A Study of Two Procedures of HIV-1 Isolation from Whole Blood Cultures

Culture techniques for isolation of HIV-1 from small amounts of whole blood (WB) treated with anticoagulant have been reported and gave results identical to those of culture of separated peripheral blood mononuclear cells. Some authors obtained much higher isolation rates when EDTA was used instead of heparin. We compared two previously described techniques for cultivation of HIV-1 from WB of adult HIV+ patients staged according to the CDC classification. In addition, we assessed the influence of the type of anticoagulant used for the collection of blood in viral replication in cell cultures from whole blood. Small volumes of WB treated with either heparin or EDTA were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMC) from healthy donors. We used two procedures for WB culture: procedure I, based on the culture of 250 μl of WB with 1 × 10⁶ PHA-PBMC from donors; and procedure II based on the culture of 500 μl of WB with 4× 10⁶ PHA-PBMC from donors. The cocultures were placed in 24-well plates and incubated for as long as 28 days in medium containing interleukin 2 (IL-2). Twice weekly half of the medium was replaced with fresh medium. In procedure II, one million fresh PHA-PBMC from donors was added on the 7th day of culture. The culture supernatant was assayed for the presence of HIV-1 p24 antigen in an enzyme immunoassay. The kinetics of HIV-1 replication in cultures of WB from 7 AIDS patients were similar using procedures I and II. In 8 HIV + patients the isolation rate was higher with heparin- than with EDTA-treated samples. The isolation rate was higher in AIDS patients (n = 8) than in others with both methods. In stage IV patients without AIDS (n = 8) we failed to isolate HIV-1 in 1 patient with procedure I, whereas we succeeded with procedure II. In stage II, HIV-I was isolated in 1 of 4 patients with both methods. HIV was isolated in cultures of WB from patients receiving zidovudine or related nucleoside analogues and in cultures of WB from untreated patients. HIV-1 could not be isolated from WB of patients with more than 400 CD4+ T lymphocytes in their peripheral blood (n=4); however, it was isolated from 14 of 16 patients with less than 400 CD4+ T lymphocytes. Our results suggest that procedure II is more sensitive than procedure I and that heparin is better than EDTA for collecting WB. We showed that the rate of HIV-1 isolation from WB increased in advanced-stage patients. Further studies are needed to define the clinical applications of WB culture.     

Ultra-Fast Analysis of Plasma and Intracellular Levels of HIV Protease Inhibitors in Children: A Clinical Application of MALDI Mass Spectrometry

HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI) - triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 µL plasma and 1×10⁶ PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.     

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10¹ to 10² bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.     

Quantification of cyclosporine A in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry using a column-switching approach

As a potential alternative to cyclosporine A (CsA) monitoring in whole blood, a sensitive and selective method was developed for quantifying this immunosuppressive drug in human peripheral blood mononuclear cells (PBMCs) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). PBMCs were isolated from whole blood by density gradient centrifugation. After purification, cell counts were performed to express CsA amounts per single cell. The pelleted cells were then lysed and CsA was extracted with methanol (MeOH) containing 27-demethoxy-sirolimus as internal standard. After evaporation of the supernatant under nitrogen, the residue was reconstituted in MeOH, further diluted with water and injected onto a column-switching unit. On-line solid-phase extraction was performed using a C8 column with an acidic aqueous mobile phase containing 5% MeOH. The analytes were transferred in the back-flush mode on a C18 column with 65% MeOH and the chromatographic separation performed with a MeOH gradient (65-90%). The detection was carried out with a single quadrupole analyzer and the sodium adducts [M+Na](+) were monitored for quantification. This sensitive method was fully validated in the range of 5-400 ng/mL. This allowed the measurement of very small CsA amounts present in cells up to 0.5 fg/PBMC in clinical samples. Trueness (95.0-113.2%), repeatability (5.1-9.9%) and intermediate precision (7.0-14.7%) were found to be satisfactory. This method represents a new potential tool for therapeutic drug monitoring of CsA and could be used in clinical conditions if the utility of intracellular measurements is confirmed in prospective clinical trials.     

Profiling gene expression in whole blood samples following an in-vitro challenge.

Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.     

Liquid chromatography analysis of enrofloxacin and ciprofloxacin in chicken blood spotted on filter-paper disks

A simple, low-cost, sensitive and selective LC method was developed for the determination of enrofloxacin and ciprofloxacin in chicken blood. The method was applied to whole blood from a chicken using dried blood spots on filter paper disks. The detection limits of enrofloxacin and ciprofloxacin (100 microl of whole blood on a disk) were 0.005 and 0.01 microg/ml, respectively. The whole procedure was verified in intra-laboratory studies (recoveries of both compounds were above 90%), and its applicability was tested with blood from the chicken receiving enrofloxacin in a single oral dose at a level of 10 mg/kg body mass. The method permits the use of a small volume of blood from a chicken and should be useful for pharmacokinetic studies.     

Determination of beta-phenylethylamine concentrations in human plasma, platelets, and urine and in animal tissues by high-performance liquid chromatography with fluorometric detection

A highly sensitive method for the determination of beta-phenylethylamine in human plasma, platelets, and urine and in mouse tissue is described. The method is based on a two-step isolation using cation-exchange columns followed by reverse phase high-performance liquid chromatography with fluorometric detection. The recovery of the amine through the whole procedure was almost complete, ranging from 99 to 101%. The calibration graph appeared linear over the range of 50 to 5000 pg/injection. Urinary excretion of beta-phenylethylamine in humans ranged from 0.93 to 51.20 ng/mg creatinine. The amine was also detectable in plasma and platelets. Of the various mouse tissues examined, the highest concentrations were found in the small intestine, followed by the blood and liver. Concentrations of about 5 ng/g wet wt were detected in brain tissue, which increased remarkably after inhibition of monoamine oxidase by pargyline.     

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g^?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N⁹‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.     

Use of a simple method for the Epstein-Barr virus transformation of lymphocytes from members of large families of Réunion Island

A simple method for the preparation of lymphoblastoid cell lines from small amounts (100 microliter) of frozen whole blood is described. A success score greater than 90% was obtained for EBV transformations using blood samples which had been collected several months before the infection. Due to the simplicity of the technique, up to 80 samples could be processed per day. This technique was used to prepared 242 permanent cell lines from 13 large families from Réunion Island showing blood group H deficiency. These cell lines are now available for genetic studies.     

Quantitative Competitive RNA PCR for Quantitation of Virion-Associated HIV-1 RNA

Quantitative competitive PCR is a highly sensitive technique that allows accurate quantitation of small amounts of RNA. We have modified the original method to include the use of an internal standard at all stages of sample analysis. In this way, the method can accommodate for variations in the recovery of viral particles and in the isolation of genomic RNA as well as provide a suitable competitive substrate during quantitative RNA PCR. We have used this method to characterize changes in virus load in plasma of HIV-1-seropositive individuals following their vaccination against opportunistic infections.     

Lipase-catalyzed synthesis of vitamin C fatty acid esters

Fatty acid esters of vitamin C (ascorbic acid) where synthesized in a mainly solid-phase system in the presence of small amounts of organic solvent (acetone or t-butanol) catalyzed by immobilized lipase B from Candida antarctica.Highest reaction rates and yields of isolated products were obtained using fatty acid vinyl esters, e.g., 6-O-palmitoyl-l-ascorbic acid was obtained in 91% isolated yield after 48 h. As vitamin C and its esters are very sensitive to oxidation, a mild extraction method for the isolation of reaction products was developed.     

Measurement of DNA breaks and oxidative damage in polymorphonuclear and mononuclear white blood cells: a novel approach using the comet assay

DNA damage is thought to play a relevant role in degenerative diseases and aging. Therefore, measuring DNA damage in living cells without artifacts is a critical issue, especially with very sensitive methods, such as the comet assay, which can detect very low levels of DNA damage. We show here that the procedures of cell subtype isolation increase DNA damage measured in human white blood cells (WBC) with the comet assay. We describe a novel and simple method to measure DNA strand breaks and oxidative damage separately in polymorphonuclear and mononuclear leukocytes, using whole blood without previous cell isolation. This method can be useful for measuring DNA damage in different subtypes of human peripheral leukocytes, avoiding the artifacts and the time involved in the cell separation procedures.     

Sensitive detection of human globin chains by microbore high-performance liquid chromatography and its forensic application

This paper describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the detection of human globin chains in blood and bloodstains. The method involves direct injection of the filtered samples of dilute hemolysates or bloodstain extracts onto a microbore C₄ reversed-phase column (2.1 mm I.D.) with UV detection at 220 nm. Microbore HPLC offers a significant improvement in sensitivity with little loss of the resolution of globin chains and only small variations in the determination of γ chain composition. The detection limit of hemoglobin (Hb) was 0.1 μg, which is equivalent to about 1 nl of fresh whole blood. Umbilical cord blood could be differentiated from adult blood in stains that were up to twenty weeks old, by the presence of γ globin chains. The present method will be useful for detection of abnormal Hbs and for the determination of γ chain composition in clinical laboratories, as well as in the practice of forensic science for the analysis of minute amounts of blood and bloodstains.     

Leukocyte counting from a small amount of whole blood using a size-controlled microcavity array

Absolute counting of total leukocytes or specific subsets within small amounts of whole blood is difficult due to a lack of techniques that enable separation of all leukocytes from limited amounts of whole blood. In this study, a microfluidic device equipped with a size-controlled microcavity array for highly efficient separation of leukocytes from submicroliters of whole blood was developed. The microcavity array can separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered over 90% of leukocytes in 1 μL of whole blood without pretreatment such as density gradient centrifugation or erythrocyte lysis. In addition, the proposed system successfully performed absolute enumeration of human CD4(+) and CD8(+) leukocytes from 1 μL of whole blood, and the obtained data showed good correlation with conventional flow cytometric analysis. Our microfluidic device has great potential as a tool for a point-of-care leukocyte analysis system.     

Risk of HIV infection from blood transfusion in Montreal

OBJECTIVES: To determine the incidence (including associated donor characteristics and time trends) of HIV infection among repeat blood donors and to estimate the risk of HIV transmission from blood transfusion in Montreal and in Canada as a whole. DESIGN: Retrospective cohort analysis. SETTING: Montreal Centre Blood Transfusion Service. PARTICIPANTS: People who donated blood at least twice after Nov. 1, 1985, and at least once from Apr. 1, 1989, to Mar. 31, 1993. INTERVENTION: Blood was screened for HIV by enzyme-linked immunosorbent assay and results were confirmed by Western blot analysis. OUTCOME MEASURES: Incidence density (the incidence rate per person-time) of HIV infection among repeat blood donors by sex, age group and region of residence, and incidence density and risk among first-time donors and for Canada as whole. RESULTS: There were 200,196 eligible donors and 432,631 person-years (PY) of observation. From 1989 to 1993, there were 18 HIV seroconversions among repeat donors. The crude incidence density was 3.3 per 100,000 PY (95% confidence interval [CI] 1.8 to 5.4 per 100,000 PY); it was 4.9 per 100,000 PY among men and 0.61 per 100,000 PY among women. Age-specific incidence per 100,000 PY was 2.5 among those 12-29 years of age, 5.1 among those 30-49, 2.9 among those 40-49, and 1.4 among those 50 and older. Based on an estimated mean "window period" (from when a donor''s blood is capable of transmitting HIV until detectable antibody appears) of 25 days, the current risk of HIV infection from repeat donors in the window period is estimated at 1 in 440,000. Inclusion of blood units from first-time donors produces an overall risk of 1 in 390,000 (95% CI 1 in 250,000 to 655,000). The estimated risk per blood unit in Canada as a whole is 1 in 913000 (95% CI 1 in 507,000 to 2,050,000). CONCLUSIONS: This "sentinel" population of repeat blood donors is subject to important trends in HIV spread. Therefore, estimating the incidence density of HIV infection in repeat donors provides insight into the epidemiologic characteristics of HIV infection at minimal expense. As a result of measures to improve blood safety, including HIV testing, the incidence of HIV infection among blood donors in Canada is low and the risk of HIV transmission from transfusion is extremely small, although not zero.     

Simplified method for isolation of white cells from whole blood suitable for direct polymerase chain reaction

A simple method for isolating mononuclear cells from whole blood is described. The procedure utilizes phytohemagglutinin to agglutinate the erythrocytes, separating white cells from whole blood in a very brief handling time. The isolated cells are readily subjected to DNA isolation simply by boiling, and the released DNA can be directly employed for the polymerase chain reaction analysis. The efficiency of this method is similar to other conventional methods, but less costly and less time-consuming. This method is particularly useful in analyzing DNA samples from the peripheral blood cells when the simplicity and low cost of the assay are preferable.     

Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens.

The present report describes an alternative method for in vitro detection of HIV-1-specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analyzed. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.     

Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid

Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their facile manipulation and low costs. Although methods involving their magnetic separation have been extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired monodispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial microbeads (NucliSENS-easyMAG, BioMérieux). Moreover, the DMSA-MNPs were successfully employed in the isolation of genomic DNA from human blood. In addition, the resulting DNA–nanoparticle complex was directly subjected to PCR amplification without prior elution, which could eventually lead to simple, rapid, sensitive and integrated diagnostic systems.     

Recovery of Small DNA Fragments from Serum Using Compaction Precipitation

Background

While most nucleic acids are intracellular, trace amounts of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including micro RNAs, can also be found in peripheral blood. Many studies have suggested the potential utility of these circulating nucleic acids in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. However, DNA circulating in blood is usually present at very low concentrations (ng/ml), and is in the form of relatively small fragments (<1,000 bp), making its isolation challenging.

Methods

Here we report an improved method for the isolation of small DNA fragments from serum using selective precipitation by quaternary ammonium compaction agents. A 151 bp fragment of double-stranded DNA from the Escherichia coli bacteriophage lambda served as the model DNA in our experiments. DNA was serially diluted in serum until undetectable by conventional polymerase chain reaction (PCR), before being enriched by compaction precipitation.

Results

Starting with concentrations two to three orders of magnitude lower than the PCR-detectable level (0.01 ng/ml), we were able to enrich the DNA to a detectable level using a novel compaction precipitation protocol. The isolated DNA product after compaction precipitation was largely free of serum contaminants and was suitable for downstream applications.

Conclusions

Using compaction precipitation, we were able to isolate and concentrate small DNA from serum, and increase the sensitivity of detection by more than four orders of magnitude. We were able to recover and detect very low levels (0.01 ng/ml) of a small DNA fragment in serum. In addition to being very sensitive, the method is fast, simple, inexpensive, and avoids the use of toxic chemicals.     

Small aggregates of platelets can be detected sensitively by a flow cytometer equipped with an imaging device: mechanisms of epinephrine-induced aggregation and antiplatelet effects of beraprost

BACKGROUND: Although cross-talks between platelets and other blood cells are important in vivo, laboratory platelet aggregation tests have been performed mainly with the use of platelet-rich plasma (PRP) as samples. Methods that enable an efficient and sensitive detection of platelet aggregates in whole blood are being developed. METHODS: A flow cytometer equipped with an imaging device, the flow imaging cytometer 2 (FIC2), was used to detect platelet aggregates in whole blood. RESULTS: The FIC2 provides a resolution that is high enough to differentiate platelet aggregates from single platelets or other blood cells. Epinephrine elicited platelet aggregate formation in hirudin plus argatroban-treated whole blood, but not in PRP. The reconstitution study revealed that a small amount of adenosine diphosphate (ADP) from erythrocytes may play an important role in epinephrine-induced platelet aggregation (in whole blood), through mediation of P2Y1 receptors. When the inhibitory effect of beraprost, an antiplatelet agent, on platelet aggregation was assessed, analysis of whole blood samples with FIC2 proved to be the most sensitive among the methods available. CONCLUSIONS: FIC2 is a promising device for detection of platelet aggregates in whole blood, with wide basic and clinical applications.