Ethylenediaminetetraacetic Acid Tubes Research Articles

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). In this study, we conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improves the stability of Aβ42 and Aβ40 across all approaches. However, the Aβ42/Aβ40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aβ42 and Aβ40, and the Aβ42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.

Trypanosoma evansi Infection in Sumba Horses in East Sumba Regency : A Study at BBVet Denpasar

Background: Sumba is one of the original habitats of the Sandalwood Ponies and the presence of pony is an important element for the community. The Sumbanese herd their ponies in the savanna and rely on nature as a source of horse feed, consequently, during the dry season, the availability of the feed decreases. These conditions can potentially reduce the health status of ponies and increase morbidity or mortality from diseases caused by Trypanosoma evansi. Purpose: This study aims to determine the occurrence and intensity of Trypanosoma evansi infection in blood samples from horses examined at the Denpasar Bali Veterinary Centre. Methods: Thirty blood samples were collected from East Sumba Regency. Three milliliters of blood were taken from the jugular vein and immediately transferred into an ethylenediaminetetraacetic acid tube. Thin blood smears were subsequently prepared and examined using the Giemsa-stained blood smears method. The preparations of blood smear were examined under microscope to determine the Trypanosoma evansi infection and the intensity was calculated on the average number of Trypanosoma evansi in 100 red blood cells. Results: The result showed that six out of 30 horses were tested positive for Trypanosoma evansi. The infection intensity ranged from two to 18 parasites with an average number of 8 in the blood smear examination method. Conclusion: The incidence of trypanosomiasis in Sumba horses varies with each season in East Sumba Regency. The prophylactic measures that should be adopted in the particular herd of horses because this is the most significant blood protozoan parasite of equines.

Evaluation of Growth-differentiation Factor-15 Level and few Physiological Parameters Patients with Diabetes Mellitus Type 2

The study included 90 subjects and is divided into two groups: The first group is the patient group, which consists of 45 patients with diabetes mellitus type two; 45 patients with diabetes mellitus type two were identified by specialized diabetes doctors by samples collection in outpatients clinic at Diabetes and Endocrine Center in Marjan Medical City in Hilla city, Babylon province from 1 November 2022 till 31 Januray 2023. The blood samples of this study were obtained from patients and controlled through drug 5 mL of blood by using medical sterile syringes from a brachial vein and placed in a gel tube and ethylenediaminetetraacetic acid (EDTA) tube. Then the gel tube was placed at room temperature for 30 minutes to coagulate the blood, and samples were centrifuged (3000 rpm/min) for 5 minutes to separate the serum from other components of the blood. The serum was withdrawn by micropipette and then placed in the eppendorf tubes in two repeaters and kept frozen at -20℃ for the determination of growth differentiation factor 15 (GDF 15), lipid profile and fasting blood glucose. The whole blood in the EDTA tube to determine HbA1C. The mean fasting blood sugar and hemoglobin A1c of diabetes patients is significantly higher at (p-value < 0.001) than that of controls. Diabetes patients had a significantly higher of serum lipid profile than the controls. Therefore, the mean of cholesterol, triglyceride, and low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) was highly significant (p < 0.001). The mean concentration of GDF-15 of patient groups was significantly increased than those matched groups of healthy control at (p < 0.001). In addition, the results also showed a highly significant increase (p < 0.001) in the mean of GDF-15 for the patient’s males and females in comparison with the control’s males and females. The correlation results indicated a statistically significant positive correlation in diabetic group between GDF-15 vs. FBS (r = 0.572, p = < 0.001) and HbA1c (r = 0.576, p = < 0.001). Positive non-significant correlations were demonstrated between GDF-15 vs. cholesterol (r = 0.131, p = > 0.05), triglyceride (TG) (r = 0.167, p = > 0.05), LDL (r = 0.38, p = > 0.05) and VLDL (r = 0.136, p = > 0.05). Negative non-significant correlation was between GDF-15 and high-density lipoprotein (HDL) (r = 0.137, p = > 0.05).

Evaluation of Collection and Processing Conditions for Gene Expression Analysis Using Human Myeloid Cells.

Background: The population of blast cells among peripheral blood mononuclear cells (PBMCs) obtained from patients is a desirable specimen for analyzing gene expression in diseases including acute myeloid leukemia. Although the enrichment of blast cells often needs to be performed at a central laboratory, acceptable conditions for sample transport from clinical sites remain to be established. Methods: We evaluated storage temperature, duration, and tube type before initiating sample processing for the analysis of cluster of differentiation (CD)33+ myeloid cells among PBMCs as an alternative to CD34+/CD33+ blast cells. Results: CD33+ myeloid cells were successfully purified by MACS. The cell viability and the RNA integrity were sustained during storage up to 48 hours before sample processing. Storage at 4°C had minimal effects on gene expression, whereas storage at room temperature induced the senescence pathway, characterized by the expression of stress-inducible genes. A CPT tube was also better than an ethylenediaminetetraacetic acid tube for minimizing gene expression change. Conclusions: Our study provided important clues for establishing a sample handling approach for gene expression analysis with purified cell fractions from human PBMCs. To keep the variation of gene expression to a minimum, samples should be delivered at 4°C within 48 hours before processing.

Kanamycin treated-ethylenediamine tetraacetic acid (EDTA)-dependent pseudothrombocytopenia in blood specimen of cats

Background: Pseudothrombocytopenia is a commonly obtained false negative result when analyzing feline platelet count by automated machine. It is related to ethylenediamine tetra-acetic acid (EDTA), a widely utilized anticoagulant in blood collection tube, resulting in EDTA-dependent pseudothrombocytopenia (EDTA-PTCP). Aim: To investigate whether treated with kanamycin enhanced the quantity of platelet aggregations in feline blood specimens collected using EDTA-PTCP. Methods: Thirty-one blood samples were obtained using EDTA tubes. The complete blood count was analyzed using automated Mindray-BC-5000Vet. Both Manual cell counts and thin blood smears were performed to estimate the amount of RBC, WBC and platelets as well as to evaluate the severity scores of platelet clumping, respectively. Comparisons were made between those pre-treated and treated with kanamycin in EDTA tube. Results: There were significantly different mean platelet counts in the samples before and after they were treated with kanamycin, both on automated (156.6  76.4 vs 260.3  115.5; p < 0.001) and manual (168.5  92.1 vs 262.8  119.6; p < 0.001) readings, with a 95% confidence interval (CI) of 0.19 (0.022–0.365). Conclusion: This study suggests that in clinical laboratory practice, kanamycin should be added to feline blood specimens with EDTA-PTCP.

Evaluating the reliability of the sickling test and peripheral blood film method for screening of sickle cell disease: a study protocol

Background: Mutations in beta-globin are the cause of a widespread condition known as sickle cell disease (SCD). Sickle cells cause organ damage, hemolysis of blood cells, weakness, and sometimes even death. SCD can be effectively managed and mortality can be reduced through early detection. This protocol’s study aims to evaluate the sensitivity and specificity of the sickling test, peripheral blood smear, and hemoglobin electrophoresis for SCD screening. Methods: In this study,75 cases of SCD will be selected. 5ml blood will be drawn into a dipotassium ethylenediaminetetraacetic acid tube for testing of SCD. Testing will involve: the sickling test, where an equal volume of K2EDTA blood will be mixed with 2% sodium metabisulfite; the peripheral blood smear, where Leishman’s stain will be placed on a thin film of blood for 2 minutes; and Hb electrophoresis, where equal parts of distilled water and packed cells will be combined and then the mixture will be centrifuged. Results: We will be comparing between the sickling test and the peripheral blood film method, and we will conclude depending upon the results as to which test is better. Conclusions: Using peripheral blood smear images, SCD can be quickly diagnosed. A peripheral smear-based differential diagnosis may be possible but requires special tests such as hemoglobin electrophoresis to confirm the diagnosis. Therefore, early diagnosis can help initiate transfusion therapy and create a better prognosis.

Exploring the role of ex vivo metabolism on blood and plasma measurements of oxytocin among women in the third stage of labour: A post hoc study.

To examine the role of ex vivo oxytocin metabolism in post-dose peptide measurements. The stability of oxytocin (Study 1) and oxytocinase activity (Study 2) in late-stage pregnancy blood was quantified using liquid-chromatography tandem mass-spectrometry (LC-MS/MS) and a fluorogenic assay, respectively. Analyses were conducted using blood from pregnant women (>36 weeks gestation) evaluated in lithium heparin (LH), ethylenediaminetetraacetic acid (EDTA) and BD P100 blood collection tubes with or without protease inhibitors. In addition, plasma oxytocin concentrations following administration of oxytocin 240 IU inhaled, 5IU intravenous or 10IU intramuscular in women in third stage of labour (TSL) were analysed using enzyme-linked immunosorbent assay (ELISA) and LC-MS/MS to understand how quantified peptide concentrations differ between these analytical methods (Study 3). Study 1: Oxytocin was stable in blood collected into EDTA tubes with or without protease inhibitors but not in LH tubes. Study 2: Blood collected into all EDTA-containing collection tubes led to near-complete inhibition of oxytocinase (≤100 min). In plasma, a 35% reduction in oxytocinase activity was observed in LH tubes with EDTA added. In plasma from late-stage pregnancy compared to nonpregnant participants, the oxytocinase activity was approximately 11-fold higher. Study 3: Plasma oxytocin concentrations from nonpregnant or women in TSL following exogenous oxytocin administration were ≤33 times higher when analysed using ELISA vs. LC-MS/MS methods. Collection of blood from late-stage pregnant women into tubes containing EDTA inhibits oxytocinase effectively stabilizing oxytocin, suggesting low concentrations of oxytocin after dose administration reflect rapid in vivo metabolism.

Blood Biomarkers of Neonatal Sepsis with Special Emphasis on the Monocyte Distribution Width Value as an Early Sepsis Index.

Background and Objectives: Early detection of neonatal sepsis is critical because it is potentially fatal. Therefore, sepsis biomarkers of sufficient sensitivity and specificity are needed. This study aimed to evaluate the utility of peripheral blood parameters as neonatal sepsis biomarkers and the diagnostic performance of the monocyte distribution width (MDW) in sepsis in a neonatal intensive care unit. Materials and Methods: A cross-sectional study was conducted from September 2019 to August 2020 at the King Saud University Medical City in Riyadh, Saudi Arabia. Samples were collected and organised as follows: 77 study cases were subdivided into two subgroups (other health complication (49) and sepsis (28)), and there were 70 controls. The causative microorganisms of neonatal sepsis were isolated. Peripheral blood samples were collected from each neonate in an ethylenediaminetetraacetic acid tube for a complete blood count and a leukocyte differential count. Moreover, the receiver operating characteristic (ROC) curve analysis was used to measure the diagnostic performance of the MDW. Results: The haematological parameters and neonatal sepsis cases had a considerable correlation. The MDW was the most significant haematological parameter. The ROC analysis of the MDW demonstrated that the area under the curve was 0.89 (95% confidence interval: 0.867 to 0.998) with a sensitivity of 89.3%, a specificity of 88.2%, and a negative predictive value of 97.2% at the cut-off point of 23. Conclusions: The use of haematological parameters is feasible and can be performed rapidly. Neonatal sepsis showed a strong correlation with leukopenia, anaemia, thrombocytopenia, and an elevated MDW value. Moreover, the ROC curve analysis confirmed the high diagnostic ability of the MDW in neonatal sepsis prediction.

Association of IL-17F Gene Polymorphism and Its Serum Level with SARS-CoV-2 Infection.

Although multiple studies have addressed the clinical outcomes of coronavirus disease, little data exist regarding the defi- nition of immune and inflammatory profiles associated with this infection. Its clinical manifestations often worsen in association with hypercytokinemia (elevated interleukin 8 and interleukin 17). We conducted this research to elucidate the effect of interleukin 17 levels and interleukin 17F gene polymorphism on the severity and outcomes of coronavirus disease. Ninety patients with confirmed coronavirus disease and 30 healthy controls were enrolled. Coronavirus disease cases were classified into nonsevere, severe, and critical according to the World Health Organization definition. Approximately 10 mL peripheral blood sample was collected from all patients and controls by venipuncture in-plane and ethylenediaminetetraacetic acid tube. Enzyme-linked immunosorbent assay kits were used for calculating serum interleukin 17 levels, whereas real-time polymerase chain reaction was used for genotyping using the 5'-nuclease allelic discrimination assay for single nucleotide polymorphisms genotyping. As regards interleukin 17 levels, there was a significant elevation of interleukin 17 in coronavirus disease cases compared to control healthy persons (P < .001). Moreover, serum interleukin 17 levels tended to be significantly higher with increased disease sever- ity (P = .004). Patients with critical diseases expressed a significant rise of interleukin 17 compared to severe (P = .03) and nonsevere cases (P = .02). We noted no significant difference between the critical, severe, and nonsevere cases regarding different interleukin 17F genotypes. Coronavirus disease is associated with elevated levels of interleukin 17, which tended to be considerably higher with disease severity. However, different interleukin 17F genotypes do not affect either the predisposition or the severity of coronavirus disease.

THE PREVALENCE OF THE THALASSEMIA TRAIT AMONG THE EAST MALAYSIAN STUDENTS IN THE FACULTY OF HEALTH SCIENCES, UNIVERSITI TEKNOLOGI MARA, UiTM SELANGOR

The thalassemia trait is an asymptomatic thalassemia syndrome that increases the risk of developing a more severe condition. This study aimed to determine the prevalence of the thalassemia trait among Sabah and Sarawak students enrolled in the Faculty of Health Sciences at Universiti Teknologi MARA, UiTM Selangor. The study was carried out over 3 months following approval from the ethics and research committees of Universiti Teknologi MARA, UiTM Shah Alam. A universal sampling method was used where the blood samples were collected from the 40 students, 16 from Sabah and 24 from Sarawak, of various ethnicities. All subjects were asked to fill out consent forms. A volunteer medical laboratory staff member assisted in taking whole blood samples from the subjects’ venous blood. The blood was then collected in a 3 mL EDTA (ethylenediaminetetraacetic acid) tube and was tested for thalassemia using a Full Blood Count (FBC) and a Full Blood Picture (FBP), as well as a Mentzer Index (MI) calculation, gel electrophoresis, and an H-inclusion test. In conclusion, the prevalence of the thalassemia trait is 7.5% (3) out of 40 volunteers among students from Sabah and Sarawak in the Faculty of Health Sciences at UiTM Selangor.

Growth traits, hematological, and ruminal fluid profile of sheep offered ensiled coffee skin replacing dried water spinach.

Developing simple, cost-efficient sheep feed will improve farmers' incomes. Including coffee skin in feed offers the most technical method of increasing sheep weight gain. This study aimed to evaluate varying proportions of ensiled coffee skin replacing dried water spinach and determine the optimal combination for the growth performance, physiological and hematological profiles, and rumen fluid of sheep. Eighty-four animals were randomly allocated to the treatments, arranged in a randomized block design using the initial weight as a block. Seven treatment diets were adjusted and a 12-animal replication was used for each treatment. The treatments were as follows: T0: 30% maize stover, 30% dried water spinach, 5% pollard, 20% coffee skin; T1: 30% maize stover, 25% dried water spinach, 5% pollard, 5% ensiled coffee skin; T2: 30% maize stover, 20% dried water spinach, 5% pollard, 10% ensiled coffee skin; T3: 30% maize stover, 15% dried water spinach, 5% pollard, 15% ensiled coffee skin; T4: 30% maize stover, 10% dried water spinach, 5% pollard, 20% ensiled coffee skin; T5: 30% maize stover, 5% dried water spinach, 5% pollard, 25% ensiled coffee skin; T6: 30% maize stover, 5% pollard, and 30% ensiled coffee skin. The sheep were reared for 70 days.The parameters observed during the early stage included growth performance (initial body weight, LW gain, final body weight, and feed intake). At the end of periods, a representative sample of ruminal fluid (approximately 150 mL) was collected from slaughtered sheep, duplicated, and then incubated for 18 h and blood samples were collected from the sheep (jugular vein) in ethylenediaminetetraacetic acid tubes. Then, used to analyze various blood biochemical parameters. The final body weights showed a linear curve increasing as the treatment increased (p < 0.05). The ensiled coffee skin tended to increase at 6 h incubation time, producing reduced methane gas (p < 0.05). However, in general, the use of ensiled coffee skin did not significantly alter the blood biochemistry of crossbreed sheep (p > 0.05). There was no significant effect on the protozoal population (p > 0.05). Increasing the level of ensiled coffee skin up to 30% replacing dried water spinach increased the final body weight of crossbreed sheep with no adverse effect.

Evaluation of sodium citrate anticoagulant for the Resolution of edta-dependent pseudo Thrombocytopenia.

EDTA-dependent pseudo thrombocytopenia (EDTA-PTCP) refers to a falsely low platelet count occurring in the presence of ethylene diamine tetra-acetic acid (EDTA) anticoagulant during blood sample collection, which results in the formation of platelet clumps in vitro. This phenomenon has significant clinical implications, including unnecessary administration of platelets. Our study aims to evaluate the efficacy of sodium citrate anticoagulant for the resolution of EDTAPTCP. This retrospective study was conducted in the haematology laboratory of Shifa International Hospital (SIH), Pakistan. Patients with pseudo thrombocytopenia (i.e. platelet count less than 150,000/ul with platelet clumps seen on peripheral smear) were included in this study if they had blood samples drawn in both EDTA and sodium citrate tubes less than 48 hours apart. Data was analyzed using IBM® SPSS Software Version 22. A total of 151 study participants were included in this study. The mean age was 48.95±20.69 years and the majority were female (52.3%). Wilcoxon signed-rank test showed that there was a statistically significant difference in platelet count measured in both tubes (Z = -3.223, p=0.001). Overall, blood samples processed in sodium citrate tubes showed lower platelet count than EDTA samples. Sodium citrate anticoagulant was able to correct EDTA-PTCP in 47 (31.1%) of the cases. Sodium citrate anticoagulant was only able to resolve one-third of our EDTA-PTCP cases. Our findings do not support the use of sodium citrate as a suitable alternative for correction of EDTA-PTCP.

Impact of pre-analytical sample handling factors on plasma biomarkers of Alzheimer's disease.

An unmet need exists for reliable plasma biomarkers of amyloid pathology, in the clinical laboratory setting, to streamline diagnosis of Alzheimer's disease (AD). For routine clinical use, a biomarker must provide robust and reliable results under pre-analytical sample handling conditions. We investigated the impact of different pre-analytical sample handling procedures on the levels of seven plasma biomarkers in development for potential routine use in AD. Using (1) fresh (never frozen) and (2) previously frozen plasma, we evaluated the effects of (A) storage time and temperature, (B) freeze/thaw (F/T) cycles, (C) anticoagulants, (D) tube transfer, and (E) plastic tube types. Blood samples were prospectively collected from patients with cognitive impairment undergoing investigation in a memory clinic. β-amyloid 1-40 (Aβ40), β-amyloid 1-42 (Aβ42), apolipoprotein E4, glial fibrillary acidic protein, neurofilament light chain, phosphorylated-tau (phospho-tau) 181, and phospho-tau-217 were measured using Elecsys® plasma prototype immunoassays. Recovery signals for each plasma biomarker and sample handling parameter were calculated. For all plasma biomarkers measured, pre-analytical effects were comparable between fresh (never frozen) and previously frozen samples. All plasma biomarkers tested were stable for ≤24 h at 4°C when stored as whole blood and ethylenediaminetetraacetic acid (EDTA) plasma. Recovery signals were acceptable for up to five tube transfers, or two F/T cycles, and in both polypropylene and low-density polyethylene tubes. For all plasma biomarkers except Aβ42 and Aβ40, analyte levels were largely comparable between EDTA, lithium heparin, and sodium citrate tubes. Aβ42 and Aβ40 were most sensitive to pre-analytical handling, and the effects could only be partially compensated by the Aβ42/Aβ40 ratio. We provide recommendations for an optimal sample handling protocol for analysis of plasma biomarkers for amyloid pathology AD, to improve the reproducibility of future studies on plasma biomarkers assays and for potential use in routine clinical practice.

“Molecular Characterization And Its Association To Ap Endonuclease-1 (Ape1) And X-Ray Cross-Complementing Group 1 (Xrcc1), As A Dna Repair Protein From Patients With The Risk Of Age-Related Cataract At A Tertiary Care Centre, Uttar Pradesh”

Introduction: Age-related cataract is the leading cause of blindness in the world. (AP)-endonuclease APE1 and the X-ray cross-complementing group 1 (XRCC1) are the key enzymes taking part in the repair of DNA damage. The association of X-ray cross-complementing group 1 (XRCC1) and AP endonuclease-1 (APE1) plays a critical role as polymorphisms of DNA repair genes decreasing the ability to repair DNA damage, leaving human body a greatly increased susceptibility to cancer or age-related diseases.&#x0D; Aim and objective: To study the Molecular characterization and its Association to AP endonuclease-1 (APE) and X-ray cross-complementing group 1 (XRCC1), as a DNA Repair protein from patients with the risk of age-related cataract at a Tertiary Care Centre, Uttar Pradesh.&#x0D; Material and methods: This was a case control study carried out in the Department of Anatomy &amp; Department of Ophthalmology of RMCH&amp;RC for a period of 2 year i.e., June 2020 to June 2022. A total of 500 clinical suspected cases were included in our study in which 250 patients were confirmed as cataract positive patients whereas 250 were the Controls. The Demographic profiles of the cases were recorded after duly obtaining consent. The 5ml of venous blood was collected in Ethylene diamine tetraacetic acid tubes.&#x0D; DNA was extracted by using Qiagen DNA Extraction kit as per manufactures guidelines. The AP endonuclease-1 (APE) and X-ray cross-complementing group 1 (XRCC1) gene was detected by the conventional PCR.&#x0D; Results: In the present study the maximum number of cases were reported in the Females as compared to the Males with the maximum age group of 51-60 years being affected the most. The Expression of (AP) –endonuclease (APE1) gene was more 68.4 % as compared to X-ray cross-complementing group 1 (XRCC1) with 62.8 %. The increase expression indicates the presence of these genes in the DNA repair damage.&#x0D; Conclusion: The Expression of (AP)-endonuclease (APE1) gene was more 68.4% as compared to X-ray cross-complementing group 1 (XRCC1) with 62.8%. The increase expression indicates the presence of these genes in the DNA repair damage. So, there should be early screening and its molecular detection of the DNA repair gene, which will help the clinician in the early diagnosis as well as early treatment of the cataract.

Molecular Characterisation of X-ray Cross-complementing Group 1 (XRCC1) Gene and Risk Factors in Senile Cataract Patients attending a Tertiary Care Hospital, Uttar Pradesh, India

Introduction: Cataract arises because of aging of the crystalline lens of the eye which prevents clear vision. X-ray Cross-complementing Group 1 (XRCC1) is a Deoxyribonucleic Acid (DNA) repair protein which is involved in Single-Strand Breaks (SSBs) and Base Excision Repair (BER) pathway which is responsible for the efficient repair of DNA damage is mainly responsible for cataract in patients. Aim: To study the prevalence, risk factors and the molecular characterisation with its special association to XRCC1 gene in senile cataract patients. Materials and Methods: This was a cross-sectional study carried out in the Department of Anatomy and Ophthalmology, Rama Medical College Hospital and Research Centre, Kanpur, Uttar Pradesh, India, from April 2021 to April 2022. A total of 500 clinical patients were included in which 250 patients were confirmed as cataract positive patients. Venous blood of 5 mL was collected in Ethylene diamine tetra-acetic acid tubes. The DNA extraction for the detection of XRCC1gene was done using Qiagen DNA extraction kit as per manufactures guidelines, which was further confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: A total of 500 clinically suspected patients were included in which 250 cases were confirmed as cataract positive patients. The ratio of females was more (n=130, 52%) compared to males (n=120, 48%) with the mean age for females with 57.6% and for males with 61.13%. Hypertension (n=173, 69.2%) was the most common disease associated with the cataract patients. The ratio of males were more (n=91, 75.8%) compared to females (n=82, 63.07%). The mean age of males was 64.40 years and that of females were 62.45 years. The other co-morbidity included diabetes (48.8%), in which males constituted 67 (55.83%) participants compared to the females with 55 (42.3%) participants. The presence of XRCC1 gene was detected in all cataract positive patients, which was also confirmed by RT-PCR. Conclusion: The polymorphisms of DNA repair genes decreased their ability to repair DNA damage, leaving human body a greatly increased susceptibility to cancer or agerelated diseases. The association of XRCC1 gene with agerelated cataract susceptibility observed in the present study supports the view that XRCC1 gene plays an important role in susceptibility to age-related cataract, so early screening and its molecular profiling will help the clinician in the early diagnosis as well as early treatment.

Pitfalls in clinical genetics.

With the increasing availability of genetic tests, more doctors are offering and ordering such tests for their patients. Ordering a genetic test appears to be a simple process of filling in paperwork, drawing 3 mL of blood in an ethylenediaminetetraacetic acid tube and receiving a test report. This is identical to sending off a full blood count. However, it is far more complex than that. There are many potential pitfalls, as shown by the increasing number of complaints and lawsuits filed against doctors and allied health staff. Furthermore, clinical genetics involves more than just ordering tests; in fact, focusing on genetic tests alone is a potential pitfall. In this review, we discuss the common pitfalls in clinical genetics and how doctors can avoid these pitfalls to ensure patient safety and to safeguard their practice.

Impact of pre‐analytical factors on blood‐based biomarkers of Alzheimer's disease

AbstractBackgroundReliable blood‐based biomarkers (BBBMs) are urgently needed to allow for robust, cost‐effective pre‐screening of patients with suspected Alzheimer’s disease (AD) before referral for detailed assessments. We evaluated the effects of storage time and temperature, plastic tube types, and freeze/thaw (F/T) cycles in fresh, never frozen and previously frozen whole blood (WB) and plasma. Effects of anticoagulants and tube transfer were only studied in previously frozen plasma.MethodWB samples from patients with clinically suspected AD were collected in ethylenediaminetetraacetic acid (EDTA) tubes and stored at room temperature (RT) or 4°C for ≤24hrs as WB or plasma. Separated plasma was transferred into polypropylene (PP) tubes to evaluate the effects of tube transfer and F/T cycles, or into low‐density polyethylene (PE‐LD) tubes to assess the effect of tube type. In addition to EDTA, lithium heparin (Li‐Hep) and sodium citrate (NaCit) tubes were used and effects of anticoagulants evaluated. Plasma levels of phosphorylated‐tau (pTau) 217, pTau181, amyloid‐β 1–42 (Aβ42), amyloid‐β 1–40 (Aβ40), apolipoprotein E4 (ApoE4), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NFL) were measured using automated Elecsys® prototype immunoassays (cobas e 601 analyzer; all Roche Diagnostics International Ltd).ResultFor all BBBMs, pre‐analytical effects were comparable between fresh, never frozen and previously frozen samples. All BBBMs were stable for ≤24hrs at 4°C in WB and EDTA plasma; Aβ42, Aβ40, and ApoE4 were unstable if stored for &gt;2hrs at RT. Up to five tube transfers, or two F/T cycles, and both tested plastic tube types were acceptable for all BBBMs. For all BBBMs, except Aβ42 and Aβ40, analyte levels were comparable between EDTA, Li‐Hep, and NaCit tubes. Aβ42 and Aβ40 were most sensitive to pre‐analytical sample handling and the effects could only be partially compensated by the Aβ42/Aβ40 ratio.ConclusionWe provide recommendations for an optimal handling protocol for blood collection and sample handling for analysis of BBBMs for AD based on measurement in fresh samples (Figure): WB and EDTA plasma should be stored at 4°C for ≤24hrs, while storage at RT should be avoided or limited to 2hrs maximum.

Hematologic Reference Intervals and Comparison of Natt-Herrick Technique and Smear-Based Leukocyte Estimation in Cockatiels (Nymphicus hollandicus)

Although cockatiels are among the most common avian species maintained as companion animals in the United States, information on standard hematologic reference values for this species is limited. The objectives of this study were to establish hematologic reference intervals (RI) for cockatiels, compare methods using both the Natt-Herrick technique (NHT) and the smear-based estimation technique (SBT), explore age and sex differences in the hematologic findings for this species, and produce the first cockatiel RI for fibrinogen concentration and thrombocyte estimate. Healthy cockatiels (60 males and 60 females, 2-11 years old) from a research colony were included in this study. Blood samples were placed in dipotassium ethylenediaminetetraacetic acid tubes, and erythrocyte counts and thrombocyte estimates were determined via automated analyzer (ADVIA 120) and SBT, respectively. Moreover, leukocyte concentrations were determined using both NHT and SBT to compare these common methods for measuring a complete blood count in cockatiels. Data were analyzed for outliers, distributions, descriptive statistics, and RI via Reference Value Adviser, a set of macroinstructions for Microsoft Excel (Microsoft, Redmond, WA, USA). Lymphocytes were the predominant leukocyte across both methods. According to the NHT, females had significantly higher concentrations of total leukocytes, heterophils, bands, lymphocytes, basophils, and total plasma protein compared with males. Significant inverse polynomial relationships were noted between total leukocyte count and age and lymphocyte counts and age for NHT. Total leukocyte count produced via NHT and SBT were compared using Passing-Bablok and Bland-Altman plots, and no significant constant or proportional biases were found. However, these methods showed wide limits of agreement. While the RI were interchangeable between methods from a clinical standpoint, the same method should be used to assess changes in an individual. The reported RI are uniquely robust given the sample size, balanced sex and age distributions, inclusion criteria, and control over sample collection.

Effect of automated feedback laboratory comments promoting good phlebotomy practice on the frequency of ethylenediaminetetraacetic acid (EDTA) contamination of serum samples.

Ethylenediaminetetraacetic acid (EDTA) contamination of serum samples is common but under-recognized unless EDTA is measured. Incorrect order of draw with closed phlebotomy (vacutainer) does not cause EDTA contamination. EDTA contamination occurs largely or solely during open phlebotomy due to syringe tip or needle-tip contamination when delivering blood into EDTA sample tubes before other sample tubes or direct transfer of blood from EDTA containing tubes to other tubes. Therefore, preference for closed phlebotomy or following the order of tube fill when open phlebotomy is used may reduce EDTA contamination. The laboratory's comments for EDTA-contaminated serum samples were amended to encourage closed phlebotomy and with open phlebotomy filling of serum tubes before EDTA and fluoride-EDTA tubes. The weekly frequency of EDTA sample contamination, normalized for weekly urea and electrolyte (U&E) requests, was studied 52 weeks before and 43 weeks after amending the comments. Median (IQR) frequency of EDTA-contaminated samples per week per 10,000 U&Es decreased by 58% [5.6 (3.1-9.2) versus 2.3 (1.1-4.4); P < 0.001] after the introduction of the new comment. Explicit automated laboratory feedback comments promoting closed phlebotomy and order of tube fill with open phlebotomy were associated with a 58% reduction in EDTA-contaminated samples and thus may play a role in improving phlebotomy practise.

Evaluating the Effects of Storage Conditions on Multiple Cell-Free RNAs in Plasma by High-Throughput Sequencing.

Background: Plasma cell-free RNAs (cfRNAs) can serve as noninvasive biomarkers for the diagnosis and monitoring of diseases. However, the delay in blood processing may lead to unreliable results. Therefore, an unbiased evaluation based on the whole transcriptome under different storage conditions is needed. Methods: Here, blood samples were collected in ethylenediaminetetraacetic acid tubes and processed immediately (0 hour), or stored at room temperature (RT) or 4°C for different time intervals (2, 6, and 24 hours) before plasma separation. High-throughput sequencing was applied to assess the effects of storage conditions on the transcript profiles and fragment characteristics of plasma cell-free mRNA, long noncoding RNA (lncRNA), and small RNAs. Results: More genes changed their expression levels with time when blood was stored at RT compared with those at 4°C. Cell-free mRNA and lncRNA were relatively stable in blood preserved at 4°C for 6 hours, while cell-free microRNA (miRNA) and piwi-interacting RNA (piRNA) remained stable at 4°C for 24 hours. After 24 hours, more contamination of the leukocyte-derived RNAs occurred at RT, possibly due to apoptosis. Meanwhile, significant changes were also observed regarding the characteristics of the RNA fragments, including fragment size, the proportion of intron, and the pyrimidine frequency of the fragmented 3' end. Fifteen tissue-enriched genes were detected in the plasma but not expressed in leukocytes. The expression level and fragment length of these genes gradually decreased during storage, suggesting the degradation of the cfRNA and the dilution of leukocyte-derived RNA with other tissue-derived cfRNA. Conclusions: Our results suggest that the contamination of leukocyte-derived RNA and the degradation of original cfRNA contribute to the changes in the cfRNA expression profiles and the fragment characteristics during short-term storage. The storage of blood at 4°C for 6 hours allows plasma cfRNA to remain relatively stable, which will be useful for further studies or clinical applications where adequate quantification or the fragment signature of cfRNA is required.