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.     

Assessment of the stability of N-terminal pro-brain natriuretic peptide in vitro: implications for assessment of left ventricular dysfunction.

Plasma concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) are raised in patients with left ventricular dysfunction. Measurement of this peptide has a potential diagnostic role in the identification and assessment of patients with heart failure. The stability of this peptide over time periods and conditions pertaining to routine clinical practice has not been reported previously. Blood samples were obtained from 15 subjects. One aliquot was processed immediately, and the remaining portions of the blood samples were stored for 24 h or 48 h at room temperature or on ice prior to processing. Plasma concentrations of NT-proBNP were measured with a novel immunoluminometric assay developed within our laboratory. Mean plasma concentrations of NT-proBNP were not significantly different whether blood samples were centrifuged immediately and stored at -70 degrees C or kept at room temperature or on ice for 24 h or 48 h. The mean percentage differences from baseline (reference standard) were +5.2% (95% confidence interval +18.2 to -7.8%) and +0.8% (+15.2 to -13.7%) after storage for 24 h at room temperature or on ice respectively, and +8.9% (+24.2 to -6. 5%) and +3.2% (+15.1 to -0.9%) for storage for 48 h at room temperature or on ice respectively. Pearson correlation coefficients for baseline NT-proBNP concentrations compared with levels at 48 h at room temperature or on ice were r=0.89 and r=0.83 respectively (both P<0.0001). Thus NT-proBNP extracted from plasma samples treated with EDTA and aprotinin is stable under conditions relevant to clinical practice.     

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.     

The stability of angiotensin I formed at room temperature in the presence of ethylenediaminetetraacetate to subsequent incubation at 37 degrees C

It has been reported that plasma renin activity values obtained from samples containing ethylenediaminetetraacetate (EDTA) and which have been allowed to remain at room temperature for 24-48 hours prior to incubation and assay are spuriously low. Since plasma renin activity is often measured in specialised centres, considerable time delays in sample transit may be difficult to avoid. We, therefore, investigated these findings using plasma samples kept at room temperature for varying times and subsequently incubated at 37 degrees C. From these studies we conclude that plasma renin activity is not significantly affected by pre-incubation at room temperature for periods up to 48 hours. However, the precision of the assay is likely to be poorer in specimens pre-incubated at room temperature. These results are supported by the finding that there is no loss of angiotensin I upon incubation and assay of samples containing pepstatin A.     

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.     

不同处理和保存条件下体外HCV RNA稳定性研究

对献血者或病人进行HCV核酸检测时，如果标本的采集、处理、保存不当，会造成病毒核酸降解，从而影响检测结果的真实性。本研究的目的是对不同抗凝剂、不同温度、不同保存时间下的HCV RNA病毒稳定性进行研究，以考察常规采供血过程中，标本采集及保存方式对NAT检测的影响。采集7例HCV RNA阳性献血者的血样，采用不同的抗凝剂、经不同的温度和不同的时间保存后，采用荧光定量PCR方法测定HCV RNA病毒含量，考察HCV RNA的稳定性。结果表明：①不同抗凝剂抗凝的全血于4℃保存48小时过程中，病毒含量下降至原滴度的42．7％；EDTA抗凝组各时间点的滴度均低于其它3组（分别相当于其它3组的67．6％-25．1％）。②ACD抗凝的全血在4、25和37℃保存48小时．病毒含量分剐下降到原滴度的53．8％、72．5％、29．8％。③ACD抗凝的全血离心分离出血浆，4℃或25℃继续保存7天，病毒含量分别下降至原滴度的70．9％和25．1％。④ACD抗凝的全血分离的血浆，反复冻融4次，病毒含量下降到原滴度的38．9％。结论：①用于核酸检测的标本应该用无菌采血管采样；②在无菌采血管采样的前提下，核酸检测标本用未抗凝血、EDTA、ACD、CPDA抗凝血均可；③采集的全血应避免放置37℃以上，ACD抗凝全血在4℃、25℃保存48小时内、37℃保存14小时内，HCV RNA病毒仍较为稳定；④分离后的血浆应避免放置25℃以上，ACD抗凝全血分离后的血浆在4℃保存7天，25℃保存3天，HCV RNA病毒仍比较稳定；⑤血浆标本应避免多次冻融，但冻融3次的血浆HCV RNA病毒含量仍然较为稳定；⑥无菌采样对维持病毒的稳定性非常重要；单纯的机械性溶血并不会明显导致病毒的降解；只要注意无菌的问题和有合适的核酸提取方法去除血红素，HCV RNA病毒实际上比以前认为的要稳定得多。     

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 storage conditions on lymphocyte phenotypes from healthy and diseased persons

Anticoagulant and room temperature storage of peripheral blood was evaluated in healthy and diseased persons (AIDS and CLL) for its effect upon lymphocyte phenotyping by flow cytometry. Whole blood was stored in either Li heparin, EDTA, or ACD for up to 96 hours. Aliquots at 5 time intervals were evaluated using CD3, CD2, CD4, CD8, and CD19 monoclonal antibodies. Blood from healthy donors stored in EDTA produced an apparent increase in T cells and decrease in B cells most evident by 96 hours storage. Blood from diseased persons stored in Li heparin up to 96 hours showed insufficient changes in phenotype to warrant a different clinical interpretation. We conclude that all three anticoagulants are adequate for short term (less than 24 hr) room temperature transport and storage of peripheral blood for flow cytometry. Li heparin or ACD are more appropriate if prolonged transport times are anticipated.     

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.     

Evaluation of three different specimen types (serum, plasma lithium heparin and serum gel separator) for analysis of certain analytes: clinical significance of differences in results and efficiency in use.

BACKGROUND: There is a lack of consensus regarding the most appropriate specimen type for analysis of many biochemistry analytes. The aim of this study was to compare renal and lipid analyte profiles and phenytoin values in plain serum (S), serum gel (G) and plasma (lithium heparin, P) tubes and to investigate the stability of these analytes after prolonged contact with cells or gel at room temperature (RT, 20 degrees C) and as aliquoted and stored at 4 degrees C. METHODS: Primary specimens were centrifuged once, maintained at RT and analysed within 2 h (T(0)) and after 24 h (T(24)) and 48 h (T(48)). For assessment of stability at 4 degrees C, two cell-free aliquots were separated from each of the primary tubes and stored at 4 degrees C and then analysed at T(24) and T(48). Differences in analyte concentrations between tubes at T(0) and following storage (at T(24) and T(48)) were evaluated for both statistical and clinical significance. RESULTS: At T(0) all analytes, except potassium, demonstrated equivalence between serum, gel and plasma tubes. Potassium and creatinine were more stable in gel tubes than in serum/plasma tubes. In contrast, phentytoin was stable in plain serum and plasma up to T(48) at RT, but showed a progressive and clinically significant decrease in concentration in gel tubes at T(24) and T(48) at RT. All analytes except CO(2) were stable up to T(48) when aliquoted and stored at 4 degrees C. CONCLUSIONS: We concluded that the serum gel tube has advantages over plain serum and plasma tubes for measurement of the analytes investigated in this study, with the exception of phenytoin. In practice, the gel tubes demonstrate enhanced analyte stability and reduce the need to aliquot specimens, with greater protection against possible contamination. Further investigation would be required to evaluate a broader range of analytes.     

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.     

Heparin plasma sampling as an alternative to EDTA for BNP determination on the Access-Beckman Coulter--effect of storage at -20 degrees C

To determine BNP, EDTA plasma is the only suitable specimen recommended by the manufacturer. Since many laboratories, especially in Europe, use heparin plasma rather than EDTA plasma for many or most of their clinical assays and in particular for determination of cardiac markers (cTnI, myoglobin), it appeared critical to evaluate the use of heparin plasma samples, in comparison to EDTA plasma, for BNP determination on a automated immunochemiluminescent analyzer. The aim of this study was first, to evaluate the use of heparin plasma samples for Biosite BNP testing on the Beckman Coulter Access Immunoassay System (n=88) and second, to evaluate the effect of storage at -20 degrees C, without protease inhibitors, on the Biosite BNP assay. We obtained acceptable imprecision results with CVs ranging from 1.7 to 11.7% regardless of the anticoagulant used. The linearity of EDTA samples was good and comparable to the results observed with heparin plasma. The concentration of BNP was categorized according to the classification of the New York Heart Association (NYHA). With EDTA fresh samples as reference anticoagulant, 90% vs. 89% of subjects were classified as "concordant" with heparin fresh vs heparin frozen plasma samples, respectively. After storage at -20 degrees C, only 86% of the values of EDTA frozen were concordant with values of EDTA fresh. No subject varied by two NYHA classes. Heparin plasma is an attractive alternative to the established EDTA samples which can be used for BNP determination. This flexibility allows the simultaneous determination of CK, CK-MB, cTnI and BNP on a single heparin specimen, which facilitates blood collection for clinicians and nursing staff in an emergency unit. In addition, our results suggest that BNP could be stored at -20 degrees C for at least one month in order to perform retrospective studies.     

Stability of the Combination of Ceftazidime and Cephazolin in Icodextrin or pH Neutral Peritoneal Dialysis Solution

♦ Introduction: The objective of this study was to investigate the stability of ceftazidime and cephazolin in a 7.5% icodextrin or pH neutral peritoneal dialysis (PD) solution.♦ Methods: Ceftazidime and cephazolin were injected into either a 7.5% icodextrin or pH neutral PD bag to obtain the concentration of 125 mg/L of each antibiotic. A total of nine 7.5% icodextrin or pH neutral PD bags containing ceftazidime and cephazolin were prepared and stored at 1 of 3 different temperatures: 4°C in a domestic refrigerator; 25°C at room temperature; or 37°C (body temperature) in an incubator. An aliquot was withdrawn immediately before (0 hour) or after 12, 24, 48, 96, 120, 144, 168 and 336 hours of storage. Each sample was analyzed in duplicate for the concentration of ceftazidime and cephazolin using a stability-indicating high-performance liquid chromatography technique. Ceftazidime and cephazolin were considered stable if they retained more than 90% of their initial concentration. Samples were also assessed for pH, colour changes and evidence of precipitation immediately after preparation and on each day of analysis.♦ Results: Ceftazidime and cephazolin in both types of PD solution retained more than 90% of their initial concentration for 168 and 336 hours respectively when stored at 4°C. Both of the antibiotics lost more than 10% of the initial concentration after 24 hours of storage at 25 or 37°C. There was no evidence of precipitation at any time under the tested storage conditions. Change in the pH and color was observed at 25 and 37°C, but not at 4°C.♦ Conclusion: Premixed ceftazidime and cephazolin in a 7.5% icodextrin or pH neutral PD solution is stable for at least 168 hours when refrigerated. This allows the preparation of PD bags in advance, avoiding the necessity for daily preparation. Both the antibiotics are stable for at least 24 hours at 25 and 37°C, permitting storage at room temperature and pre-warming of PD bags to body temperature prior to its administration.     

Stability of coagulation factors in plasma prepared after a 24‐hour room temperature hold

BACKGROUND: The manufacture of fresh‐frozen plasma (FFP) requires that plasma be frozen within 8 hours of collection and 24‐hour frozen plasma requires 1 to 6°C refrigeration before freezing. Manufacture of plasma after a room temperature hold for 24 hours, while convenient, could compromise clotting factor levels. STUDY DESIGN AND METHODS: Pairs of FFP and 24‐hour room temperature–frozen plasma (PLT‐rich plasma [PRP]‐24HRTFP) were manufactured from PRP after a room temperature hold for 8 and 24 hours, respectively. Additional whole blood (WB) donations were kept at room temperature for 24 hours before plasma manufacture (WB‐24HRTFP). The frozen plasma products were stored at −18°C, thawed, and then stored at 1 to 6°C, with coagulation factor assays performed for up to 7 days. RESULTS: On the day of thaw, Factor (F)VIII was lower in PRP‐24HRTFP by 13% (p = 0.002) but not in WB‐24HRTFP (p = 0.3) compared to FFP. All other clotting factors were within normal range. During the postthaw period FVIII and FV declined 25 and 6%, respectively, in WB‐24HRTFP and 23 to 50% in the paired products; however, the difference between both types of 24HRTFP and FFP is insignificant by Day 7 (p > 0.05). Other clotting factors either were unchanged or showed minimal reduction (<15%). CONCLUSION: Plasma manufactured after a 24‐hour room temperature hold contains coagulation factors comparable to FFP except for a possible reduction of up to 20% in FVIII. This plasma appears suitable as a transfusable product and extension of liquid storage to 7 days merits consideration.     

Warm storage of whole blood for 72 hours

BACKGROUND: In field emergency medicine, fresh whole-blood units are stored at room temperature up to 24 hours or occasionally longer. Few data exist on the integrity and in vitro functional properties of whole blood stored warm beyond 24 hours. STUDY DESIGN AND METHODS: Ten citrate phosphate dextrose solution whole-blood units were collected and divided into two equal volumes. One-half of each unit was stored at 19 degrees C and the other half was stored at 25 degrees C, encompassing the accepted range for room temperature storage. At 6, 24, 48, and 72 hours, aliquots were collected from each unit and whole blood analyzed for cell counts, gases, and clotting function with thromboelastography, red cells for intracellular analytes, platelet (PLT)-rich plasma for aggregometry, and the supernatant for hemoglobin, potassium, glucose, lactate, and plasma clotting studies. RESULTS: Whole-blood units stored at room temperature maintained cellular counts and coagulation activity for up to 72 hours. Units stored at 19 degrees C demonstrated greater RBC adenosine triphosphate and 2,3-diphosphoglycerate (DPG) content and stronger responses in PLT aggregation studies when compared with 25 degrees C storage. No significant hemolysis was observed, and no bacterial growth was detected. CONCLUSION: Storage of whole blood at room temperature for 72 hours leads to marked reductions in pH and DPG, but the observed reduction in PLT function and plasma coagulation factor activity was surprisingly modest compared to literature values. These findings should prompt additional investigation, given their potential importance for whole blood processing and field-expedient transfusion.     

Effect of Storage up to 48 Hours on Response to Transfusions of Platelet Rich Plasma

The effect of in vitro storage of platelet rich plasma (PRP) on the circulating platelet count following platelet transfusion was evaluated in patients with thrombocytopenia and acute leukemia. PRP was obtained by plasmapheresis using ACD anticoagulant. There was little or no decline in response to PRP up to seven hours after donation whether kept at room temperature or at 4 C. In order to study the effect of storage at 4 C. for 24 and 48 hours, a protocol was designed which limited donor-patient variables. PRP stored 24 and 48 hours was 62 per cent and 37 per cent, respectively, as effective as fresh PRP in elevating the platelet count in the recipient one hour after transfusion.
Stored platelets are less than 5 per cent as effective as fresh PRP in maintaining elevation of platelet count 20 hours after transfusion. These studies define, quantitatively, the effect of short-term storage on response to transfusions of platelet rich plasma.     

Measurement of Elecsys NT-proBNP in serum, K2 EDTA and heparin plasma

OBJECTIVE: There is controversial evidence on a matrix influence on the measurement of NT-proBNP. DESIGN AND METHODS: We compared results of Elecsys NT-proBNP measurement on serum, K2 EDTA plasma and lithium heparin plasma. RESULTS: Samples collected in K2 EDTA showed a marginally significant underestimation when compared to serum and heparin, whereas no significant difference was observed between serum and heparin plasma. CONCLUSIONS: Serum, heparin and K2 EDTA plasma may be suitable for NT-proBNP measurement.     

Simultaneous determination of mycophenolate and its metabolite mycophenolate-7-o-glucuronide with an isocratic HPLC-UV-based method in human plasma and stability evaluation

Objectives: Mycophenolic acid (MPA) is an immunosuppressive agent which is commonly used in a fixed dose regime in solid organ transplantation. For clinical trials and therapeutic drug monitoring measuring plasma concentrations is necessary. Also, stability issues have to be addressed.

Methods: We describe an isocratic, RP-based HPLC-UV method for simultaneous determination of MPA and its major metabolite Mycophenolic acid 7-o Glucuronide (MPAG) in human plasma. Pre-analytics included protein precipitation with acetonitrile. The method was validated according to EMA/FDA guidelines. Patient lithium-heparin plasma and blood was used for evaluation of short-term (72?hours at room temperature?=?RT) and long-term stability (2 years at ?80?°C) without acidification.

Results: Linearity was assessed in the concentration range of 0.5–40.0?μg/mL for MPA and 5.0–350.0?μg/mL for MPAG, respectively. For MPA coefficient of variation was <7.0% (lower limit of quantification?=?LLOQ: 10.8%), for MPAG <9.6% (LLOQ: 10.6%). Bias ranged between ?1.9 and?+1.5% for MPA and for MPAG between ?4.3 and ?0.3%. The method showed agreement with a reference method for both analytes. MPA remained stable for 7?h (?1.6 to?+8.4% change to the initial concentration) and MPAG for 24?h (?1.8 to ?11.5% change) at RT in lithium heparin blood. After 2 years of storage at ?80?°C MPA, MPAG concentrations and 95% CIs remained within ±15% of the initial value.

Conclusion: The presented assay is applicable for clinical studies. Blood samples were stable for 7?hours at RT and plasma for 2 years stored at ?80?°C.     

Stability of urinary fractionated metanephrines and catecholamines during collection, shipment, and storage of samples

BACKGROUND: Measurements of 24-h fractionated urinary metanephrines and catecholamines are used for the diagnosis of pheochromocytoma, but adequate information is needed regarding collection, storage, and shipment conditions. METHODS: Spot urine samples were collected from 8 healthy volunteers. Aliquots were immediately frozen at -20 degrees C, or acidified to pH 4 and then frozen either directly or after 24 h at room temperature. The remaining urine was left at room temperature for 24 h and then split into one portion that was acidified and one portion that was not. Aliquots were either frozen or allowed to stand at room temperature for an additional 24, 48, 72, 96, and 168 h before freezing. We also tested the efficacy of adding Na(2)EDTA and Na(2)S(2)O(5), as an alternative to acidification for preservation of the catecholamines. RESULTS: No clinically relevant degradation (<5%) was observed for the fractionated metanephrines under any of the storage conditions. In contrast, in approximately 50% of the untreated samples catecholamines were partially degraded during the first 24 h at room temperature. Immediate acidification, however, prevented degradation, whereas acidification after 24 h prevented further decay. Addition of Na(2)EDTA and Na(2)S(2)O(5) fully prevented degradation of catecholamines during the first 24 h in 4 of 5 cases. In the remaining case, degradation did not exceed 10%. CONCLUSION: Preservation of samples for measurements of urinary fractionated metanephrines is not necessary if samples are assayed or frozen within 1 week, which is an important advantage if transport of samples is necessary. In contrast, urinary catecholamines require preservation measures during collection.     

Effect of delayed refrigeration on plasma factors in whole blood collected in CPDA-2

Extension of the time within which whole blood may be separated into components offers logistic advantages for the operation of remote mobile drawing teams. We evaluated the effect of an 8-hour hold of whole blood at room temperature before preparation of components. Plasma coagulation activity and opsonic factor content were studied in 14 units drawn into the anticoagulant-preservative solution citrate-phosphate-dextrose-adenine (CPDA-2). At the time of collection, an additional 7-ml aliquot was drawn into 1 ml of CPDA-2, the plasma separated and frozen immediately. Components were prepared from whole blood units allowed to rest undisturbed at 22 +/- 1 degrees C for 8 hours. After 8 hours, a significant decrement of about 10 percent was found in the concentration of fibrinogen, plasminogen, fibronectin, and activity of Factor V. Factor VIII activities (VIIIAHF and VIIIAGN) were not significantly different after 8 hours. Our results indicate that room temperature storage for 8 hours before component processing has minimal effects on potentially labile plasma protein factors using CPDA-2 anticoagulant-preservative solution.