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.     

Better stability of total homocysteine measurement with sodium fluoride than with EDTA

The total homocysteine (tHcy) plasma concentration increases 10% per hour when whole blood is collected on ethylene diamine tetraacetate (EDTA) and stored at room temperature. The aim of this study was to investigate the stability of tHcy plasma concentration during 24 hours of storage at room temperature in two different collection tubes: EDTA and sodium fluoride (NaF). The evolution of tHcy plasma concentration was also compared in two different populations: healthy individuals (controls) and patients with end-stage renal failure, known to have increased plasma tHcy concentrations. Plasma was separated from erythrocytes at 0, 2, 6 and 24 hours. tHcy was measured with a competitive immunoassay on Immulite 2000 (Diagnostic Products Corporation). Plasma tHcy concentration started to rise significantly on EDTA after two hours of storage in patients and controls in comparison to baseline (defined as time: 0 hour). It remained stable on NaF during the first two hours and started to rise significantly after six hours of storage for both populations. In conclusion, NaF tubes should be preferred to EDTA tubes for tHcy determination in routine clinical chemistry laboratories.     

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.     

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.     

Can fluoride-oxalate and sodium citrate stabilise homocysteine levels after blood collection?

The concentration of homocysteine (Hcy) rises rapidly after the collection of blood. This feature requires blood to be collected into the anticoagulants EDTA or heparin and the plasma to then be immediately separated; alternatively, the blood may be kept on ice and centrifuged within 1 hour. The use of chemical preservatives has been proposed as a means of stabilising Hcy levels in whole blood after collection. The objective of this study was to determine whether the commonly available fluoride-oxalate (Fl-Ox) and sodium citrate (Na-Cit) containers could stabilise Hcy levels in blood. Our results showed that when blood was collected into potassium EDTA (K-EDTA) tubes, Hcy levels rose from initial levels, on standing at room temperature (approximately 25 degrees C), by an average of 21% after 3 hours and 32% after 5 hours. The initial Hcy levels of blood collected into Fl-Ox and Na-Cit containers, however, were lower, at averages of 89% and 91%, respectively, compared to that of the same samples when collected into K-EDTA tubes. Hcy in these samples subsequently rose on standing, and after 5 hours was, on the average, 10 and 13% higher, respectively, compared with the initial levels in K-EDTA tubes. We conclude that Fl-Ox and Na-Cit do not stabilise Hcy in blood after collection and should not be used as preservatives.     

不同抗凝剂对肝移植受者FK506血药浓度测定的影响及临床意义

目的探讨不同抗凝剂对肝移植受者他克莫司（FK506）血药浓度测定的影响及临床意义。方法采集肝移植受者静脉全血34份,使用乙二胺四乙酸二钾（EDTA-K2）、枸橼酸钠和肝素锂抗凝剂分别抗凝同一份血样本,在IMx型免疫分析仪上用微粒子酶免疫分析法（MEIA）测定FK506血药浓度。结果肝素锂组与EDTA组差异无统计学意义（P=0.660）,呈正相关（r=0.982 8）。枸橼酸钠组与EDTA组差异有统计学意义（P=0.000）,呈正相关（r=0.961 3）。枸橼酸钠组与肝素锂组差异有统计学意义（P=0.000）,呈正相关（r=0.939 8）。结论肝素锂组与EDTA组几乎无偏差,但是枸橼酸钠组分别与EDTA组和肝素锂组存在一定程度的负偏差。枸橼酸钠对MEIA测定FK506血药浓度有一定的影响,应优先考虑EDTA或肝素锂抗凝剂采集全血样本。     

Optimal conditions for collection of blood samples for assay of α2-macroglobulin trypsin complex-like substance (MTLS)

We have developed an enzyme-linked immunosorbent assay (ELISA) for the specific quantification of α2-macroglobulin-trypsin complex-like substance (MTLS). To exclude artifacts in measured values of MTLS, the conditions for collection of blood samples are critical. In the present study, we have determined the optimal conditions for blood collection and investigated the role of MTLS as a clinical tool for diagnosis in pancreatitis. Results obtained are as follows: (1) MTLS levels of all sera were more than 10-fold higher than the corresponding plasma; (2) MTLS levels of heparinized plasma were the lowest among plasma with three anticoagulants (sodium citrate, sodium EDTA and heparin); (3) some kinds of blood collection tubes containing heparin were not suitable for the sampling; (4) MTLS values of plasma obtained by blood collection tubes containing Trasylol® and sodium EDTA were demonstrated more stable and lower than those obtained by heparin tubes; and (5) under these conditions, we can exclude elevation of MTLS values caused by inappropriate blood sampling and find the time course of the elevation reflecting clinical course of a patient with acute pancreatitis and a patient after endoscopic retrograde cholangiopan-creatography (ERCP). The optimal conditions for collection of blood samples were as follows. Blood sampling should be performed by blood collection tubes containing Trasylol® (50 μl/ml blood) and sodium EDTA (1.5 mg/ml blood). The samples were immediately stored at 4°C and centrifuged at 3,000 rpm for 15 min. The plasma were stored in plastic tubes at 4°C until assayed. © 1996 Wiley-Liss, Inc.     

Pre-analytical conditions affecting the determination of the plasma homocysteine concentration.

In the past decade, moderately elevated homocysteine concentration has achieved wide-spread recognition as an independent risk factor for vascular diseases, such as stroke and peripheral vascular disease, as well as for an impaired nutritional status. In general, EDTA plasma is used for the determination of homocysteine. However, from the pre-analytical point of view it is important, that, when plasma is not separated from blood cells within 30 minutes, homocysteine levels increase in samples significantly by about 10% per hour. This 10% increase is very important, because the normal range is between 5 and 15 micromol/l and moderately elevated homocysteine concentrations above 15 micromol/l may signify an increased risk of vascular disease. These preliminary cut-off points show that there is only a small difference between normal and moderately elevated homocysteine concentrations. Most blood samples are obtained outside the hospital, and in these cases homocysteine concentrations will be falsely elevated, if no precautions are taken, such as immediate centrifugation and separation of plasma and cells. This aspect is critical both for clinical studies and in patient care outside the hospital. But even in the hospital it is difficult to separate plasma and cells within 30 minutes. In the past, different approaches were adopted to solve this problem. Potential stabilisers were sodium fluoride (4 g/l) and 3-deazaadenosine (100 micromol/l). Sodium fluoride initially increased the homocysteine concentration, which dropped below the initial values after 72 h. On the other hand, 3-deazaadenosine stabilised homocysteine concentrations for 24 h, but increased it within 72 h by roughly 10%. However, this stabiliser is restricted to HPLC technology but does not work reliably with immunoassays. Lysis of blood stabilised homocysteine, but homocysteine concentrations were systematically lower requiring totally new reference ranges. In addition, acidic citrate (0.5 mol/l) was evaluated, which seems to stabilise plasma homocysteine concentrations at ambient temperatures for several hours. However, small but systematic deviations at baseline are observed. This stabilisation procedure does not interfere with immunoassays. Because immunoassays will be the future method of choice for robust and easy to perform homocysteine measurements, because they easily allow the analyses of high sample numbers, homocysteine stabilisation in whole blood is still an important matter. It must be solved before homocysteine determinations are introduced as a general screening for vascular risk factors in non-specialist laboratories.     

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.     

Osteocalcin concentrations in plasma prepared with different anticoagulants

We investigated the effects on plasma osteocalcin concentrations of different anticoagulants used to collect the blood samples. Plasma osteocalcin concentrations measured by enzyme immunoassay and radioimmunoassay are influenced by the nature of the anticoagulants used. The most significant difference between concentrations found in plasma and serum was seen with oxalate/fluoride anticoagulant, which reduced osteocalcin concentrations to 37.3% of serum values. This is probably related to increased hemolysis with this anticoagulant compared with osteocalcin concentrations in plasma prepared with other anticoagulants. Samples prepared with sodium citrate (0.105 mol/L) or lithium heparin gave values 92.4% and 83.6% of those obtained with matched serum samples. Osteocalcin concentrations were relatively stable in plasma and serum at -20 degrees C for two freeze/thaw cycles. In blood from 100 patients there was a good correlation between osteocalcin concentrations in serum and plasma (lithium heparin) (r2 = 0.831); the slope and intercept (+/- SE) were 0.924 +/- 0.04 and 4.92 +/- 1.25 micrograms/L, respectively. However, in 10 patients, serum osteocalcin concentrations were two- to threefold higher than those in matched plasma samples.     

Stability of total plasma homocysteine in perinatology

BACKGROUND: Typical assay methods for total homocysteine in human plasma involve EDTA-containing whole blood. Unfortunately, rapid increases of the plasma homocysteine concentrations due to cellular export are observed when the EDTA-containing samples are not stored on ice and processed shortly after collection. This is a cumbersome procedure in perinatal settings, whereby delivery usually takes place at unpredictable times. METHODS: The stability of homocysteine was assessed from six placental and neonatal blood samples collected in citrate buffer. Samples were stored at 4 degrees C and tested at regular intervals for the first 24 h. RESULTS: There was no statistical difference in homocysteine concentrations as observed over the study storage period (mean coefficient of variation [CV] 4.9%). CONCLUSIONS: Citrated samples can be left in a refrigerator for at least 24 h with no effect on the plasma homocysteine concentrations.     

FACTORS INFLUENCING THE PROTEIN BINDING OF BUMETANIDE USING AN EQUILIBRIUM DIALYSIS TECHNIQUE

Factors that influence the plasma protein binding of bumetanide were evaluated using equilibrium dialysis. It took approximately 12 h of incubation to reach an equilibrium between plasma and isotonic phosphate buffer of pH 7.4 containing 3% dextran using a Spectrapor 2 membrane (mol. wt cut-off = 12,000-14,000) in a water-bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. Bumetanide was fairly stable in both 4% human serum albumin (HSA) and in the isotonic phosphate buffer of pH 7.4 for up to 24 h. The binding of bumetanide to 4% HSA was constant (87.5 +/- 1.73%) at bumetanide concentrations ranging from 0.1 to 100 micrograms/ml. The extents of binding were 72.0, 83.3, 88.5, 90.2, 91.3 and 91.4% at albumin concentrations of 0.5, 1, 2, 3, 4 and 5 g/100 ml, respectively, and increased with a decrease in incubation temperature; the values bound were 94.6, 90.3 and 89.3% when incubated at 4, 22 and 37 degrees C, respectively. The binding of bumetanide was independent of the buffer composition used, the quantities of AAG (up to 0.32%), heparin (up to 40 units/ml), sodium azide (up to 0.5%) and anticoagulants (EDTA, heparin and citrate). The free fraction of bumetanide in rabbit plasma (2.91%) was significantly higher than in humans (1.98%) or rats (1.85%).     

Alpha 1-antitrypsin: the effect of anticoagulants on the trypsin inhibitory capacity, concentration and phenotype

The effects of anticoagulants on the determination of both trypsin inhibitory capacity and the concentration of alpha 1-antitrypsin measured by radial immunodiffusion, and on the alpha 1-antitrypsin phenotype were investigated. These results were compared with those obtained for serum. The following anticoagulants were investigated: sodium citrate; sodium oxalate; buffered citrate; potassium oxalate/sodium fluoride; sodium heparin; and potassium EDTA. It was found that plasmas from all of the anticoagulants, except sodium heparin, resulted in apparently significant decreases of both trypsin inhibitory capacity and concentration of alpha 1-antitrypsin measured by radial immunodiffusion, relative to serum. These decreases were not simply due to dilution by anticoagulants. Using both acid starch gel electrophoresis followed by immunofixation and isoelectric focusing in agarose, no interference was found in the phenotype determination. It is concluded that serum should be used to measure the trypsin inhibitory capacity or the concentration of alpha 1-antitrypsin by radial immunodiffusion, although plasma is also suitable provided that sodium heparin is used as the anticoagulant. The alpha 1-antitrypsin phenotype can be determined with either serum or any of the plasma. None of the anticoagulants employed in this study was present in excess.     

Influence of anticoagulants on the measurement of S100B protein in blood

OBJECTIVE: Evaluate anticoagulants influence on plasma S100B levels. DESIGN AND METHODS: Blood were collected from 18 healthy adult subjects using: no anticoagulants, EDTA, heparin, and citrate. S100B levels were determined using LIA-mat assay. RESULTS: Heparin plasma and citrate increased plasma S100B levels (p < 0.001), whereas EDTA had no effect (p = 0.24). Heparin plasma samples were highly (r2 = 0.97, p < 0.001), citrate samples were moderately (r2 = 0.49, p < 0.001), and EDTA samples were not (r2 = 0.22, p = 0.059) correlated with serum samples. CONCLUSIONS: When anticoagulant is required, heparin plasma should be the primary choice for measurement of S100 B levels.     

Limited preanalytical requirements for insulin measurement

OBJECTIVES: To evaluate the stability of insulin in serum and plasma under conditions relevant for the transport of samples from outpatient clinics to a centralized laboratory. DESIGN AND METHODS: Venous blood samples were taken from 15 volunteers and processed either immediately or after storage at room temperature for 24 h. The effect of EDTA or sodium fluoride on insulin results was investigated. RESULTS: Insulin was found to be stable in EDTA-containing tubes at room temperature for at least 24 h, whereas significantly lower concentrations were found if samples without additives or potassium fluoride as an additive were used. CONCLUSIONS: If shipment of patients' samples for insulin measurement from an outpatient clinic to a centralized laboratory can be performed within 24 h, centrifugation and freezing of plasma is not required. These findings facilitate a widespread application of insulin measurements to characterize and follow-up individual insulin sensitivity.     

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.     

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.     

Factors which influence blood platelet migration.

Migration of human blood platelets in vitro was investigated by a modification of the capillary-tube migration chamber technique used to study the migration inhibition factor of macrophages. Platelets were packed in capillary tubes and incubated in autologous platelet-free plasma (PFP). The extent of migration was quantified by planimetry (measurement of the area of platelet migration visible by stereomicroscopy). Among the various anticoagulants employed, sodium citrate was most suitable for studying platelet migration. Optimal migration occured at 22 degrees to 37 degrees C and pH 7.2 to 7.4. Migration was inhibited by metabolic inhibitors such as iodoacetic acid, sodium fluoride, and 2,4-dinitrophenol, and inhibition was proportional to the dose of the agent added to the incubation medium. Mobility was also inhibited by cytochalasin B, which disrupts cellular microfilaments, at 1 microgram/ml PFP, but not by colchicine, a microtubule inhibitor, even at 40 microgram/ml of PFP. Light and electron microscopy showed that this inhibition was not ascribable to platelet clumping. These observations suggest that platelet mobility is an active process. The possible significance of platelet migration in hemostasis is discussed.     

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.     

Anticoagulants affect matrix metalloproteinase 9 levels in blood samples of stroke patients and healthy controls

ObjectivesMatrix metalloproteinase-9 (MMP-9) represents a promising marker for acute stroke management. In clinical studies MMP-9 has been quantified by ELISA using differing protocols. We aimed to establish a valid protocol by evaluation of preanalytics.Design and methodsBlood from stroke patients (n = 28) and healthy controls (n = 28) was drawn into tubes containing different anticoagulants (EDTA, citrate, lithium-heparin (heparin) and heparin with proteinase inhibitors) and processed after 0, 60 and 240 min. MMP-9 plasma protein and mRNA from mononuclear leukocytes were determined.ResultsIn regard to anticoagulants used, samples showed different MMP-9 protein baseline values and kinetics. Stable MMP-9 protein concentrations were only measured from EDTA samples. Particularly in samples with proteinase inhibitors protein and mRNA concentrations increased over time. Kinetics did not differ between patients and controls.ConclusionsPreanalytics plays a key role for determination of MMP-9. EDTA seems to be a valid anticoagulant for MMP-9 protein measurement.