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A school and community outbreak of influenza A.

In May 1995 a department of public health medicine was informed of an outbreak of respiratory and gastrointestinal illness in a local school. Eighty-three pupils and staff were affected out of a total of 247 people--an attack rate of 34%. The outbreak was investigated, control measures were instigated, and the outbreak subsided. Pupils and staff were surveyed and faecal specimens were collected. Blood specimens from a sample of pupils were examined serologically. No organisms were isolated from faecal specimens. Nine of the 18 blood specimens taken showed raised antibody titres against influenza A. This labour intensive investigation revealed a community outbreak of influenza A. Investigations in schools can be useful in community surveillance.

Antibodies, Viral↗

Free erythrocyte porphyrins in the detection of undue absorption of Pb and of Fe deficiency.

A fluorometric assay for free erythrocyte porphyrins (FEP) is described in detail, the direct test being used for the analysis of blood specimens collected with anticoagulants and the spot test for analysis of blood specimens collected on filter paper. A method for determining the amount of blood absorbed by different lots of filter paper is described.

Anemia, Hypochromic↗

Comparison of blood specimens from plain and gel vacuum blood collection tubes.

This study was set in the Division of Laboratory Medicine, Chulalongkorn Hospital. All 2,000 blood specimens were randomly collected using evacuated blood collection by plain or gel vacuum tubes. After collection, each specimen was considered and judged using criteria of specimen rejection to determine how proper the specimen presentations were. All data were reviewed, collected and interpreted. It revealed that there were only 20 (1%) improper specimens and all were improper in quality. There was no significant difference between the ratio of improper specimens in both groups (P > 0.30). From this study, it revealed that efficacy of both types of vacuum tubes was not different. The new gel vacuum tube seems to be an effective tool in the evacuated blood collection system due to its advantage in reduction of time in specimen processing.

Blood Specimen Collection↗

Blood collection, shipment, processing, and storage.

As part of the AACR Annual Meeting Methods Workshop "Sample Collection, Processing, and Storage for Large-Scale Studies: Biorepositories to Support Cancer Research," blood specimen collection, processing, storage, and dissemination issues were discussed. Whole blood and blood fractions comprise a major portion of biospecimen collections for population-based studies. Although procedures for collecting, processing, storing, and shipping blood components are generally standardized and well documented, several important factors need to be considered before a new study involving blood collection is initiated. Blood is collected from study participants for a variety of purposes, including as a source of DNA, and for a variety of laboratory analyses that may be done on whole blood or blood fractions, including serum, plasma, and lymphocytes. Blood or blood fractions may be shipped to laboratories and biorepositories either at ambient temperature, cooled or frozen, depending on the intended analyses. Blood processing may include fractionation, cryopreservation to preserve the viability of lymphocytes, or purification of nucleic acids. The proper storage conditions depend on a variety of factors, including the intended analyses and whether the specimens will be used within a short period or need to be stored for longer periods. As for all laboratory and biorepository procedures, blood collection, shipment, processing, and storage should be conducted under a strict quality assurance program, including standard operating procedures and regular quality control reviews.

Blood Preservation↗

Integrated and automatic mixing of whole blood: an evaluation of a novel blood gas analyzer.

BACKGROUND: A homogeneous whole blood specimen is essential to produce quality results from blood gas analysis, however achieving an adequately mixed specimen can be difficult due to the absence of any dead space in a blood gas syringe. This study evaluated the efficacy of an automatic mixing feature incorporated into the ABL800 FLEX blood gas analyzer and estimated the systematic error in tests performed by the instrument. METHODS: Quantitative measurements of pH, PCO(2), PO(2), Na(+), K(+), Ca(+), glucose, lactate, and total hemoglobin were performed on 388 whole blood specimens collected into standard blood gas syringes or specific syringes designed to work in conjunction with the ABL800 FLEX. One hundred eighty specimens were manually or automatically mixed following horizontal storage for 10, 20, or 30 min and total hemoglobin was used as in indicator of specimen homogeneity. Two hundred eight specimens were used to estimate the systematic error of tests performed by the ABL800 FLEX. RESULTS: Manual mixing produced significantly more variation in paired hemoglobin measurements compared to automatic mixing at all three time points (p<0.0001). All estimates of systematic error were very low and clinically insignificant. Quantitative results showed excellent agreement with a reference analyzer. CONCLUSIONS: In contrast to manual mixing, automatic mixing by the ABL800 FLEX consistently produces a homogeneous specimen.

Adult↗

Immunophenotypic characterization of tumor infiltrating lymphocytes and peripheral blood lymphocytes isolated from melanomatous and non-melanomatous Sinclair miniature swine.

Selectively-bred Sinclair miniature swine exhibit a high incidence of congenital malignant melanoma which either proves fatal (10-15% of tumor-bearing piglets) or spontaneously regresses with a biphasic immunological phenomenon (85-90%) and no recurrence of malignancy. Mononuclear leukocytes were isolated from cutaneous melanomas and peripheral blood specimens collected from melanomatous (tumor-bearing) Sinclair swine during second-phase regression, and from peripheral blood specimens collected from non-melanomatous (tumor-free) Sinclair swine and control Hanford swine. Leukocyte identities were determined with single- and dual-parameter indirect immunofluorescence assays via flow cytometry. Assays for the specific surface antigens CD45, CD2, CD4, CD8, CD1, MHC class II, and N1 were employed to develop immunophenotypic profiles within the gated lymphocyte clusters from each TIL and PBL suspension. Significantly more CD8+ T-lymphocytes were identified in TIL suspensions than in peripheral blood leukocyte (PBL) suspensions (P < and = 0.05), regardless of breed or tumor status. Conversely, PBL suspensions contained significantly higher percentages of CD4+ T-lymphocytes than the levels found in TIL suspensions (P < and = 0.05). Virtually all TIL were MHC class II+, whereas the percentages of PBL expressing this antigen were markedly lower (P < and = 0.05). The percentages of T-lymphocytes co-expressing CD4 and CD8, a normal subset unique to swine, were generally consistent in all TIL and PBL suspensions examined. The results of this study have firmly established the immunophenotypic identities of cells associated with the second-phase regression phenomenon of this melanoma and have identified specific variations in the leukocyte profiles of the respective TIL and PBL suspensions.

Animals↗

AMPLICOR MTB polymerase chain reaction test for identification of Mycobacterium tuberculosis in positive Difco ESP II broth cultures.

The reliability of the Roche AMPLICOR polymerase chain reaction (PCR) Mycobacterium tuberculosis (MTB) test for identification of M. tuberculosis complex (MTBC) in Difco ESP II broth cultures was evaluated by testing aliquots from all cultures, regardless of specimen type, that were signaled positive by the ESP instrument. Polymerase chain reaction results were compared to those obtained by usual laboratory protocol, whereby MTBC was identified by DNA probe (Gen Probe, Inc., San Diego, California, USA), testing sediment from broth samples or colonies on a solid medium. Of the 242 signal-positive ESP II cultures (from 182 patients) evaluated, 98 (40.5%) contained mycobacteria, including 26 MTBC. On initial testing, 27 samples were positive by PCR; 22 of these were MTBC culture positive, and 5 were culture negative for mycobacteria. The sensitivity, specificity, and positive and negative predictive values of PCR were 84.6, 97.7, 81.5, and 98.1%, respectively. Based on review of the medical records of the patients for whom PCR and culture results were discrepant, one PCR-positive culture negative was a true PCR positive; thus the actual sensitivity, specificity, and positive and negative predictive values of PCR were 85.2, 98.1, 85.2, and 98.1%. Two of the PCR-negative, MTBC culture-positive samples were blood specimens collected in an Isolator tube; and these tubes contain sodium polyanetholsulfonate, which seems to inhibit PCR. If blood specimens are excluded from analysis, the sensitivity, specificity, and positive and negative predictive values for PCR are 92.0, 97.6, 85.2, and 98.8%, respectively. The mean times from specimen receipt to identification of MTBC were 19 (+/- 2) days (range, 6 to 39 days) for ESP II plus AMPLICOR MTB PCR and 25 (+/- 2) days (range, 8 to 42 days) for DNA probe testing of sediment from broth or colonies on solid media (p < .05). These data indicate that the AMPLICOR MTB PCR test is a rapid, reliable method for identification of MTBC in positive ESP II cultures, excluding blood specimens collected in Isolator tubes.

Bacteriological Techniques↗

Comparison of Bayer Advia Centaur immunoassay results obtained on samples collected in four different Becton Dickinson Vacutainer tubes.

BACKGROUND: Medicines and Healthcare products Regulatory Agency's (MHRA's) Medical Device Alert MDA/2004/048 described bias in some endocrine test results obtained on a few immunoassay platforms, particularly the Bayer Advia Centaur instrument, when using blood specimens collected into Becton Dickinson (BD) Vacutainer SSTII Advance tubes. As users of BD tubes and the Advia Centaur instrument, we addressed our concerns about the quality of the results that we had previously reported by undertaking an independent study. METHOD: We compared the results of 15 immunoassays performed on Bayer Advia Centaur using blood specimens collected into four different BD Vacutainer tubes (plain, old and newly released BD SSTII Advance, and BD PSTII). RESULTS: Compared with plain tubes, old SSTII Advance tube results showed no bias for testosterone, CA15-3, follicle-stimulating hormone and folate assays, but gave a positive bias for cortisol and a negative bias for vitamin-B12. Compared with plain tubes, BD PSTII tubes gave no significant bias for thyroid function tests, prolactin, parathyroid hormone, and CA125, but gave a negative bias for steroid assays, and a positive bias for gonadotrophins. The results obtained using new BD SSTII Advance tubes were generally comparable with those on plain tubes. CONCLUSIONS: Only for cortisol did our findings support the bias described by MHRA. Based on our results, apart from vitamin-B12 and possibly cortisol, there may have been no significant influence on clinical decisions as a result of using the old BD SSTII Advance specimen tubes. New BD SSTII Advance tubes and plain tubes give generally comparable results. BD PSTII tubes should not be used for steroid hormone measurements on the Bayer Advia Centaur instrument.

Adult↗

Experimental basis of standardized specimen collection: effect of posture on blood picture.

22 subjectively healthy females were supine, sat in an armchair and stood while specimens of peripheral venous blood were collected after at least 15 min in each position without using a tourniquet. The albumin, haemoglobin, erythrocyte concentration and the haematocrit increased significantly when the subjects assumed a more erect position, probably as a result of increased hydrostatic pressure. The leucocyte count did not rise, and there was a statistically significant drop in the lymphocyte concentration when changing from supine to sitting. However, the leucocyte concentration rose significantly from supine to sitting or standing. When interpreting laboratory data, the difference in the behaviour of different cell species should be kept in mind. However, on the whole this study supports the stipulation contained in international recommendations that the posture of the subject should be standardized before collection of peripheral blood for haematological tests.

Adult↗

Five-year thyrotropin screening for congenital hypothyroidism in Ramathibodi Hospital.

OBJECTIVES: To detect newborns with congenital hypothyroidism (CH) and to treat the affected infants as early as possible. STUDY DESIGN: Cord blood thyrotropin (TSH) screening for CH in Ramathibodi Hospital began in 1993. From October 1993 to December 1998, 35,390 neonates were screened. The infants with elevated TSH level of greater than 30 mU/L were recalled for verification of CH. Confirmation tests included total thyroxine, free thyroxine and TSH level. Thyroid scan and uptake were performed in some affected infants. RESULTS: Twelve infants with CH were detected resulting in an incidence of one in 2,949 live-births. All affected infants were asymptomatic at birth. Of 12 infants with CH, one premature neonate had a delayed TSH elevation and was diagnosed as having primary hypothyroidism at 2 months of age. The recall rate for validation of CH based on a cut-off value at serum TSH level of greater than 30 mU/L is 1.1 per cent. If the cut-off value of serum TSH level was raised to greater than 40 mU/L, the recall rate would decrease to 0.43 per cent. None of the affected infants had cord blood TSH level of less than 50 mU/L except one premature patient. Therefore, beginning in January 1997, the cut-off value of TSH was raised to 40 mU/L or greater. Pitfalls in this program include incomplete blood-specimen collection and incomplete follow-up. To strengthen the program, improvements were made in the follow-up system from 1996 onward. Therefore, the coverage for blood-specimen collection progressively increased from 84 per cent in 1994 to 96 per cent in 1998. Simultaneously, the patients' return after recalls also increased from 38 per cent to 100 per cent. CONCLUSIONS: The incidence of CH in Ramathibodi Hospital is approximately 1:3,000 live-births. The optimal cord blood TSH level for recall is 40 mU/L or greater. The intensification of follow-up strategy resulted in better response to recall and earlier treatment in the affected infants.

Congenital Hypothyroidism↗

Home collection kits for HIV testing: evaluation of three strategies for dealing with insufficient dried blood specimens.

Home collection kits allow individuals to obtain a blood specimen at home and send it to a laboratory for HIV testing. In preliminary studies, 15% of kit users submitted specimens considered to be insufficient for analysis. The current Public Health Service policy requires the laboratory to reject all such specimens entirely and request a second specimen, even though some specimens are sufficient to perform an enzyme immunoassay (EIA) but not a Western blot (WB) test. Using decision analysis, we evaluated three strategies to handle specimens sufficient to perform an EIA but insufficient to perform a WB analysis: current recommendation, or baseline, for which no test is preformed and "quantity not sufficient" is reported; alternative 1 , for which an EIA is done and reported as "negative" or "quantity not sufficient" (if initially reactive); and alternative 2, for which an EIA is done and reported as "negative" or "a reactive screening test" (if initially reactive). Baseline strategy requires all consumers with an initial specimen sufficient for EIA only to submit a second specimen, but either alternative requires fewer than 3% to submit a second specimen. Although 80% of consumers with an initial specimen sufficient for EIA learn their test results only with the baseline strategy, more than 99% learn their test results with either alternative. With the scenario of high (2%) HIV prevalence, 91% of consumers who would be told "reactive screening test" are truly infected. At a low (0.1%) HIV prevalence, 33% of consumers who would be told "reactive screening test" are truly infected. When a specimen is sufficient for EIA only, it is preferable to perform the EIA instead of rejecting the specimen, because many persons can get results from the initial specimen and because many fewer are required to submit a second specimen. Current policy should be reexamined in light of these findings.

Blood Specimen Collection↗

The use of Vacutainer tubes for collection and storage of blood for coagulation testing.

A study has been performed to evaluate the suitability of Vacutainer tubes in blood specimen collection for coagulation tests and to compare them with the conventional syringe technique employed in UK hospitals. Blood was collected from healthy volunteers, an ante-natal group and patients on long-term oral anticoagulants. Samples were stored at two different temperatures; 4 degrees C and ambient room temperature (RT). Prothrombin times, factor VII assays and APTT were performed at baseline and after 2 h and 4 h storage. There was significant activation of the extrinsic system in the blood samples collected by Vacutainer when stored at 4 degrees C which became more significant on prolonged storage. The effect was less pronounced when the Vacutainer tubes were stored at RT. In contrast, the blood collected by the syringe method did not show these changes with the exception of the ante-natal specimens where a lesser degree of activation than in the Vacutainer tubes was observed after 4 h at 4 degrees C. The activation of the Vacutainer samples at 4 degrees C is considered undesirable and could be of clinical significance in oral anticoagulant dosage.

Blood Coagulation Tests↗

Laboratory screening of hemoglobinopathies.

A procedure for the laboratory screening of hemoglobinopathies is described. The procedure employs hemoglobin electrophoresis followed by solubility testing to confirm presence of sickling hemoglobins. All initial screening tests are carried out on whole blood specimens collected on filter paper. The use of filter paper facilitates collection and results in considerable saving in time and mailing cost. The results of the first year screening confirm the findings of earlier studies that blood specimens collected on filter paper are satisfactory for electrophoresis and solubility tests. All tests are relatively easy to perform using readily available commercial reagents. The procedure is particularly suitable for public health laboratories in carrying out hemoglobinopathy screening programs.

Electrophoresis, Agar Gel↗

A study to implement early diagnosis of HIV infection in infants born to infected mothers.

A protocol for detecting HIV DNA from specimens collected on filter papers and the effect of storage temperatures on determination of HIV DNA from dried blood spots has been developed and optimized. Blood specimens collected from HIV-1 infected and normal persons were spotted onto blood collection cards (Whatman BFC 180). The HIV DNA was extracted by phenol-chloroform-isoamyl alcohol and was detected for C2V4 of HIV-1 env by nested polymerase chain reaction (nested PCR). One set was stored at -20 degrees C for 14 weeks, another at 37 degrees C for 1 week and then kept at -20 degrees C for 13 weeks and a third set at 25 degrees C for I week and then -20 degrees C for 13 weeks. The dried blood spots from each set were detected for the HIV DNA every 2 weeks for 14 weeks. The C2V4 region of HIV env DNA was determined from small amounts of the dried blood collected on the filter papers. The nested PCR procedure could detect as few as 5 copies of HIV proviral DNA, and HIV DNA could be detected from specimens with viral loads of 2x 10(4) copies/ml. HIV DNA could be detected from specimens collected at all temperatures tested for at least 14 weeks. Therefore, laboratory diagnosis of HIV infection can be done by PCR on dried blood spots. These techniques will be useful as a tool for studying the epidemiology of HIV infection among populations of interest such as mother to child infection using newborn screening specimens.

Blood Specimen Collection↗

Slight hyperinsulinaemia but no hypoglycaemia in pertussis patients.

Because of the central rôle postulated for Pertussis Toxin in the pathogenesis of whooping cough, and the well-established ability of this toxin to alter insulin and glucose levels in animal blood, a study of insulin and glucose levels in hospitalised pertussis patients and in controls was made. With blood specimens collected in heparin-fluoride anticoagulant, the geometric mean plasma-insulin level (13.3 microU/ml) in a series of 24 pertussis patients was slightly, but statistically significantly, higher than that (8.9 microU/ml) in a series of 27 non-pertussis controls with other infectious diseases (p less than 0.02). Portions of the same blood specimens collected in lithium-heparin anticoagulant yielded higher mean plasma-insulin values of 15.5 and 11.4 microU/ml respectively, with no significant difference between them (p less than 0.05). Mean plasma glucose concentrations were not significantly different between the two groups, and hypoglycaemia was not detected in any pertussis patient. There were no statistically significant differences between pertussis and control children in the mean levels of plasma calcium, magnesium or phosphate.

Blood Glucose↗

A practical community-based approach to the diagnosis of dengue virus infections.

A study is described in which private physicians participated in the surveillance and diagnosis for dengue disease within a community. Using simple, economical methods it was shown that a significant proportion of specimens submitted were positive for dengue infection. Of 610 fingerprick blood specimens collected on filter paper strips, 7% to 33% were positive by serology. Of these specimens, 423 (69%) were single specimens and 92 (22%) of the single specimens were positive by serology. Of 153 fingerprick blood specimens collected in heparinized, glass capillary tubes, 15% to 50% yielded a dengue virus isolate. When a positive result was based on serology and/or virus isolation, an overall positive rate of 39% was obtained for one of the participating clinics. Inclusion of thrombocytopenia and leukopenia in selection of cases significantly improved the positive rate. It is argued that this simple and practical community-based approach will result in more accurate surveillance of dengue transmission within an endemic area and thus aid in the development of long-term strategies for control of disease.

Dengue↗