Small Volumes Of Whole Blood Research Articles

Stability of infectious HIV in clinical samples and isolation from small volumes of whole blood.

To evaluate the stability of infectious HIV in clinical samples and the efficiency of isolating it from small volumes of whole blood. Titres of infectious HIV were measured in peripheral blood mononuclear cells and plasma 2, 24, and 48 hours after peripheral blood had been collected from 10 HIV positive adult volunteers. Volumes of whole blood (1 ml to 5 microliters), collected from a further five volunteers, were used to determine the minimum volume from which HIV could be isolated. Infectious HIV was isolated by co-culture with phytohaemagglutinin stimulated umbilical cord mononuclear cells. Geometric mean titres of infectious HIV seemed to be more stable in peripheral blood mononuclear cells than in plasma. HIV was recovered from all 10 peripheral blood mononuclear cell samples during the 48 hours after sample collection, but from only four plasma samples. HIV could occasionally be isolated from 5 microliters of whole blood and reliably from 200 microliters. HIV can be isolated from peripheral blood mononuclear cells and plasma for up to 48 hours after sample collection. Isolation of HIV from small volumes of whole blood has applications for the diagnosis and management, of HIV positive children.

A rapid, specific assay for superoxide release from phagocytes in small volumes of whole blood.

A rapid, specific assay was developed to measure the release of superoxide anion from stimulated phagocytic cells in small volumes of whole blood. The assay is based on the chemiluminescence that results when lucigenin (bis-N-methyl acridinium nitrate) is reduced by superoxide. Heparinized whole blood, at volumes from 0.2 mL to a single drop from a 20-gauge needle, was mixed with lucigenin and either a soluble or particulate stimulus (phorbol myristate acetate or opsonized zymosan particles, respectively) in a standard volume of 1 mL. The chemiluminescence was measured at 3-minute intervals for a 30-minute period in a luminometer capable of automated operation. Characteristic plots of chemiluminescence versus time were obtained. This assay is rapid and simple, obviates the need to isolate leukocytes from whole blood, is specific for superoxide, can be performed with both soluble and particulate stimuli, and is sensitive enough to detect characteristic responses with as little as a single drop of blood. Patients with chronic granulomatous disease were readily identified using this assay. The ability to use very small blood volumes makes the assay suitable for use in prenatal diagnosis. The main disadvantage of this assay compared to the often-employed slide nitroblue tetrazolium test, with respect to testing for chronic granulomatous disease, is that carriers of x-linked chronic granulomatous disease are not readily detected with this chemiluminescence assay, as they are with the nitroblue tetrazolium test.

Rapid Immunometric Measurement of C-Reactive Protein in Whole Blood

We examined an instrument-free test for C-reactive protein (CRP) in whole blood. The NycoCard CRP Whole Blood test uses a cell-solubilizing dilution liquid, a membrane-bound antibody that binds CRP, and a gold-conjugated antibody for making visible the bound CRP. We obtained essentially identical dose-response curves in citrate-, heparin-, and EDTA-treated blood. CVs were 6.7-12.5% within series and 10.1-14.7% between series. The detection limit was 12 mg/L. Intralipid added to blood increased measured CRP by 10-20%, whereas no change was seen with added bilirubin, added serum amyloid P component, or the presence of rheumatoid factor. In 234 patients' blood samples the results of the NycoCard Whole Blood test correlated well (r = 0.96) with those of a turbidimetric serum method. The test allows reliable measurement of CRP from a small volume of whole blood (25 microL) without using expensive equipment; it should be useful for decentralized testing in hospital departments, emergency units, and primary health care centers.

Comparison of a Noninstrumented Immunoassay for Carbamazepine to High Performance Liquid Chromatography and Fluorescence Polarization Immunoassay

The accuracy, precision, and potential clinical utility of a new whole blood, noninstrumented immunochromatographic assay (AccuLevel) for carbamazepine (CBZ) was evaluated in a multicenter trial including 100 pediatric and 205 adult patients. The AccuLevel assay, a fluorescence polarization immunoassay (FPIA), and high-performance liquid chromatography (HPLC) were used to determine CBZ concentration in samples from 111 female and 194 male patients aged 2-72 years (median 25 years). Mean +/- SD plasma CBZ concentrations in all patients were 7.4 +/- 3.0 micrograms/ml with the AccuLevel assay and 7.5 +/- 2.9 micrograms/ml with FPIA. In 204 patients, the mean concentration determined by the HPLC assay was 7.7 +/- 3.0 micrograms/ml, whereas concentrations determined by the AccuLevel and FPIA assays were 8.0 +/- 3.1 and 8.1 +/- 3.1 micrograms/ml, respectively. Concentrations determined by the AccuLevel and FPIA assays were significantly higher than those quantified by HPLC (p less than 0.05), but not different from each other. In addition, the AccuLevel assay was highly correlated with FPIA (r = 0.97) and HPLC (r = 0.98). Coefficients of variation for the AccuLevel assay at 8 micrograms/ml ranged from 6.8 to 7.5% for the three institutions. We conclude that the AccuLevel assay is a simple, reliable method for determining CBZ concentration in a small volume of whole blood and is an acceptable alternative for assessment of CBZ therapy and individualization of CBZ dosage in the physician's office or emergency room.

Sex identification in chickens by means of the presence of the W chromosome-specific repetitive DNA units.

The W chromosome specific repetitive DNA units were detected easily by the XhoI digestion of genomic DNA, purified from a female whole blood sample, but could not be detected in male. Since the red blood cells of the chicken are nucleated, it is possible to obtain enough amounts of DNA preparation from a small volume of whole blood without sacrificing the animal. The detection of the female-specific repetitive DNA units is simple, reliable and efficient. By applying this technique, the identification of the sex in chickens was easily achived.

Generation and measurement of interleukin‐1, interleukin‐2, and mitogen levels in small volumes of whole blood

AbstractWhole blood assays that allow simultaneous performance of IL‐1 and IL‐2 production as well as lymphocyte mitogen responsiveness are described. The advantages to these techniques are: (1) small volume of sample required (1 ml of whole blood or less), (2) time required to perform the assays (10 min), and (3) use of whole blood rather than isolated cells in the evaluation of immune competency which is more reflective of the in vivo condition. The techniques described lend themselves to application in the clinical setting.

Phagocytosis-connected oxygen consumption by peripheral leukocytes from patients with systemic lupus erythematosus.

The phagocytic activity of peripheral blood leukocytes was assayed by means of oxygen consumption in 28 patients with systemic lupus erythematosus (SLE) and 22 normal individuals. The method in which zymosan was added to a small volume of whole blood enabled us to assay quantitatively and rapidly both the phagocytosis-connected oxygen consumption and the opsonizing time. The degree of oxygen consumption by phagocytes from SLE patients was not significantly different from that in normals. Opsonizing time of zymosan by autologous serum in SLE patients did not differ from that in normals either. It should be emphasized, however, that the opsonizing time of zymosan in SLE patients was inversely correlated with serum complement level, especially C3 level (coefficient of correlation = -0.76, p less than 0.001). This raises the possibility that the phagocytosis may be delayed in SLE patients with low titers of C3.

Measuring platelet and leucocyte aggregation/adhesion responses in very small volumes of whole blood

A technique is described by which leucocyte and platelet aggregation may be measured in 100 μl (or less) of citrated whole blood. The blood is first diluted in a balanced salt solution and aggregation responses measured with an electronic aggregometer. Leucocytes may be aggregated with substances such as the chemotactic peptide f-met-leu-phe, and platelets with the conventional agents such as ADP or collagen. The response of the leucocytes to f-met-leu-phe is very sensitive to the inhibitory activity of mepacrine. The aggregation of platelets, but not leucocytes, is blocked by low concentrations of indomethacin. The technique is useful for drug screening, diagnostic purposes, and investigating platelet and leucocyte pharmacology and physiology.

Simplified neutrophil hexose monophosphate shunt assay on whole blood.

Neutrophil function was determined in resting and phagocytic cells by an assay of hexose monophosphate shunt activity performed on small volumes of whole blood. This new micromethod clearly differentiated between zymosan-stimulated and unstimulated hexose monophosphate shunt activity. Furthermore, the whole blood microassay was able to measure the large quantities of 14CO2 that previously have been measured by hexose monophosphate shunt assays performed on erythrocyte-free leukocyte preparations. Thus, because of its simplicity and accuracy, the microassay described in this report can be used for routine evaluation of those metabolic activities of neutrophils associated with phagocytosis and bactericidal activity.

A method for the collection of small volumes of whole blood in the field and their preparation for serological studies

A method for the collection of small volumes of whole blood in the field and their preparation for serological studies

Leukocyte migration inhibition of tumor antigen and purified protein derivative reactivity in guinea pigs sensitized to line 10 hepatocarcinoma and BCG.

Agarose microdroplet leukocyte migration inhibition (LMI) assays were performed to measure reactivity against line 10 hepatocarcinoma antigens and purified protein derivative (PPD) with the use of peripheral blood leukocytes from line 10 and/or BCG-sensitized syngeneic guinea pigs. The assay was quite sensitive and detected leukocyte migration inhibition with concentrations as low as 12.6 ng protein/ml of the crude sonicate of the line 10 tumor and 0.1 pg PPD. Specificity was shown by lack of reactivity in leukocytes of line 10 and/or BCG-sensitized animals with antigen preparations of L2C leukemia cells or normal syngeneic liver. Furthermore, leukocytes from normal control guinea pigs failed to react with any antigen. The results also suggested antigen cross-reactivity between line 10 tumor and BCG. Leukocytes from guinea pigs sensitized to only BCG became LMI reactive to the line 10 sonicate as well as PPD. No reactivity was observed with leukocytes of the animals in simultaneous tests with a sonicate of guinea pig L2C leukemia cells. The results demonstrated the usefulness of this microassay in detection of LMI reactivity with low antigen concentrations and small volumes of whole blood.

A simple and efficient method for measuring the uptake of radiolabelled compounds by leukocytes in whole blood

A liquid scintillation counting procedure is described for measuring the uptake of radiolabelled compounds by leukocytes in large or small volumes of whole blood. Color and chemical quenching as well as other technical difficulties noted previously by investigators attempting to process whole blood were eliminated by selectively disrupting the erythrocytes. This was readily accomplished by washing the blood with 3% acetic acid. After bleaching the leukocyte pellet with hydrogen peroxide, the sample was prepared for counting using methods in which the cells were dissolved or oxidized. Examples demonstrating the utility of the proposed technique are presented in which DNA, RNA and protein synthesis of leukocytes from patients with uncontrolled leukemia were measured by the uptake of tritiated thymidine, uridine and leucine, respectively. Also noted is the feasibility of employing a multiple automated sample harvester and whole blood microcultures. The advantages realized by these methods and their potential clinical and experimental utility are discussed.

A direct micromethod for the preparation of blood platelets and thrombocytes for electron microscopy

A micromethod for the preparation of small volumes of whole blood (down to .05ml) for ultrastructural studies is described. It uses standard haematological equipment with minimal centrifugation, and by employing immediate and brief pre-fixation in glutaraldehyde, avoids the use of anticoagulants, thus ensuring preservation of undistorted morphology and ultrastructure in all cell types. A large number of small compact buffy coat preparations can be produced rapidly and consistently, in which stratification is maintained during subsequent processing; this allows examination of the stratification at either the light or electron microscope level. The method makes haematological studies in small animals possible. Platelets and thrombocytes from a wide range of mammalian and avian species, having large or small blood volumes, have been examined.