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Cryobiology Dec 2022Leptospires are preserved by frequent sub-culturing in semisolid media due to the challenge of low recovery by freezing or liquid nitrogen methods. The present study...
Leptospires are preserved by frequent sub-culturing in semisolid media due to the challenge of low recovery by freezing or liquid nitrogen methods. The present study evaluated three liquid EMJH medium compositions (Medium A: Leptospira medium base EMJH, Leptospira enrichment EMJH, 5-fluorouracil (3%), rabbit serum (1%) and calf serum (1%); Medium B: same as Medium A but without 5-fluorouracil; Medium C: same as Medium B but with the addition of sodium pyruvate) for the revival of leptospires after storage at -80 °C. A total of 18 Leptospira serovars cultured in Medium A was aliquoted into cryogenic vials and directly stored at -80 °C. A hundred microlitre from each serovar culture stored at -80 °C was sub-cultured on a selected time over a period of 30 months into Media A, B and C. Regrowth on Media B and C showed a better and faster recovery (89-100%) (p-value <0.05) compared to Medium A (67-100%). Leptospires can be stored longer at -80 °C and a good recovery could be obtained when sub-cultured on EMJH medium without 5-fluorouracil.
Topics: Animals; Rabbits; Cryopreservation; Leptospira; Freezing; Fluorouracil; Nitrogen; Culture Media
PubMed: 36179819
DOI: 10.1016/j.cryobiol.2022.09.007 -
Metabolites Jun 2020Metabolomics can be significantly influenced by a range of pre-analytical factors, such as sample collection, pre-processing, aliquoting, transport, storage and thawing.... (Review)
Review
Metabolomics can be significantly influenced by a range of pre-analytical factors, such as sample collection, pre-processing, aliquoting, transport, storage and thawing. This therefore shows the crucial need for standardizing the pre-analytical phase with the aim of minimizing the inter-sample variability driven by these technical issues, as well as for maintaining the metabolic integrity of biological samples to ensure that metabolomic profiles are a direct expression of the in vivo biochemical status. This review article provides an updated literature revision of the most important factors related to sample handling and pre-processing that may affect metabolomics results, particularly focusing on the most commonly investigated biofluids in metabolomics, namely blood plasma/serum and urine. Finally, we also provide some general recommendations and best practices aimed to standardize and accurately report all these pre-analytical aspects in metabolomics research.
PubMed: 32503183
DOI: 10.3390/metabo10060229 -
Annals of Laboratory Medicine May 2015The identification of in vitro hemolysis (IVH) using a hematology analyzer is challenging because centrifugation of the specimens cannot be performed for cell counts. In...
BACKGROUND
The identification of in vitro hemolysis (IVH) using a hematology analyzer is challenging because centrifugation of the specimens cannot be performed for cell counts. In the present study, we aimed to develop a scoring system to help identify the presence of hemolysis in anticoagulated blood specimens.
METHODS
Thirty-seven potassium EDTA anticoagulated blood specimens were obtained, and each specimen was divided into 3 aliquots (A, B, and C). Aliquots B and C were mechanically hemolyzed by aspirating 2 and 5 times, respectively, using a 27-gauge needle and then tested; aliquot A was analyzed immediately without any hemolysis. After the cells were counted, aliquots B and C were centrifuged and the supernatants were tested for the hemolytic index and lactate dehydrogenase levels.
RESULTS
The 4 hematologic parameters were selected and scored from 0 to 3 as follows:< 34.0, 34.0-36.2, 36.3-38.4, and ≥38.5 for mean cell hemoglobin concentration (MCHC, g/dL); <0.02, 0.02, 0.03, and ≥0.04 for red blood cell ghosts (10(12)/L); <0.13, 0.13-0.38, 0.39-1.30, and ≥1.31 for difference value (g/dL) of measured hemoglobin and calculated hemoglobin; and <0.26, 0.26-0.95, 0.96-3.34, and ≥3.35 for difference value (g/dL) of MCHC and cell hemoglobin concentration mean. The hemolysis score was calculated by adding all the scores from the 4 parameters. At the cutoff hemolysis score of 3, the IVH of aliquots B and C were detected as 64.9% and 91.9%, respectively.
CONCLUSIONS
The scoring system might provide effective screening for detecting spurious IVH.
Topics: Anticoagulants; Blood Specimen Collection; Edetic Acid; Hemoglobins; Hemolysis; Humans
PubMed: 25932443
DOI: 10.3343/alm.2015.35.3.341 -
Biochemia Medica Oct 2021The current study aimed to assess the interference of haemolysis on complete blood count (CBC) using Abbott Alinity hq system, and to determine which haemolysis levels...
INTRODUCTION
The current study aimed to assess the interference of haemolysis on complete blood count (CBC) using Abbott Alinity hq system, and to determine which haemolysis levels affect the reliability of sample results.
MATERIALS AND METHODS
Blood samples obtained from 25 volunteers in K3-EDTA tubes were divided into four aliquots. The first aliquot was not subjected to any intervention. The second, third and fourth aliquots were passed through a fine needle 2, 4 and 6 times, respectively. Complete blood count was performed by multi-angle polarized scatter separation technology and haemolysis index (HI) was assessed from the plasma samples separated by centrifugation. Five groups were formed according to the HI values. The percentage biases between the results of non-haemolysed and haemolysed groups were compared with the desirable bias limits from The European Federation of Clinical Chemistry and Laboratory Medicine database and reference change values (RCVs).
RESULTS
In groups 1 to 4, the effects of haemolysis on CBC parameters were acceptable comparing to the analytical bias except for lymphocytes (7.26%-7.42%), MCH (2.59%), and MCHC (0.47%-2.81%). Results of group 5 (gross haemolysis) showed decreases in HCT(- 4.56%), RBC (- 4.07%) count and increase in lymphocyte (11.60%) count higher than the analytical performance specifications. Moreover, variations in MCH (4.65%) and MCHC (5.24%) were exceeding the RCVs.
CONCLUSIONS
Gross haemolysis (haemoglobin concentration > 10 g/L) is likely to produce unreliable CBC results on non-pathological samples. Further studies including pathological specimens are needed.
Topics: Blood Cell Count; Hematologic Tests; Hemolysis; Humans; Laboratories; Reproducibility of Results
PubMed: 34658647
DOI: 10.11613/BM.2021.030706 -
The International Journal of... Oct 2023With an increased demand for rapid, diagnostic tools for TB and drug resistance detection, Truenat MTB-RIF assay has proven to be a rapid point of care molecular test....
With an increased demand for rapid, diagnostic tools for TB and drug resistance detection, Truenat MTB-RIF assay has proven to be a rapid point of care molecular test. The present study aimed to establish a proof of concept of using Trueprep-extracted DNA for line-probe assay (LPA) testing. A total of 150 sputum samples (MTB-positive at Truenat sites) were divided into two aliquots. One aliquot was used for DNA extraction using the Trueprep device and MTB testing. The second aliquot of the sample was subjected to GenoLyse DNA extraction. DNA from both the Trueprep and GenoLyse methods was subjected to first-line (FL) and second-line (SL) LPA testing. Of 139 Trueprep-extracted DNA, respectively 135 (97%) and 105 (75%) had interpretable results by FL and SL-LPA testing. Of 128 GenoLyse-extracted DNA, all 128 (100%) had interpretable FL-LPA results and 114 (89%) had interpretable SL-LPA results. The results obtained in this study indicate that Trueprep-extracted DNA can be used in obtaining valid LPA results. However, the study needs to be conducted on a larger sample size before our recommendations can be used for policy-making decisions.
Topics: Humans; Rifampin; Mycobacterium tuberculosis; Tuberculosis, Multidrug-Resistant; Point-of-Care Testing; Sputum; Sensitivity and Specificity
PubMed: 37749831
DOI: 10.5588/ijtld.23.0003 -
International Journal of Pharmaceutical... 2023Tablet formulations fail to meet the needs of patients unable to swallow tablets such as pediatric, elderly, and patients that must receive medications via feeding...
Tablet formulations fail to meet the needs of patients unable to swallow tablets such as pediatric, elderly, and patients that must receive medications via feeding tubes. Our objective was to develop and test a new, simple device (XTEMP-R) and the methodology for converting tablets into a homogeneous suspension for medication administration. We developed a new device comprised of a flexible receptacle, a tight-fitting cap, and a suction cup bottom to convert tablets into liquid preparations. Tuberculosis treatment drugs, TBAJ-876 and TBI-223, were dispersed within the device utilizing water and commonly available suspending vehicles. We investigated the effectiveness of the XTEMP-R device in dispersing tablets. This was accomplished by visual observations, determining the fineness of dispersion, and measuring the total drug recovery from the dispersions in XTEMP-R. We investigated the accuracy and reproducibility of delivering aliquots from these suspensions by determining the dose reproducibility upon suspension and upon redispersion after 24 hours. The effectiveness of the device was also evaluated using commercially available tablets of acetaminophen, amlodipine, glimepiride, metformin, and valsartan. The suspensions were visually uniform without any large particles. The suspensions passed through a #18 sieve confirming that the particles were less than 1000 µm. The average total dose recovery of three suspensions each was determined to be 101.3% and 99.2% for TBI-223 and TBAJ-876, respectively. Reproducibility from aliquots of 2 mL each was 98.9% to 99.7% for three replicates of TBI-223 suspensions, and 102.6% to 103.2% for TBAJ-876 suspensions. Aliquots tested after 24 hours confirmed uniform redispersibility. We have demonstrated that XTEMP-R can be utilized to prepare homogeneous suspensions conveniently and efficiently in less than 10 minutes without any drug loss. Aliquots for partial dose delivery can be withdrawn accurately. These findings demonstrate that XTEMP-R can be used to accurately deliver doses of suspensions for patients who cannot swallow tablets.
Topics: Humans; Child; Aged; Reproducibility of Results; Suspensions; Drug Compounding; Acetaminophen; Tablets; Administration, Oral
PubMed: 37267527
DOI: No ID Found -
Current Eye Research Aug 2022Substance P is a sensory neuropeptide increasingly used as a biomarker for ocular and systemic neuropathic conditions. Due to the limited studies on tear storage...
PURPOSE
Substance P is a sensory neuropeptide increasingly used as a biomarker for ocular and systemic neuropathic conditions. Due to the limited studies on tear storage conditions compared to other bodily fluids including blood and urine, the aim of this study was to investigate whether different storage durations at 4 °C can impact on substance P concentrations prior to storage at -80 °C. This is important to assess potential practical limitations in the handling and storage of tear fluid essential.
METHODS
Tears were collected and pooled from both eyes of 31 healthy participants using the flush tears method. The samples were centrifuged and aliquoted into three sets of microcentrifuge tubes with each stored at 4 °C for <2 h, 4 h or 6 h (Timepoints 1, 2 or 3). After each respective storage duration, the aliquoted samples were than stored at -80 °C before analysis, within 6 months. Tears were analyzed for the concentration of substance P and the total protein content (TPC).
RESULTS
Substance P concentrations across the three timepoints were not significantly different ( > 0.05), including Timepoint 1 (Median [interquartile range]: 10.7 ng/ml [1.6-37.9]), Timepoint 2 (10.9 ng/ml [1.6-32.6]) and Timepoint 3 (5.2 ng/ml [1.3-25.2]). There were also no significant differences in TPC concentrations measured at the three timepoints, including Timepoint 1 (3.1 mg/ml [1.7-3.8]), Timepoint 2 (2.9 mg/ml [1.9-4.1]) and Timepoint 3 (2.7 mg/ml [1.6-3.7]).
CONCLUSIONS
While the levels of substance P were stable while stored at 4 °C prior to proper -80 °C storage and analysis, future research should investigate the impact of other storage conditions such as ambient room temperature to optimize the feasibility of using tears for biomarker purposes in clinical settings.
Topics: Biomarkers; Eye Proteins; Healthy Volunteers; Humans; Specimen Handling; Substance P; Tears
PubMed: 35485451
DOI: 10.1080/02713683.2022.2067565 -
Vox Sanguinis Feb 2022Alloantibodies to human platelet antigen-15b (anti-HPA-15b) have been detected in mothers with foetal-neonatal alloimmune thrombocytopenia and in multiply transfused...
BACKGROUND AND OBJECTIVES
Alloantibodies to human platelet antigen-15b (anti-HPA-15b) have been detected in mothers with foetal-neonatal alloimmune thrombocytopenia and in multiply transfused patients. Assays used to detect this antibody, which aids in disease diagnosis, can be unreliable and vary in sensitivity. The objective was to generate a stable, lyophilized anti-HPA-15b preparation and evaluate its suitability as a World Health Organization (WHO) reference reagent for use in the quality control of platelet alloantibody detection assays. Results from an international collaborative study to evaluate the preparation were used to assign a minimum potency at which laboratories can be expected to detect the antibody.
MATERIALS AND METHODS
Recalcified plasma containing anti-HPA-15b was aliquotted, lyophilized and coded 18/220. Twenty-five laboratories in 16 countries tested doubling dilutions of the reconstituted material in glycoprotein-specific assays such as the monoclonal antibody-specific immobilization of platelet antigen assay and reported the last positive (or endpoint) dilution.
RESULTS
Twenty-four laboratories (96%) detected antibodies with HPA-15b specificity in preparation 18/220. Reported endpoint dilutions were normally distributed with a modal dilution of 1 in 16 and ranged from 1 in 2 to 1 in 128. Only two laboratories (8%) failed to detect anti-HPA-15b at 1 in 8 dilution.
CONCLUSIONS
When diluted 1 in 8, most laboratories detected anti-HPA-15b in preparation 18/220 using HPA-15bb platelets but not with HPA-15aa platelets. The participants agreed this to be an appropriate dilution for assignment as the minimum potency. In October 2020, the WHO Expert Committee on Biological Standardization approved 18/220 as an International Reference Reagent.
Topics: Antigens, Human Platelet; Blood Platelets; Humans; Indicators and Reagents; Infant, Newborn; Isoantibodies; Thrombocytopenia, Neonatal Alloimmune; World Health Organization
PubMed: 34164825
DOI: 10.1111/vox.13167 -
Cureus Jun 2023We aim to find the time in which a thawed citrate plasma sample that was preserved can be analyzed for routine coagulation testing without losing precision.
OBJECTIVE
We aim to find the time in which a thawed citrate plasma sample that was preserved can be analyzed for routine coagulation testing without losing precision.
METHODS
Whole blood samples from 30 healthy volunteers were collected in 3.2% sodium citrate vacutainer and centrifuged to separate platelet-poor plasma. Each sample was then aliquoted, one aliquot was used immediately for prothrombin time (PT)-international normalized ratio (INR) and activated partial thromboplastin time (APTT), four were stored at -20°C, and four were stored at -80°C for 24 hours. After 24 hours, the aliquots were taken out and thawed at 37°C in water bath and analyzed after 15, 30, 60, and 120 minutes.
STATISTICAL ANALYSIS
Data were presented as mean with standard deviation (SD). Repeated measures ANOVA with Tukey post-hoc test was performed for multiple comparisons. All analysis was done using GraphPAD Prism 8.0 software (GraphPad Software, San Diego, California, USA). Results: In the case of PT and INR, no statistically significant difference was found between the mean values after thawing for 120 minutes when compared with the mean baseline value. However, the APTT showed a statistically significant difference (p = 0.0232) after 30 minutes of thawing when the sample was stored at -20°C. Furthermore, a statistically significance difference (p = 0.0001) was found after 60 minutes of thawing when the samples were stored at -80°C.
CONCLUSION
Plasma samples for the PT and INR may be accepted for assessment up to 120 minutes, when stored at -20°C and -80°C for 24 hours. In the case of APTT, the plasma sample can be used for assessment up to 30 minutes after thawing when stored at -20°C and up to 60 minutes when stored at -80°C.
PubMed: 37425605
DOI: 10.7759/cureus.40023 -
Journal of Visualized Experiments : JoVE Sep 2016The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary...
The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.
Topics: Flow Cytometry; Granulocytes; Humans; Monocytes; Phagocytosis; Respiratory Burst; Staphylococcal Infections; Staphylococcus aureus
PubMed: 27684595
DOI: 10.3791/54264