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.     

Serum creatine kinase MM sub-band determination by isoelectric focusing: a potential method for the monitoring of myocardial infarction

Fresh myocardium homogenates analyzed by thin-layer isoelectric focusing revealed the presence of two prominent creatine kinase (CK; EC 2.7.3.2) sub-bands, MMO (pI 7.10) and MM1 (pI 6.88), in approximately equal proportion. While these forms represented together as much as 85% of the cellular MM fraction, they accounted only for viz. 2.2 and 27.7% of the total serum MM activity when measured 8 h before the CK peak in patients with myocardial infarction. Incubation of the isolated MMO and MM1 with normal human serum demonstrated that the former turned to MM1 within 5 h at 37°C; further changes affecting MM1 gave rise to other sub-bands, MM2 (pI 6.70), MM3 (pI 6.45), and MM4 (pI 6.25). In our patient population, these three forms represented more than 75% of the serum CK-MM activity at the CK peak; hence, soon after the enzyme release, the serum MM isoenzyme mainly consists of degradation products arising from the labile MMO and MMl. Among the two cellular forms, MMO was the best related to the total enzyme activities and the most efficient for differentiating the patients with left ventricular failure from the others during the entire survey period (F = 3.8, p < 0.05). Because its presence in the blood provides evidence for a very recent CK release from the tissues, serum CK-MMO determinations might be proposed for following the extension of the lesion after a myocardial infarct.     

Factors influencing analysis of prolyl endopeptidase in human blood and cerebrospinal fluid: increase in assay sensitivity

Prolyl endopeptidase (EC 3.4.21.26) (PEP) is present in nearly all investigated mammalian cells and biological fluids and might be involved in the degradation of physiologically important neuropeptides. To be able to investigate the variation of PEP in blood and cerebrospinal fluid (CSF) in human disease, the factors influencing analysis of PEP in these body fluids must be determined. The purpose of the present work was to study the influence of storage conditions, anticoagulation additives, freezing and thawing and substrate solvent on determination of PEP in blood plasma/serum and CSF. It was found that the PEP activity was about 10% higher in plasma (with EDTA and heparinate for anticoagulation) than in serum. Storage at room temperature (20°C) caused a rapid decline in enzyme activity, which was smaller but still considerable at 4°C. Storage at ?20°C and ?70°C did not decrease the PEP activity. Freezing and thawing of plasma/serum samples showed that the first freeze‐thawing cycle produced a 20% reduction in enzyme activity but little further decrease was observed during subsequent cycles of freeze‐thawing. In conclusion, PEP activity should preferably be measured within one hour after sampling using EDTA‐ or heparinate plasma. For long‐term storage, samples should be immediately frozen and stored at ?20°C or colder. The selection and amount of the organic solvent used to dissolve the fluorogenic substrate strongly influenced the sensitivity of the assay. By developing an optimal solvent system an increase in assay sensitivity of about 400% could be obtained, which for the first time allowed measurement of the PEP activity in CSF.     

Whatman 3 MM dried blood spots for identifying alpha-thalassemia-1

The present study was undertaken to assess the feasibility of whole dried blood spots to identify alpha-thalassemia-1 by PCR technique. Logistical constraints associated with sample collection and processing make the measurement of alpha-thalassemia-1 at community laboratories difficult. Dried blood spot samples provide a relatively convenient and minimally invasive means for collecting and transporting samples. We present Whatman 3 MM paper for spotting blood samples used for the first time for alpha-thalassemia-1 detection. Measurements of fresh EDTA whole blood and Whatman 3 MM dried blood spots prepared from venous blood were performed in 60 subjects. Whatman 3 MM dried blood spots results were precise and reliable. There was no actual difference in comparison to fresh EDTA whole blood samples. Whatman 3 MM dried blood spots for alpha-thalassemia-1 detection using the PCR technique showed no changes for 6 months.     

Serum growth factor stability in different eye drop packaging systems during storage

Serum eye drops (SED) have shown beneficial effects in patients suffering from dry eye syndrome and are manufactured for an increasing number of patients in Australia every year. Previous studies have examined the stability of serum growth factors during storage in either experimental vessels not used as the final packaging system or in eye drop bottles. To ensure the quality and safety of SED product manufactured in Australia, the stability of growth factors in serum packaged into two different systems during storage at different temperatures was examined. Healthy blood donors provided a whole blood donation, from which serum was prepared, diluted to 20% and dispensed into either a tube or a vial packaging system. The stability of growth factors, fibronectin and total protein in tube segments was comparable to matched vials samples during storage at −30 °C, 4 °C, 22 °C and 37 °C, with the exception of EGF and fibronectin in 20% SED stored in tube segments, which were more sensitive to storage conditions at 4 °C and 22 °C when compared to vials. Additionally, the growth factor, fibronectin and total protein concentration in both tube segments and vials was stable during storage at −30 °C for at least 9 months. This study highlights the impact of different manufacturing procedures on serum growth factor stability during storage.     

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.     

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.     

The Quick and Owren prothrombin time methods for oral anticoagulant therapy do not agree well using the International Normalized Ratio (INR) units

Abstract

Natriuretic peptides are a laboratory tool with significant implications for the diagnosis and prognosis of heart failure (HF). The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommended that assays must be examined for sample stability because there appears to be assay dependent. We aimed to evaluate the in vitro stability of B-type natriuretic peptide (BNP) under different handling conditions and using a BNP assay from Fujirebio Diagnostics (Tokyo, Japan). BNP concentrations were measured in plasma EDTA samples from 11 subjects to evaluate the in vitro stability at room temperature and at 4?°C and in 10 subjects to check the in vitro stability of samples stored at –20?°C during 1 and 3 months. Stability limit was defined according to Spanish Society of Laboratory Medicine (SEQC-ML) recommendations. At room temperature and 4?°C, BNP concentrations decreased progressively in samples collected in both groups, remaining stable within four hours from collection. BNP concentrations also were stable within four hours from collection in whole blood at room temperature. Finally, at –20?°C, BNP concentrations remained stable in both groups at 1 and 3 months, respectively. According to our results, BNP, stored at room temperature or at 4?°C, should be assayed in the first four hours after collection. Besides, BNP was shown to be stable in whole blood for at least four hours at room temperature. If the testing cannot be performed within the first four hours, the plasma should be frozen and kept at –20?°C for up to 3 months.     

Plasma ascorbic acid preparation and storage for epidemiological studies using TCA precipitation

Objectives:To introduce a procedure to validate an ascorbic acid method using trichloroacetic acid (TCA) for plasma stabilization at different storage temperatures.Methods:EDTA and heparin plasma were precipitated with TCA (1:5) containing 0.54 mol/L EDTA, or without. Samples were stored at ? 20 °C and ? 70 °C and their stability was tested at room temperature for 24 h.Results:A significant 40% loss (p < 0.001) of plasma ascorbic acid was found when EDTA samples with added EDTA were stored at ? 20 °C for 2–4 weeks compared with storage at ? 70 °C. Ascorbic acid in heparin plasma without added EDTA was most unstable and samples left at room temperature for 24 h lead to almost a total loss of ascorbic acid. Addition of EDTA to the TCA solution improved stability of samples of both plasma types at room temperature.Conclusion:The recommended procedure for ascorbic acid determination in plasma stabilized with TCA is immediate storage at ? 70 °C and inclusion of EDTA into the TCA solution.     

Studies on the change of electrophoretic mobility and the decay of catalytic activity of brain-type creatine kinase isoenzyme (CK-BB) following incubation at 37 °C

Incubation of CK-BB in serum (1:3, v/v) at 37°C for 3 h caused a change of its electrophoretic mobility and decay of its catalytic activity. Similar effects were observed following incubation in water. Incubation in saline somehow preserved the electrophoretic mobility but not the catalytic activity. No effect was noted when incubated at 4°C for 3 h. Further study on the rate of decay revealed that the decay in albumin solution (1:50, v/v) is quite similar to that in serum. More dramatic decay was noted when incubated in water and less when incubated in saline. It was further shown that the higher the incubation temperature (4°C, 25°C or 37°C) the faster the decay. The rate of decay of CK-MM was much slower in all conditions of incubation. Determination of isoenzyme activities by means of an immunoprecipitation method again demonstrated that CK-BB lost a great deal of its catalytic activity following incubation at 37°C for 1 h, and hence falsification of the isoenzyme pattern.     

The Effect of Heparin on Serum Lipoproteins

Abstract

The aim of the investigation was to find a method for the conservation of 5-hydroxytryptamine (5-HT, serotonin) in blood. The effect of ascorbic acid, carbon monoxide, hydrogen sulphide, and cyanide on blood frozen at -20°C was studied. It was demonstrated that 30 mg ascorbic acid and 10 mg EDTA per 10 ml blood could keep 5-Hydroxytryptamine unchanged in the blood for at least a month when frozen at -20°C. No additional effect could be obtained by the known stabilizing effect of CO and not by H₂S or NaCN. The method is simple and well adapted for routine work. The method of Halevy et al. was used, slightly modified, for the determination of 5-Hydroxytryptamine. Normal values, accuracy, and recoveries of the method were presented.     

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.     

Long‐term stability of serum components in the Janus Serum Bank

Objective. Biobank material is frequently used in epidemiological studies, but long‐term storage of serum at ?25°C may reduce the quality of the samples. Knowledge about the stability of components in biological samples is fundamental for the interpretation of such studies. Material and methods. We investigated the stability of seven biological components in serum samples stored at ?25°C for 25 and 2 years compared with 1‐month‐old samples. Specimens from 130 blood donors from each group were randomly selected among men without a cancer diagnosis during the follow‐up time. We compared the distribution, dispersion and localization of medians and means, and established reference intervals of the components. Results. The study demonstrated non‐significant and numerically small differences in the levels of sodium, calcium, iron and creatinine over time. Differences between mean values for uric acid (?7.6%), potassium (+26.4%) and bilirubin (?59.4%) between fresh and 25‐year‐old samples indicated that sample handling before freezing and degradation during long‐term storage may introduce a considerable bias for vulnerable components. Conclusions. There is large variation in level stability between different serum components in serum stored at ?25°C. Differences in sample handling before freezing may introduce bias on vulnerable components. The present study supports a routine of careful matching of cases and controls on storage time in epidemiological studies when biobank material is used.     

Proper zinc evaluation in clinical practice: Effect of sample type and it's stability

Zinc (Zn) is a ubiquitous element that plays a vital role in the growth and development of human body. Traditionally, Zn has been measured in plasma samples using “trace element” dedicated sampling tubes. However, the recent increase in its assessment leads to the need to use others than plasma samples and the use of sera could be a justified and valid alternative. We evaluated the differences between plasma and serum, for Zn quantification. 307 blood paired samples from patients enrolled for the treatment of obesity-related pathology at our out-patient department were assessed. The quantification of Zn was performed by Flame atomic absorption spectroscopy. Using 123 serum samples randomly selected from our own biobank and stored at −80 °C for 5 years, we further investigated the long-term stability of serum Zn. The mean result for Zn was 77.8 ± 13.2 μg·dL⁻¹ and 77.4 ± 12.8 μg·dL⁻¹, respectively for plasma and for serum, (p = 0.43). Bland-Altman analyses demonstrated excellent concordance of the assay in the two different blood matrices. The mean difference ± SD between serum and plasma matrices was 0.32 ± 3.40 μg·dL⁻¹. The assessment of serum Zn long-term stability indicated a significant change after 5 years storage. The mean value was 74.2 ± 10.9 μg·dL⁻¹ for fresh samples and 83.3 ± 10.9 μg·dL⁻¹ after 5 years of storage at −80 °C, corresponding to a mean difference of +9.1 μg·dL⁻¹(+10,9%, p < 0.05). The increase in Zn values described after long-term storage has to be considered though that should not have a significant clinical value. In conclusion, the routine measurement of Zn can be made in an accurate way using a serum sample, without the need for a specific tube for trace elements assessment.     

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.     

Dynamics of interaction between complement-fixing antibody/dsDNA immune complexes and erythrocytes. In vitro studies and potential general applications to clinical immune complex testing.

Soluble antibody/3H-double-stranded PM2 DNA (dsDNA) immune complexes were briefly opsonized with complement and then allowed to bind to human erythrocytes (via complement receptors). The cells were washed and subsequently a volume of autologous blood in a variety of media was added, and the release of the bound immune complexes from the erythrocytes was studied as a function of temperature and time. After 1-2 h, the majority of the bound immune complexes were not released into the serum during blood clotting at either 37 degrees C or room temperature, but there was a considerably greater release of the immune complexes into the plasma of blood that was anticoagulated with EDTA. Similar results were obtained using various conditions of opsonization and also using complexes that contained lower molecular weight dsDNA. Thus, the kinetics of release of these antibody/dsDNA immune complexes differed substantially from the kinetics of release of antibody/bovine serum albumin complexes that was reported by others. Studies using the solution phase C1q immune complex binding assay confirmed that in approximately half of the SLE samples that were positive for immune complexes, there was a significantly higher level of detectable immune complexes in plasma vs. serum. Freshly drawn erythrocytes from some SLE patients exhibiting this plasma/serum discrepancy had IgG antigen on their surface that was released by incubation in EDTA plasma. Thus, the higher levels of immune complexes observed in EDTA plasma vs. serum using the C1q assay may often reflect the existence of immune complexes circulating in vivo bound to erythrocytes.     

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.     

Preanalytical stability of HIV-1 and HCV RNA: impact of storage and plasma separation from cells on blood donation testing by NAT

Background: The aim of this study was to evaluate the optimal preanalytical conditions prior to nucleic acid amplification technology (NAT) for human immunodeficiency virus‐1 (HIV‐1) or Hepatitis C virus (HCV) RNA in pools of 96 plasma specimens with regard to storage temperature, time and plasma separation in a blood donation environment. Study design and methods: Changes in viral nucleic acid concentration of HIV‐1 and HCV were observed for 5 days according to the Paul‐Ehrlich‐Institute's (PEI) guidelines that demand 95%‐detection limit of at least 10 000 IU mL^?1 for HIV‐1 RNA and 5000 IU mL^?1 for HCV RNA within a single donor blood specimen. Ninety‐five per cent detection limits of HIV‐1 RNA over 3 days after storage at either 5 or 21 °C were evaluated by using standardised HIV‐1 RNA‐positive plasma. Results: HCV RNA in whole blood samples proved to be more stable than HIV‐1 RNA. Whole blood storage at 21 °C was shown to decrease the detectability of HIV‐1 RNA even after only 18 h. Plasma samples once used for NAT at time 18 h did not alter viral stability up to 48 h after donation. Ninety‐five per cent detection limits of HIV‐1 RNA were securely below 10 000 IU mL^?1 for 24 h after whole blood storage at 5 °C. Conclusions: These results may lead to a discussion around the most suitable preanalytical conditions in blood donation environments. Contrary to the current PEI guidelines that allow storage of whole blood specimens up to 18 h at 21 °C, these results suggest that immediate storage in a 5 °C container after blood donation is more suitable and would permit storage of whole blood up to 24 h prior to the separation of plasma from cells.     

Effect of local cold provocation on systolic blood pressure and skin blood flow in the finger

Demonstration of increased vascular cold reactivity in patients with Raynaud’s syndrome is difficult. For medico‐legal reasons, it is important to get objective measures of vasospasm in these patients. Evaluation of the degree of vasospasm also provides prognostic information which is useful for patient management. In this study, we compare two methods of arterial circulation measurement. The laser Doppler scanning is a new method, which uses the recently developed laser Doppler perfusion imaging (LDPI) instrument. The aim of the present study was to compare the effect on finger skin blood flow measured with LDPI with changes in finger systolic blood pressure during local cold provocation. The effect of such provocation, skin blood flow and systolic blood pressure, were studied in 15 healthy controls. Six patients with known traumatic vasospastic disease (TVD) were also tested with both methods. Finger skin blood flow was measured with LDPI on the distal phalanx of the index finger of the left hand, every minutes during 6 min of local heating at 40°C followed by local cooling for 3 min at 15°C and then for 3 min at 10°C. Finger systolic blood pressure was measured with strain‐gauge method before and after local cooling to 10°C with a cuff perfused with water of desired temperature. The test was performed in the same finger within a week of the laser Doppler scanning. Local finger cooling to 15°C and 10°C caused a significant decrease in blood flow, most marked at 10°C. There was, however, no correlation between the decrease in blood flow and blood pressure. In the TVD‐patients decreases in skin blood flow were similar compared with the healthy controls. In contrast, the changes in systolic blood pressure, were outside normal range (systolic quotient <0·65) in five of the six patients (83%), and also in 11 of the 15 healthy controls (73%). In conclusion, there is no correlation between the decrease in finger skin blood flow and systolic blood pressure during local cold provocation. For diagnosis of traumatic vasospastic disease (TVD), local cold‐induced changes in finger systolic blood pressure seems superior to changes in skin blood flow, but the ideal clinical method for demonstrating increased cold‐induced vasospasm is, however, still lacking.