Measurement of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 after delayed separation of whole blood samples

Objectives:Epidemiological studies benefit from unbiased blood specimens collected with minimal cost and effort of blood collection and storage. We evaluated the stability of IGF-1 and IGFBP-3 in whole blood samples stored at room temperature to justify delays in blood processing.Design and methods:Total IGF-1 and IGFBP-3 levels were measured in EDTA plasma (n = 12), heparin plasma (n = 12) and serum (n = 10) samples of healthy volunteers after blood processing delays up till 14 days. Stability of measured IGF-1 and IGFBP-3 levels was tested by paired t-test and a linear mixed effect model.Results:Longitudinal analysis showed that IGF-1 levels were not significantly affected by blood processing delays in EDTA tubes (p = 0.18) and IGFBP-3 levels were marginally stable (p = 0.06). In heparin plasma and serum, however, IGF-1 increased over time of delayed processing and IGFBP-3 levels tended to decrease (p < 0.01).Conclusion:Total IGF-1 and IGFBP-3 levels are stable in whole blood collected in EDTA tubes at room temperature up till 7 days, allowing a delay in blood processing to reduce costs in large multi-center studies.     

Long-term stability of soluble ST2 in frozen plasma samples

ObjectiveThe aim of this study was to investigate the long-term in vitro stability of soluble ST2 (sST2).Design and methodsEDTA plasma samples were drawn from 15 individuals with various diseases. The Presage^TM ST2 assay was used for measurement of sST2 concentrations directly after blood collection and after storing plasma samples for 18 months at ?20 °C and ?80 °C. The default criterion for analyte stability was set at 95%.ResultssST2 concentrations in the 15 individuals ranged from 12 U/mL to 140 U/mL. Directly after blood collection, the mean (± SD) sST2 concentration was 51 ± 37 U/mL, and absolute analyte recoveries were 50 ± 35 U/mL and 51 ± 34 U/mL after storage of samples for 18 months at ?20 °C and ?80 °C, respectively. Relative analyte recoveries after 18 months of storage at ?20 °C and ?80 °C were 99 ± 5% and 101 ± 7%.ConclusionsST2 is stable for at least 1.5 years in plasma samples stored at ?20 °C and ?80 °C.     

Evaluation of a novel colorimetric assay for free oxygen radicals as marker of oxidative stress

BackgroundFree oxygen radicals play an important role in the pathogenesis of many diseases including cardiovascular disease, diabetes, hypertension, cancer and aging. Several methods were developed for the direct or indirect measurement of oxygen free radical and its byproducts. The free oxygen radicals monitor (FORM) is a novel point-of-care system for the rapid measurement of free oxygen radicals (FORT) in blood. We have carefully evaluated the use of this assay for batch analysis of plasma samples in a research environment with respect to factors affecting its performance, including storage temperature and duration.MethodsWe determined the effect of storage, hemolysis, variability and reproducibility of the FORT in blood and plasma.ResultsPlasma FORT correlated significantly with hsCRP (P < 0.0001) and CHOL/HDL ratio (P < 0.02). While hsCRP results have shown agreater range of assay variability (27.82%–53.92%), FORT measurements in the same samples have less assay variability (5.63%–9.61%). Collected whole blood can be kept on ice for up to 7 h prior to plasma isolation without affecting FORT values. Storage of plasma has no effect on FORT when stored at 4 °C for up to 3 weeks (R² = 0.685). Comparable values can be obtained in samples stored for up to 3 months at ? 80 °C (R² = 0.5888) but not at ? 20 °C.ConclusionsThe day to day variability of FORT, as assessed by multiple measures in a group of controls over time, is minimal. FORT assay isstable when stored at ? 80 °C for a couple of months or at 4 °C for a few weeks. FORT correlation with hsCRP and other lipid markers provides an interesting insight and a novel link between oxidative stress, inflammation and lipid metabolism.     

The use of a cytokine panel to define the long-term risk stratification of heart failure/death in patients presenting with chest pain to the emergency department

Objective:To determine if a cytokine panel could be informative regarding subsequent heart failure(HF)/death.Design and methods:In 216 subjects presenting with chest pain to an emergency department in 1996, EDTA plasma (? 70 °C) was thawed for IL-6, MCP-1, IL-10, VEGF, EGF measurement.Results:Subjects with any three cytokines elevated were at higher risk for HF/death compared to those with ≤ two cytokines elevated.Discussion:A cytokine panel might be useful for risk stratification for HF/death.     

Long-term stability of biochemical markers in pediatric serum specimens stored at -80 °C: a CALIPER Substudy

ObjectivesPediatric serum samples collected from healthy children in the CALIPER (Canadian Laboratory Initiative in Pediatric Reference Interval) project are stored at ? 80 °C for various periods of time. This study aimed to determine the stability of chemistry, protein, and hormone analytes under these conditions.Design and methodsSerum samples collected from children of 0–18 years of age attending outpatient clinics were pooled into a single pool or into age-group specific pools. Following baseline measurement, each pool was aliquoted and kept frozen at ? 80 °C until analysis. Samples were analyzed for 57 biochemical markers at monthly intervals over a 10–13 month period and each aliquot was subject to one freeze–thaw cycle before analysis. The analysis was performed on VITROS® Chemistry System, COBAS INTEGRA® 400 Plus and IMMULITE® 2500. Values obtained at monthly intervals were compared to baseline measurements and examined for trends over time.ResultsA majority of analytes measured in this study showed no significant time-dependent change relative to baseline or trend over time after up to 13 months of storage. PTH showed up to 27.2% decline after 10 months of storage with most of the decline evident after 2 months. Most analytes showed variability over time, which is thought to reflect assay variability rather than changes in analyte stability.ConclusionsThe study shows stability for a majority of analytes stored in serum at ? 80 °C after up to 13 months of storage. Samples do not require immediate testing for reference interval determination for the selected analytes with possible exception of PTH.     

Influence of pre-analytical factors on α-galactosidase A, arylsulfatase B and α-glucosidase activities measured on dried blood spots on filter paper

ObjectivesTo analyze the effect of blood collection and storage conditions on activity of α-galactosidase A, arylsulfatase B and α-glucosidase.Design and methodsBlood was collected in EDTA, heparin, or direct spotting on filter paper and stored at different temperatures (? 20, 4, 25 and 37 °C) and storage times (3, 10, 17 and 180 days). The influence of filter paper size was also assessed (3.0 and 1.2 mm).ResultsNo statistically significant difference was observed between the three collection methods. α-Glucosidase A activity significantly decreased after the 10th day, while arylsulfatase B activity only differed significantly after the 180th day, and α-galactosidase A activity remained constant throughout this storage time. Excellent correlation coefficients were observed for the two filter paper sizes used.ConclusionsBoth paper sizes may be employed. Filter paper specimens should be transported under refrigeration as soon as possible after blood collection.     

Clotting factor activity in thawed Octaplas® LG during storage at 2–6 °C for 6 days from a quality assurance point of view

IntroductionOctaplas® LG is a second-generation solvent/detergent-treated plasma that offers an additional safety benefit by prion elimination. The stability of clotting factors of the new S/D plasma after thawing has not been investigated yet. This study intended to measure the time course of fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI through storage at 2–6 °C over 6 days.Materials and methodsWe investigated 20 plasma bags (five bags per blood group) and measured fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI immediately after thawing and after 2, 4, 6, 24, 48, 72, 96, 120 and 144 h storage at 2–6 °C. Five separate plasma bags were thawed and stored at 2–6 °C for microbiological assessment. After 6 days samples were drawn for blood cultures that were incubated for six more days.ResultsAfter 6 days FII, FIX and PC showed no significant changes. FV (?16%, p < 0.001), FVII (?19%, p < 0.001), FVIII (?19%, p < 0.001), FXI (?13%, p < 0.0001) and fPS (?4%, p < 0.0007) decreased significantly. PI levels were stable at 56%. The microbiological investigation showed no bacterial contamination.ConclusionsIn Octaplas® LG plasma clotting factors decreased slightly through storage of 6 days. PI levels were remarkably higher and stable over time in the new Octaplas® LG. Stability of stored Octaplas® LG was limited by the decrease of FVIII to 53%, which may warrant storage up to 24 h from a quality assurance point of view. This could result in reduced plasma wastage and costs for healthcare givers.     

Pleural fluid: Are temperature and storage time critical preanalytical error factors in biochemical analyses?

BackgroundBiochemical analysis of fluid is the primary laboratory approach in pleural effusion diagnosis. Standardization of the steps between collection and laboratorial analyses are fundamental to maintain the quality of the results. We evaluated the influence of temperature and storage time on sample stability.MethodsPleural fluid from 30 patients was submitted to analyses of proteins, albumin, lactic dehydrogenase (LDH), cholesterol, triglycerides, and glucose. Aliquots were stored at 21°, 4°, and?20 °C, and concentrations were determined after 1, 2, 3, 4, 7, and 14 days. LDH isoenzymes were quantified in 7 random samples.ResultsDue to the instability of isoenzymes 4 and 5, a decrease in LDH was observed in the first 24 h in samples maintained at ? 20 °C and after 2 days when maintained at 4 °C. Aside from glucose, all parameters were stable for up to at least day 4 when stored at room temperature or 4 °C.ConclusionsTemperature and storage time are potential preanalytical errors in pleural fluid analyses, mainly if we consider the instability of glucose and LDH. The ideal procedure is to execute all the tests immediately after collection. However, most of the tests can be done in refrigerated samples, excepting LDH analysis.     

Evaluation of the ARCHITECT urine NGAL assay: Assay performance,specimen handling requirements and biological variability

Objectives:NGAL (Neutrophil Gelatinase-Associated Lipocalin) has emerged as a new biomarker for the identification of acute kidney injury. Reliable clinical evaluations require a simple, robust test method for NGAL, and knowledge of specimen handling and specimen stability characteristics. We evaluated the performance of a new urine NGAL assay on the ARCHITECT analyzer.Methods:Assay performance characteristics were evaluated using standard protocols. Urine specimen storage requirements were determined and biological variability was assessed in a self-declared apparently healthy population.Results:Assay performance data showed good precision, sensitivity and lot-to-lot reproducibility. There was good short term 2–8 °C sample stability, however, long term storage samples must be kept at ? 70 °C or colder. The largest variance component in a biological variance study was within-day.Conclusions:The ARCHITECT NGAL assay proved to be a precise and reproducible assay for the determination of urine NGAL.     

Determination of creatine and guanidinoacetate by GC-MS: study of their stability in urine at different temperatures

ObjectivesEvaluation of a GC-MS method using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) as the silylating agent for GC-MS. Study of the stability of creatine and guanidinoacetate in urine.Design and methods22 urines were kept at RT, 4 °C and ? 30 °C for 15 days.ResultsMTBSTFA produces a single chromatographic peak in contrast with other derivatizing agents. Creatine concentration increases at room temperature (326% on average), and at 4 °C (75%). However, detection decreases after freezing (? 37%). Guanidinoacetate is stable, but decreases after freezing (? 37%). Sonication before analysis is crucial to obtain repetitive results.ConclusionsA modified GC-MS method has been validated and the conditions for preservation of the urine have been established.     

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.     

Effects of anticoagulants on human plasma soluble corin levels measured by ELISA

BackgroundRecently, soluble corin was detected in human plasma. In patients with heart failure, plasma corin levels were lower than that of normal controls. In this study, we analyzed experimental conditions for measuring plasma or serum corin by an immunoassay.MethodsSerum and plasma corin levels were measured by ELISA. Effects of different anticoagulants (EDTA, heparin and sodium citrate) on plasma corin levels were examined.ResultsCorin levels in serum were similar to that in plasma with heparin (950 ± 305 vs. 929 ± 301 pg/ml, n = 40, p = 0.73), but were significantly higher than those in plasma with sodium citrate (735 ± 237 pg/ml, p < 0.01) or EDTA (716 ± 261 pg/ml, p < 0.001). Native and recombinant human corin proteins were stable in human plasma with EDTA at 4 °C or underwent freezing-and-thawing. In 348 healthy Chinese individuals, plasma corin levels ranged from 216 to 1663 pg/ml. The levels were higher in males than that in females (842 ± 283 vs. 569 ± 192 pg/ml, p < 0.001).ConclusionSoluble corin was stable in plasma samples. Plasma soluble corin levels vary depending on anticoagulants used. Samples containing heparin had significantly higher levels of corin than that in samples with EDTA or sodium citrate.     

Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection

ObjectivesTo assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA).Design and methodsIsolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA-induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca²⁺]_i) was measured using Fluo-3 AM, a calcium-sensitive dye.ResultsEDTA provided the highest number of isolated neutrophils/mL of blood (1.7 × 10⁶ ± 1.5 × 10⁵) when compared with citrate (0.46 × 10⁶ ± 0.95 × 10⁵) and heparin (0.66 × 10⁶ ± 0.15 × 10⁵). EDTA originated less degree of PMA-induced activation (370 ± 30%) relatively to citrate (830 ± 98%) and heparin (827 ± 77%). [Ca²⁺]_i was lower with EDTA (122 ± 11 nM) when compared with citrate and heparin (150 ± 13 and 230 ± 30 nM).ConclusionThe anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.     

Atrial natriuretic peptide stability

ObjectiveAtrial natriuretic peptide (ANP) is a key regulator in the homeostasis of water excretion and has emerged as an important prognostic marker for symptomatic chronic heart failure (CHF). The stability of ANP represents a crucial factor in assessing its use as a cardiac biomarker. Accordingly, we assessed the stability of ANP in blood samples collected from healthy controls and CHF subjects for a 12 month period.MethodsBlood samples from 10 healthy controls and 12 symptomatic CHF subjects with left ventricular systolic dysfunction were drawn. Determination of plasma ANP was performed by a standardized radioimmunoassay protocol.ResultsThe ANP levels of healthy subjects were 68.5 ± 11.6 pg/mL at baseline and 69.9 ± 17.2 pg/mL at 12 months (p = 0.71). The ANP concentrations of CHF subjects were 199.25 ± 44.8 pg/mL at baseline and 197.83 ± 47.4 pg/mL at 12 months (p = 0.70) respectively.ConclusionANP is a stable molecule with no evidence of degradation when stored at ? 80 °C.     

Butyrylcholinesterase activity is reduced in haemodialysis patients: Is there association with hyperhomocysteinemia and/or oxidative stress?

Objectives:To quantify serum butyrylcholinesterase activity in haemodialysis patients and to evaluate if the homocysteine levels and/or oxidative stress biomarkers have an effect on butyrylcholinesterase.Materials and methods:Blood samples were collected from patients and healthy subjects (control). The plasma homocysteine and TBARS levels; serum butyrylcholinesterase activity; blood δ aminolevulinic acid dehydratase (ALA-D) activity and methahaemoglobin were analyzed. The mortality of the patients was also evaluated after 3 years.Results:The homocysteine was increased and butyrylcholinesterase decreased compared to control (p < 0.05). TBARS and methahaemoglobin were increased and ALA-D decreased (p < 0.05). The following correlations were found: homocysteine with butyrylcholinesterase (? 0.44); methahaemoglobin (0.41); ALA-D (? 0.68); and TBARS (0.66). The partial correlation between homocysteine with butyrylcholinesterase, withdrawn the effect of TBARS, was ? 0.30; all p < 0.05. Moreover, it was observed that 22% of the patients died due to cardiovascular problems.Conclusion:Thus, our findings support a direct association between the reduction of butyrylcholinesterase by the increase of homocysteine and an indirect effect by increase in oxidative stress.     

Effect of preanalytical variables on myeloperoxidase levels

BackgroundMyeloperoxidase (MPO) levels have prognostic value in cardiovascular events, but information about preanalytical variables is scarce. This study evaluated the effect of different sample types and storage conditions on MPO measurements.MethodsPlasma and serum samples [heparinized plasma (MPO-Hep), EDTA plasma (MPO-EDTA), and serum with (MPO-Gel) and without separator gel (MPO-Serum)] from 40 volunteers were assayed for MPO by ELISA (Bioxytech® MPO-EIA? kit). To evaluate MPO stability, samples were stored at 18–25 °C, at 2–8 °C and at ? 20 °C and ? 80 °C for predetermined periods.ResultsMPO levels ranged from 16 to 69 ng/ml and were higher in patients with heart disease compared to healthy volunteers (35.0 vs. 24.9 ng/ml; P = 0.03). There were no statistical differences between MPO-Hep, MPO-Gel and MPO-Serum (P > 0.05), and MPO-Hep showed a good correlation with MPO-Gel and MPO-Serum (r = 0.775 and r = 0.792; P < 0.001). No correlation between MPO-Hep and MPO-EDTA was found (r = 0.21; P = 0.20), and mean MPO-EDTA value was 1.8 times higher than MPO-Hep (51.4 vs. 28.7 ng/ml; P < 0.001). Mean differences (ng/ml) between MPO-Hep and MPO-EDTA, MPO-Gel and MPO-Serum were 24.7 (19.5–30.0), 0.225 (? 2.54–2.99), and 1.55 (? 1.16–4.26), respectively.ConclusionsEDTA had a significant effect on MPO results. MPO-Hep values correlate well with MPO-Gel and MPO-Serum. MPO levels seem to be stably frozen at ? 20 °C or ? 80 °C over 6 months depending on preanalytical handling.     

The preparation and storage of dried-blood spot quality control materials for lysosomal storage disease screening tests

ObjectiveWe aimed to prepare dried-blood spot (DBS) quality control (QC) materials for lysosomal storage disease (LSD) screening tests and to determine optimum blood and DBS storage conditions.MethodsWe compared enzyme activities of five LSD markers in adult blood, umbilical-cord blood, and leukocyte-reduced blood. We measured activities in liquid blood and DBSs after predetermined intervals at controlled temperatures and humidities.ResultsLysosomal-enzyme activity levels in umbilical-cord blood mimicked those in newborn screening samples. Lysosomal-enzyme activities in leukocyte-reduced blood were lower than in LSD-positive patient samples. Enzyme activities were stable in refrigerated liquid blood for 32 days and in frozen DBSs stored at low humidity for a year. Activity losses from DBSs after 34 days at 37 ± 1 °C were 35%–66% in low humidity and 61%–100% in high humidity.ConclusionsUmbilical-cord blood is the preferred matrix for LSD-normal DBS QC materials. Leukocyte-reduced blood is lysosomal enzyme-deficient. Failure to control humidity during DBS storage results in loss of lysosomal-enzyme activities.     

Persistent deep mechanical hyperalgesia induced by repeated cold stress in rats

Chronic muscle pain of the neck, shoulder and low back is quite common and often related to a stressed condition. In this study we tried to make a model of long-lasting muscle mechanical hyperalgesia based on one type of stress, repeated cold stress (RCS) (Kita T, Hata T, Yoneda R, Okage T. Stress state caused by alternation of rhythm in environmental temperature, and the functional disorders in mice and rats. Folia Pharmacol Jpn 1975;71:195–210). We first validated a method of measuring the muscle mechanical nociceptive threshold through skin, with surface anesthesia of the skin covering the muscle. We found that a pressure test using a Randall–Selitto analgesiometer equipped with a larger probe (? 2.6 mm) can measure the deep mechanical withdrawal threshold even under the presence of cutaneous punctuate hyperalgesia. RCS was performed by changing the temperature from 22 °C to either 4 °C (RCS at 4 °C) or ?3 °C (RCS at ?3 °C) every 30 min, and then maintained at 4 °C/?3 °C from 17:30 to 10:00 the next day. RCS at 4 °C for 5 days induced bilateral deep mechanical hyperalgesia lasting 2–3 weeks without cutaneous punctuate hyperalgesia. Deep mechanical hyperalgesia observed after RCS at ?3 °C lasted longer (～6 weeks) and was severer than RCS at 4 °C. Bilateral cutaneous punctuate hyperalgesia was also observed with RCS at ?3 °C. Intramuscular injection of lidocaine confirmed that the muscle was hyperalgesic. RCS might serve as a useful model for study of the mechanism of chronic muscle pain and its treatment.     

Effects of temperature and light on the stability of bilirubin in plasma samples

BackgroundAlthough it is known that bilirubin is photo-sensitive, detailed effects of both temperature and artificial light exposure on bilirubin stability in plasma have not been well investigated. We determined the impact of temperature and artificial light on bilirubin stability in plasma.MethodsPlasma total and direct bilirubin were analyzed using a diazo method. The aliquots of 38 samples were stored at 3 °C and 22 °C with light protection for 2, 4, 8, and 24 h respectively before analysis. The aliquots of 20 samples with normal bilirubin and additional 20 with elevated bilirubin were exposed to artificial light for 2, 4, 8, 24, and 48 h at 22 °C, and total and direct bilirubin were measured. The differences between the baselines and subsequent measurements were analyzed with analysis of variance.ResultsThe baseline total bilirubin was 9.6 ± 8.1 mg/dl (mean ± SD) and the concentrations were 9.6 ± 8.2, 9.0 ± 7.4, 9.0 ± 7.5, and 8.8 ± 7.5 mg/dl at 3 °C and 9.5 ± 8.1, 9.0 ± 7.4, 9.6 ± 8.1, and 9.5 ± 8.0 mg/dl at 22 °C after 2, 4, 8, and 24 h (p > 0.05, n = 38). The baseline direct bilirubin was 1.3 ± 1.2 mg/dl and the concentrations after 2, 4, 8, and 24 h were 1.4 ± 1.2, 1.4 ± 1.2, 1.5 ± 1.2, and 1.3 ± 1.1 mg/dl at 3 °C and 1.4 ± 1.1, 1.3 ± 1.1, 1.3 ± 1.1, and 1.3 ± 1.0 mg/dl at 22 °C (p > 0.05, n = 19). In samples with elevated bilirubin exposed to light at 22 °C, the baseline total and direct bilirubin concentrations were 10.2 ± 1.7 mg/dl and 5.0 ± 1.9 mg/dl, respectively. After 2, 4, 8, 24, and 48 h, total bilirubin concentrations were 10.1 ± 1.8, 10.0 ± 1.8, 10.0 ± 1.8, 9.3 ± 2.0 (p > 0.05, n = 20), and 8.4 ± 2.3 (p < 0.01, n = 20) mg/dl and direct bilirubin concentrations were 4.9 ± 1.8, 4.9 ± 1.9, 4.8 ± 1.8, 4.2 ± 1.6 (p > 0.05, n = 20), and 3.5 ± 1.5 (p < 0.01, n = 20) mg/dl. For samples with normal bilirubin levels under the same conditions, the average baseline total and direct bilirubin concentrations were 0.7 ± 0.1 mg/dl and below the lower limit of quantification (LLOQ), respectively. After 2, 4, 8, 24, and 48 h, the average total bilirubin concentrations were 0.7 ± 0.1, 0.6 ± 0.1, 0.6 ± 0.1 (p > 0.05, n = 20), 0.5 ± 0.1, and 0.4 ± 0.1 mg/dl (p < 0.01, n = 20) and direct bilirubin concentrations were still below LLOQ.ConclusionsBilirubin in plasma is stable in refrigerator or at room temperature without light exposure for at least 24 h. In normal laboratory environment, a delay of up to 8 h in the measurement of bilirubin left unprotected from light at room temperature does not significantly affect the results. Under these conditions, the changes in bilirubin concentration are not clinically significant until 24 h (direct bilirubin) and after 48 h (total bilirubin).