Amount Of Whole Blood Research Articles

An integrated system using immunomagnetic separation, polymerase chain reaction, and colorimetric detection for diagnosis of Plasmodium falciparum.

An integrated system for sample preparation and DNA detection of the malaria parasite using immunomagnetic separation in combination with the polymerase chain reaction (PCR) and colorimetric analysis is described. A cocktail of three monoclonal antibodies towards merozoite surface antigen-1 was used for magnetic capture of parasites from microliter amounts of whole blood. A sensitivity down to a parasitemia of 10(-6)% was achieved using cultured parasites as a model. The integrated approach was evaluated in a field study. A total of 410 blood samples from patients attending malaria clinics in Trat province and Kanchanaburi province in Thailand were analyzed. The samples were processed by immunomagnetic separation and transferred to central laboratory for PCR-based detection. Microscopic examinations on blood smears were done in parallel; 53% were positive using the DNA-based assay, while only 32% were positive using conventional microscopic analysis. This field study suggests a possible model for large-scale testing of malaria with an increased sensitivity compared with conventional methods.

Rapid calibration‐free determination of lead in microliter amounts of whole blood

AbstractA calibration‐free method using portable instrumentation and thus suitable for field screening of lead in whole blood is described. Whole blood (2.5 μL) is diluted first with distilled water (5.0 μL) and then with a matrix‐modifying solution (17.5 μL) containing 0.50M HCl, 600 mg/L HgII, 5% v./v. Triton X‐100 and l00 μg/L of cadmium(II), the latter being used for performance check. A 12 μL subsample is then electrolyzed for 2 min. on a vibrating glassy carbon electrode whereby 90% of the lead(II) ions are reduced to amalgamated lead. Stripping oxidation is then performed with a constant current of 40μA. In two consecutive stripping oxidations the chemical contribution (chemical current) to the oxidation is determined facilitating calculation of the total amount of lead(II) in the sample using Coulombs law subsequent to correction for nonquantitative electrolysis. Results obtained on analysis of certified blood standards deviated less than 14 μg/L on the 3 sigma level in the concentration range 22–118 μg/L and by less than 13% on the 3 sigma level in the range 220–616 μg/L.

A Prospective Study of the Application of Transfusions in a Field Hospital during the War in Croatia

Use of blood products are recommended worldwide as a substitution for whole blood. In the case of war wounds, because of their specificity, this is not possible. We studied prospectively the use of blood and blood products during management of the wounded in a field hospital during the war in Croatia. During a 6-month period, 178 soldiers out of a total number of 593 were hospitalized, and of this number 64 (36%) received transfusions. Localization of the wound, and speed and quality of first aid had significant effects on the average amount of blood used. Consequently, norms for blood use in wartime in west European countries and NATO are lower than what we used for each wounded patient. Ensuring an adequate amount of whole blood still remains one of the primary tasks of the transfusion service in wartime.

Development of a simple, rapid and accurate in vitro whole blood technique for the detection and semi-quantification of FIV cellular viremia

A new, simple, rapid and accurate culture technique is described for a semi-quantitative analysis of cellular viremia in FIV-infected cats. This assay can be carried out with small amounts of whole blood, and is based on the detection of FIV core gag antigen, which is released in culture supernatants. The amount of core antigen produced is measured with an enzyme-linked immunoassay using specific monoclonal antibodies. This whole blood technique (WB method) was compared with a culture method using isolated peripheral blood mononuclear cells (PBMC method). FIV could be detected in whole blood of all experimentally infected cats, but not from uninfected cats. This assay offers a number of advantages (small blood samples required, no leukocyte separation and lymphocyte purification procedures) and its reproducibility is very good. It provides a convenient in vitro cellular assay for viral semi-quantitation, well adapted for monitoring efficacy of prototype FIV vaccines or experimental antiviral drugs. Also, it could facilitate the study of the pathogenesis of FIV-related progressive immunodepression. Finally, it offers an alternative to serological techniques for diagnostic purposes in several circumstances: early viremia, maternal antibodies···

Criteria for safe hepatic resection

To lower morbidity after hepatic resection, the authors examined the influence of predictor variables including: age, sex, preoperative risk factors, serum total bilirubin level, plasma retention rate of indocyanine green at 15 minutes, underlying liver disease, operative blood loss, operation time, amount of whole blood transfused, vascular occlusion time, surgical procedure employed, and extent of hepatic resection. Between January 1990 and December 1992, 172 patients underwent hepatic resection based on our own criteria for hepatectomy, including the presence or absence of ascites, serum total bilirubin level, and the plasma retention rate of indocyanine green at 15 minutes in patients with chronic liver disease. The morbidity rate was 37.2%, and the hospital and operative mortality rates were 2.3% and 0.6%. The multiple logistic model revealed that the risk of morbidity was increased by longer operation time, major hepatic resection, and preoperative cardiovascular disease. Shortening the operation time without increasing operative blood loss and further modalities for making major hepatectomy safer are future problems to be addressed.

Stable-isotope dilution GC-MS for determination of toluene in submilliliter volumes of whole blood.

An accurate and sensitive yet simple protocol for the analysis of toluene in submilliliter quantities of whole blood using stable-isotope dilution GC-MS has been developed and evaluated for use in pharmacokinetic studies of toluene exposure. The method involves the use of toluene-d8, a relatively inexpensive chemical used as a solvent for nuclear magnetic resonance spectroscopy, as an internal standard and extraction with pentane. This procedure allows for the determination of toluene in whole blood down to approximately 5 ng/mL and is suitable for pharmacokinetic measurements and biomonitoring of exposure to toluene.

Intravascular ultrasound characterization of thrombi of different composition

An in vitro model was designed to test the hypothesis that thrombi of varying composition have different echogenic patterns. Thrombi were prepared in specially designed tubes, mounted on a holder that allows introduction of an intravascular ultrasound catheter rotated inside a subselective sheath. The thrombi were made by the addition of thrombin to whole blood, plateletrich plasma, and to mixtures of whole blood and platelet-rich plasma with increasing concentration of whole blood relative to the volume of the mixture in the following ratios: 1:5, 2:5, 3:5 and 4:5. Sixty-six thrombi prepared from 11 blood samples of healthy subjects were studied and compared with control tubes filled with saline solution. Platelet-rich thrombi showed low echogenicity similar to saline solution. Whole blood thrombi appeared uniformly “speckled.” Mixtures of whole blood and platelet-rich plasma showed a gradual increase in echogenicity with an increasing amount of whole blood in the mixture. Quantitative videodensitometry compared the gray scale intensity of each image relative to background saline. The mean value of echogenicity of platelet-rich thrombi was 0.9 ± 1.2, and the mean value of whole blood thrombi was 13 ± 5.3. Platelet-rich thrombi are echolucent, and the main echogenic reflectance of thrombi originates from red blood cells. The ultrasound intensity is in linear relation to the amount of red blood cells in the thrombus.

Determination of lead in microliter amounts of whole blood by stripping potentiometry

AbstractLead in whole blood is determined using a commercial potentiometric stripping analyzer in combination with a portable personal computer and a new electrode design permitting rotation of the sample. A 70 μL blood sample is added to 200 μL distilled water contained in a disposable test tube. After mixing, 400 μL of a matrix‐modifying solution containing hydrochloric acid, mercury (II) ions, Triton X‐100, and bismuth (III) as an internal standard, is added to the sample which is then transferred to the sample holder. Lead amalgam is deposited on the glassy carbon electrode by means of a pulsed potential cycle for 3 minutes after which lead is reoxidized by means of a constant current. The complete electroanalytical procedure, including result evaluation by means of the internal standard, and graphical display is under computer control. For ca. every 15th sample, the instrument is recalibrated, if necessary, using two whole blood standards. The relative precision is ca. 10% and the detection limit 3.5 μg/L. The stripping potentiometry results agree well with the results obtained by inductively coupled plasma mass spectrometry in the analysis of whole blood from individuals occupationally exposed to lead and with the results obtained by graphite‐furnace atomic spectroscopy in the analysis of whole blood from 141 school children.

A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood

Phagocytes play an important role in host defence against microorganisms and different techniques are needed to evaluate their functional activities. Here we describe a rapid, simple and reliable one step procedure to measure the phagocytosis rate and oxidative burst activation of both polymorphonuclear leukocytes (PMN) and monocytes, by means of flow cytometry using only small quantities of whole blood. The two species of bacteria employed as test microorganisms, Staphylococcus aureus and Candida albicans showed a somewhat different behaviour. We found that the present method could be used as an alternative test in the diagnosis of chronic granulomatous disease (CGD). Moreover we were able to analyse, in a one step procedure, defective phagocyte functions in a group of paediatric patients suffering from recurrent microbial infections.

Development of simian immunodeficiency virus isolation, titration, and neutralization assays which use whole blood from rhesus monkeys and an antigen capture enzyme-linked immunosorbent assay

Assays that use rhesus macaque whole blood and an antigen capture enzyme-linked immunosorbent assay for the simian immunodeficiency virus (SIV) p27 core protein were developed for the isolation of SIV from the blood of infected animals, the titration of infectivity of SIV inocula, and the quantitation of virus neutralizing antibodies in serum. These assays required small amounts of whole blood, were adaptable to a microtiter format, and used substrates mainly of rhesus macaque origin.

Blood Component Induction of Cerebral Vasospasm

Abstract The role of blood components in cerebral vasospasm was evaluated using an in vivo canine model. An intracisternal injection of 5 ml of washed red blood cells (RBCs) and platelet-rich plasma (PRP) resulted in acute vasospasm of the basilar artery as seen by angiography. This was comparable with the degree of vasospasm induced by an injection of the same amount of whole blood. Repeated injections of blood components or whole blood on Days 1 and 3 induced chronic vasospasm. as shown by angiography on Day 7. Results clearly indicate that chronic vasospasm was produced by RBCs and PRP. and it was in a dose-dependent manner with increasing concentrations of RBCs (hematocrit: 30, 50. and 70%). The vasospasm induced by both components closely reproduced that seen with whole blood. Neither acute nor chronic vasospasm induced by blood component injection was associated with clot formation in the subarachnoid space, as confirmed by an autopsy. These results suggest that the extravasation of RBCs and PRP into the subarachnoid space produces cerebral vasospasm by mechanisms independent of blood clot formation.

Blood component induction of cerebral vasospasm

The role of blood components in cerebral vasospasm was evaluated using an in vivo canine model. An intracisternal injection of 5 ml of washed red blood cells (RBCs) and platelet-rich plasma (PRP) resulted in acute vasospasm of the basilar artery as seen by angiography. This was comparable with the degree of vasospasm induced by an injection of the same amount of whole blood. Repeated injections of blood components or whole blood on Days 1 and 3 induced chronic vasospasm, as shown by angiography on Day 7. Results clearly indicate that chronic vasospasm was produced by RBCs and PRP, and it was in a dose-dependent manner with increasing concentrations of RBCs (hematocrit: 30, 50, and 70%). The vasospasm induced by both components closely reproduced that seen with whole blood. Neither acute nor chronic vasospasm induced by blood component injection was associated with clot formation in the subarachnoid space, as confirmed by an autopsy. These results suggest that the extravasation of RBCs and PRP into the subarachnoid space produces cerebral vasospasm by mechanisms independent of blood clot formation.

Chemiluminescence properties of human, canine and rat polymorphonuclear cells

Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two-step discontinuous Percoll gradients with distinct different densities were used. The chemiluminescence properties of the isolated PMN and of phagocytes in small quantities of whole blood were compared in luminol-enhanced assays after stimulation with various agents: non-opsonized zymosan (3.5 g/l), phorbol myristate acetate (PMA, 2.8 x 10(-6) mol/l), calcium ionophore A 23187 (10(-5) mol/l) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 3.5 x 10(-6) mol/l). The isolated cells of the three species responded to all of the various stimuli. Species-related sensitivity could be ordered: human greater than canine greater than rat. Response to the various agents in the human cells can be ranked: PMA greater than or equal to A 23187 greater than zymosan greater than FMLP; for the dog: A 23187 greater than PMA greater than zymosan greater than FMLP; and for the rat: zymosan greater than or equal to PMA greater than FMLP greater than or equal to A 23187. Time course and peak maximum response were different upon stimulation in the absence and presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.

Micro extraction of DNA from whole blood and amniocytes.

We describe methods for extracting genomic DNA from a small amount of whole blood or cultured amniocytes. Nuclear DNA was extracted from whole blood spotted on blotting paper. Relatively large molecules of DNA with the average amount of 7-9 micrograms was extracted from 1 ml of blood spotted and stored for at most two years, being roughly 1/3 of that extracted directly from fresh whole blood. The estimated minimum amount of whole blood that gives a suitable autoradiogram of Southern hybridization was 0.3 ml. Another series of amounts of whole blood or an amniocyte suspension were molded in low-melting agarose into an 100 microliter gel block. The DNA extracted from a block that was made from at least 0.25 ml of whole blood, or from 1.25 x 10(5) amniocytes (equivalent to 1/8 of the number of confluent cells in a 25 cm2 culture flask) resulted in one suitable Southern analysis. Both methods described here are applicable to the diagnosis of newborns and/or fetuses at risk of a genetic disease and to the diagnosis of a patient from whom a large amount of blood material is difficult to obtain. These methods also make a long-way transportation of the materials possible.

A preliminary study on the use of enzyme-linked immunosorbent assay (ELISA) for detection of Trichinella spiralis infections in dogs

An enzyme-linked immunosorbent assay (ELISA) was developed to detect Trichinella spiralis infections in dogs using rabbit anti-canine IgG-horseradish peroxidase prepared according to the improved periodate method and an antigen purified from T. spiralis larvae by Sephadex G-200 chromatography. Sixty-six canine sera were tested for trichinosis by the ELISA and it showed a detection rate which was significantly higher than that by trichinoscopy. This antigen of T. spiralis appeared not to cross-react with the sera of dogs infected with Ancylostoma caninum or Taenia spp. A comparison of ortho-phenylenediamine and 5-amino-2-hydroxybenzoic acid as substrates in the ELISA did not reveal a significant difference. Pieces of filter paper saturated with a defined quantity of whole blood can be substituted for serum as a source material for the test. The relationship between worm burden and the adsorbance value in ELISA is discussed.

Increase in ovarian blood volume during ovulation in the gonadotropin-primed immature rat.

This study quantifies ovarian blood volume in Wistar rats by measuring the optical density (414 nm) of hemoglobin in ovarian extracts and comparing this measurement to the optical density of known amounts of whole blood. Immature rats were primed with pregnant mare's serum gonadotropin (PMSG), 10 IU s.c., at 23 days of age. On Day 25, the ovulatory process was initiated by human chorionic gonadotropin (hCG), 10 IU s.c., and ova began to appear in the oviducts 10 h later. At 2-h intervals, the ovaries were extirpated and homogenized in 1.0 ml of 0.05 M tris (hydroxymethyl)aminomethane buffer (pH 7.4) for 30 s. Homogenates were centrifuged for 20 min and the supernatant fluids were analyzed with a Gilford RESPONSE UV/VIS spectrophotometer. The hemoglobin in these ovarian extracts had the same peak absorbance of 414 nm characteristic of oxyhemoglobin in whole blood taken by cardiac puncture of the rats. There was a linear relationship between the absorbance and the volume of whole blood in the samples. The volume of blood per ovary from groups of 8 rats was 0.60 +/- 0.07 microL at 0 h after hCG. The volume increased to 1.37 +/- 0.26 microL at 4 h after hCG and reached a peak of 4.55 +/- 0.72 microL at 10 h. Indomethacin treatment (0.3-10.0 mg/rat, s.c.) partially inhibited this 7-fold increase in ovarian blood volume. In conclusion, the increase in ovarian blood volume during ovulation may reflect the vasodilation and hyperemia that are characteristic of inflamed tissues.

Separation and chemiluminescence properties of human, canine and rat polymorphonuclear cells

Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two-step discontinuous Percoll gradients with distinct densities were used. The purity of the preparations was 99.2%, 98.4% and 97.9%, respectively and the corresponding recoveries were 80.1; 66.3% and 69%. The chemiluminescence properties of the isolated PMN and of phagocytes in small quantities of whole blood were compared in luminol-enhanced assays after stimulation with various agents: non-opsonized zymosan (3.5 g/l), phorbol myristate acetate (PMA, 2.8 × 10 −6 M), calcium ionophore A 23187 (10 −5 M) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 3.5 × 10 −6 M). The isolated cells of the three species responded to all of the various stimuli. Species-related sensitivity followed the order: human > canine > rat. Responses to the various agents in the human cells was ranked: PMA ≥ A 23187 > zymosan > FMLP; for the dog: A 23187 > PMA > zymosan > FMLP; and for the rat: zymosan ≥ PMA > FMLP ≥ A 23187. Time courses and peak maximum responses were different following stimulation in the absence or presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.

How Much Blood for the World?

Based on data from 96, and estimates from 32 countries with populations of over 1 million, it was calculated that the total amount of whole blood collected annually in the world in the early 1980s was about 75 million units. Of this, one-third was collected by Red Cross and Red Crescent blood programmes. The index of donations per 1,000 population was on average 50.2 in industrial market countries, 9.5 in middle-income countries and 1.1 in low-income countries. For planning purposes donation rates in relation to health services are better than the population index. It seems that roughly 10 annual donations per acute hospital bed, or 0.40 donation per patient admission to these hospitals would be sufficient to provide adequate quantitites of blood and blood products for modern haemotherapy.

How much blood for the world?

Based on data from 96, and estimates from 32 countries with populations of over 1 million, it was calculated that the total amount of whole blood collected annually in the world in the early 1980s was about 75 million units. Of this, one-third was collected by Red Cross and Red Crescent blood programmes. The index of donations per 1,000 population was on average 50.2 in industrial market countries, 9.5 in middle-income countries and 1.1 in low-income countries. For planning purposes donation rates in relation to health services are better than the population index. It seems that roughly 10 annual donations per acute hospital bed, or 0.40 donation per patient admission to these hospitals would be sufficient to provide adequate quantities of blood and blood products for modern haemotherapy.

Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000.

A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.