Storage Of Whole Blood Samples Research Articles

Influence of human peripheral blood samples preprocessing on the quality of Hi-C libraries.

The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

Biochemical and blood gas alterations in buffalo (Bubalus bubalis) whole blood stored in CPDA-1 and CPD/SAG-M bags.

The objective of this study was to evaluate the biochemical and blood gas alterations of whole blood of buffaloes that was stored in citrate-phosphate-dextrose with adenine (CPDA-1) and CPD/SAG-M blood bags for 42 days. Prospective study. Ten male buffaloes were used in this study. A total volume of 900mL of blood was collected from each buffalo so that 450mL was stored in CPDA-1 and 450mL was stored in CPD/SAG-M bags at 2-6°C for 42 days. The stored blood was evaluated at 7 time points (D): D0 (immediately after blood collection) and 7 (D7), 14 (D14), 21 (D21), 28 (D28), 35 (D35), and 42 (D42) days after collection. Blood gas, biochemical, and microbiological parameters were monitored. The overall blood pH decreased from 6.997 ± 0.05 at D0 to 6.784 ± 0.09 at D42, differing from baseline from D14 onward (P<0.05). There were increases in partial pressure of oxygen (pO2 ), partial pressure of carbon dioxide (pCO2 ), lactate, and potassium (K) and decreases in the concentrations of sodium, bicarbonate, glucose, and pH (P<0.05) during storage in both bags but no alterations in total protein concentration. Most of the variables were consistently similar between the 2 types of blood bags (P>0.05) evaluated, with the exception of pCO2 , HCO3, cholesterol, and total protein, which had higher values in the CPDA-1 bag (P<0.05). The K, pO2 , and lactate had the highest alterations during storage, with increases from baseline to D42 of 563%, 317%, and 169%, respectively. In general, no significant changes of clinical importance were observed after storage of whole blood samples from buffaloes for 42 days in the 2 types of blood bags that are indicated for use with this species.

Shelf life of whole blood samples in a biobank and the yield of deoxyribonucleic acid during genetic testing

Aim. To study the effect of the shelf life of frozen whole blood samples in a biobank on the amount of released deoxyribonucleic acid (DNA).Material and methods. The study included whole blood samples placed in tubes with the anticoagulant EDTA (ethylenediaminetetraacetic acid at a concentration of 1,8 mg/ml) from participants in the epidemiological study ESSE-RF-1 and ESSE-RF-2 and cohort studies conducted at the National Medical Research Center for Therapy and Preventive Medicine. The samples were stored in the biobank of the National Medical Research Center for Therapy and Preventive Medicine at temperature from -22О C to -32О C. The shelf life from blood collection to DNA extraction ranged from several weeks to 11 years. DNA was extracted using QIAamp DNA Blood Mini Kit (250) and 96 Blood Kit (Qiagen, Germany). Statistical analysis was performed using the R 3.6.1 software. To analyze the association of blood storage time with the logarithm of DNA concentration, a linear regression was used.Results. The analysis included data on the DNA concentration of 5405 samples. Multivariate regression showed that the blood shelf life was significantly associated with a decrease in concentration by 3,92% (3,16-4,68) for each year of storage (p &lt;0,0001). For 509 samples, the DNA concentration was measured twice, immediately after isolation and after 4,5 years of DNA storage at -32О C. During storage, the concentration of DNA increased by an average of 2% (p=0,046).Conclusion. Long-term storage of whole blood samples at temperature from -22О C to -32О C is associated with a decrease in the DNA yield. Long-term storage of the isolated DNA at a temperature of -32О C is not associated with a decrease in its concentration.

Impact of Delayed Whole Blood Processing Time on Plasma Levels of miR- 1 and miR-423-5p up to 24 Hours

Circulating cell-free miRNAs hold great promise as a new class of biomarkers due to their high stability in body fluids and association with disease stages. However, even using sensitive and specific methods, technical challenges are associated with miRNA analysis in body fluids. A major source of variation in plasma and serum is the potential cell-derived miRNA contamination from hemolysis. The study aimed to evaluate the effect of the delayed whole blood processing time on the concentrations of miR-1 and -423-5p. Ten blood samples were incubated for 0, 3 and 24 hours at room temperature prior to processing into plasma. For each time point, hemolysis was assessed in plasma by UV spectrophotometry at 414nm wavelength (λ414). Circulating levels of miR-1 and -423-5p were measured by RT-qPCR; miR-23a and -451 were also analyzed as controls. A significant hemolysis was observed only after 24h (λ414 0.3 ± 0.02, p < 0.001). However, only small changes in miR-1 and -423-5p levels were observed up to 24h of storage at room temperature (Ct 31.5 ± 0.5 to 31.8 ± 0.6for miR-1, p = 0.989; and 29.01 ± 0.3 to 29.04 ± 0.3, p = 0.614 for - 423-5p). No correlation was observed between hemolysis and the levels of miR-1 and -423-5p. Our data indicate that the storage of whole blood samples at room temperature for up to 24h prior to their processing into plasma does not appear to have a significant impact on miR-1 and - 423-5p concentrations.

Biobanking in Precision Medicine

Precise treatment of a disease, with a concerted analysis at the genomic and phenotypic level, is a paradigm shift from the one-size-fits-all treatment practiced for centuries. As part of the Precision Medicine Initiative, a million cohort samples will be collected for research and development of unique genetic markers in the study of the relationship of genomic markers to cancer. The million cohort samples are only as good as the conditions under which the samples are collected and stored. The purpose of this review is to discuss an economically viable biobanking solution for tissue, blood, and nucleic acid storage for such an endeavor! Tissue biopsy and whole blood are two common types of tissues collected for the Precision Medicine Initiative. Tissue samples can be stored as formalin-fixed paraffin-embedded (FFPE) blocks at ambient for decades, but fresh tissue samples although limited in sample size are better suited for downstream application. Postmortem collection of tissue is a good alternative to fresh tissue samples if the samples can be acquired in a timely manner before cold ischemia sets in. Blood is the preferred tissue sample for the Precision Medicine Initiative as it is easy to collect compared to other tissue types. Energy and space limitations are going to be crucial for storing a million samples for decades. Dry storage at ambient temperature is an alternative to the ultra-low-temperature storage of samples. Dry storage of whole blood samples as dried blood spots (DBS) or of the isolated components such as nucleic acids at ambient is ideal. In this review, we discuss the ambient temperature storage of blood samples and of nucleic acid. The million cohort biobanked blood and tissue samples will be crucial references for decades to come as new discoveries are made and new markers identified. Collection of blood samples at ambient as DBS and storage of the associated nucleic acid at ambient will be the key to the long-term success of biobanking of this large cohort.

Quality Assessment of Platelet-Rich Fibrin-Like Matrix Prepared from Whole Blood Samples after Extended Storage.

The platelet-rich fibrin–like matrix (PRFM) is usually prepared onsite and immediately used for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without quality deterioration, we developed a method for preparation of PRFMs from short-term-stored whole blood (WB) samples. In this study, to evaluate the practical expiration date of storage, we extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl2 in glass tubes and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth Factor-BB (PDGF-BB) levels were examined by scanning electron microscopy (SEM), flow cytometry, and an Enzyme-Linked ImmunoSorbent Assay (ELISA), respectively. Long-term storage had no significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the coagulation activity may diminish, we propose that PRFMs can be prepared—without evident loss of quality—from WB samples stored for up to 7 days by our previously developed method.

Platelet-rich fibrin prepared from stored whole-blood samples

BackgroundIn regenerative therapy, self-clotted platelet concentrates, such as platelet-rich fibrin (PRF), are generally prepared on-site and are immediately used for treatment. If blood samples or prepared clots can be preserved for several days, their clinical applicability will expand. Here, we prepared PRF from stored whole-blood samples and examined their characteristics.MethodsBlood samples were collected from non-smoking, healthy male donors (aged 27–67 years, N = 6), and PRF clots were prepared immediately or after storage for 1–2 days. Fibrin fiber was examined by scanning electron microscopy. Bioactivity was evaluated by means of a bioassay system involving human periosteal cells, whereas PDGF-BB concentrations were determined by an enzyme-linked immunosorbent assay.ResultsAddition of optimal amounts of a 10% CaCl2 solution restored the coagulative ability of whole-blood samples that contained an anticoagulant (acid citrate dextrose) and were stored for up to 2 days at ambient temperature. In PRF clots prepared from the stored whole-blood samples, the thickness and cross-links of fibrin fibers were almost identical to those of freshly prepared PRF clots. PDGF-BB concentrations in the PRF extract were significantly lower in stored whole-blood samples than in fresh samples; however, both extracts had similar stimulatory effects on periosteal-cell proliferation.ConclusionsQuality of PRF clots prepared from stored whole-blood samples is not reduced significantly and can be ensured for use in regenerative therapy. Therefore, the proposed method enables a more flexible treatment schedule and choice of a more suitable platelet concentrate immediately before treatment, not after blood collection.

Effects of time and temperature on 48 routine chemistry, haematology and coagulation analytes in whole blood samples.

Background Phlebotomy for the purpose of blood analysis is often performed at remote locations, and samples are usually temporarily stored before transport to a central laboratory for analysis. The circumstances during storage and shipment may not meet the necessary requirements. If analysed anyway, false results may be generated. We therefore examined the influence of precentrifugation time and temperature of the most frequently requested tests in whole blood. Methods Healthy volunteers donated blood in which 48 analytes were tested. Routine chemistry was performed in lithium heparin tubes, haematology in ethylenediaminetetraacetic acid tubes, coagulation in citrate tubes and glucose in sodium fluoride tubes. One tube was measured directly. The others were kept at different temperatures (4, 8, 20 or 30℃) and stored for 4, 6, 8 or 24 h before analysis. Additionally, some analytes were examined at 12, 16, 24 and 28℃. The mean percentage deviation was compared with different decision levels, including the total allowable error. Results When using the total allowable error as an acceptable limit, most of the investigated analytes remained stable. However, bicarbonate is unstable at almost all tested time-points and temperatures. Calcium, lactate dehydrogenase, potassium and sodium are particularly affected at low temperatures, while phosphate is mainly affected at and above room temperature after 8 h. Conclusion We established the influence of time and temperature on a broad range of analytes, which may be applied to set the limits in transportation and storage of whole blood samples.

A stable-isotope HPLC–MS/MS method to simplify storage of human whole blood samples for glutathione assay

BackgroundGlutathione is the principal non-protein tripeptide thiol present in most mammalian cells and plays an important role in the redox status of biological systems. The accurate assessment of reduced glutathione (GSH) status as a reliable index of oxidative stress is of research and clinical significance. GSH undergoes rapid oxidation after sample collection and this presents a challenge. MethodsValidation of an HPLC–MS/MS assay is reported. Storage stability using four variants of a methanolic precipitation with addition of stable isotope internal standard at collection is compared to l-serine borate/EDTA with perchloric acid precipitation (SBPE). ResultsPrecipitation with methanol and addition of stable isotope on sample collection, combined with storage in solution at −70°C showed superior storage stability to SBPE and other variants of the methanolic precipitation method up to 99 days. ConclusionsThe combination of stable isotope with methanolic precipitation at collection, with assay by HPLC–MS/MS provides superior results after storage of whole blood samples for at least 99 days.

Blood and serum samples collection and storage for further selenium measurements

The aim of the study was to determine the right storage and transport conditions for blood and serum samples intended for later selenium measurements. Blood and serum samples were stored in different temperature conditions (+4°C and -20°C). After 3, 7 and 14 days the selenium level was measured in each sample and the results were compared with the initial selenium level. Selenium was quantitatively measured using graphite furnance atomic absorption spectrometry (GF-AAS) with Zeeman correction (AAnalyst 600, PerkinElmer). Storage of whole blood samples in -20°C occurred to be inappropriate. Centrifugation of serum for the measurement was not possible even after the shortest time of incubation. However, samples stored at +4°C were measured and analyzed. After the period of 3, 7 and 14 days the selenium level in the sample was higher than initially (7%, 11% and 24% respectively). On the other hand, neither the temperature nor the time of storage of centrifuged serum samples had any influence on selenium level. In all analyzed cases the selenium level did not differ more than 10% of initial level. The experiment clearly showed how blood or serum samples should be stored before the measurement to obtain correct and repeatable results of selenium level. The experiment will be also very helpful in creation of a set of rules for laboratories in different places in Poland and Europe for proceeding with samples after collection to assure the highest quality of results.

Influence de la température de conservation sur le dosage d’HbA1c par méthode de chromatographie liquide haute performance

We have evaluated the conservation prior to HbA(1c) determination of three whole blood samples stored at -80 degrees C, -20 degrees C, 4 degrees C and 20 degrees C, for a maximal duration of one year. HbA(1c) was measured by an ion-exchange high performance liquid chromatography (HPLC) method (Variant II). We have analyzed the HbA(1c) value and the quality of the chromatographic separation for each sample. Storage of whole blood samples at -80 degrees C is good for at least one year. Storage at 4 degrees C is correct for two weeks, without major sample degradation. A more important and earlier degradation occurs at -20 degrees C. The conservation at 20 degrees C (room temperature) is very short. In conclusion, the temperatures of 4 and -80 degrees C are of interest for whole blood storage before HbA(1c) measurement, respectively for short and long term conservations. The temperatures of 20 and -20 degrees C are not recommended.

Measurements of brain-derived neurotrophic factor: Methodological aspects and demographical data

Although numerous studies have dealt with changes in blood brain-derived neurotrophic factor (BDNF), methodological issues about BDNF measurements have only been incompletely resolved. We validated BDNF ELISA with respect to accuracy, reproducibility and the effect of storage and repeated freezing cycles on BDNF concentrations. Additionally, the effect of demographic characteristics in healthy subjects on BDNF was verified. Whole blood and serum was collected from 206 healthy subjects and a subgroup was genotyped for BDNF Val66Met polymorphism. The effect of age, gender, BDNF genotype and thrombocyte count on whole blood BDNF was assessed. The BDNF ELISA measurement was accurate, 91.6 ± 3.0%, and showed high reproducibility, whereas inter-assay and intra-subject variations were modest, 8.4 ± 5.2% and 17.5 ± 14.1%, respectively. Storage of whole blood samples at 4 °C significantly decreased BDNF concentration, while repeated freezing cycles and storage at −20 °C was without any effect. Storage at −20 °C of serum, but not whole blood, was associated with a significant decrease in BDNF concentration. Women had significantly higher whole blood BDNF concentrations than men (18.6 ± 1.3 ng/ml versus 16.5 ± 1.4 ng/ml), and showed a right-skewed BDNF concentration distribution. No association between whole blood BDNF concentrations and thrombocyte count, age, or BDNF genotype was found. In conclusion, the BDNF ELISA assay determines whole blood BDNF accurately and with high reproducibility. Female gender is associated with higher whole blood BDNF concentrations whereas age, thrombocyte count and BDNF Val66Met polymorphism were un-associated.

Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry

Given the role of folate in many disorders, intracellular distribution of folate vitamers is of potential clinical importance. In particular, accumulation of non-methyltetrahydrofolates due to altered partitioning of folate metabolism at the level of methylenetetrahydrofolate is of interest. We describe a positive-electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows determination of erythrocyte folate vitamer distribution by accurately measuring both 5-methyltetrahydrofolate (5-methylTHF) and non-methyl folate vitamers. Whole blood lysates are deconjugated in ascorbic acid solutions, deproteinized, purified using folate-binding protein affinity columns, concentrated by solid-phase extraction (SPE) and evaporation, and separated on a C18 column within 6 min. The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4 nmol/L (signal-to-noise >10). Intra- and inter-assay CVs for 5-methylTHF were 1.2% and 2.8%, respectively. Intra- and inter-assay CVs for non-methylTHF as a group were 1.6% and 1.5%, respectively. Recovery results were 97-107%. We measured 8-72% non-methyl folate vitamers in volunteers (n=5) with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype. Concentrations ranged from 117 to 327 nmol/L and 23 to 363 nmol/L for 5-methylTHF and non-methylTHF vitamers, respectively. We measured 0-2% non-methylTHF vitamers in MTHFR 677 CC genotype volunteers. In addition, we found that storage of whole-blood samples in ascorbic acid at low pH resulted in 53-90% loss of the non-methylTHF fraction. This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.

Standardized quantification of circulating peripheral tumor cells from lung and breast cancer

Detection and quantitation of circulating tumor cells from solid epithelial tumors could become a valuable tool for therapy monitoring if the procedure can be standardized. In the present work we assessed the influence of pre-analytical handling, storage and white blood cell isolation on analysis of a population of spiked tumor cell-line cells and intrinsically present epithelial cells in the peripheral blood of breast and lung cancer patients and the sensitivity of their detection. Sucrose density separation did not enrich epithelial cells, and even depleted them, leading to a gross underestimation of their numbers (3/13 positive, between 2.9 and 50 cells/mL) in comparison to red blood cell lysis (13/13 positive, between 77,200 and 800 cells/mL). Short-term storage of whole blood samples for up to 7 days had little influence on the number of epithelial cells recovered. The effectiveness of magnetic bead enrichment was dependent on the number of relevant cells and the volume used for enrichment. Red blood cell lysis and fluorochrome-labeled antibody staining in a no-wash procedure with subsequent laser scanning cytometry allowed the detection of circulating epithelial cells in 92% of breast and lung cancer patients. Two examples of how this method can be applied for the longitudinal analysis in individual patients are shown, with an increase in numbers preceding relapse and a decrease paralleling tumor reduction. The proposed simple and easy method allows close monitoring, which may help in real-time analysis of the response of solid tumors, especially their systemic component, to therapy and hopefully will contribute to more individually tailored therapy.

Stabilization of mRNA Expression in Whole Blood Samples

Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgene Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis. We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 degrees C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR). Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors. Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.

Addition of Sodium Fluoride to Whole Blood Does Not Stabilize Plasma Homocysteine But Produces Dilution Effects on Plasma Constituents and Hematocrit

Interest in total plasma homocysteine (Hcy) measurements has increased with the availability of evidence that even mild hyperhomocysteinemia is an independent risk factor for cardiovascular disease (1). Consequently, the variability of current Hcy results has been the subject of considerable research (2)(3)(4). Whole blood stored at room temperature after phlebotomy shows an increase in Hcy concentrations of ∼10% per hour, apparently because of Hcy synthesis and release from erythrocytes (5)(6). Previous studies have shown that the best method for avoiding falsely high Hcy results is prompt postphlebotomy centrifugation and separation of the plasma from the red blood cells (6) or storage of whole blood samples at 0 °C if prompt centrifugation is not feasible (3). The use of anticoagulants as preservatives in whole blood has also been advocated (2)(3)(4); among these, sodium fluoride (NaF) in particular has been described as useful when added to whole blood at suggested concentrations of up to 4 g/L (95 mmol/L) (4)(7). Many laboratories, therefore, now use NaF as a preservative in the blood collection tubes used for Hcy assays. We measured plasma Hcy, using a modified version of the HPLC method with fluorescence detection introduced by Refsum and co-workers (8), after the addition of pure NaF (2.5 g/L, 60 mmol/L) to heparinized whole blood. The studies were approved by the University of Washington Institutional Review Board and involved the donation of blood from men and women after informed consent. We found minimal inhibition of the time-dependent increase in Hcy concentrations. We also investigated the preservative abilities of Na2EDTA (1.5 g/L, 4.0 mmol/L), EGTA (1.5 g/L, 3.9 mmol/L), and thymol (1.0 g/L, 6.6 mmol/L), again added in pure form to heparinized whole …

Effects of processing and storage on the measurement of nitrogenous compounds in ovine blood

Four experiments were conducted to evaluate effects of processing and storage of sheep blood samples on measurements of ammonia N (AMN), urea N (URN), total protein (TP), albumin (ALB), total globulin (GLB), and the non-albumin non-globulin protein fraction (NAG). Experiment 1 compared the effects of using different anticoagulants. Blood samples ( n = 15) were collected via jugular venipuncture into one of four evacuated tubes: no anticoagulant, serum; sodium heparin, plasma; sodium fluoride and potassium oxalate, plasma; tripotassium-ethylenediaminetetraacetic acid (EDTA), plasma. Anticoagulant affected ( P < 0.0001) metabolite values, with TP, ALB, and GLB being higher in heparinized plasma, and URN, NAG, and AMN highest in serum. In addition, blood packed-cell volume was 12% and 15% higher ( P < 0.0001) in samples collected in heparin and EDTA compared with samples collected in sodium fluoride and potassium oxalate. Experiment 2 investigated the effects of repetitive freezing and thawing (0–3 thaw cycles) of plasma samples ( n = 15 blood samples collected in tubes containing sodium fluoride and potassium oxalate). Repetitive freezing and thawing decreased ( P < 0.0001) URN and NAG by 21% and 54%, respectively, the latter as a result of a 4–13% increase ( P < 0.0006) in ALB and GLB values. Experiment 3 evaluated the effects of storage length (0–26 weeks) and freezing temperatures (−20 °C vs. − 80 °C) on plasma samples ( n = 16–20 blood samples collected in tubes containing sodium fluoride and potassium oxalate). All nitrogenous metabolites were affected by length of storage ( P < 0.0001). Plasma AMN, URN, TP, and NAG were affected (0.0001 < P < 0.10) by storage temperature, and AMN, TP, GLB, and NAG measurements exhibited a temperature × day interaction ( P < 0.05). In general, TP and NAG values declined with storage length, whereas URN, GLB, and AMN values increased. Plasma AMN increased 74% by Week 1 and 160% by Week 9, and samples stored at −20 °C were 14% higher than samples stored at −80 °C ( P < 0.0001). Experiment 4 assessed the effects of storage of whole blood samples ( n = 15 blood samples collected for serum) at 4 °C for 0–7 days. Serum URN and GLB decreased ( P < 0.0001), and TP and AMN increased (0.0001 < P < 0.02) with increasing refrigeration time. Standardized analytical procedures must be followed to obtain reliable nitrogenous metabolite measurements.

Optimal collection of blood samples for the measurement of tumor necrosis factor α

We have examined how delayed separation of plasma from cells affects the recovery of recombinant human tumor necrosis factor α (rhTNFα) from whole blood. Storage of heparinized whole blood samples at room temperature for 1 hr results in a significant (p = 0.036) fall in recovery of plasma TNFα from 788 ± 119 pg/mL to 472 ± 77 pg/mL, measured by specific enzyme-linked immunosorbent assay (ELISA). Storage of whole blood samples at 4°C for 1 hr reduces but does not prevent the fall in recovery of plasma TNFα: 725 ± 82 pg/mL at time 0, 472 ± 81 pg/mL after 1 hr, p = 0.038. Recovery of bioactive TNFα (cytotoxocity for L929 cells) after 1 hr at room temperature is also significantly reduced from 576 ± 139 pg/mL to 450 ± 154 pg/mL, p = 0.036. Studies with 125I-rhTNFα confirmed the fall in plasma activity and revealed a rapid commensurate increase in 125I-rhTNFα activity in the cell fractions. We recommend that clinical samples for the measurement of cytokines should be kept at 4°C and separated rapidly (within half an hour) before storing the plasma at −70°C.

Changes in haematological values and plasma electrolytes following storage of whole blood samples collected from horses

Changes in haematological values and plasma electrolytes following storage of whole blood samples collected from horses

Determination of serum ionised calcium by ion-exchange electrode in normal subjects

Serum ionised calcium concentration [Ca 2+] was measured with a calcium selective electrode in 65 normal people. A mean value of 1.06 mmol/1 (± 0.04 S.D.) with an actual range of 0.97 to 1.13 mmol/1 was obtained. Serum samples refrigerated at 4° C for 24 h were satisfactory for analysis. Storage of whole blood samples for 6 h at room temperature before separation caused a very small error (+0.04 mmol/1). Mean results and ranges were similar in males and females and there was no significant correlation with age. No significant change in serum [Ca 2+] was found following a normal meal. Serum [Ca 2+] and total serum calcium showed a very slight correlation ( r = 0.35). The method is reproducible and sensitive.