The estimation of whole blood viscosity by a porous bed method.

A significant impediment in determining the relative contribution of whole blood viscosity to the pathogenesis of cardiovascular and cerebrovascular disease has been the lack of an uncomplicated method to measure whole blood viscosity. To address this problem, a simplified porous bed viscometer has been developed to measure whole blood viscosity. Whole blood is passed through a porous bed of branching channels with a mean pore diameter of 69.6 +/- 20.2 microns and an estimated mean shear rate of 19.6 seconds-1. The effects of sample collection, sample storage, and temperature are described. The mean whole blood viscosity of 242 healthy persons was 22.7 +/- 5.3 seconds, which, when corrected to centipoise using Darcy's equation, corresponds to an apparent viscosity of 5.7 +/- 1.3 cp. There was a significant difference in the whole blood viscosity of normal men and women related to their different packed cell volumes. Platelets and granulocytes influenced whole blood viscosity in proportion to their contribution to the total packed cell volume. Fibrinogen levels did not significantly influence measured whole blood viscosity, which is consistent with the disaggregating conditions and the mean shear rate of the instrument. The porous bed viscometer is a convenient means to measure whole blood viscosity and it should be useful as a screening test for clinical and epidemiologic studies.     

Reliable estimation of hemoglobin A2 concentration by electrophoresis with densitometry.

Elevated hemoglobin A2 (Hb A2) levels can be identified conveniently by densitometry after electrophoresis on cellulose acetate strips. Because a recent report questioned the accuracy of this technic, the method was re-evaluated by paired comparison with microcolumn chromatography. Analysis of 100 patient specimens showed high correlation (r = +0.84), but an average Hb A2 concentration 0.7% higher by densitometry than by chromatography (P less than 0.001). With upper limits set at 4.5%, and 3.8%, respectively, results were divided into "normal" and "high" for each method. Concordant results were obtained in 97 of the 100 cases (82 normal, 15 high). Another densitometer of improved design was used for paired analysis of 50 additional specimens, 25 normal and 25 with beta-thalassemia trait. The two groups were well separated by both procedures, and Hb A2 levels were similar (r = +0.92, P greater than 0.6). This study demonstrates that it is possible, with carefully controlled technics and properly calibrated instruments, to use electrophoresis with densitometry as a reliable means of identifying abnormal Hb A2 levels.     

Facilitated diffusion of carbon dioxide in whole blood and hemoglobin solutions

The values of effective permeability (Krogh's diffusion coefficient) for carbon dioxide have been measured in horizontal stationary layers of whole blood and hemoglobin solutions in quasi-steady state, with the goal of understanding the specific nature of facilitated diffusion of carbon dioxide occurring in these media. The average partial pressure of carbon dioxide within the layer ranged from 0.74 kPa (5.6 mm Hg) to 15.7 kPa (118 mm Hg). Facilitation effects were significant in hemolysed blood and in hemoglobin solutions at low pCO₂; the facilitation factor was up to 2.3. Facilitation effects were considerably less for intact blood; the facilitation factor of intact blood with hematocrit 45% was 0.3. The presence of the red cell membrane appears to have a negative effect on facilitation of carbon dioxide transport.     

Prediction of glucose in whole blood by near-infrared spectroscopy: influence of wavelength region, preprocessing, and hemoglobin concentration

Measurement accuracy for predicting glucose in whole blood was studied based on near-infrared spectroscopy. Optimal wavelength regions, preprocessing, and the influence of hemoglobin were examined using partial least-squares regression. Spectra between 1100 and 2400 nm were measured from 98 whole blood samples. In order to study the influence of hemoglobin, which is the most dominant component in blood, 98 samples were arranged such that glucose and hemoglobin concentrations were distributed in their physiological ranges. Samples were grouped into three depending on hemoglobin level. The results showed that glucose prediction was influenced by hemoglobin concentrations in the calibration model. It was necessary for samples used in the calibration model to represent the entire range of hemoglobin level. The cross-validation errors were the smallest when the wavelength regions of 1390 to 1888 nm and 2044 to 2393 nm were used. However, prediction accuracy was not very dependent on preprocessing methods in this optimal region. The standard error of glucose prediction was 25.5 mgdL and the coefficient of variation in prediction was 11.2%.     

Error in noninvasive spectrophotometric measurement of blood hemoglobin concentration under conditions of blood loss

This paper discusses a current misinterpretation between different parameters of hemoglobin concentration measurement and its amplification under conditions of blood loss. The paper details the distinction between microcirculatory hematocrit and the hematocrit of the macrocirculation to analyze clinical use of real-time patient hemoglobin concentration measurement by noninvasive point-of-care devices such as the Rainbow Pulse CO-Oximetry™ (Masimo Corp., Irvine, CA). The hemoglobin concentration or hematocrit values have clinical significance such as for diagnosing anemia or as indicators to when a blood transfusion is needed. The device infers hemoglobin concentration from spectrophotometry of the fingertip and therefore the measured absorption is due to hemoglobin present in capillaries as well as in larger vessels, and the device accordingly reports the hemoglobin concentration as ‘total hemoglobin’ in a proprietary SpHb parameter. SpHb and macro hemoglobin concentration are different parameters. However, the numerical resemblance of SpHb values to values of macro hemoglobin concentrations, combined with the widely used unspecified term “Hb” in the medical setting, suggests that SpHb values are often interpreted by the clinician as macro hematocrit values.The claim of this paper is that under conditions of blood loss the portion of the SpHb total hemoglobin measure that is contributed from microcirculation increases, due to the decrease of macro hematocrit while microcirculatory hematocrit remains constant when above a critical value. The device is calibrated from phlembotomy drawn blood (from a vein in the arm), which is the gold standard in blood collection, and hence this changing contribution of microcirculatory hemoglobin to the SpHb value would distort the gap between macro hemoglobin and total hemoglobin, SpHb. The hypothesis is that if clinicians indeed interpret the SpHb values as macro hemoglobin values then there is an unreported discrepancy between SpHb to macro hemoglobin concentrations during blood loss due to the increasing effect of microcirculatory hemoglobin measurement on the mixed parameter, SpHb.     

Chemiluminescence of whole blood. 1. A simple and rapid method for the estimation of phagocytic function of granulocytes and opsonic activity in whole blood

Luminol-dependent chemiluminescence of whole blood (whole blood CL) was developed to estimate the phagocytic function of granulocytes and the serum opsonic activity simultaneously. Whole blood (0.1 ml) was examined directly and results were obtained within 20 minutes. Phagocytic function of granulocytes can be estimated from the peak CL of whole blood and the number of granulocytes in a specimen, and the opsonic activity from the amount of time the peak CL is shown after the addition of nonopsonized CL inducer. Thus this method, using several types of CL inducer, offers much information concerning the functions of the phagocytic system in whole blood.     

The determination of INR in stored whole blood.

AIMS: To examine the reliability of international normalised ratio (INR) determination on samples stored as whole blood for up to two days at room temperature. METHODS: The INR of 40 patients receiving oral anticoagulants was determined on fresh blood and on samples stored for 24 and 48 hours, using five locally calibrated prothrombin time systems. These incorporated Manchester reagent, Recombiplastin, IL PT Fibrinogen HS Plus, Manchester combined capillary prothrombin time reagent, and a freeze dried in-house reference rabbit brain thromboplastin, RBT 1010. In addition, factors II, V, VII, and X were determined on samples obtained from 18 of these patients before and after incubation at room temperature. RESULTS: The INR of the samples changed by differing amounts during storage, depending on which system was employed. Although the mean change after 24 hours storage was relatively small, there were individual samples that changed by > 0.5 INR with all systems. These changes would lead to adjustment in dosage of certain patients. After 48 hours these effects were greater with all systems except that employing Recombiplastin. There were only small reduction in the measured factors by 48 hours. CONCLUSIONS: After storage of samples for only 24 hours, some patients' INR changed sufficiently to affect dosage. In view of these observations, the practice of storing whole blood samples for INR determination cannot be recommended.     

Evaluation of methods and protocols for hemoglobin screening of prospective whole blood donors

Fifty-nine volunteers were screened, as if for blood donation, with the use of simultaneous fingerstick (FS), earlobe (EL), and venipuncture (VP) samples tested by copper sulfate density and two instruments for rapid assay, Statcrit and HemoCue. The "true" hemoglobin was determined on the venipuncture sample by cyanmethemoglobin assay. Eligibility (pass/fail) was established for each site/result with the use of standard and investigational cut-offs. Results were used to predict the performance of two-method protocols beginning with copper sulfate followed by FS or EL using Statcrit or HemoCue. Individually, the method sensitivities for hemoglobin below cut-off were low (12.5-62.5%) with the use of standard cut-offs. Assuming 5% prevalence of low hemoglobin, two-method protocols would inappropriately pass more than half of those with low hemoglobin, whereas the proportion of deferred donors with adequate "true" hemoglobin ranged widely (2.8-72.3%). For some methods, investigational cut-offs achieved improved sensitivity with no effect on specificity. Despite standard hemoglobin "requirements" for blood donation, the ability of the investigated methods to correctly classify donors is poor and varies considerably with method and protocol. With cut-offs as specified, the standards for FS and EL samples are not equivalent; the EL cut-offs are too low and the method is inherently less sensitive. Standardization may be better achieved by specifying both required hemoglobin and minimum performance for screening methods.     

The use of Fourier-transform infrared spectroscopy for the quantitative determination of glucose concentration in whole blood

Fourier-transform infrared transmission spectroscopy has been used for the determination of glucose concentration in whole blood samples from 28 patients. A 4-vector partial least-squares calibration model, using the spectral range 950-1200 cm(-1), yielded a standard-error-of-prediction of 0.59 mM for an independent test set. For blood samples from a single patient, we found that the glucose concentration was proportional to the difference between the values of the second derivative spectrum at 1082 cm(-1) and 1093 cm(-1). This indicates that spectroscopy at these two specific wavenumbers alone could be used to determine the glucose concentration in blood plasma samples from a single patient, with a prediction error of 0.95 mM.     

Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood.

An indirect (I) enzyme-linked immunosorbent assay (ELISA) and a competitive (C) ELISA, using a group-specific monoclonal antibody against bluetongue virus (BTV), are described for the detection of antibodies to BTV in cattle and sheep sera. The performance of these assays in detecting anti-BTV antibody in sequential serum samples and eluates from whole blood (WB) dried on filter paper from three calves and four sheep experimentally infected with type 10 BTV was evaluated. The C-ELISA was superior to the I-ELISA in the detection of anti-BTV antibody in the sera and WB samples from both cattle and sheep early after infection with BTV. BTV antibodies were demonstrable by C-ELISA in all the bovine and ovine sera and WB eluates by 9 days postinfection; whereas the I-ELISA results for sheep sera and WB eluates were similar, anti-BTV antibody was not detected in bovine serum and WB eluates until 26 and 14 days postinfection, respectively. While both ELISAs proved reliable, under the present test conditions involving detection of early postinfection reactions of experimentally infected animals, the C-ELISA was always as sensitive or more sensitive than the standard agar gel immunodiffusion test, the modified complement fixation test, and the plaque neutralization tests in the detection of anti-BTV antibodies. Unlike observations with the immunodiffusion test, no reaction was seen between BTV antigen and bovine epizootic hemorrhagic disease virus antiserum in either ELISA. The results suggest that either ELISA may be suitable for routine diagnostic testing and may have the potential to replace other tests for detection of anti-BTV group-specific antibodies and that the C-ELISA may have the most potential.     

Microfluidic aqueous two phase system for leukocyte concentration from whole blood

Leukocytes from a whole blood sample were concentrated using a microfluidic aqueous two phase system (μATPS). Whole blood was simultaneously exposed to polyethylene glycol (PEG) and dextran (Dex) phase streams and cells were partitioned based on their differential affinity for the streams. The laminar flow characteristic of microfluidic devices was used to create zero, one, and two stable interfaces between the polymer streams. Three different patterns of three polymer streams each were evaluated for their effectiveness in concentrating leukocytes: immiscible PEG-PEG-Dex, immiscible Dex-PEG-Dex, and miscible PEG-PBS-Dex. The most effective configuration was the Dex-PEG-Dex stream pattern which on average increased the ratio of leukocytes to erythrocytes by a factor of 9.13 over unconcentrated blood.     

The mixed lymphocyte response in whole blood: technical aspects.

Experiments were conducted to standardize the response of lymphocytes in whole blood in mixed culture with allogeneic lymphocytes. The following conditions were found suitable: (1) A culture period of 6-8 days. (2) The ratio of stimulator to responder lymphocytes should be 4-8, but may vary from one batch of stimulator cells to another. (3) Tests may be performed in culture tubes with 50-100 microliter blood in a total volume of 2 ml (macrotechnique) or in microplates with 10 microliter of blood in a volume of 0.2 ml (microtechnique). (4) Serum supplement is not required. (5) Results should be expressed as counts/min (cpm) per a given number of responder lymphocytes. Stimulation indices are less reliable.     

Automated quantitation of hemoglobin-based blood substitutes in whole blood samples.

It is necessary to develop methods for accurate monitoring of cell-free hemoglobin in circulation. Routine monitoring of circulating cell-free hemoglobin will be useful for evaluating the efficacy of blood substitute administration andfor determining the clearance rates of the blood substitute from circulation. In addition, discriminating between cell-free hemoglobin and cell-associated hemoglobin will enable accurate determination of RBC indices, mean cell hemoglobin and mean corpuscular hemoglobin concentration, in individuals receiving hemoglobin-based blood substitutes. As colorimetric methods used by hematology analyzers to quantitate the hemoglobin value of a blood sample cannot distinguish between cell-associated and cell-free hemoglobin, it is currently not feasible to quantitate the levels of hemoglobin substitutes in circulation. The advent of a technology that measures volume and hemoglobin concentration of individual RBCs provides an alternative strategy for quantitating the cell-associated hemoglobin in a blood sample. We document that the combined use of cell-based and colorimetric hemoglobin measurements provides accurate discrimination between cell-associated and cell-free hemoglobin over a wide range of hemoglobin levels. This strategy should enable rapid and accurate monitoring of the levels of cell-free hemoglobin substitutes in the circulation of recipients of these blood substitutes.     

Pattern of hemoglobin concentration in untreated Burkitt lymphoma patients in Ibadan.

Hemoglobin concentration is studied in 128 untreated Burkitt lymphoma patients in Ibadan. While there is no significant diagnostic pattern in hemoglobin concentration, Burkitt lymphoma patients show marked anemia as compared with healthy adult Nigerians. The degree of involvement of malarial parasitemia in producing anemia in Burkitt lymphoma patients has yet to be determined.