scholarly journals Importance of Preanalytical Factors in Measuring Cr and Co Levels in Human Whole Blood: Contamination Control, Proper Sample Collection and Long-Term Storage Stability

2020 ◽  
Author(s):  
Yuliya L Sommer ◽  
Cynthia D Ward ◽  
Joaudimir Castro Georgi ◽  
Po-Yung Cheng ◽  
Robert L Jones
Keyword(s):  
Stainless Steel ◽  
Whole Blood ◽  
Consumer Products ◽  
Blood Collection ◽  
Sample Collection ◽  
Blood Samples ◽  
Contamination Control ◽  
Human Whole Blood

Abstract A number of errors with potentially significant consequences may be introduced at various points in the analytical process, which result in skewed, erroneous analytical results. Precautionary procedures such as contamination control, following established sample collection protocols, and having a complete understanding of the long-term stability of the elements of interest can minimize or eliminate these errors. Contamination control is critical in the quantification of Cr and Co in human whole blood. Cr and Co levels in most biological samples are low, but these elements occur naturally in the environment and are often found in commercial and consumer products, which increases the risk of contamination. In this paper, we demonstrated that lot screening process in which we pre-screen a sub-set of manufactured lots used in collecting, analyzing and storing blood samples is a critical step in controlling Cr and Co contamination. Stainless steel needles are often utilized in blood collection but are considered as a potential source of introducing metal contamination to the patient sample. We conducted two studies to determine if there is a possibility of Cr or Co leaching into the human whole blood from the needles during blood collection. We analyzed blood collected from 100 donors and blood collected in vitro in the laboratory from designated vessel containing spiked blood with higher levels of Cr and Co. Two blood tubes were consecutively collected through one needle. In both studies, Cr and Co concentration levels in the two consecutively collected tubes were compared. Based on the results from donor and in vitro blood collection studies, we concluded that there was no Cr and Co leaching from the limited sets of stainless steel needles used in these studies. Furthermore, we demonstrated that Cr and Co human whole blood samples are stable for 1 year stored at temperatures of −70, −20 and 4°C and 6 months at room temperature.

scholarly journals Validation of Blood Collection Procedures for the Determination of Circulating Vascular Endothelial Growth Factor (VEGF) in Different Blood Compartments

2001 ◽  
Vol 16 (2) ◽  
pp. 87-96 ◽  
Author(s):  
R. Dittadi ◽  
S. Meo ◽  
F. Fabris ◽  
G. Gasparini ◽  
D. Contri ◽  
...  
Keyword(s):  
Platelet Count ◽  
Whole Blood ◽  
Relevant Information ◽  
Sampling Procedure ◽  
Systematic Evaluation ◽  
Blood Collection ◽  
Sample Collection ◽  
Analytical Phase

Aims of the study. Studies on circulating VEGF have reported mixed results, possibly due to a lack of standardization of the pre-analytical phase. The aim of our investigation was to standardize the sampling procedure for the determination of VEGF in different blood fractions. Basic procedures. We evaluated various clotting times for obtaining serum in 30 subjects, as well as different procedures for the preparation of plasma Edinburgh anticoagulant mixture (EDTA, PGE1, theophylline) and CTAD. VEGF was also assayed in lysed whole blood. In vitro platelet activation was monitored by measuring the levels of PF4. VEGF and PF4 were measured using commercially available enzyme-linked immunoassays. Main findings. Clotting time increased the release of VEGF, which reached a plateau between 2 and 4 hours. The percent increase of VEGF at 2 hours ranged from 118% to 4515% (median 327%) compared to samples centrifuged within 10 min from withdrawal. VEGF was not different and PF4 was very low or undetectable in Edinburgh plasma and CTAD plasma, while it was significantly higher in sodium citrate plasma. VEGF in CTAD plasma was not correlated with platelet count or leukocytes. Serum VEGF did not correlate with the leukocyte number, but it correlated significantly with the platelet count. Principal conclusions. The procedures for sample collection described above are highly standardized and easy to perform in a routine setting. We therefore suggest systematic evaluation of VEGF in CTAD plasma, in serum (clotting for 2 hours at room temperature) and in whole blood, until prospective controlled clinical studies will have clarified in which blood compartment(s) VEGF provides clinically relevant information.

Rapid Radioimmunoassay For Fibrinopeptide A In Human Plasma

1981 ◽  
Author(s):  
B J Woodhams ◽  
P B A Kernoff
Keyword(s):  
Human Plasma ◽  
Degradation Products ◽  
Clinical Samples ◽  
Blood Collection ◽  
Sample Collection ◽  
Normal Range ◽  
Blood Samples ◽  
Fibrin Degradation Products ◽  
Sequential Samples

The originally-described method for assay of fibrino- peptide A (FPA) in human plasma includes alcohol precipitation and dialysis steps which are complex, time consuming, and limit the applicability of the assay. The purpose of this work was to devise an assay for FPA which could be more easily applied to clinical studies, and to use this assay to study some of the factors which might cause artefactual results on blood samples obtained from patients. A rapid method for FPA assay has been developed which is simple, robust and sensitive and allows results to be obtained within 2.5 hrs. of blood collection. The assay depends on the use of bentonite to absorb fibrinogen from plasma, and gives a normal range for plasma FPA (0.3 - 1.5 pmol/ml) which is similar to that measured using the originally-described method. There is an equally good correlation between results obtained by the two methods on samples obtained from patients with various levels of circulating FPA and/or fibrinogen/fibrin degradation products (FDPs). Following venepuncture, FPA levels in sequential samples of blood drawn through 19-gauge ‘Butterfly’ needles became progressively lower in successive samples, indicating that a larger than usual discard of blood is necessary if basal levels are to be accurately assessed. Different antisera showed differences in affinity for FPA and cross reaction with des amino tyrosyl FPA, but such differences are unlikely to cause problems when assaying clinical samples. Generation of FPA from whole blood in vitro is completely suppressed by heparin/Trasylol anticoagulant in conventionally-used concentration, and we therefore find no evidence in favour of the suggestion that this anticoagulant mixture is unsuitable for sample collection for FPA assay.

scholarly journals Sample Aging Profoundly Reduces Monocyte Responses in Human Whole Blood Cultures

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
H. W. Grievink ◽  
M. Moerland
Keyword(s):  
Whole Blood ◽  
Intracellular Cytokine Staining ◽  
Drug Effects ◽  
Blood Cultures ◽  
Blood Collection ◽  
Innate Immune Responses ◽  
Cytokine Release ◽  
Human Whole Blood ◽  
The Public

Human whole blood cultures are widely used for the investigation of physiological pathways and drug effectsin vitro. Detailed information on the effect of “sample aging” (the time span between blood collection and experimental start) on the experimental outcome is not readily available in the public domain. We studied the effect of sample aging on the ability of immune cells to respond to cell-specific immune triggers (LPS, PMA/ionomycin, and SEB). Sample aging at room temperature profoundly inhibited the LPS-induced monocytic cytokine release in minimally diluted whole blood cultures. The reduction ranged from 20–50% after 30 minutes to 80–100% after 10 hours and differed between cytokines (IL-1β, IL-2, IL-6, IFNγ, and TNFα). Sample storage at 4°C or 37°C even worsened this. PMA/ionomycin- and SEB-induced cytokine release, both mainly T-cell-driven, were also reduced by sample aging but to a lesser extent (20–50% after 24 hours). Intracellular cytokine staining revealed that the number of LPS-responding cells was not impacted by sample aging and reduced LPS responsivity could also not be explained by apoptosis or downregulated TLR4 expression. Thus, we speculate that sample aging induces an inhibitory pathway downstream from TLR4 in monocytes. These results underline the importance of quick sample handling when investigating innate immune responses in whole blood, especially for monocyte responses.

Stability of antioxidant vitamins in whole human blood during overnight storage at 4°C and frozen storage up to 6 months

2018 ◽  
Vol 88 (3-4) ◽  
pp. 151-157 ◽  
Author(s):  
Scott W. Leonard ◽  
Gerd Bobe ◽  
Maret G. Traber
Keyword(s):  
Ascorbic Acid ◽  
Whole Blood ◽  
Healthy Subjects ◽  
Frozen Storage ◽  
Blood Collection ◽  
Plasma Samples ◽  
Optimal Conditions ◽  
Plasma Ascorbic Acid ◽  
Blood Samples ◽  
Sample Handling

Abstract. To determine optimal conditions for blood collection during clinical trials, where sample handling logistics might preclude prompt separation of erythrocytes from plasma, healthy subjects (n=8, 6 M/2F) were recruited and non-fasting blood samples were collected into tubes containing different anticoagulants (ethylenediaminetetra-acetic acid (EDTA), Li-heparin or Na-heparin). We hypothesized that heparin, but not EDTA, would effectively protect plasma tocopherols, ascorbic acid, and vitamin E catabolites (α- and γ-CEHC) from oxidative damage. To test this hypothesis, one set of tubes was processed immediately and plasma samples were stored at −80°C, while the other set was stored at 4°C and processed the following morning (~30 hours) and analyzed, or the samples were analyzed after 6 months of storage. Plasma ascorbic acid, as measured using HPLC with electrochemical detection (LC-ECD) decreased by 75% with overnight storage using EDTA as an anticoagulant, but was unchanged when heparin was used. Neither time prior to processing, nor anticoagulant, had any significant effects upon plasma α- or γ-tocopherols or α- or γ-CEHC concentrations. α- and γ-tocopherol concentrations remained unchanged after 6 months of storage at −80°C, when measured using either LC-ECD or LC/mass spectrometry. Thus, refrigeration of whole blood at 4°C overnight does not change plasma α- or γ-tocopherol concentrations or their catabolites. Ascorbic acid is unstable in whole blood when EDTA is used as an anticoagulant, but when whole blood is collected with heparin, it can be stored overnight and subsequently processed.

Monitoring of r-Hirudin Anticoagulation during Cardiopulmonary Bypass – Assessment of the Whole Blood Ecarin Clotting Time

1997 ◽  
Vol 77 (05) ◽  
pp. 0920-0925 ◽  
Author(s):  
Bernd Pötzsch ◽  
Katharina Madlener ◽  
Christoph Seelig ◽  
Christian F Riess ◽  
Andreas Greinacher ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Whole Blood ◽  
Heart Surgery ◽  
Ex Vivo ◽  
Open Heart Surgery ◽  
Clotting Time ◽  
Blood Samples ◽  
Alternative Approach ◽  
Ecarin Clotting Time

SummaryThe use of recombinant ® hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor Ila assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin- spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 µg/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin- spiked blood samples obtained from 50 healthy blood donors. CV- values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 µg/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

scholarly journals Isolation and characterization of circulating pro-vascular progenitor cell subsets from human whole blood samples

2021 ◽  
Vol 2 (1) ◽  
pp. 100311
Author(s):  
Daniella C. Terenzi ◽  
Ehab Bakbak ◽  
Justin Z. Trac ◽  
Mohammad Al-Omran ◽  
Adrian Quan ◽  
...  
Keyword(s):  
Whole Blood ◽  
Progenitor Cell ◽  
Blood Samples ◽  
Human Whole Blood ◽  
Isolation And Characterization ◽  
Whole Blood Samples

The distribution of BrKα/RbKα peak intensity ratio in human whole blood samples

1994 ◽  
Vol 42 (3) ◽  
pp. 231-241 ◽  
Author(s):  
C. Shenberg ◽  
S. Spiegel ◽  
S. Chaitchik ◽  
P. Jordan ◽  
M. Kitzis ◽  
...  
Keyword(s):  
Whole Blood ◽  
Intensity Ratio ◽  
Peak Intensity Ratio ◽  
Blood Samples ◽  
Human Whole Blood ◽  
Peak Intensity ◽  
Whole Blood Samples

In vitro effects of temperature on red blood cell deformability and membrane stability in human and various vertebrate species

2021 ◽  
pp. 1-10
Author(s):  
Adam Attila Matrai ◽  
Gabor Varga ◽  
Bence Tanczos ◽  
Barbara Barath ◽  
Adam Varga ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Mechanical Stability ◽  
Cell Deformability ◽  
Blood Samples ◽  
Effects Of Temperature ◽  
The Way ◽  
Rbc Deformability

BACKGROUND: The effects of temperature on micro-rheological variables have not been completely revealed yet. OBJECTIVE: To investigate micro-rheological effects of heat treatment in human, rat, dog, and porcine blood samples. METHODS: Red blood cell (RBC) - buffer suspensions were prepared and immersed in a 37, 40, and 43°C heat-controlled water bath for 10 minutes. Deformability, as well as mechanical stability of RBCs were measured in ektacytometer. These tests were also examined in whole blood samples at various temperatures, gradually between 37 and 45°C in the ektacytometer. RESULTS: RBC deformability significantly worsened in the samples treated at 40 and 43°C degrees, more expressed in human, porcine, rat, and in smaller degree in canine samples. The way of heating (incubation vs. ektacytometer temperation) and the composition of the sample (RBC-PBS suspension or whole blood) resulted in the different magnitude of RBC deformability deterioration. Heating affected RBC membrane (mechanical) stability, showing controversial alterations. CONCLUSION: Significant changes occur in RBC deformability by increasing temperature, showing inter-species differences. The magnitude of alterations is depending on the way of heating and the composition of the sample. The results may contribute to better understanding the micro-rheological deterioration in hyperthermia or fever.

scholarly journals AB0095 PRECLINICAL CHARACTERIZATION OF CJ-15314, A HIGHLY SELECTIVE JAK1 INHIBITOR, FOR THE TREATMENT OF AUTOIMMUNE DISEASES

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1347.2-1347
Author(s):  
S. Y. Ki ◽  
H. Shin ◽  
Y. Lee ◽  
H. R. Bak ◽  
H. Yu ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Autoimmune Diseases ◽  
Whole Blood ◽  
Inflammatory Diseases ◽  
Selective Inhibition ◽  
Janus Kinase ◽  
Dependent Manner ◽  
Human Whole Blood

Background:Janus kinases (JAK1, JAK2, JAK3, and TYK2) play critical roles in mediating various cytokine signaling, and has been developed as a target for autoimmune diseases such as RA. Tofacitinib, oral Pan-JAK inhibitor, demonstrated efficacy in RA patients, but its widespread use is limited by safety issues. Baricitinib, JAK1/2 inhibitor, is also known to interfere with the hematopoiesis system, such as anemia and thrombocytopenia associated with suppression of JAK2 signals. Therefore, it is necessary to develop a new potent compound that selectively inhibits JAK1 over JAK2, 3Objectives:To identify the pharmacological characteristic based on efficacy of CJ-15314 as potent and selective JAK1 inhibitor for treatment of autoimmune disease.Methods:In vitro, cell-based, kinase panel, Kd value and human whole blood assay were performed to determine the inhibition potency and selectivity for JAK subfamily kinases. In vivo therapeutic potential was evaluated by RA model including rat Adjuvant-Induced Arthritis (AIA) and collagen-induced arthritic (CIA). To confirm the possibility of further expansion into the autoimmune disease, BioMAP® Diversity PLUS® Panel was performed by discoverX.Results:In vitro assay, CJ-15314 inhibited JAK kinase family in a concentration-dependent manner with IC50 values of 3.8 nM against JAK1, Selectivity for JAK1 over JAK2, 3 was approximately 18, 83 fold greater for CJ-15314. In 1mM ATP condition, CJ-15314 has been confirmed to have the highest JAK1 selectivity over competing drugs, under 1 mM ATP condition that reflects the physiological environment in the body. Similarly, Kd values has also confirmed the selectivity of JAK1, which is 10 fold higher than JAK2, 3. Accordingly, in human whole blood assays, CJ-15314 is 11 fold more potent against IL-6 induced pSTAT1 inhibition through JAK1 (IC50 value: 70 nM) than GM-CSF-induced pSTAT5 inhibition (JAK2) whereas baricitinib and filgotinib exhibited only 2 fold and 7 fold respectively.In vivo efficacy model, CJ-15314 inhibited disease severity scores in a dose dependent manner. In the rat AIA model, CJ-15314 at 30 mg/kg dose showed 95.3% decrease in arthritis activity score, 51.2% in figotinib at 30 mg/kg, 97.7% showed baricitinib at 10 mg/kg. CJ-15314 showed superior anti-arthritic efficacy than filgotinib. CJ-15314 also minimally affected anemia-related parameters but not bricitinib end of the 2-week treatment. In the rat CIA model, like 10 mg/kg of bricitinib, 30 mg/kg of CJ-15314 also has a similar effect, with a significant reduction in histopathological scores.In biomap diversity panel, CJ-15314 inhibited the expression of genes such as MCP-1, VCAM-1, IP-10, IL-8, IL-1, sTNF-α and HLA-DR confirming the possibility of expansion into other diseases beyond arthritis.Conclusion:CJ-15314 is a highly selective JAK1 inhibitor, demonstrates robust efficacy in RA animal model and is good candidate for further development for inflammatory diseases.* CJ-15314 is currently conducting a phase I trial in south Korea.References:[1]Clark JD et al. Discovery and development of Janus kinase (JAK) inhibitors for inflammatory diseases. J Med Chem. 2014; 57(12):5023-38.[2]Burmester GR et al. Emerging cell and cytokine targets in rheumatoid arthritis. Nat Rev Rheumatol. 2014; 10(2):77-88[3]Jean-Baptiste Telliez et al. Discovery of a JAK3-selective inhibitor: functional differentiation of JAK3-selective inhibition over pan-JAK or JAK1-selective inhibition. ACS Chem. Biol., 2016; 11 (12):3442-3451Disclosure of Interests:so young Ki Employee of: CJ healthcare, hyunwoo shin Employee of: CJ healthcare, yelim lee Employee of: CJ healthcare, Hyoung rok Bak Employee of: CJ healthcare, hana yu Employee of: CJ healthcare, Seung Chan Kim Employee of: CJ healthcare, juhyun lee Employee of: CJ healthcare, donghyun kim Employee of: CJ healthcare, Dong-hyun Ko Employee of: CJ Healthcare, dongkyu kim Employee of: CJ healthcare

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