Molecular Detection of Sepsis-Causing Pathogens Directly From Whole Blood Specimens

AbstractBlood specimens from primary care centres are normally transported to central laboratories by mail. This necessitates centrifugation and separation, especially since the potassium ion concentration in whole blood changes during storage at ambient temperature. Thus, because of the growing awareness of and concern for pre-analytical contributions to the uncertainty of measurements, we investigated 27 components and their stability under controlled temperature conditions from 17 to 23°C. We found that storage of whole blood can be prolonged by up to 8–12h for all components examined, including potassium ions, when stored at 20±0.2°C. We conclude that this opens the possibility for establishing a pick-up service, by which whole blood specimens stored at 20–21°C can be collected at the doctor's office, making centrifugation, separation and mailing superfluous. In addition, the turn-around time from sample drawing to reporting the analytical result would be shortened. After investments in thermostatted boxes and logistics, the system could reduce costs for transporting blood samples from general practice centres to central laboratories.

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Occurrence of adenovirus infection and clinical impact in paediatric stem cell transplant recipients

Microbiologia Medica ◽

10.4081/mm.2016.5850 ◽

2016 ◽

Vol 31 (3) ◽

Author(s):

Gabriele Bianco ◽

Cristina Costa ◽

Andrea Piceghello ◽

Francesca Sidoti ◽

Mareva Giacchino ◽

...

Keyword(s):

Stem Cell ◽

Whole Blood ◽

Limit Of Detection ◽

Antiviral Agent ◽

Clinical Impact ◽

Cell Transplant ◽

Adenovirus Infection ◽

Hematopoietic Stem ◽

Organ Specific ◽

Blood Specimens

In this study, the occurrence and clinical impact of adenovirus (AdV) infection was investigated in paediatric hematopoietic stem cell transplantation (HSCT) recipients. A number of 603 specimens (including whole blood, respiratory and other samples) from 181 patients were tested by real-time polymerase chain reaction; clinical outcome was investigated. Overall, 118/603 (19.6%) specimens from 21/181 (11.6%) patients resulted positive to AdV (including 17.3, 29.9, 17.6, and 15.8% of total number of whole blood, respiratory, urine and other specimens, respectively). On whole blood specimens, viral loads ranged from <600 (limit of detection) to >5×10<sup>6</sup> copies/mL, with a median value 2×104. Multiple specimens were positive in patients in which viral load on whole blood was high. Adenoviral positivity on whole blood was associated to poor prognosis, as death occurred in three of ten (30%) patients with persistent positivity on whole blood specimens, also despite the administration of an antiviral agent (cidofovir). Adenovirus infection can account for systemic and/or organ-specific signs/symptoms in approximately 10% of paediatric HSCT recipients. At moment, there is no indication for routine monitor of AdV in these patients, although AdV aetiology of infectious transplant complications should be taken in account.

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Detection of Pneumocystis carinii DNA in Blood Specimens from Human Immunodeficiency Virus-Infected Patients by Nested PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.37.1.127-131.1999 ◽

1999 ◽

Vol 37 (1) ◽

pp. 127-131 ◽

Cited By ~ 21

Author(s):

Meja Rabodonirina ◽

Laurent Cotte ◽

André Boibieux ◽

Karine Kaiser ◽

Martine Mayençon ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Whole Blood ◽

Nested Pcr ◽

Large Subunit ◽

Pneumocystis Carinii ◽

Proteinase K ◽

Rrna Gene ◽

Immunodeficiency Virus ◽

Blood Specimens ◽

Large Subunit Rrna

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp.hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

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Stability of 5-fluorouracil in whole blood and plasma.

Clinical Chemistry ◽

10.1093/clinchem/33.12.2299 ◽

1987 ◽

Vol 33 (12) ◽

pp. 2299-2300 ◽

Cited By ~ 21

Author(s):

R F Murphy ◽

F M Balis ◽

D G Poplack

Keyword(s):

Whole Blood ◽

Enzymatic Degradation ◽

Room Temperature ◽

Parent Drug ◽

Plasma Samples ◽

Blood Specimens ◽

The Stability ◽

Frozen Plasma

Abstract We studied the stability of 5-fluorouracil (5-FU) in plasma and whole blood kept at room temperature and on ice for 1 to 24 h. At room temperature, there was a steady loss of 94% of the parent drug over 24 h in whole blood and 52% in plasma. In the presence of an excess of uracil, 5-FU was stable for 24 h, suggesting that the loss of 5-FU is the result of enzymatic degradation. 5-FU is more stable in whole blood and plasma when samples are kept cold. For blood and plasma samples maintained on ice, the loss was only 30% and 10% of the parent drug in the respective samples over 24 h. Frozen plasma samples (-20 degrees C) were stable for five weeks. Blood specimens collected for quantifying 5-FU should be immediately placed on ice, and the plasma should be separated and frozen as promptly as possible.

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Colorimetric determination of potassium in whole blood, serum, and plasma.

Clinical Chemistry ◽

10.1093/clinchem/31.9.1464 ◽

1985 ◽

Vol 31 (9) ◽

pp. 1464-1467 ◽

Cited By ~ 6

Author(s):

S T Wong ◽

J Spoo ◽

K C Kerst ◽

T G Spring

Keyword(s):

Whole Blood ◽

Colorimetric Method ◽

Direct Determination ◽

Ion Pair ◽

Photometric Method ◽

Colorimetric Determination ◽

Two Dimensional ◽

Subsequent Formation ◽

Blood Specimens

Abstract This spectrophotometric method for the direct determination of potassium in serum or plasma is based on the selective complexing of potassium by a specific macrocyclic polyether, with the subsequent formation of an ion-pair with a colored anion. The colored anion is extracted into an organic solvent, clarified by centrifugation, and then measured at 415 nm. The absorbance of the chromogen varies linearly with [K+] to at least 15 mmol/L. Results of this colorimetric method (y) correlate well with the results obtained by a flame-photometric method (y = 1.04x - 0.22, r = 0.97, n = 81), with CVs ranging from 2 to 4%. We observed no interferences from lipemia, added bilirubin, or various electrolytes. We also evaluated the use of this reagent in a new automated blood analyzer developed by Abbott, a two-dimensional centrifugal system (Clin Chem 31:1457-1463, 1985). Potassium determined with this system (y) correlated well with results by flame photometry: y = 1.02x + 0.02 (r = 0.94, n = 168). With this system one can use whole-blood specimens in measuring potassium.

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Clinical, Virologic, and Immunologic Characteristics of Zika Virus Infection in a Cohort of US Patients: Prolonged RNA Detection in Whole Blood

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy352 ◽

2018 ◽

Vol 6 (1) ◽

Cited By ~ 8

Author(s):

Hana M El Sahly ◽

Rodion Gorchakov ◽

Lilin Lai ◽

Muktha S Natrajan ◽

Shital M Patel ◽

...

Keyword(s):

T Cells ◽

Zika Virus ◽

Whole Blood ◽

Neutralizing Antibodies ◽

Intracellular Cytokine Staining ◽

Dengue Infection ◽

Nonstructural Proteins ◽

Zikv Infection ◽

Rna Detection ◽

Blood Specimens

Abstract Background Clinical, virologic, and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. Methods US subjects with suspected ZIKV infection were enrolled. Clinical data and specimens were prospectively collected for ZIKV RNA detection and serologic and cellular assays. Confirmed ZIKV infection (cases) and ZIKV-negative (controls) subjects were compared. Dengue-experienced and dengue-naïve cases were also compared. Results We enrolled 45 cases and 14 controls. Commonly reported symptoms among cases and controls were maculopapular rash (97.8% and 81.8%), fatigue (86.7% and 81.8%), and arthralgia (82.2% and 54.5%), respectively. The sensitivity (94%) and duration of infection detection (80% positivity at 65–79 days after disease onset) by polymerase chain reaction were highest in whole-blood specimens. ZIKV-neutralizing antibodies had a half-life of 105 days and were significantly higher in dengue virus–experienced cases than naïve ones (P = .046). In intracellular cytokine staining assays, the ZIKV proteins targeted most often by peripheral blood mononuclear cells from cases were structural proteins C and E for CD4+ T cells and nonstructural proteins NS3, NS5, and NS4B for CD8+ T cells. Conclusions ZIKV RNA detection was more frequent and prolonged in whole-blood specimens. Immunoglobulin G (IgG) and neutralizing antibodies, but not IgM, were influenced by prior dengue infection. Robust cellular responses to E and nonstructural proteins have potential vaccine development implications.

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Three commercial polyclonal immunoassays for cyclosporine in whole blood compared: 1. Results with patients' specimens

Clinical Chemistry ◽

10.1093/clinchem/36.1.115 ◽

1990 ◽

Vol 36 (1) ◽

pp. 115-118 ◽

Cited By ~ 7

Author(s):

M J Strassman ◽

G L Lensmeyer ◽

D A Wiebe ◽

I H Carlson

Keyword(s):

Whole Blood ◽

Single Sample ◽

Transplant Recipients ◽

Post Transplant ◽

Transplant Patients ◽

Analytical Results ◽

Analytical Recovery ◽

Blood Specimens ◽

The Impact

Abstract We assessed the performance of three commercially available polyclonal immunoassays for apparent cyclosporine in 120 whole-blood specimens collected from transplant recipients just before their next dose of cyclosporine (CsA). The assays were (a) Abbott's TDx fluorescent polarization immunoassay for CsA and its metabolites in whole blood; (b) the Sandoz radioimmunoassay (RIA); and (c) Incstar's Cyclo-Trac RIA. Mean respective CVs were 3.8%, 9.3%, and 24.3%. Analytical recovery was nearly 100% for concentrations up to 1000 micrograms/L for Incstar and up to 1500 micrograms/L for Abbott and Sandoz; linearity was compromised at greater concentrations. We also quantified the parent CsA concentrations by HPLC. Moreover, to follow day-to-day fluctuations in patients' "cyclosporine" concentrations with each method and to assess the impact these differences have on interpretation of the analytical results, we assayed serial specimens from six post-transplant patients. These showed significantly dissimilar, but parallel, results among the methods for any single sample. Occasionally, however, a result would not fit the established trend. Biases observed among the assays can be explained in part by the nonspecific antisera cross-reacting with CsA metabolites. Most important, we demonstrate that patients' results are not reliably interchangeable among the methods.

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Oxyhemoglobin measurement of whole blood specimens in a silicon microfabricated cuvette

10.1117/12.269966 ◽

1997 ◽

Author(s):

Caicai Wu ◽

Mark R. Holl ◽

Margaret Kenny ◽

Paul Yager

Keyword(s):

Whole Blood ◽

Blood Specimens

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Robotic Automation of Cyclosporine Analysis in Whole Blood

Clinical Chemistry ◽

10.1093/clinchem/38.8.1440 ◽

1992 ◽

Vol 38 (8) ◽

pp. 1440-1443 ◽

Cited By ~ 6

Author(s):

J W Holman ◽

R A Felder

Keyword(s):

Whole Blood ◽

Solid Phase ◽

Internal Standard ◽

Sample Tube ◽

Organ Rejection ◽

Analytical Recovery ◽

Analytical Imprecision ◽

Blood Specimens ◽

Methods Analytical ◽

Robotic Automation

Abstract Cyclosporin A (CsA) is currently the most selective immunosuppressant used clinically to prevent organ rejection after transplantation. We used a reprogrammable robot, the Benchmate (Zymark Inc., Hopkinton, MA), to automate much of the sample preparation involved in solid-phase extraction of CsA. Lysed whole-blood specimens containing previously added internal standard were placed on the Benchmate in the specimen-holding area, and a C18 Bond-Elut column was placed in the top of the sample tube. Specimen extraction from this point was handled automatically by the Benchmate, after which we manually injected the sample into the HPLC system for quantification. Analytical recovery of CsA in two concentrations of calibrator was similar for the manual and Benchmate methods. Analytical imprecision for the Benchmate was less than for manual extraction: within-run imprecision (CV) was less than 8% at either concentration. Between-run imprecision, determined by having the Benchmate extract CsA from two specimens each day for 20 days, was less than 9%. Patients' specimens were extracted manually (x) or by using the Benchmate robot (y); the results, compared by linear regression, agreed well: (y = 0.99 (SD 0.02)x + 2.6 (SD 2.8) micrograms/L (Sy[x = 7.59 micrograms/L, n = 27). We conclude that the Benchmate usefully reduces the manual steps necessary to extract specimens before HPLC analysis for CsA.

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Effect of lot-to-lot variability in filter paper on the quantification of thyroxin, thyrotropin, and phenylalanine in dried-blood specimens.

Clinical Chemistry ◽

10.1093/clinchem/34.1.53 ◽

1988 ◽

Vol 34 (1) ◽

pp. 53-58 ◽

Cited By ~ 25

Author(s):

W E Slazyk ◽

D L Phillips ◽

B L Therrell ◽

W H Hannon

Keyword(s):

Filter Paper ◽

Whole Blood ◽

Neonatal Screening ◽

Intact Cell ◽

High Concentration ◽

Mean Values ◽

Screening Programs ◽

Blood Specimens

Abstract We prepared whole-blood pools to contain various concentrations of phenylalanine (Phe), thyroxin (T4), and thyrotropin (TSH) and applied them to six different lots of Schleicher & Schuell Grade 903 filter paper, two of which represented extremes for serum-absorbancy. Individual measured T4 values showed minimal overlap among all pools for each individual filter-paper lot and for all lots combined, but Phe values overlapped considerably among the high-concentration pools within and among lots. Individual TSH values also showed considerable overlap among the high-concentration pools for all lots combined, but little overlap within each lot. Maximum differences in mean observed values among lots ranged from 6% to 36% for all analytes. Assay results from hemolyzed blood specimens generally were lower than from intact-cell blood specimens for T4 and TSH, but slightly higher for Phe. Maximum among-lot differences in mean values ranged from 13% to 29% for all analytes when each tested lot was used for assay calibration. Lot-to-lot differences in measured values were not strongly related to serum absorbancy values. We conclude that routinely encountered within- and among-lot filter paper variability, as measured by serum-absorbancy, is not alone sufficient to cause gross quantification errors in neonatal screening programs.

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