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1.
The development of monoclonal antibodies combined with flow cytometry has revolutionized the analysis of lymphocyte subsets. These newer methods using the Q-prep leucocyte preparation system require only 1–2 ml of blood as compared to 10 ml required traditionally. One of the main impediments in the use of this superior technology in Indian laboratories has been the high cost of reagents. This study evaluated methods to reduce the cost of assays. In the first experiment from 26 healthy subjects, 2ml venous blood samples in EDTA (ethylenediamine tetra-acetate) were obtained. Each sample was divided into two equal portions, one portion was stained using diluted monoclonal antibody, whereas the other portion was stained using standard concentrations of antibodies. In the second experiment, blood samples from 12 subjects were again divided into 2 portions; one portion of each pair was processed using commercial Q-prep reagents while the other portion was processed using our own reagents. In the first experiment, which evaluated use of a diluted antibody against the standard recommended concentrations, a 5-tube panel that estimated CD3, CD4, CD8, CD20 was used. In the second experiment CD3, CD4 and CD8 were estimated. The total cost per sample for a 5-panel estimation was however reduced from $39.11 to $1.10. Given the proven advantages of using a whole blood stain-lyse method for T cell subset estimations, its use should be encouraged in developing country settings. With the suggested methods the whole blood Q-prep could be performed at appreciably reduced costs, without loss in precision.  相似文献   

2.
Approaches to the stabilization of tumor markers vary depending on the lability of the tumor marker. Thus while merely freezing the serum promptly at ?70°C may be adequate for some analytes such as gastrin, some other polypeptide hormones such as vasoactive intestinal peptide require collection of blood in EDTA and prompt freezing of plasma to protect the analyte from metal oxidation. In addition to collection of blood in EDTA an addition of a proteolytic enzyme inhibitor such as aprotinin may be necessary to preserve labile polypeptide hormones such as ACTH, somatostatin etc. Analytes such as parathyroid hormone related protein (PTH-rp) are so labile that additives such as leupeptin and pepstatin have to be added to EDTA and aprotinin and the plasma refrigerated to achieve stability for up to 24 hrs. Catecholamines also require stabilization with additives such as EGTA and glutathione and prompt freezing of plasma at ?70°C. Special precautions are required in handling tumor tissue intended for hormone receptor measurements since they are not only extremely heat labile but are also subjected to alteration during specimen preparation.  相似文献   

3.
Determination of ammonia level in blood is important, especially in the diagnosis of hepatic disorders. An indigenously purified enzyme was used in the standardisation of the assay. The assay is a two reagent system, requires five minutes for completion and can be performed at temperature between 25–27°C. Performance of the assay was assessed by linearity, imprecision, functional sensitivity and interference studies. Lyophilised reagent I and reagent II were found stable for at least one year. The plasma level of ammonia for the controls was 13.7±7.3 μMol/L, whereas for subjects of hepatic disorders, it was 69.1±32.4 μMol/L (P<0.001). The functional sensitivity was between 2–1000 μMol/L. Within-run coefficient of variation was between 1.1–2.0% and between-run coefficient of variation was between 1.9–3.7%. The mean recovery after dilution was 99.6%. The present method can estimate ammonia up to 1000 μMol/L without dilution of sample. Assay time of five minute may be shortened to one minute. This method is suited for routine clinical use in treatment of hepatic disorders.  相似文献   

4.
Fructose developed a pinkish orange chromogen on treatment with o-cresol: 70% sulphuric acid at 32°C for 15 minutes with a λ max of 500nm. Fructose was 185 times more chromogenic than glucose. Total carbohydrate and fructose values in protein-free filtrate of normal serum samples were in the range, 55.4–86.3 mg/dl and 1.55–3.29 mg/dl, respectively. In diabetes, the observed values were 197–354 mg/dl and 2.91–6.81 mg/dl, respectively.  相似文献   

5.

Introduction

The collected and shipped blood samples are exposed to a various extra-analytical factors prior to analysis. The aim of the study was to determine the stability of analytes in serum gel tubes and plain tubes exposed to a range of storage temperatures and times after centrifugation.

Materials and methods:

Fifteen healthy volunteers were recruited and venous blood was collected into four tubes, two with and two without gel separator. Analyzing the baseline samples in 30 min, all were stored at 4ºC or 24ºC for 6, 12, 18, 24, 30, 36, 48 and 72 hours and 1 week. Sixteen biochemical anaytes were measured on each sample. Variations remained under the desirable bias considered as clinically insignificant.

Results:

On day three, most analytes remained stable including albumin, protein, creatinine, cholesterol, triglycerides, gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LD) regardless of tube types. Glucose concentration decreased markedly (P = 0.001) beginning from the first hours of storage in plain serum. The stability maximized for the analytes including glucose, total bilirubin, urea nitrogen (BUN), uric acid stored at 4 ºC in gel tubes. Aspartate aminotransferase (AST) activity increased significantly (P = 0.002) up to 48-h, however bias was not significant clinically. High density lipoprotein (HDL) concentration was stable in gel tubes at 24 ºC, in plain tubes at 4 ºC stored up to 36-h.

Conclusion:

Serum gel or non-gel tubes might be used interchangeably for 11 analytes chilled or at 24 ºC, whereas some restrictions must be applied for glucose, AST, BUN, HDL, and uric acid.  相似文献   

6.
The present preliminary study was performed to find out stability of total prostate specific antigen (PSA) and free prostate specific antigen (FPSA) in serum of healthy males as well as in patients of benign and malignant disorders of prostate at various freezing and nonfreezing temperatures and at different duration of time. The results of our study indicated long-term stability of both the analytes in frozen serum. Serum total and free PSA were stable only for three to four days in regular refrigerators in unfrozen states. Clotted blood kept at room temperature (25°C–30°C) did not cause change in concentrations of both the analytes for twenty four hours.  相似文献   

7.

Introduction

Measurements of blood ethanol concentrations must be accurate and reliable. The most important factors affecting blood ethanol stability are temperature and storage time. In this study, we aimed to compare ethanol stability in plasma samples at -20 °C for the different storage periods.

Materials and methods

Blood samples were collected from intoxicated drivers (N = 80) and initial plasma ethanol concentrations were measured immediately. Plasma samples were then stored at -20 °C and re-assessed after 2, 3, 4, or 5 months of storage. Differences between the initial and stored ethanol concentrations in each group (N = 20) were analyzed using Wilcoxon matched-pairs test. The deviation from the initial concentration was calculated and compared with Clinical Laboratory Improvement Amendments (CLIA’88) Proficiency Testing Limits. Relationships between the initial concentrations and deviations from initial concentrations were analyzed by Spearman’s correlation analysis. For all statistical tests, differences with P values of less than 0.05 were considered statistically significant.

Results

Statistically significant differences were observed between the initial and poststorage ethanol concentrations in the overall sample group (P < 0.001). However, for the individual storage duration groups, analytically significant decreases were observed only for samples stored for 5 months, deviations from the initial concentrations exceeded the allowable total error (TEa). Ethanol decreases in the other groups did not exceed the TEa.

Conclusion

According to our results, plasma ethanol samples can be kept at -20 °C for up to 3-4 months until re-analysis. However, each laboratory should also establish its own work-flow rules and criterion for reliable ethanol measurement in forensic cases.Key words: preanalytical phase, ethanol, stability, storage temperature  相似文献   

8.
Accurate monitoring of blood cyclosporin C2 levels is vital to prevent over immunosuppression and acute renal toxicity in patients who receive organ transplant. The matrix used to dilute patients’ C2 samples prior to the assay affected the final measured values. Hence there was a need to develop a method of dilution that would accurately estimate C2 levels when cyclosporin levels were beyond the calibration range of the method employed. Whole blood, cyclosporin free hemolysate and cell and protein free supernatant obtained after pretreatment of normal blood were used to dilute patients’ C2 samples. C2 was measured in 188 patients using the supernatant method of dilution. C2 was correlated with Co and dose of cyclosporin received by the patient. The use of cell and protein free supernatant obtained after pretreatment of normal blood as a C2 diluent detected higher levels of C2 in the sample. Measured C2 correlated significantly with Co and the cyclosporin dose received by the patient. The uniformly aqueous cell and protein free supernatant ensures uniform dilution of the patients’ C2 sample and measures higher cyclosporin levels.  相似文献   

9.

Introduction:

Studies about vitamin D [25(OH)D] stability in plasma are limited and preanalytical variables such as tube type may affect results. We aimed to evaluate effect of storage conditions, sample type and some preanalytical variables on vitamin D concentration.

Materials and methods:

Blood samples from 15 healthy subjects were centrifuged at different temperatures and stored under different conditions. Serum and plasma 25(OH)D difference, effect of centrifugation temperature and common storage conditions were investigated.

Results:

There was no difference between serum and plasma vitamin D concentration. Centrifugation temperature had no impact on vitamin D concentration. 25(OH)D is stable under common storage conditions: 4 hours at room temperature, 24 hours at 2–8 °C, 7 days at −20 °C, 3 months at −80 °C.

Conclusion:

Vitamin D does not require any special storage conditions and refrigeration. Both serum and plasma can be used for measurement.  相似文献   

10.
Separation and quantitative estimation of the isoenzymes of lactate dehydrogenase(LD) in serum were accomplished with capillary electrophoresis system. An uncoated fused silica capillary column 50 cm long, 75μm I.D. and substrate containing running buffer including L-lactic acid and NAD+ were used for the separation of serum LD isoenzymes. The resulting product of “NADH” was detected at 340 nm. Injection of 10 nL of five fold diluted serum sample were performed by pressure injection within 2 seconds. The isoenzymes were separated at 10 kV of voltage for 5 min, by turning off the voltage applied for 30 min incubation at 24°C for reaction between substrate and isoenzymes, and applying voltage of 30 min. Under these conditions, the isoenzymes of LD were detected by a NADH generated as isoenzyme of LD-5 emerged at 20 min, LD-1 peak at 23.5 min with close to baseline separation of the other isoenzymes which emerge between LD-5 to LD-1, after the emergence to LD-1 peak, followed another peak, termed “sample shock”: The results obtained by the proposed method correlated well with those by gel electrophoresis systemes (r=0.92∼0.98) for each five LD isoenzymes, respectively. Within-run precision CVs for 5 replicate analysis were 3.01 (LD-3, mean 14.6%)%∼7.82% (LD-4, mean 4.22%.), respectively.  相似文献   

11.

Introduction

Glycolysis affects glucose determination in vitro. The placement of sample tubes in ice-water slurry with plasma separation within 30 minutes is recommended, or alternatively the use of a glycolysis inhibitor. The aim of our two-steps study was to evaluate which Terumo tube is best for glucose determination in routine clinical setting.

Materials and methods

In the first study, blood from 100 volunteers was collected into lithium heparin (LH), NaF/Na heparin (FH) and NaF/citrate buffer/Na2EDTA (FC-Mixture) tubes. LH sample was treated as recommended and considered as reference, while FH and FC-Mixture samples were aliquoted, maintained at room temperature (RT) for 1, 2 and 4 hours; centrifuged and plasma analysed in triplicate. In the second study, samples from 375 volunteers were collected in LH, FH and FC-Mixture tubes and held at RT before centrifugation from 10 to 340 minutes, depending on each laboratory practice. Samples were analysed in one analytical run.

Results

In the first study, FH glucose concentrations were 5.15 ± 0.66 mmol/L, 5.05 ± 0.65 mmol/L and 5.00 ± 0.65 mmol/L (P < 0.001) in tubes stored at RT for 1, 2 and 4 hours, respectively. Mean biases in all time points exceeded the analytical goal for desirable bias based on biological variation criteria. FC-Mixture glucose concentrations were 5.48 ± 0.65 mmol/L, 5.46 ± 0.6 mmol/L and 5.46 ± 0.64 mmol/L in tubes stored at RT for 1, 2 and 4 hours, respectively. Mean biases for FC-Mixture glucose in all time points reached optimal analytical goals. In the second study, the biases for LH and FH glucose compared to reference FC-Mixture glucose exceeded the preset analytical goals, regardless of the blood collection to centrifugation time interval.

Conclusions

FC-mixture tubes glucose concentrations were preserved up to 4h storage at RT. We confirmed that NaF alone does not allow immediate glycolysis inhibition in real life pre-centrifugation storage conditions (up to 340 minutes). FC-Mixture should be used exclusively for glucose determination in laboratories unable to implement the recommended blood samples’ treatment.Key words: glucose, pre-analytical phase, sodium fluoride, citrate acidification, stability  相似文献   

12.

Introduction:

The contamination of serum or lithium heparin blood with ethylenediaminetetraacetic acid (EDTA) salts may affect accuracy of some critical analytes and jeopardize patient safety. The aim of this study was to evaluate the effect of lithium heparin sample contamination with different amounts of K2EDTA.

Materials and methods:

Fifteen volunteers were enrolled among the laboratory staff. Two lithium heparin tubes and one K2EDTA tube were collected from each subject. The lithium-heparin tubes of each subject were pooled and divided in 5 aliquots. The whole blood of K2EDTA tube was then added in scalar amount to autologous heparinised aliquots, to obtained different degrees of K2EDTA blood volume contamination (0%; 5%; 13%; 29%; 43%). The following clinical chemistry parameters were then measured in centrifuged aliquots: alanine aminotranspherase (ALT), bilirubin (total), calcium, chloride, creatinine, iron, lactate dehydrogenase (LD), lipase, magnesium, phosphate, potassium, sodium.

Results:

A significant variation starting from 5% K2EDTA contamination was observed for calcium, chloride, iron, LD, magnesium (all decreased) and potassium (increased). The variation of phosphate and sodium (both increased) was significant after 13% and 29% K2EDTA contamination, respectively. The values of ALT, bilirubin, creatinine and lipase remained unchanged up to 43% K2EDTA contamination. When variations were compared with desirable quality specifications, the bias was significant for calcium, chloride, LD, magnesium and potassium (from 5% K2EDTA contamination), sodium, phosphate and iron (from 29% K2EDTA contamination).

Conclusions:

The concentration of calcium, magnesium, potassium, chloride and LD appears to be dramatically biased by even modest K2EDTA contamination (i.e., 5%). The values of iron, phosphate, and sodium are still reliable up to 29% K2EDTA contamination, whereas ALT, bilirubin, creatinine and lipase appear overall less vulnerable towards K2EDTA contamination.  相似文献   

13.
The quality control sera commonly available in market does not provide the value of CKMB. The CKMB kit of Randox laboratories contains two lyophilized control sera. But the stability of the control serum after reconstitution with 2ml of distilled water is 5 days at 2–8 degree Celsius, 8hrs at 25 degree Celsius and 4 weeks at-20 degree Celsius when frozen once. Hence stability after reconstitution is not sufficient to fulfill the daily need of a laboratory. In quest of a good internal quality assessment (IQA) sample trial run has been performed at 37 degree Celsius using external quality assessment (EQA) samples obtained from Randox international quality assessment sample (RIQAS). The trial run was found to be successful.  相似文献   

14.
Currently available method(s) for assaying pyrroline-5-carboxylate (P5C), an important intermediate metabolite of ornithine, proline and glutamate metabolic pathways, are cumbersome or not sensitive enough for microanalysis. The present study involving the synthesis of P5C followed by purity check, molecular mass (amu =113.1) determination by mass spectrometry and spectral characterization of P5C-ninhydrin derivative (λ max: 510 nm) confirmed the authenticity of the preparation. Studies on the effect of pH on spectral characteristics of P5C ninhydrin derivative demonstrated a significant change with respect to λ max (620 nm) and several ∼ 12 fold increase in molar extinction coefficient (ε: 1.96 × 105) in alkaline conditions (pH:7.0–8.0) as compared to the reported Molar ε of 1.65 × 104 at max λ 510 nm in ethanolic solution. The modified method, with the improved sensitivity, is adopted for the assay of ornithine amino transferase activity in WBC’s/platelets lysate(s) from human blood.  相似文献   

15.
As the oxygen tension of inspired air falls with increasing altitude in normal subjects, hyperventilation ensues. This acute respiratory alkalosis, induces increased renal excretion of bicarbonate, returning the pH back to normal, giving rise to compensated respiratory alkalosis or chronic hypocapnia. It seems a contradiction that so many normal people at high altitude should permanently live as chronic acid–base patients. Blood gas analyses of 1,865 subjects at 3,510 m, reported a P aCO2 (arterial carbon dioxide tension ± SEM) = 29.4 ± 0.16 mmHg and pH = 7.40 ± 0.005. Base excess, calculated with the Van Slyke sea level equation, is −5 mM (milliMolar or mmol/l) as an average, suggesting chronic hypocapnia. THID, a new term replacing “Base Excess” is determined by titration to a pH of 7.40 at a P aCO2 of 5.33 kPa (40 mmHg) at sea level, oxygen saturated and at 37°C blood temperature. Since our new modified Van Slyke equations operate with normal values for P aCO2 at the actual altitude, a calculation of THID will always result in normal values—that is, zero.  相似文献   

16.
This study was undertaken to evaluate the role of serum neuron specific enolase (NSE) in prediction of disability and neurological worsening in hypertensive ischemic cerebrovascular stroke. 80 hypertensive ischemic stroke patients diagnosed by a neurologist as per WHO definition along with radiological findings suggestive of cerebrovascular stroke and differentiating from hemorrhagic stroke and 60 controls having essential hypertension coming to hospital because of regular checkup or headache but with no neurological disease were included in the study. Neurological disability was assessed by NIHSS at the time of admission (within 72 h from the onset of stroke) and on 7th day after admission and cases were categorized into mild, moderate and severe disability. Venous blood samples were drawn within 72 h from the onset of symptoms. The samples were processed as per the laboratory protocol. The serum NSE samples were analyzed using an enzyme immunoassay based on the sandwich technique. We observed raised serum NSE in hypertensive ischemic stroke (17.4 ± 5.4 ng/ml) with significant association between different hypertensive groups than in hypertensive controls (9.1 ± 0.75 ng/ml). Greater degree of disability was observed in hypertensive stroke patients with raised serum NSE and hypertensive patients with mean serum NSE level of 22.9 ± 3.6 ng/ml and dyslipidemia had greater probability of neurological worsening as compared to those with mean serum NSE level of 12.7 ± 1.2 ng/ml. Serum NSE levels can serve as a peripheral indicator of neuronal damage and assist in the prediction of disability and clinical outcome in hypertensive cerebrovascular ischemic stroke patients.  相似文献   

17.
This study evaluated the types and frequencies of pre-examination errors recorded in the chemical pathology laboratory at the University Hospital of the West Indies, Jamaica. This was a retrospective analysis of errors recorded over a three year period. Data analysis was done on an average of 519,084 samples collected and tested per year. Samples included blood, urine, stool and other fluids. Pre-examination errors were identified and recorded following visual inspection of the samples and corresponding request forms by laboratory staff, then subsequently by the Senior Medical Technologist. Errors were generally classified as inappropriate sample (58 %), inappropriate form (23.4 %), inappropriate sample volume (9.3 %) and inappropriate sample tube (9.3 %). Over 90 % of recorded pre-examination errors were related to blood samples while urine samples accounted for 6.8 % error. Pre-examination errors were lower at this study location than elsewhere. Measures aimed at reducing instances of these errors are recommended for improved laboratory quality output.  相似文献   

18.

Background:

Presently the necessity of fasting time for coagulation tests is not standardized. Our hypothesis is that this can harm patient safety. This study is aimed at evaluating whether a light meal (i.e. breakfast) can jeopardize laboratory coagulation tests.

Materials and methods:

A blood sample was firstly collected from 17 fasting volunteers (12 h). Immediately after blood collection, the volunteers consumed a light meal. Then samples were collected at 1, 2 and 4 h after the meal. Coagulation tests included: activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (Fbg), antithrombin III (AT), protein C (PC) and protein S (PS). Differences between samples were assessed by Wilcoxon ranked-pairs test. The level of statistical significance was set at P < 0.05. Mean % differences were determined and differences between and baseline and 1, 2 and 4h samples were compared with reference change value (RCV).

Results:

A significantly higher % activity of AT was observed at 1 h and 4 h after meal vs. baseline specimen [113 (104–117) and 111 (107–120) vs. 109 (102–118), respectively; P = 0.029 and P = 0.016]. APTT at 2 h was found significantly lower than baseline samples [32.0 (29.9–34.8) vs. 34.1 (32.2–35.2), respectively; P = 0.041]. The results of both Fbg and PS tests were not influenced by a light meal. Furthermore, no coagulation tests had significant variation after comparison with RCV.

Conclusion:

A light meal does not influence the laboratory coagulation tests we assessed, but we suggest that the laboratory quality managers standardize the fasting time for all blood tests at 12 hours, to completely metabolize the lipids intake.  相似文献   

19.
IntroductionA specific sequence is recommended for filling blood tubes during blood collection to prevent erroneous test results due to carryover of additives. However, requirement of this procedure is still debatable. This study was aimed to investigate the potassium ethylenediaminetetraacetic acid (K-EDTA) contamination in blood samples taken after a tube containing the additive during routine workflow. The study was also carried out to examine the effect of order of draw on potassium results, regardless of K-EDTA contamination.Materials and methodsIn 388 outpatients, to determine the probability of K-EDTA cross-contamination, blood was drawn sequentially into a serum tube, followed by a tube containing K-EDTA, and by another serum tube. In another 405 outpatients, to evaluate the effect of order of draw blood unrelated to K-EDTA contamination, two serum tube were successively collected. Potassium was measured on Cobas 6000 c501 analyser (Roche Diagnostic GmbH, Mannheim, Germany) by indirect ion selective electrode method.ResultsOf paired samples collected before and after a K-EDTA tube, 24% had a potassium difference of above 0.3 mmol/L. However, no EDTA contamination was detected in these samples as well as 95% confidence intervals (CI) of limits of agreement for calcium were within the allowable error limits based on reference change values. Interestingly, of blood samples drawn successively, 24% had also a difference greater than 0.3 mmol/L for potassium.ConclusionIncorrect order of draw using closed blood collection system does not cause K-EDTA contamination, even in routine workflow. However, regardless of K-EDTA contamination, order of draw has significant influence on the potassium results.  相似文献   

20.
Our study aimed at comparing lead and zinc protoporphyrin (ZPP) levels in capillary and venous blood samples in a small population and to employ an easier method of sample collection for a major screening program in school children in major Indian cities. An awareness program on lead and its effects was conducted in two different schools. A total of thirty urban school children from South India, with an age group between 4–12 years consented for dual blood sampling and reported for the study. Venous and capillary blood samples were obtained simultaneously. Blood lead and zinc protoporphyrin (ZPP) levels were estimated using ESA Lead Analyzer and Haematofluorometer respectively. A significant correlation between capillary and venous ZPP (r=0.98) and lead (r=0.99) was observed. Rank sum test showed that there is no statistically significant difference between capillary and venous ZPP (P=0.891) and lead (P=0.672) values. This pilot study recommends that screening for lead may be done using capillary blood samples since significant correlation is observed between capillary and venous blood measurements. Obtaining samples using this mode is a non-invasive, less expensive, quick and easy method in children. Appropriately performed capillary sampling may be considered as an acceptable alternative to venipuncture for screening of blood for lead poisoning both in children and adults.  相似文献   

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