiotensin-converting enzyme (ACE), few studies have evaluated the ideal conditions for measuring human plasma ACE activity, specifically when using Z-phenylalanine-histidyl-leucine as substrate. This study, performed in volunteer patients, assessed the reproducibility of human plasma ACE activity measured by fluorimetry with Z-phenyl-histidyl-leucine as the substrate. After blood centrifugation, plasma was stored under different conditions until processing. The following sources of variability were evaluated: (1) the interval to centrifugation of blood after collection, (2) the temperature and (3) safe time for storing the plasma after cold centrifugation, (4) the effect of fasting. Plasma ACE activity was 20.6+/-7.7 U/mL, 20.9+/-8 U/mL, and 20.5+/-7.9 U/mL (n = 25) when samples were centrifuged immediately, after 1 hour of blood sampling, and after 3 hours of blood sampling, respectively (not significant). In plasma kept at -20 degrees C, ACE activity was not different after 1 week (17.4+/-4.3 U/mL) nor after 1 month (17.9+/-4 U/mL), whereas baseline ACE was 16.7+/-4.3 U/mL (n = 10). In plasma stored at -80 degrees C, ACE activity was 15.5+/-5.7 U/mL after 1 month (baseline 15+/-5.3 U/mL; not significant; n = 12). No evidence for hydrolysis of the reaction product of ACE (his-leu dipeptide) was observed in plasma samples kept for 1 month at -20 degrees C or at -80 degrees C (by high-performance liquid chromatography analysis). In plasma obtained before breakfast, ACE activity was 12.8+/-7.1 U/mL, and it was 12.3+/-7.5 U/mL 2 hours afterwards (not significant; n = 12). Thus, to determine human plasma ACE activity by fluorimetry with reliability, with Z-phenylalanine-histidyl-leucine used as a substrate, there is a safe interval of at least 3 hours before blood centrifugation at -4 degrees C. Plasma may be kept at -20 degrees C or at -80 degrees C for at least 4 weeks before final processing. Fasting does not influence its enzymatic activity.     

Serum cystatin C in renal transplant patients

BACKGROUND: Waiting temperature before centrifugation and anticoagulants used, markedly effect total homocysteine concentrations. The aim of this study was to investigate the effect of different anticoagulants and temperature on plasma homocysteine levels. METHODS: We studied total homocysteine concentrations in 23 healthy subjects. Blood was drawn in K(3)EDTA, sodium citrate- or sodium fluoride-containing tubes, and kept at 0 degrees C or 22 degrees C for 3 h. Total homocysteine measurements were performed with fluorescence polarization immunoassay (FPIA) method. We compared all results with baseline EDTA values (samples put on crushed ice and centrifuged immediately) recommended in literature for reference handling. RESULTS: At 22 degrees C, the tubes containing sodium citrate and sodium fluoride showed significantly higher total homocysteine concentrations than their respective baseline values (p=0.000). However, sodium fluoride tubes were not significantly different than baseline EDTA levels. Waiting 3 h at 0 degrees C did not effect sodium citrate and EDTA plasma total homocysteine concentrations when compared to baseline EDTA, but sodium fluoride-containing plasma levels were significantly decreased (p=0.000). CONCLUSIONS: According to our results, the most available and practical temperature and anticoagulant for total homocysteine determination is sodium fluoride at room temperature up to 3 h.     

Measurement of the noncomplexed free fraction of tissue inhibitor of metalloproteinases 1 in plasma by immunoassay

BACKGROUND: We previously found differences in total concentrations of tissue inhibitor of metalloproteinases 1 (TIMP-1) in plasma from donors and cancer patients. Because TIMP-1 can exist in more than one molecular form, a new immunoassay to specifically detect free TIMP-1 was developed and concentrations were determined in plasma from healthy donors and colorectal cancer (CRC) patients. METHODS: We established and validated an immunoassay for the specific measurement of free TIMP-1 that uses a polyclonal anti-TIMP-1 antibody for capture and a monoclonal anti-TIMP-1 antibody that binds only free TIMP-1 for detection of antigen. Plasma samples from healthy donors and CRC patients were assayed for free TIMP-1. Total TIMP-1 was measured by our previously published assay. RESULTS: The mean (SD) concentrations of free TIMP-1 were similar in citrate [55.5 (11.5) microg/L] and EDTA plasma [58.9 (13.3) microg/L] from 76 donors (r(2) = 0.82). In 154 donors, the ratio of free TIMP-1 [mean (SD), 64.5 (18.0) microg/L] to total TIMP-1 [83.8 (19.8) microg/L] in EDTA plasma was 0.77. Plasma concentrations of free and total TIMP-1 correlated significantly to age (free, r(2) = 0.19; total, r(2) = 0.27; P <0.0001), increasing 50% over an age span of 45 years. Free and total TIMP-1 were significantly increased in CRC patients (P <0.0001), whereas the ratio of free to total TIMP-1 (mean, 0.58) was significantly lower than in donors. CONCLUSIONS: Most of the TIMP-1 in donor plasma is present in its free form, and free TIMP-1 increases with age. Free and total TIMP-1 are increased in CRC patient plasma, but the ratio of free to total TIMP-1 is significantly lower in these patients than in donors.     

Factor VII in plasma of women taking oral contraceptives. Lack of cold activation under blood bank conditions

Factor VII in plasma from about 15 percent of healthy subjects undergoes activation when samples are kept in plastic tubes at 4 degrees C. In women taking oral contraceptives, this phenomenon is observed much more frequently. If this phenomenon occurred under blood bank conditions as well, the transfusion of such plasma from donors taking oral contraceptives to patients afflicted by trauma could enhance thromboembolism. Plasma packs of 72 female donors taking oral contraceptives were separated and stored at 4 degrees C for 24 hours in the blood bank. No significant change in factor VII:C level was observed: The initial level was 110.2 +/− 6.2 U per dl (mean +/− SEM), and the 24-hour level, 97 +/− 3.3 U per dl. Among the 72 donors, 10 were identified as cold activators; their factor VII:C level increased from 85.6 +/− 2.5 U per dl (mean +/− SEM) to 222.0 +/− 7.5 U per dl when their plasma samples were kept in plastic tubes for 24 hours at 4 degrees C. In contrast, the factor VII:C level in the plasma packs kept simultaneously in the blood bank at 4 degrees C was only 101.1 +/− 9.0 U per dl at 24 hours. Thus, it appears that plasma from donors taking oral contraceptives can be used safely even when they are not frozen immediately.     

The effect of long-term storage on measured plasma lactate concentrations and prospective lactate results from a multicenter trial of antiretroviral therapy.

Plasma lactate measurements are typically performed in real time, limiting their usefulness in multicenter or longitudinal studies. To determine the stability of lactate specimens, blood was drawn in sodium fluoride/potassium oxalate tubes from 13 volunteers before and after 5 min of handgrip exercise to intentionally increase lactate concentrations. Plasma was stored at -70 degrees C. Aliquots were assayed in real time and after 1, 3, 6, 9, 12, 18, and 24 months. Real-time lactate concentrations measured at baseline ranged from 0.52 to 2.23 mmol/L before and from 2.91 to 11.04 mmol/L after handgrip exercise. Using a linear mixed model, the estimated change from baseline at month 24 was 1.67% (95% confidence interval, -0.70% to 4.03%) for pre-exercise samples and 0.39% (95% CI, -1.13% to 1.91%) for post-exercise samples. Stored serial specimens from 232 HIV-infected subjects in a multicenter trial of antiretroviral therapy were also assayed centrally. Among those, median plasma lactate increased from baseline to 64 weeks by 0.4 mmol/L with zidovudine+lamivudine treatment and by 0.6 mmol/L with didanosine+stavudine (each p<0.001 from baseline; p=0.04 for difference between groups over time). When performed as in this study, frozen storage with central batch lactate analysis is appropriate for prospectively collected samples in multicenter trials.     

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.     

Simultaneous quantification of ghrelin and desacyl-ghrelin by liquid chromatography-tandem mass spectrometry in plasma, serum, and cell supernatants

BACKGROUND: A sensitive method specific for ghrelins is needed for investigations of this gastrointestinal peptide. Our aim was to develop and validate a quantitative mass spectrometry (MS) method to measure ghrelin and desacyl-ghrelin simultaneously. METHODS: After deproteinization by precipitation, we performed reversed-phase separation with a rapid 2-column online extraction design coupled to a quadrupole mass spectrometer for electrospray ionization MS detection. Chromatography was performed on a C(18) monolithic column, with ammonium acetate buffer/methanol as the mobile phase and a chromatographic run time of 6 min/sample. The 4-fold-charged ions were used for multiple reaction monitoring experiments. RESULTS: The method was linear with injections of 0.01-10 ng. Limits of detection and quantification were 0.02 and 0.07 microg/L for ghrelin, respectively, and 0.03 and 0.35 microg/L for desacyl-ghrelin. Intra- and interday imprecision (CVs) were 9%-4% and 12%-6% at concentrations of 0.33-5.93 microg/L for ghrelin, respectively, and 16%-6% and 15%-8% at concentrations of 1.12-10.02 microg/L for desacyl-ghrelin. The mean (SD) recoveries in plasma of added ghrelin and desacyl-ghrelin were 95.8% (12%) and 101% (1.2%), respectively. Using kinetic modeling, we determined the mean (SD) periods of half-change (t(1/2)) of ghrelin to be 156 (16) min in EDTA plasma and 49 (1) min in Li-heparin plasma. Bland-Altman analysis showed that the median differences between EIA and liquid chromatography-tandem mass spectrometry (MS/MS) for desacyl-ghrelin were -40% for plasma/serum samples and 85% for cell supernatants and for ghrelin were 6% for enriched plasma samples and 44% for cell supernatants. CONCLUSION: Our HPLC-MS/MS procedure has excellent selectivity and sufficient limit of quantification to allow the monitoring of concentration-time profiles in biological matrices.     

pK1' and bicarbonate concentration in plasma

Values for pK1' were determined from pH measured at 37 degrees C with three blood-gas analyzers and from calculated pco2 values in 443 freshly separated plasmas, tonometered at 37 degrees C. Plasma was taken from healthy volunteers, seriously ill patients, and hyponatremic patients. pK1' values varied by considerably more than 0.06 in healthy volunteers as well as in very ill patients, and bicarbonate concentrations ([HCO3]p) calculated by blood-gas analyzers based on the pK1' value of 6.1 could be in error by some +/- 60%. pK1' was similarly determined for tonometered (37 degrees C) replicate dilutions of plasma samples. By adding weighed amounts of dry NaCl and NaHCO3 to the diluted samples we increased the Na+ concentration to approximately 150 mmol/L and bicarbonate concentrations to values ranging from approximately 2.5 to approximately 52.5 mmol/L. pK1' values decreased when [HCO3]p was increased in dilutions of plasma kept at constant ionic strength. At any given [HCO3]p, pK1' values were higher at high than at low values of pco2.     

Measurement of acetate in human blood by gas chromatography: effects of sample preparation, feeding, and various diseases.

We measured acetate concentrations in whole blood, serum, and plasma by a modification of a previously described method involving vacuum distillation and gas chromatography. The mean acetate concentration of fresh venous plasma from 27 normal subjects was 51 +/- 5 mumol/L (95% confidence limits ranged from 0 to 103 mumol/L). The acetate concentrations of serum and plasma incubated for 2 h at either 4 degrees C or 27 degrees C were the same. The acetate concentration of whole blood incubated at 27 degrees C was significantly greater than that of blood incubated at 4 degrees C. This change may have resulted from the production of acetate by erythrocytes or from the hydrolysis of acetate esters. Storage of plasma at -20 degrees C for 24 h significantly increased acetate concentrations from 26 +/- 6 mumol/L to 63 +/- 4 mumol/L. After the subjects consumed a standard breakfast, venous plasma acetate concentrations increased from 58 to 97 mumol/L at 30 min. Acetate concentrations in arterial plasma exceeded those in venous plasma. Plasma acetate concentrations were not significantly altered in patients with malignancy or diabetes mellitus, but severe liver disease and severe acidosis were both associated with increased acetate concentrations. These preliminary observations suggest that plasma acetate concentrations may be altered in several disease states.     

Centrifugal analysis for plasma kallikrein activity, with use of the chromogenic substrate S-2302

The amidolytic activity of activated kallikrein in plasma can be measured by use of the chromogenic substrate, S-2302 (H-D-Pro-Phe-Arg-pNA). Plasma prekallikrein was activated to kallikrein by exposure to 50 mg/L dextran sulfate in acetone/water (35/65 by vol) at 0 degrees C for 15 min. The acetone slows anti-kallikrein activity and increases the kallikrein activity by 30%. The 37 degrees C reaction mixture contained 0.54 mmol of S-2302 substrate per liter of Tris buffer (pH 7.5 at 37 degrees C). We monitored the change in absorbance at 405 nm for 60 s. The specificity of the substrate for kallikrein was demonstrated by using plasma deficient in prekallikrein (Fletcher trait) diluted with pooled normal human plasma. We recommend collecting blood specimens with sodium citrate as the anticoagulant and with use of a double-syringe technique and all-plastic containers. Plasma kallikrein activity with Chromozym-PK (Bz-Pro-Phe-Arg-pNA) as substrate (y-axis) compared with S-2302 as substrate (x-axis) gave the relation: y = 0.28x + 0.82 (r = 0.94). Day-to-day analytical variation was 2.4% for a pooled plasma with a mean value of 85.9 mukat/L. The mean (and 2 SD) for 50 healthy adults was 86.4 (32.4) mukat/L.     

Effect of temperature on protein binding of cortisol

OBJECTIVES: Biological effects of cortisol are substantially determined by protein binding of the hormone. The aim of our study was to characterize temperature effects on cortisol protein binding by use of an equilibrium dialysis method. DESIGN AND METHODS: Serum samples obtained from ten healthy volunteers were submitted to equilibrium dialysis. Each sample from the individuals was incubated for 16 h at 37 degrees C, 38 degrees C, 39 degrees C, 40 degrees C and 41 degrees C, respectively. In the dialysate samples obtained, cortisol concentrations were measured by immunoassay. RESULTS: For samples incubated at 37 degrees C, a mean dialysate cortisol concentration of 0.41 microg/dL (SD=0.14) was found. Gradual increase of dialysate cortisol concentration was observed with increasing incubation temperatures. For samples incubated at 41 degrees C, a mean dialysate cortisol of 0.75 microg/dL (SD=0.24) was found. Thus, the mean percentage of free-to-total cortisol increased by about 80% from 3.7% (SD=1.1) at 37 degrees C to 6.7% (SD=1.8) at 41 degrees C. CONCLUSIONS: The results of our in vitro experiments suggest that during fever the free-to-total ratio of cortisol is increased substantially compared to normal conditions, and that administration of antipyretic drugs might potentially be associated with substantial changes in the bioavailability of cortisol.     

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.     

Preanalytical handling of samples for measurement of plasma lactate in HIV patients

Lactic acidosis is a feared side effect of nucleoside analog treatment, one of the cornerstones in the management of HIV infection. Precise and reliable lactate measurements are prerequisites for the diagnosis of hyperlactatemia. The effects of venous stasis, time to measurement and storage temperature on p-lactate levels, p-glucose levels, anion gap and pH were investigated. Ten HIV patients (n=8 on highly active antiretroviral therapy) and 4 healthy control subjects were studied. Blood was drawn without stasis at time 0 and with stasis for 2 and 8 min into heparin-preserved test tubes. The tubes were placed at a room temperature (25 degrees C) and on crushed ice and consecutively monitored for up to 360 min. The mean increases in p-lactate in blood kept in test tubes at 25 degrees C, measured from 0 to 60 min and from 240 to 360 min, were increased in HIV patients compared with controls (0.78 mmol/Lh +/- 0.02 vs. 0.63 mmol/Lh +/- 0.05, (p=0.009) and 0.65 mmol/Lh +/- 0.03 vs. 0.53 mmol/Lh +/- 0.05, (p=0.042)). It was found that placing the tubes on crushed ice rather than keeping them at 25 degrees C controlled glycolysis and lactate production measured over a 6-h period (0.033 mmol/Lh +/- 0.006 vs. 0.32 mmol/Lh +/- 0.01, (p<0.0001) and 0.064 mmol/Lh +/- 0.008 vs. 0.64 mmol/Lh +/- 0.02, (p<0.0001)). The total increases in lactate levels in the test tubes placed on crushed ice for 4 h and in those kept at 25 degrees C for 15 min were comparable (0.28 +/- 0.03 mmol/L vs. 0.20 +/- 0.03). Compared with storage at 25 degrees C, keeping the test tubes on crushed ice also preserved pH and anion gap over a 6-h measurement period (pH: 0.026 +/- 0.004 vs. 0.12 +/- 0.01 and anion gap: -0.8 +/- 0.4 mmol/L vs. 4.1 +/- 0.4). Two minutes of venous stasis had no influence on p-lactate levels (0.02 +/- 0.04 mmol/L, p=0.70), whereas 8 min of stasis increased p-lactate levels by 0.11 +/- 0.04 mmol/L, p=0.009. It is concluded that major errors in measurements of p-lactate, anion gap and pH can be prevented by placing test tubes on crushed ice for up to 4 h until measurement.     

Quantitative analysis of circulating plasma DNA as a tumor marker in thoracic malignancies

BACKGROUND: Increased plasma DNA has been found in cancer patients and may have potential as a tumor marker. The objectives of this study were to develop a controlled, quantitative PCR (QPCR) assay to measure plasma DNA and then evaluate plasma DNA concentrations as a tumor marker in patients with thoracic malignancies. METHODS: We developed a QPCR assay for DNA, using the human beta-actin gene. Plasma samples were analyzed from 58 patients with esophageal cancer (EC; 20 banked samples and 38 prospectively collected samples) and 25 patients with lung cancer (LC; all prospectively collected). Control groups consisting of 51 patients with gastroesophageal reflux disease (GERD; 23 banked samples and 28 prospectively collected) and 11 healthy volunteers were also analyzed. RESULTS: The assay had an experimental variability <4%. In our banked samples, the mean concentration of plasma DNA in EC was 819.0 microg/L (range, 46.2-4738.0 microg/L) vs 432.0 microg/L (6.0-2888.0 microg/L) in GERD (P = 0.02). However, the prospectively collected samples had lower DNA concentrations, and there was no difference between cancer patients and controls. The mean DNA concentration was 10.6 microg/L (range, 7.0-14.0 microg/L) in healthy volunteers and 10.5 microg/L (range, 4.0-23.5 microg/L) in GERD controls vs 13.0 microg/L (range, 4.5-46.5 microg/L) in EC and 14.6 microg/L (range, 3.0-30.0 microg/L) in LC. CONCLUSIONS: Our data indicate that plasma DNA concentrations are of limited diagnostic value when samples are prospectively collected and uniformly handled. This is in contrast to previously published results. Qualitative analysis of DNA may be needed if plasma nucleic acids are to be used as a diagnostic tool in cancer screening.     

Measurement of midregional proadrenomedullin in plasma with an immunoluminometric assay

BACKGROUND: Adrenomedullin (ADM) is a potent vasodilatory peptide, and circulating concentrations have been described for several disease states, including dysfunction of the cardiovascular system and sepsis. Reliable quantification has been hampered by the short half-life, the existence of a binding protein, and physical properties. Here we report the technical evaluation of an assay for midregional pro-ADM (MR-proADM) that does not have these problems. METHODS: MR-proADM was measured in a sandwich immunoluminometric assay using 2 polyclonal antibodies to amino acids 45-92 of proADM. The reference interval was defined in EDTA plasma of 264 healthy individuals (117 male, 147 female), and increased MR-proADM concentrations were found in 95 patients with sepsis and 54 patients with cardiovascular disease. RESULTS: The assay has an analytical detection limit of 0.08 nmol/L, and the interassay CV was <20% for values >0.12 nmol/L. The assay was linear on dilution with undisturbed recovery of the analyte. EDTA-, heparin-, and citrate-plasma samples were stable (<20% loss of analyte) for at least 3 days at room temperature, 14 days at 4 degrees C, and 1 year at -20 degrees C. MR-proADM values followed a gaussian distribution in healthy individuals with a mean (SD) of 0.33 (0.07) nmol/L (range, 0.10-0.64 nmol/L), without significant difference between males or females. The correlation coefficient for MR-proADM vs age was 0.50 (P < 0.001). MR-proADM was significantly (P < 0.001) increased in patients with cardiovascular disease [median (range), 0.56 (0.08-3.9) nmol/L] and patients with sepsis [3.7 (0.72-25.4) nmol/L]. CONCLUSIONS: MR-proADM is stable in plasma of healthy individuals and patients. MR-proADM measurements may be useful for evaluating patients with sepsis, systemic inflammation, or heart failure.     

Distribution of ethanol and water between plasma and whole blood; inter- and intra-individual variations after administration of ethanol by intravenous infusion

Hospital patients (n = 17) received 0.216 mmol/min kg body weight of ethanol as an intravenous infusion over 60 min. At fixed time intervals during and after administration, two consecutive samples of whole blood (5 ml each) were taken from a cubital vein. The plasma fraction was obtained by centrifugation of one of the tubes. The concentrations of ethanol in whole blood and plasma were determined by headspace gas chromatography and the water contents of the specimens by desiccation. The mean plasma:whole blood ratio of ethanol was 1.10:1, range 1.03:1 to 1.24:1 (n = 159). The components of variance (SD) within subjects and between subjects were 0.0293 (CV 2.7%) and 0.0165 (CV 1.5%) respectively. The 95% confidence interval ranged from 1.03:1 to 1.16:1 for a single new determination in a subject from the same population. The average water content in plasma and whole blood were 91.8% w/w (SD 0.49) and 80.1% w/w (SD 1.03) respectively. Neither the mean plasma:blood ratio of ethanol nor the water content of the specimens depended on sampling time after ethanol administration.