Ethylenediaminetetraacetic Acid Tubes Research Articles

Distribution of Genetic Polymorphisms of Genes Implicated in Thiopurine Drugs Metabolism.

Thiopurine-S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) are crucial enzymes involved in the metabolism of thiopurine drugs. Significant interethnic variation in the expression of TPMT and ITPA is caused by single nucleotide polymorphisms of genes encoding these proteins. The aim of this study was to describe the distribution of TPMT and ITPA polymorphisms in healthy Tunisian subjects and to establish the metabolizer status of thiopurine drugs in this population. A total of 309 healthy Tunisian subjects were recruited among blood donors of Fattouma Bourguiba Hospital of Monastir. A written informed consent was obtained from all subjects. Whole blood samples were collected from every subject in ethylenediaminetetraacetic acid tubes. TPMT (c.238 G > C, c.460 G > A and c.719A > G) and ITPA (c.94C > A and IVS2+21A > C) mutations were genotyped using polymerase chain reaction-restriction fragment length polymorphism. The observed frequencies of TPMT*3A and TPMT*3C alleles were both 0.8%. The phenotype distribution of TPMT was bimodal: 96.8% of subjects were extensive metabolizers and 3.2% were intermediate metabolizers. Genotyping of ITPA revealed frequencies of 9% and 3% for IVS2+21A > C and c.94C > A mutations, respectively. Accordingly, a trimodal phenotype distribution was found: 75.4% of the subjects were extensive metabolizers, 23.4% were intermediate metabolizers, and 1.2% wereslow metabolizers. Combination of TPMT and ITPA genotyping has revealed that a quarter of the Tunisian Population carries polymorphisms that reduce the metabolic activities of these enzymes.

Ethylenediaminetetraacetic acid (EDTA) anticoagulant affects the refractometric measurement of total protein concentration in equine synovial fluid

SummaryThe objectives of this study were to (i) determine the effect of ethylenediaminetetraacetic acid (EDTA) concentration on the refractometric measurement of the total protein (TP) concentration of equine synovial fluid and (ii) assess the effect of time and temperature on the refractometric measurement of TP concentration. Synovial fluid was retrieved aseptically from horses presenting to Liphook Equine Hospital either (a) immediately after euthanasia or (b) from those requiring arthrocentesis as part of their clinical investigation. Samples were aliquoted into commercially available (i) plain tubes (0.5 mL; control) and (ii) EDTA tubes (at different volumes to obtain serial dilutions of EDTA:synovial fluid). TP concentration was measured by two independent observers using a calibrated hand‐held refractometer. Samples from a separate group of horses were stored for 24 h at 4°C and 20°C before analysis to assess the effects of time and temperature. Results were compared using repeated measures ANOVA. TP concentration was significantly higher at all EDTA concentrations compared to control samples, with the greatest effect seen with higher concentrations (P<0.0001 for samples with ≥3.17 mg EDTA/mL). Storage time or temperature did not significantly affect TP concentration. Refractometric measurement of small volumes of synovial fluid in commercially available EDTA tubes leads to artificially elevated TP concentrations. Delays in sample analysis and alterations in storage conditions did not significantly affect TP concentration within the parameters studied. The magnitude of overestimation of synovial total protein concentration could result in misclassification of suspect synovial sepsis cases where EDTA tubes are used.

The Effects of Storage and Additives on Postmortem HbA1c Measurements.

HbA1c is used in forensic toxicology to identify undiagnosed diabetes mellitus (DM) and those with poor glycemic control prior to death. HbA1c is typically measured in whole blood collected in tubes containing ethylenediaminetetraacetic acid (EDTA). The effect of other additives, including sodium fluoride (NaF), is unclear. Furthermore, the assessment of short- and long-term stability of HbA1c has produced conflicting results. In this study, we collected paired postmortem blood samples in EDTA and NaF tubes (n=142) to assess their comparability for HbA1c measurement. Stability was assessed by measuring HbA1c at baseline, 2, 3, and 4weeks postcollection (stored at 4°C) and at 2, 4, 6, and 12months postcollection (stored at -20°C). We found no significant difference in HbA1c between the two preservatives at any of the time points indicating NaF is a suitable preservative for HbA1c measurement. We also determined that DM status, postmortem interval, and decomposition had no effect on stability.

Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays.

Background A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles. Methods Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 °C or 22 °C for 1-36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at -80 °C and thawing at 22 °C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins. Results Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 °C or 22 °C, respectively. Some increases became noticeable after 8 h delay at 4 °C but already after 1 h at 22 °C. For samples stored at 4 °C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 °C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 °C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles. Conclusions Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Implication of foam sclerosant inactivation by human whole blood in a laboratory setting.

Background During sclerotherapy, it has been recommended to confirm intravenous placement of the needle by aspirating blood into the sclerosant syringe. This may inactivate some, or all of the sclerosant. Aims To quantify the volume of human blood needed to completely inactivate 1 ml of sodium tetradecyl sulphate, and comparing fresh blood and blood that has been stored in an ethylenediaminetetraacetic acid tube. Methods A series of manual titrations were carried out following a procedure developed at STD Pharmaceutical Products Ltd (Hereford, UK) and listed in the British Pharmacopeia. Three percent of sodium tetradecyl sulphate stock solutions were made with increasing volumes of blood and titrated against benzethonium chloride to determine the active concentration (% w/v) of sodium tetradecyl sulphate remaining in the solution. Results A calculated approximation showed 0.3 ml of blood is required to fully inactivate 1 ml of 3% sodium tetradecyl sulphate when made into a foam. A comparison was made between the use of fresh blood and blood stored in ethylenediaminetetraacetic acid tubes. Blood stored in ethylenediaminetetraacetic acid tubes showed more inactivation of sodium tetradecyl sulphate, but this was not significant at the P ≤ 0.05 level. Conclusion The data from our study have shown that a minimum of 0.3 ml of fresh blood is required to inactivate 1 ml of 3% sodium tetradecyl sulphate as a foam and it is not significantly affected by storing blood in an ethylenediaminetetraacetic acid tube. Our methodology suggests that during foam sclerotherapy treatment, blood should not be aspirated into the syringe to confirm position, and that ultrasound guidance is more appropriate for needle placement.

CTC-mRNA (AR-V7) Analysis from Blood Samples-Impact of Blood Collection Tube and Storage Time.

Circulating tumour cells (CTCs) are an emerging resource for monitoring cancer biomarkers. New technologies for CTC isolation and biomarker detection are increasingly sensitive, however, the ideal blood storage conditions to preserve CTC-specific mRNA biomarkers remains undetermined. Here we tested the preservation of tumour cells and CTC-mRNA over time in common anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) and acid citrate dextrose solution B (Citrate) blood tubes compared to preservative-containing blood tubes. Blood samples spiked with prostate cancer cells were processed after 0, 24, 30, and 48 h storage at room temperature. The tumour cell isolation efficiency and the mRNA levels of the prostate cancer biomarkers androgen receptor variant 7 (AR-V7) and total AR, as well as epithelial cell adhesion molecule (EpCAM) were measured. Spiked cells were recovered across all storage tube types and times. Surprisingly, tumour mRNA biomarkers were readily detectable after 48 h storage in EDTA and Citrate tubes, but not in preservative-containing tubes. Notably, AR-V7 expression was detected in prostate cancer patient blood samples after 48 h storage in EDTA tubes at room temperature. This important finding presents opportunities for measuring AR-V7 expression from clinical trial patient samples processed within 48 h—a much more feasible timeframe compared to previous recommendations.

Pseudothrombocytopenia induced by incubation at 37 ℃ in ethylenediaminetetraacetic acid tubes

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant currently used for cell blood counts. Several studies showed that exposure of platelets (PLTs) to EDTA for a short period of time promote tyrosine phosphorylation of certain proteins, events that are related to activation. Spuriously low PLT counts were observed in a patient with cold agglutinins after the blood being warmed at 37 ℃ in EDTA tubes. Some cloudy polymers were found on peripheral blood smear after incubation at 37 ℃ for 15 minutes. These data suggest that PLT were activated after being warmed to 37 ℃ with EDTA. This process initiated coagulation and generated fibrin and PLT polymers from PLT agglutination and release. This case report hence describes a very rare phenomenon, which produces a spuriously low PLT counts caused by warming of EDTA tubes at 37 ℃.

The frequency of aberrant lymphoid antigens expression in 202 Iraqi patients with de novo acute myeloid leukemia

Background: Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered, particularly useful for identifying acute myeloid leukemia (AML) with lymphoid marker expression. The incidence of the aberrant phenotypes in AML is still controversial; incidences as high as 88% have been reported. Objectives: To evaluate the occurrence of aberrant lymphoid phenotypes and to correlate their presence with various French-American-British classification (FAB subtypes), 202 cases of newly diagnosed AML were analyzed for lymphoid markers CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, and CD79a. Materials And Methods: Whole blood or bone marrow aspirate of 202 patients with de novo AML was collected in ethylenediaminetetraacetic acid tube and analyzed by flow cytometry using a large panel of fluorochrome-labeled monoclonal antibodies. Identification of blast cells was performed using forward scatter versus side scatter (SSC) parameters and CD45 intensity versus SSC dot plots. An antigen was considered positively expressed when at least 20% of the gated cells expressed that antigen. Results: Eighty-five patients (42%) with de novo AML expressed lymphoid-associated antigens. All AML subtypes demonstrated lymphoid-associated antigens except M7. T-cell aberrancy was the most common comprising 32.2% of the total aberrancy. The most frequently lymphoid antigen aberrantly expressed was CD7 (25.7%), followed by CD4 (22.4%) and CD19 (7.9%). Conclusion: A large number of AML cases showed aberrant lymphoid phenotypes. These lymphoid phenotypes might be associated with different leukemia subtypes. T-cell markers are more common than B-cell markers. CD7 was the most common lymphoid marker aberrantly expressed in AML.

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

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

Diagnosing Salmonella enterica Serovar Typhi Infections by Polymerase Chain Reaction Using EDTA Blood Samples of Febrile Patients From Burkina Faso.

Globally, there are an estimated 22 million cases of Salmonella enterica serovar Typhi infection each year. However, this figure is likely to be an underestimate due to the low sensitivity of blood culture in S. Typhi diagnosis. The aim of this study was to diagnose S. Typhi by conventional polymerase chain reaction (PCR) using patient's blood preserved with ethylenediamine tetraacetic acid (EDTA). From April 2012 to September 2013, typhoid fever surveillance was conducted in Polesgo and Nioko, 2 dry slum areas in Ouagadougou, Burkina Faso. Blood culture was performed for febrile patients using an automated blood culture system. Additional blood was collected in EDTA tubes from those patients and preserved at -80°C. DNA was extracted from EDTA blood and PCR was performed to identify presence of S. Typhi. Randomly selected PCR products were further sequenced to identify S. Typhi-specific amplicons. Of 1674 patients, S. Typhi was isolated from 18 (1.1%) individuals by blood culture. EDTA blood was collected from 1578 patients, of which 298 EDTA samples were tested by PCR. Salmonella Typhi-specific DNA was identified in 44 (14.8%) samples. The sensitivity of S. Typhi-specific PCR from EDTA blood was 89% (74%-100%) among the blood culture-positive cases. Sixteen S. Typhi-positive PCR products were sequenced, and 13 retrieved the sequence of a S. Typhi-specific amplicon. These findings suggest that blood culture-based diagnoses of S. Typhi underestimate the burden of typhoid fever in Burkina Faso. PCR could be considered as an alternative method for the identification and diagnosis of S. Typhi in blood samples.

Evaluation of Glucose-6-Phosphate Dehydrogenase stability in stored blood samples.

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the commonest cause of neonatal jaundice in Malaysia. Recently, OSMMR2000-D G6PD Assay Kit has been introduced to quantitate the level of G6PD activity in newborns delivered in Universiti Kebangsaan Malaysia Medical Centre (UKMMC). As duration of sample storage prior to analysis is one of the matters of concern, this study was conducted to identify the stability of G6PD enzyme during storage. A total of 188 cord blood samples from normal term newborns delivered at UKMMC were selected for this study. The cord bloods samples were collected in ethylene-diamine-tetra-acetic acid (EDTA) tubes and refrigerated at 2-8 °C. In addition, 32 out of 188 cord blood samples were spotted on chromatography paper, air-dried and stored at room temperature. G6PD enzyme activities were measured daily for 7 days using the OSMMR2000-D G6PD Assay Kit on both the EDTA blood and dried blood samples. The mean value for G6PD activity was compared between days of analysis using Student Paired T-Test. In this study, 172 out of 188 cord blood samples showed normal enzyme levels while 16 had levels corresponding to severe enzyme deficiency. The daily mean G6PD activity for EDTA blood samples of newborns with normal G6PD activity showed a significant drop on the fourth day of storage (p < 0.005) while for samples with severely deficient G6PD activity, significant drop was seen on third day of storage (p = 0.002). Analysis of dried cord blood showed a significant reduction in enzyme activity as early as the second day of storage (p = 0.001). It was also noted that mean G6PD activity for spotted blood samples were lower compared to those in EDTA tubes for all days (p = 0.001). Thus, EDTA blood samples stored at 2-8 °C appeared to have better stability in terms of their G6PD enzyme level as compared to dried blood samples on filter paper, giving a storage time of up to 3 days.

Monocyte Expression Levels of Cytokines in Patients With Fibromyalgia Syndrome

Objectives: This study aims to determine monocyte expression levels of interleukin 1 beta (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha in fibromyalgia syndrome (FMS) patients and healthy females. Patients and methods: A total of 80 female FMS patients and 50 healthy females were evaluated. Twelve of the FMS patients who did not meet the 1990 American College of Rheumatology criteria and 10 healthy controls were excluded. After exclusion 68 participants as FMS patient (mean age 41.41±7.14 years; range 18 to 55 years) and 40 participants as control group (mean age 39.12±10.9 years; range 18 to 55 years) were included in the study. For both groups, a detailed form was filled out which included information on age, body mass index, marital status, educational status, visual analog scale, fibromyalgia impact questionnaire, Hamilton anxiety scale, Hamilton depression scale, modified fatigue impact scale, and Nottingham health profile. Whole blood peripheral blood monocyte cells were drawn in ethylenediaminetetraacetic acid tubes from patient and control groups between 08:30 to 9:30 hours. Tumor necrosis factor alpha, IL-6, IL-8, and IL-1β expressions were evaluated using fluorescence-activated cell sorting flow cytometry device. Results: No statistically significant difference was detected between the two groups in terms of average age, body mass index, and other demographic data (p>0.05). Visual analog scale, Hamilton anxiety scale, Hamilton Depression Scale, fibromyalgia impact questionnaire, modified fatigue impact scale total score and Nottingham health profile total-subscore values were statistically significantly higher in the patient group than the control group (p 0.05). Conclusion: Although FMS is not traditionally considered an inflammatory disorder, evidence for elevated inflammatory processes has been noted in some studies. Our results do not support any role of the inflammatory cytokines in the pathogenesis of FMS.

Preanalytical Stability of Gemcitabine and its Metabolite 2′, 2′-Difluoro-2′-Deoxyuridine in Whole Blood—Assessed by Liquid Chromatography Tandem Mass Spectrometry

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) and metabolite (2',2'-difluoro-2'-deoxyuridine, dFdU) quantification is warranted for individualized treatment strategies. Analyte stability is crucial for the validity of such quantification. We therefore studied the impact of the time interval from blood sampling to separation of plasma on gemcitabine stability. Blood from gemcitabine-treated patients was drawn into tetrahydrouridine (THU)-spiked heparin and ethylenediaminetetraacetic acid tubes and kept on ice until separation. Plasma was separated sequentially up to 24 h after sampling and dFdC and dFdU were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The change in plasma concentrations over time was compared with the highest imprecision for concentrations above the lower limit of quantification of the LC-MS/MS method. Analyte concentrations decreased slightly over time, but for samples stored for 4 h on ice, the decline was smaller than the expected analytical imprecision. After 24 h, the maximum decline was 14.0%, which exceeded the expected analytical imprecision. dFdC and dFdU stabilities were acceptable for at least 4 h when THU-spiked whole blood samples were kept on ice. This is within the scope of routine sampling procedures. Further, variations in separation time intervals within this time frame are negligible when interpreting drug concentrations.

Comparative study of the influence of EDTA and sodium heparin on long term storage of cattle DNA.

Blood collection in heparin tubes for cytogenetic, and ethylenediaminetetraacetic acid (EDTA) tubes for molecular genetics applications respectively, are routine practices everywhere. If blood samples are required for cytogenetics as well as DNA work, two samples from each animal are usually collected, which leads to wastage of time and money. The present study tried to explore the possibilities of collecting a single blood sample in a heparinised tube for use in both applications. Two blood samples were collected from the same animals; one in a heparin tube and the other in an EDTA tube. DNA was extracted and stored at the same temperature and for the same durations. Comparative studies revealed that the DNA samples extracted from blood using these two different coagulants give more or less the same quality of results especially for polymerase chain reaction (PCR) based applications in cattle. The purpose of the present study was to establish the possibility of using heparin blood for chromosomal studies as well as for molecular biology. Such a practice will obviously save time and money in collecting samples in duplicate.

Relevance of EDTA carryover during blood collection.

The order of draw is regarded as a preanalytical issue to prevent carryover of additives during blood collection. Our objective was to prove the theory of ethylenediaminetetraacetic acid (EDTA) carryover for a closed vacuum system and the influence of EDTA on concentrations of selected biomarkers. To test the carryover of EDTA, a blood collection with tripotassium EDTA (K3EDTA) and subsequent non-additive tubes was simulated using distilled water as substitute for blood. EDTA concentrations were measured by tandem mass spectrometry. Then we added increasing concentrations of EDTA to heparinized blood and measured routine biomarkers, thereby simulating a carryover of EDTA whole blood and pure EDTA, respectively. Additionally, we tested for EDTA contamination and biomarker alteration in samples collected from 10 healthy volunteers by a syringe with subsequent transfer into sample tubes. No EDTA contamination was detected in samples collected subsequent to a K3EDTA tube when adhering to guidelines of blood sampling. Magnesium, calcium, and potassium levels were altered by artificial K3EDTA whole-blood contamination as well as when adding 1 μL pure K3EDTA. Iron values were altered at EDTA concentrations of 4.4 mmol/L. All other parameters remained unaffected. A slight EDTA carryover was observed in syringe collection and subsequent transfer into EDTA and heparin tubes, however, without any biomarker alteration. An EDTA carryover during blood collection using a closed vacuum system is highly unlikely. Even if carryover of EDTA whole blood occurs, an absolute volume larger than 10 μL would be necessary to alter test results. However, contamination of samples with preloaded pure K3EDTA solution by severe neglect of current recommendations in blood collection may significantly alter testing results.

Detection of sCD21 in Human Blood Using ELISA is Influenced by Ethylenediaminetetraacetic Acid (EDTA) Containing Blood Collection Tubes.

CD21 is a 145 kDa membrane glycoprotein expressed mainly on B-cells and follicular dendritic cells. It is involved in activation, survival and proliferation of B-cells. CD21 can be cleaved to give soluble CD21 (sCD21), which is constantly shed in healthy people whereas the level of sCD21 is low in patients suffering from various autoimmune diseases. Blood was collected from 12 healthy donors into Heparin, EDTA and gel-separation tubes. Furthermore, plasma from 6 healthy donors was combined and EDTA or EGTA was subsequently added to analyze its influence on sCD21 levels measured by ELISA. Differences in sCD21 levels were observed dependent on blood collection tubes used. sCD21 levels measured by ELISA were significantly higher if heparin/gel containing tubes were used compared to EDTA blood collection tubes. Upon addition of EDTA/EGTA to plasma drawn using Heparin blood collection tubes, sCD21 levels decrease to amounts found in EDTA-containing blood collection tubes suggesting calcium as the responsible factor rather than magnesium. EDTA influences sCD21 levels determined by ELISA and therefore sCD21 measurements should be carried out using one type of blood collection tube throughout the experiment/medical examination.

The authors' reply.

We thank Dr. Kayadibi and colleagues (1) for their comments regarding our paper on hyperhomocysteinemia. (2) As it is known, vitamin B9, also called folate or folic acid, is one of the eighth forms of vitamin B. (3, 4) So, in the current study by Monfared et al. (2), folic acid is obviously considered as one of the forms of vitamin B, so the authors did not separately mention folic acid, and it is considered as an exclusion criteria. In addition, if despite adequate vitamin supplementation, adequately lowering plasma homocysteine has failed, the patients should be evaluated for vitamin deficiency, renal impairment, and thyroid dysfunction. It should be remembered that a (low) “normal” vitamin B12 level does not exclude intracellular vitamin B12 deficiency. Serum methylmalonic acid (MMA) and holotranscobalamin II levels are more reliable markers of vitamin B12 deficiency compared to the serum vitamin B12 concentration (5). Although Solomon demonstrated that high serum folate levels (>20 ng/mL) are associated with higher MMA, at the same time, homocysteine levels and cobalamin levels (201–300 pg/mL) are low normal. It should be mentioned that its effect is age dependent (≥60 years). In the case of having a normal range of serum folate, the effects would not progress (suggesting a threshold effect), but it would be reversed by cobalamin therapy (6). In our study, hyperhomocysteinemic patients had a mean folate level of 16.26±4.06 ng/mL (normal levels) with mid-normal cobalamin levels (405.56±169.28 pg/mL). It seems that, as Kayadibi et al. (1) proposed, evaluation of the functional deficiency of vitamin B12 by means of MMA, as an early functional marker of tissue vitamin B12 deficiency, could provide more accurate results. Further studies should consider this point. It is recommended by Kayadibi and colleagues (1) that multiple linear regression analysis should be performed with sex adjustment to eliminate tHcy differences that may be because of sex differences, so homogeneous distribution of sex would be achieved. As it is shown, in the univariate analysis, a significant association was found between tHcy and age (P=0.02), male sex (P=0001), serum creatinine (P=0.0001), blood urea nitrogen (P=0.0001), estimated creatinine clearance (P=0.001), the history of glomerulonephritis before transplantation (P=0.05), body mass index (P=0.02), serum uric acid (P=0.003), serum folate (P=0.0001), cyclosporine A dose (P=0.01), and CsA trough level (C0; P=0.02) (2). Then, in the multivariate analysis, multiple linear regression models were used by stepwise (entry=0.05, removal=0.1) method for determining associated factors of Hcy levels after adjusting all the demographic (including sex) and clinical variables (2). In response to tHcy analysis, fasting blood sample was collected into an ethylenediaminetetraacetic acid tube which was used for measuring plasma homocysteine of each patient. To separate the plasma, the blood sample was centrifuged immediately after collecting. Then the separated plasma was deeply frozen (−20°C). By this method, we tried to minimize the production and release of tHcy by red blood cells after obtaining the blood sample. (2) Congruent to the conclusion of Kayadibi and colleagues, we considered that measuring MMA, in addition to tHCY, folate, and vitamin B12, could reveal more accurate results. Ali Monfared 1 Seyyede Zeinab Azimi1 Ehsan Kazemnezhad1 Masoud Khosravi1 Mohammadkazem Lebadi1 Ebrahim Mirzajani2 Mohammad Najafi Ashtiani2 1 Urology Research Center School of Medicine Guilan University of Medical Sciences Rasht, Iran 2 School of Medicine Guilan University of Medical Sciences Rasht, Iran

Assessment of free fetal DNA concentration in maternal plasma during the first trimester of pregnancy: comparative study between EDTA and PPT tubes – pilot study

Objective: To compare ethylenediamine tetraacetic acid (EDTA) tubes and plasma preparation tubes (PPT) for evaluating maternal plasma during the first trimester of pregnancy.Methods: A cross-sectional study was conducted on 24 male fetuses in women between 6 and 14 weeks of pregnancy. Blood samples (10 mL) were collected and stored in EDTA and PPT tubes. Subsequently, the samples were centrifuged and sent for free fetal DNA extraction by means of the polymerase chain reaction (PCR) technique. The reactions were performed in a real time PCR machine for detecting the amplification products. The genome region chosen for performing the PCR reactions was a target specific for the Y chromosome, in which the DYS-14 marker was amplified only when the DNA was of male sex. The free fetal DNA concentration was given by the threshold cycle (TC). To compare the tubes, the paired Student t-test was used.Results: The mean gestational age was 11.08 ± 2.30 weeks (range: 6–14). The mean TC for PPT was 30.08 ± 1.05 (range: 27.08–32.61) and for EDTA, 30.23 ± 0.96 (range: 28.01–32.09), but without statistical significance (p = 0.357).Conclusion: We did not observe any statistically significant difference in free fetal DNA concentration between the EDTA and PPT tubes.

Evaluation of Protease Inhibitors Containing Tubes for MS-Based Plasma Peptide Profiling Studies

Peptide profiling of biological fluids is a promising tool for biomarker discovery. Blood is an ideal entity for proteomic studies but it is subjected to a proteolytic activity that sets up just at the moment of phlebotomy. Intending to prevent this proteolytic activity, tubes containing protease inhibitors (PI) have been developed. In this study, we evaluated the effect on plasma peptide profile of using tubes containing PI and the evolution of this effect over time. Blood samples from ten subjects were drawn into conventional tubes containing ethylenediaminetetraacetic acid (EDTA) and tubes containing PI. Samples were processed at time "zero" and after 1, 2, 4, and 8 hr. Plasma peptide profiles were analyzed by magnetic bead based technology coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry readout. When comparing plasma peptide profile of blood samples collected into tubes containing PI with samples collected into conventional EDTA tubes, differences in the area of 13 peaks were detected at time "zero"; after 8 hr these differences tended to disappear. Moreover, bradykinin and C3- and C4-derived peptides were produced promptly after blood extraction when samples were collected into conventional EDTA tubes, and the use of PI prevented their generation. Considering that time taken to process blood samples affects their peptide profile and a decrease in PI's effect occurs over time, it may be concluded that the use of tubes containing PI for blood collection may be advantageous in the context of research, but may have some limitations regarding clinical practice.

Preanalysis Storage Conditions Influence the Measurement of Brain-Derived Neurotrophic Factor Levels in Peripheral Blood

Background: Brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays a pivotal role in regulating neuronal function throughout life, and this factor is regarded as a potential biomarker of mental disorders. However, previous studies have suggested that plasma BDNF levels are more variable than serum BDNF levels. Methods: We determined the influence of time and temperature on the measurement of peripheral blood BDNF levels. Blood samples were aliquoted into four types of tubes, including tubes containing heparin, ethylenediaminetetraacetic acid (EDTA), and citrate for plasma, and anticoagulant-free tubes for serum. The samples were stored at 4 or 25°C for 0, 1, 2, 4, 6, 24 or 48 h, and the plasma and serum BDNF levels were measured using enzyme-linked immunosorbent assay. Results: There were interindividual and interanticoagulant compound variability in the plasma BDNF levels. The measured plasma BDNF levels increased over time, whereas the serum BDNF levels remained unchanged. Furthermore, the BDNF levels detected in plasma stored in heparin tubes at 4°C and those for samples stored in EDTA tubes at 25°C were much higher than those of the other samples. Conclusion: This study indicates that measurements of plasma BDNF levels are dependent not only on the anticoagulant compounds but also on the storage time and temperature conditions used after blood sampling.