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 degrees C) caused a rapid decline in enzyme activity, which was smaller but still considerable at 4 degrees C. Storage at -20 degrees C and -70 degrees 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 degrees 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.     

Analysis of prolonged storage on coagulation Factor (F)V,FVII, and FVIII in thawed plasma: is it time to extend the expiration date beyond 5 days?

BACKGROUND: According to AABB standards, fresh‐frozen plasma (FFP) should be thawed at 30 to 37°C and expire after 24 hours. An increase in the aggressive management of trauma patients with thawed plasma has heightened the risk of plasma waste. One way to reduce plasma waste is to extend its shelf life, given that the full range of therapeutic efficacy is maintained. We evaluated the effect of prolonged storage at 1 to 6°C on the activity of Factor (F)V, FVII, and FVIII in plasma thawed at 37 or 45°C. STUDY DESIGN AND METHODS: Group O plasma from healthy donors (n = 20) was divided into 10 pairs and frozen and stored at not more than ?18°C. One sample from each pair was thawed at 37 or 45°C, and all were stored at 1 to 6°C. Samples were analyzed for FV, FVII, and FVIII activity on Days 0, 5, 10, 15, and 20. RESULTS: Plasma thawing time was 17% less at 45°C than at 37°C. No differences were observed between thawing groups in coagulation activity of FV, FVII, and FVIII during the 20‐day storage period (p > 0.12). In both groups, the activity of FV and FVIII decreased over time but remained within a normal range at 10 days. CONCLUSION: Although levels of plasma clotting factors are reduced in storage, therapeutic levels of FV and FVIII are maintained in thawed plasma stored for up to 10 days at 1 to 6°C. Thawing of FFP at 45°C decreases thawing time but does not affect the activity of FV, FVII, and FVIII.     

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.     

Storage of thawed plasma for a liquid plasma bank: impact of temperature and methylene blue pathogen inactivation

BACKGROUND: Rapid transfusion of fresh‐frozen plasma (FFP) is desired for treating coagulopathies, but thawing and issuing of FFP takes more than 40 minutes. Liquid storage of plasma is a potential solution but uncertainties exist regarding clotting factor stability. We assessed different storage conditions of thawed FFP and plasma treated by methylene blue plus light (MB/light) for pathogen inactivation. STUDY DESIGN AND METHODS: Fifty thawed apheresis plasma samples (approx. 750 mL) were divided into three subunits and either stored for 7 days at 4°C, at room temperature (RT), and at 4°C after MB/light treatment. Clotting factor activities (Factor [F] II, FV, FVII through FXIII, fibrinogen, antithrombin, von Willebrand factor antigen, Protein C and S) were assessed after thawing and on Days 3, 5, and 7. Changes were classified as “minor” (activities within the reference range) and “major” (activities outside the reference range). RESULTS: FFP storage at 4°C revealed major changes for FVIII (median [range], 56% [33%‐114%]) and Protein S (51% [20%‐88%]). Changes were more pronounced when plasma was stored at RT (FVIII, 59% [37%‐123%]; FVII, 69% [42%‐125%]; Protein S, 20% [10%‐35%]). MB/light treatment of thawed FFP resulted in minor changes. However, further storage for 7 days at 4°C revealed major decreases for FVIII (47% [12%‐91%]) and Protein S (49% [18%‐95%]) and increases for FVII (150% [48%‐285%]) and FX (126% [62%‐206%]). CONCLUSION: Storage of liquid plasma at 4°C for 7 days is feasible for FFP as is MB/light treatment of thawed plasma. In contrast, storage of thawed plasma for 7 days at RT or after MB/light treatment at 4°C affects clotting factor stability substantially and is not recommended.     

Effect of freeze–thaw cycles on serum measurements of AFP,CEA, CA125 and CA19‐9

AFP, CEA, CA125 and CA19‐9 are commonly used serum tumour markers (TMs) in clinical practice, although their quantification by immunoassay may be influenced by pre‐analytical sample handling. Though the effect of repetitive freeze–thaw cycles is generally recognized, it is not clear in detail. The present study measured (CLIA) these TMs in serum samples freshly separated and after each of five freeze–thaw cycles, in which the samples were frozen at ?40°C for 10 months at cycle 4 and 2?h at other cycles. Statistical analysis with the General Linear Model for Repeated Measures revealed significant decreases in the measurements of the four TMs, with the least decrease of 6.8?% for CA125 and the most decrease of 18.2?% for CA19‐9 after the last cycle, and an overwhelming single cycle decrease of mean 7.7?% at cycle 4 for AFP, CEA and CA125, of 7.5?% and 9.3?% at cycles 4 and 5 for CA19‐9. So it seems that measurements of AFP, CEA and CA125 are more readily affected by long‐term frozen storage compared with frequent freezing–thawing, while CA19‐9 is relatively unstable under both conditions.     

Long‐term stability of salivary cortisol

The measurement of salivary cortisol provides a simple, non‐invasive, and stress‐free measure frequently used in studies of the hypothalamic‐pituitary‐adrenal axis activity. In research projects, samples are often required to be stored for longer periods of time either because of the protocol of the project or because of lack of funding for analysis. The aim of the present study was to explore the effects of long‐term storage of samples on the amounts of measurable cortisol. Ten pools of saliva were collected on polyester Salivette^® tampons from five subjects. After centrifugation the samples were either stored in small vials or spiked to polyester Salivette tampons before analysis for cortisol using Spectria RIA kits. The effects of storage were evaluated by a linear regression model (mixed procedure) on a logarithmic scale. No effects on cortisol concentrations were found after storage of saliva at 5°C for up to 3 months or at ?20°C and ?80°C for up to one year. In contrast, concentrations of cortisol were found to decrease by 9.2% (95% confidence interval (CI): 3.8%; 14.3%) per month in samples stored at room temperature. Repeated freezing and thawing of samples up to four times before analysis did not affect the measured concentrations of cortisol. The coefficient of residual variation (CVresid) for samples stored on Salivette tampons were twice the CVresid for samples stored in separate vials after centrifugation. In conclusion, centrifuged saliva samples for analysis of cortisol may be stored at 5°C for up to 3 months or at ?20°C or ?80°C for at least one year. However, long‐term storage at room temperature cannot be recommended. Repeated cycles of freezing and thawing did not appear to affect the concentrations of cortisol.     

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.     

Current databases on biological variation: pros,cons and progress

Abstract

GP73 is a transmembrane glycoprotein that increases in viral and non-viral liver diseases, especially in hepatocellular carcinoma. This study aims to evaluate the effect of sample type and storage conditions on GP73 concentration. Twenty subjects were enrolled in this study. Serum and citrated plasma samples were collected. Both were subjected to different time intervals and storage temperature. Baseline GP73 concentrations ranged from 1.7 to 16.9?ng/mL in serum samples, and from 1.1 to 15.3?ng/mL in citrated plasma (Mann–Whitney U test, p?=?.1). The acceptable change limit for GP73 was 6.1%. As the highest value of the median percentage deviation was ?5.3% in both sample types at different storage condition so, deviations were within the accepted limits. But there were considerable variations in the GP-73 concentrations after 2 cycles of freezing and thawing at ?20?°C. This study shows that both serum and citrated plasma can be used for the measurement of GP73 concentration. GP73 seems to be stable under common storage conditions, but it may be unstable with frequent cycles of freezing and thawing.     

Effect of freeze-thawing on the long-term stability of calcium levofolinate in 5% dextrose stored on polyolefin infusion bags

Background: Calcium levofolinate infusions could be prepared in advance by a centralized intravenous additive service (CIVAS) to improve safety and time management. Objective: To investigate the effect of freezing, microwave thawing and long‐term storage at 5 ± 3 °C on the stability of calcium levofolinate in 5% dextrose solution. Methods: Solutions of 250 mL of 5% dextrose in polyolefin bags (n = 5) containing approximately 400 mg of calcium levofolinate were prepared under aseptic conditions and frozen for 95 days at ?20 °C. The solutions were then thawed using microwaves and stored at 5 ± 3 °C for 1 month. The calcium levofolinate concentrations were measured by high performance liquid chromatography (HPLC). Visual inspection was performed and pH was measured periodically during the storage at 5 ± 3 °C. Stability of the solution was defined as a concentration remaining superior to 90% of the initial concentration by regression analysis as recommended by the Food and Drug Administration (FDA). Results: No colour change or precipitation in the solutions was observed. Calcium levofolinate infusions were stable when stored at 5 ± 3 °C during 1 month after freeze‐thaw treatment. Throughout this period, the lower confidence limit of the estimated regression line of concentration‐time profile remained above 90% of the initial concentration. Slight change in pH values from 6·52 ± 0·01 to 6·50 ± 0·01 during storage time did not affect retention time on HPLC and has no clinical consequence, the solutions remaining in the acceptable range for perfusion (4 ≤ pH ≤ 10). Conclusion: Under the conditions of this study, calcium levofolinate in 5% dextrose infusion may be prepared, frozen in advance by CIVAS, and then microwave thawed before use. Such treatment extends long‐term stability and releases pharmacist’s time for major activities such as checking medication order errors.     

不同标本类型间髓过氧化物酶水平的检测分析

目的探讨不同标本类型间髓过氧化物酶(MPO)检测结果的差异性、抗凝剂的选择及检测结果的比对。方法同时采集165例健康体检人群含乙二胺四乙酸二钾(EDTA-K2)、肝素钠两种抗凝剂的血浆标本及不含抗凝剂的普通生化管的血清标本,分别检测3种标本类型中MPO水平,并对各组检测结果进行统计学分析。结果同一例体检者不同抗凝剂的血浆标本间及与不含抗凝剂的血清标本间MPO检测结果差异有统计学意义(P0.05)。结论不同标本类型对血液标本中MPO水平检测结果差异较大,建议各个实验室检测时根据不同抗凝剂制订相应的参考区间;由于EDTA-K2抗凝血浆不受体外白细胞中MPO释放的影响,推荐采用EDTA-K2抗凝血作为检测MPO水平的首选。     

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.     

Insect cold hardiness: the role of mitogen‐activated protein kinase and Akt signalling in freeze avoiding larvae of the goldenrod gall moth,Epiblema scudderiana

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of cold hardiness to survive the winter. Here we report that protein kinase‐dependent signal transduction featuring mitogen‐activated protein kinase (MAPK) signalling cascades (extracellular signal regulated kinase, c‐jun N‐terminal kinase and p38 MAPK pathways) and the Akt (also known as protein kinase B, or PKB) pathway could be integral parts of the development of cold hardiness by E. scudderiana. We used Luminex technology to assess the protein levels and phosphorylation status of key components and downstream targets of those pathways in larvae in response to low temperature acclimation. The data showed that MAPK pathways (both total protein and phosphorylated MAPK targets) were inhibited after 5°C acclimation, but not ?15°C exposure, as compared with the 15°C control group. However, total heat shock protein 27 (HSP27) levels increased dramatically by ～12‐fold in the ?15°C acclimated insects. Elevated HSP27 may facilitate anti‐apoptotic mechanisms in an Akt‐dependent fashion. By contrast, both 5 and ?15°C acclimation produced signs of Akt pathway activation. In particular, the inhibitor phosphorylated Glycogen Synthase Kinase 3a (p‐GSK3) levels remained high in cold‐exposed larvae. Additionally, activation of the Akt pathway might also facilitate inhibition of apoptosis independently of GSK3. Overall, the current study indicates that both MAPK and Akt signal transduction may play essential roles in freeze avoidance by E. scudderiana.     

Influence of air exposure and storage condition on serum ionized magnesium level

The aim of this study was to evaluate whether reporting serum level of ionized magnesium (iMg) is appropriate when affected by various conditions such as exposure to air and delayed measurement. Serum levels of pH, iMg and normalized magnesium (nMg, normalized or adjusted concentration of iMg to pH?7.40) from 28 inpatients were measured at intervals of 3?min after exposing the samples to air at room temperature. Serum from 30 inpatients was stored in closed tubes at 4°C and ?20°C and iMg and nMg levels were measured after 2 days. It was found that serum iMg and nMg concentrations exposed to air were decreased by 0.0023?mmol/l and 0.0001?mmol/l per minute, respectively. nMg did not display any significant changes compared with iMg at 0?min, whereas iMg showed significant changes, which exceeded between‐day precision. For the stored serum, only iMg of serum at ?20°C showed no statistically significant changes (p?=?0.169). It is concluded that to report the result as iMg, the sample should be kept anaerobically, and if exposed to air, the result should be reported as nMg. For storage, iMg of serum kept anaerobically at ?20°C is reliable.     

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.     

Platelet apoptosis and activation in platelet concentrates stored for up to 12 days in plasma or additive solution

Background: Several studies suggest that apoptosis of platelets occurs during storage of platelet concentrates (PC). We sought to determine whether storage of PC in additive solution alters levels of apoptosis during storage beyond the current shelf life (5–7 days). Study design and methods: Pooled buffy coat PC (n = 7) were prepared in either 100% plasma or 70% Composol and stored at 22 °C for 12 days. A third arm of the study stored PC in 100% plasma at 37 °C, which is thought to induce apoptosis. PC were tested for mitochrondrial membrane potential, annexin V binding, microparticles, caspase‐3/7 activity and decoy cell death receptor 2, as well as standard platelet quality tests. Results: Composol units remained ≥pH 6·88, with 36% lower lactate and higher pH vs plasma by day 12 (P < 0·001). Platelet function was better maintained, and activation and apoptotic markers tended to be lower in Composol units towards the end of storage. However, levels of all apoptosis markers assessed were not significantly different in units stored in Composol. Storage at 37 °C saw stronger correlation of apoptotic markers with standard quality tests compared to 22 °C, but loss of correlation of caspase‐3/7 activity with other apoptosis markers. Conclusion: We conclude that storage of platelets in 70% Composol vs 100% plasma does not increase the rate of platelet apoptosis. Our data agree with other studies suggesting that platelet apoptosis is sequential to high levels of activation, but share a significant degree of overlap.     

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.     

Coagulation factor levels in plasma frozen within 24 hours of phlebotomy over 5 days of storage at 1 to 6 degrees C

BACKGROUND: The use of plasma frozen within 24 hours after phlebotomy (FP24) is likely to increase as male donors become the predominant source of plasma products. This study was performed to investigate the levels of clotting factors in thawed plasma (TP) prepared from FP24 during 5 days of storage at 1 to 6°C. STUDY DESIGN AND METHODS: Five units of A, B, and O and 3 units of AB FP24 were obtained from the local blood provider. They were thawed and maintained at 1 to 6°C for a total of 5 days. Within 6 hours of thawing and every 24 hours thereafter for 5 days, each unit was assayed for the following clotting factors: Factor (F)II, FV, FVII, FVIII, F IX, FXI, FXII, antithrombin (AT), protein C (PC), and protein S (PS). ADAMTS‐13 was assayed on Days 2, 4, and 5. Time is expressed as mean hours or days (standard deviation). RESULTS: On average the units were frozen 21.3 (3.8) hours after phlebotomy and had been frozen for a mean of 30.1 (32.3) days before thawing. The activities of all procoagulant factors including FVIII, along with AT, PC, and ADAMTS‐13, were well maintained in their normal range during the 5‐day storage. The activity of PS was slightly below the normal range by Day 5. CONCLUSIONS: The activity of all factors assayed, except for PS, were within their normal range during the 5‐day storage period. These results show comparable factor assay levels in TP prepared from fresh‐frozen plasma and FP24.     

Mannitol clearance for the determination of glomerular filtration rate–a validation against clearance of 51Cr‐EDTA

We studied the agreement between plasma clearance of mannitol and the reference method, plasma clearance of ⁵¹Cr‐EDTA in outpatients with normal to moderately impaired renal function. Forty‐one patients with a serum creatinine <200 μmol l^?1 entered the study. ⁵¹Cr‐EDTA clearance was measured with the standard bolus injection technique and glomerular filtration rate (GFR) was calculated by the single‐sample method described by Jacobsson. Mannitol, 0·25 g kg^?1 body weight (150 mg ml^?1), was infused for 4–14 min and blood samples taken at 1‐, 2‐, 3‐ and 4‐h (n = 24) or 2‐, 3‐, 3·5‐ and 4‐h after infusion (n = 17). Mannitol in serum was measured by an enzymatic method. Plasma clearance for mannitol and its apparent volume of distribution (Vd) were calculated according to Brøchner‐Mortensen. Mean plasma clearance (±SD) for ⁵¹Cr‐EDTA was 59·7 ± 18·8 ml min^?1. The mean plasma clearance for mannitol ranged between 57·0 ± 20·1 and 61·1 ± 16·7 ml min^?1 and Vd was 21·3 ± 6·2% per kg b.w. The between‐method bias ranged between ?0·23 and 2·73 ml min^?1, the percentage error between 26·7 and 39·5% and the limits of agreement between ?14·3/17·2 and ?25·3/19·9 ml min^?1. The best agreement was seen when three‐ or four‐sample measurements of plasma mannitol were obtained and when sampling started 60 min after injection. Furthermore, accuracy of plasma clearance determinations was 88–96% (P30) and 41–63% (P10) and was highest when three‐ or four‐sample measurements of plasma mannitol were obtained, including the first hour after the bolus dose. We conclude that there is a good agreement between plasma clearances of mannitol and ⁵¹Cr‐EDTA for the assessment of GFR.     

Serum ferritin: Physiological and methodological studies

Some aspects of normal ferritin physiology have been investigated as well as methodological problems concerning test sample handling etc. No circadian rhythm was found in 11 subjects. The day-to-day variation showed a mean of 9% in 22 subjects, but with considerable individual variation. The ferritin content in erythrocytes was about 0.045 fg/cell, and in leucocytes about 10 fg/cell. Hemolysis of test samples up to a hemoglobin concentration of 3 g/1 in the serum did not significantly change the ferritin concentration. This means that hemolysis of test samples is usually no problem in clinical practice. Serum samples could be stored at ?20°C for a year or freeze-thawed six times without change in ferritin concentration. Heparin- and sodium citrate plasma gave the same results as serum, but EDTA plasma gave 23% (mean) lower values. A moderate amount of alcohol, corresponding to 15 cl of whisky, gave no rise within 56 h in serum ferritin levels in four subjects.