EDTA Vacutainer Tubes Research Articles

Opportunities to improve storage and transportation of blood specimens for CD4 testing in a rural district in Zimbabwe using BD vacutainer CD4 stabilization tubes: a stability and diagnostic accuracy study.

BackgroundCD4+ T-cell testing of blood specimens collected in standard EDTA Vacutainer tubes and transported at ambient temperature, must be completed within 48 hours with the BD FACSCount™ flow cytometer, restricting specimen collection in remote clinics with no on-site testing and limited specimen transport services. We conducted a study in Buhera District, Zimbabwe, to assess the stability and accuracy of CD4+ T-cell results of samples collected in Stabilization Tubes (ST) and stored at ambient temperature for varying time periods.MethodsPaired EDTA and ST samples were collected from 51 HIV-positive patients aged 18 years and older. CD4+ T-cell testing was done on arrival in the laboratory (Day 0). ST samples were retested on Days 3, 5, and 7. Nineteen ST samples were stored for an additional week and retested on Day 14.ResultsThere was a strong correlation between absolute CD4+ T-cell counts measured in the EDTA Day 0 reference sample and Day 7 ST sample (Spearman's rho: 0.9778; mean difference: -4.9 cells/μL and limits of agreement (LOA): 98.5 and 88.7 cells/μL); and the reference sample and Day 14 ST sample (Spearman's rho: 0.9632; mean difference 5.1 cells/μL and LOA: -99.6 and 109.8 cells/μL. Using a 350 cells/μL threshold, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were all 100% on Day 7, and 83.3%, 100%, 100% and 92.9% on Day 14. Using a 500 cells/μL threshold, the sensitivity, specificity, PPV and NVP were 100%, 88.5%, 88.5% and 100% on Day 7 and 88.9%, 80.0%, 80.0% and 88.9% on Day 14.ConclusionsCD4 ST can be used and stored up to 7 days as a reliable alternative to standard EDTA tubes in settings where CD4+ T-cell testing within 48 hours is not feasible. Despite the small sample size, results suggest that ST may be stored up to 14 days at room temperature for CD4 testing, without compromising accuracy. However, further studies with larger sample sizes are needed to confirm this preliminary finding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0553-9) contains supplementary material, which is available to authorized users.

Effect of bovine colostrum administration on plasma protein profile, growth, and survival in Red kid

This study evaluated the effect of colostrum from Azawak cows on plasma protein profile, growth, and survival in Red kids from Niger. Forty (40) newborn kids were allocated to one of two treatment-groups: control (free access to water and the mother) and colostrum (free access to water and the mother, but with additional 50mL of colostrum/animal/day of birth and 25mL/animal/day from the 2nd to 15th day of age). Blood samples were collected into EDTA vacutainer tubes by jugular puncture at 10 and 30 days of age. Total protein was quantified by the Biuret method. The agarose gel electrophoresis was used to determine the serum levels of albumin, α-globulin, β1-globulin, β2-globulin, γ-globulin and the albumin/globulin ratio. The animals from the colostrum group showed higher body weight and average daily gain when compared to the control group (P<0.001). The average concentration in protein at the both sampling times reached higher values in the colostrum than in the control group. At D10, the colostrum group tended (P<0.07) to show higher levels of α-globulin and had higher values (P<0.05) for β1-globulin. At D30, total protein and β2-globulins were higher in colostrum group. Colostrum from Azawak cows seems to have positive effects on some plasma proteins levels and on growth rate in Red kids.

Longitudinal Analysis of Maternal Plasma Apolipoproteins in Pregnancy: A Targeted Proteomics Approach

Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4-5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III(2) and Apo C-III(1) (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r(2)>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III(0) showed no correlation (r(2) = 0.01-0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.

Association of Plasma Cell-Free Hemoglobin with Physiological Measures of Nitric Oxide Responsiveness and Estimated Pulmonary Artery Systolic Pressure: Requirement for Rigorously Optimized Blood Processing Variables

AbstractAbstract 3253 Background:Accurate plasma hemoglobin (PHb) measurements are needed to investigate a model proposing a link between intravascular hemolysis and decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD). Artifactual hemolysis hypothetically can produce excessive noise that might obscure the signal and lead to misinterpretation of data. We systematically studied possible blood processing variables that could cause artifactual hemolysis to obscure in vivo levels of free hemoglobin in adults with SCD. Methods:We systematically compared the effects on PHb measured by ELISA of anticoagulant type, Vacutainer materials and processing time on the same blood specimens from healthy controls and adults with SCD divided into aliquots that were then processed using a variety of in vitro conditions. We analyzed the results of PHb measurements from archived specimens from 467 SCD patients. We evaluated in greater detail plasma specimens collected and processed at bedside from 44 patients with SCD under optimal conditions: drawn from indwelling wide-bore brachial artery catheters by minimal negative pressure into heparinized syringes, gently transferred to tubes for centrifugation within 5 minutes, evaluating the associations of PHb levels from these optimally processed samples with relevant in vivo physiological measurements. Results:Analysis of specimens from healthy control subjects (n=10) and adults with SCD (n=10) for each experiment, split into aliquots to test each variable under defined conditions yielded significant evidence indicating that: (1) arterial catheter syringe specimens are superior to conventional Vacutainer venipuncture specimens; (2) heparin is superior to EDTA anticoagulant; (3) glass tubes are superior to plastic tubes; (4) processing in 2 hours or less is superior to longer processing times. Comparison of 467 archived EDTA plasma specimens processed under comparably variable conditions provided results consistent with the bench top results, confirming the importance of these processing variables on research specimens. Conventional venipuncture EDTA Vacutainer tubes with conventional processing yielded median PHb levels four-fold that of arterial catheter heparin syringe specimens processed at bedside, suggesting that on average 75% of the PHb in conventional specimens may represent background noise and only 25% of the PHb level may represent in vivo free hemoglobin.Blood specimens were collected and processed by the optimal conditioned defined by the preceding experiments from 44 adults with sickle cell anemia. This set of specimens yielded a median PHb level of 6.07μM (CI95=5.27–7.03μM). PHb correlated significantly with blunting of vasodilation in vivo after brachial artery infusion of each of three graded doses of the NO donor sodium nitroprusside (SNP), an indicator of vascular NO responsiveness; at doses of 0.8μg/min(r=−0.46 p=0.0022), 1.6μg/min (r=−0.41, p=0.0082), and 3.2μg/min (r=−0.46, p=0.0024). Quartile analysis revealed a significant difference in forearm blood flow response across all doses of SNP among the highest, middle, and lowest PHb quartiles (p=0.0386). PHb also correlated significantly with tricuspid regurgitant velocity (R=0.37, p=0.0147), suggesting a significant association with Doppler echocardiography estimates of pulmonary artery pressure. A limited number of patients from this specimen pool (n=7) subsequently underwent clinically indicated right heart catheterization. There was a trend in this small subset correlating PHb with mean pulmonary artery pressure (r=0.61, p=0.1482). Conclusions:Plasma hemoglobin levels collected and processed under optimal conditions show statistically significant associations with in vivo physiological measures of nitric oxide responsiveness and echocardiographically estimated pulmonary artery pressure, with supportive trends from right heart catheterization in a limited subgroup of patients with SCD. This supports a role for plasma hemoglobin as a biomarker of nitric oxide resistance and pulmonary hypertension in SCD. Conventional phlebotomy into Vacutainer tubes introduces a high background level of artifactual ex vivo hemolysis in SCD specimens that is capable of obscuring these associations, especially if processing is delayed 2 hours or more. Disclosures:No relevant conflicts of interest to declare.

A study on speciation and antifungal susceptibility pattern of Candida isolates from HIV patients with oropharyngeal candidiasis and correlation with CD4 count

INTRODUCTION : Infection with Human Immunodeficiency Virus (HIV) and its end stage Acquired Immunodeficiency Syndrome (AIDS) is the major public challenge of Modern times, with over 25 million persons already dead and over 30 million Living with HIV / AIDS, the majority of whom are without access to therapy. Opportunistic infection continue to cause morbidity and mortality in Patients with human immunodeficiency virus infection throughout the world. Although the incidences of opportunistic infections have been reduced Around the globe by Highly Active Anti Retroviral Therapy (HAART), the situation remains the same in most developing countries including India. This is due to the fact that some patients do not have access to the treatment and others do not have sustained response to antiretroviral drugs for multiple reasons including poor adherence, drug toxicities, drug interactions or initial acquisition of a drug resistant strains. Oropharyngeal candidiasis is the most common opportunistic infection Observed in AIDS patients, occurring in an estimated 80 to 95% of these patients when the CD4 T lymphocyte counts are below 200 cells /mm3. Increased retroviral replication and an associated decline in immune defences render these patients more susceptible to oropharyngeal candidiasis. Considering the above the facts, the present study was conducted to determine the species distribution and antifungal susceptibility profiles of the Candida isolates from HIV patients with Oropharyngeal Candidiasis. AIMS AND OBJECTIVES : 1. To isolate Candida species from HIV patients with oropharyngeal Candidiasis. 2. To speciate the Candida isolates. 3. To determine the antifungal susceptibility pattern of the isolates by disk diffusion and microbroth dilution method. 4. To find out the correlation of various Candida isolates and their antifungal susceptibility pattern to the CD4 count of the patients. MATERIALS AND METHODS : Statistical analysis were carried out using statistical package for Social sciences and Epi- software by statistician .The proportional data of this cross sectional study were tested using Pearson’s Chi Square Analysis test and Binomial Proportion test. Study group includes 150 HIV positive patients presenting with clinical picture of oral candidiasis. Inclusion Criteria: 1. HIV positive patients: Seropositivity status of the patients were determined as per NACO guidelines. 2. Patients with clinical picture of candidiasis on the oral /pharyngeal mucosa. 3. Patients above 18 years of age group. 4. Both Males and Female were included in the study. 5. Not on any antifungals or corticosteroids 30 days prior to the study. Patients were included in the study only after getting informed written consent. All patients satisfying the inclusion criteria were only once documented and were assigned serial numbers. Patients were interviewed by structured questionnaire and their hospital records were used to know about the previous episodes of Oropharyngeal Candidiasis, use of any antifungal agents and past medical conditions.Oropharyngeal specimens were collected by firmly swabbing the lesion site with two steile cotton swabs under strict aseptic precautions. Samples were transported immediately to the laboratory and subjected to various mycological tests. Blood was collected in EDTA vacutainer tubes from each patients for enumeration of their CD4 count using BD FACS Counter. CONCLUSION : In conclusion, Candida albicans is the most frequently isolated species from HIV seropositive patients with primary as well as recurrent Candidiasis. Non- albicans Candida species are emerging as important pathogens with increasing rates of azole resistance and with increased immunosuppression. Since oral candidiasis may be considered as indirect marker of immunosuppression in HIV patients, regular oral check up of these patients could be an indicator of immunosuppression. Further the increasing rates of resistance particularly among the non- albicans Candida spp. emphasizes the need for speciation and determination of antifungal susceptibility pattern of the oral candida isolates from HIV patients with oropharyngeal candidiasis.

Human Platelet Alloantigens (HPA)1, HPA2 and HPA3 SNPs in Tunisian Sickle Cell Disease Patients

Abstract 4852Sickle Cell Disease (SCD) is a complex disease with various complications such as stroke, vaso-occlusive crisis (VOC), acute chest syndrome and leg ulcers. Sickle cell anemia (SCA; homozygous hemoglobin SS) is the most common form of SCD. Genetic variations and/or environmental modifiers may modulate clinical presentation of SCD. Few studies have examined hemoglobinopathies in Tunisia, North Africa. However, recently, frequencies of beta-thalassemia and sickle cell trait were estimated at 2.21% and 1.89%, respectively (Fattoum, 2006). In order to identify genetic factors that may predispose patients to SCD complications in this population, a pilot case control study was designed to assess polymorphisms in Human Platelet Antigen (HPA) Genes. HPA polymorphisms were recently associated with severe coronary artery disease in the general population in Tunisia (Abboud et al, 2010) and VOC presentation in SCA patients from Bahrain (Al-Subaie et al, 2009). We present here a study conducted in collaboration with the Department of Clinical Hematology at the Aziza Othmana Hospital in Tunis (Tunisia). The National Medical Ethics committee of Tunisia as well as the Institutional Review Board (IRB) of Morehouse School of Medicine (MSM) approved the study. Blood samples, clinical history and DNA samples were collected from SCD adult patients and healthy controls after informed consent. Previously validated questionnaires for genetic risks in patients with SCD (courtesy of Dr Telen, Duke University) were adapted to French. The Helena test kit was used to generate hemoglobin variant data in conjunction with cellulose acetate electrophoresis. Blood samples were collected in EDTA vacutainer tubes and genomic DNA was isolated,stored at −80°C and then shipped to MSM. Single nucleotide polymorphisms, SNPs (Table 1) were genotyped using PCR-RFLPs and compared with different clinical sub-phenotypes such as, onset age, strokes, cardiac problems, splenectomies, etc. as defined in the questionnaire. Pearson Chi-Square was used for comparison and a P<0.05 value was considered significant. A total of 98 DNA samples were collected and 54 questionnaires were filled (Table 2). Age of patients at time of sample collection ranged from 19 to 61 (n = 49) with a mean ± sd (standard deviation) of 29 ± 7. The reported age at onset ranged from 1 to 30 (n = 44) with a mean ± sd of 12 ± 9. No significant differences were found in the HPA alleles and genotype frequencies in SCD versus healthy controls. There was significant association between HPA1 polymorphism with patient defined cardiac problems in all SCD patients (P = 0.002) as well as in the SS sub-group separately (P = 0.01). There was significant association between HPA1 and reported age of onset in SCD patients (P = 0.05) as well as in the non-SS sub-group alone (P = 0.04). The HPA1 variant was linked to self-reported age of disease onset and heart complications in adult SCD patients in Tunisia. This present study is one of the first genetic studies in a seldom-studied group of Tunisian adult SCD patients. These results show that identification of biomarkers of SCD disease severity may be possible using a validated self-reporting instrument. This kind of approach could help to improve early diagnosis of at risk patients and enable development of early interventions.Table 1:SNPs screened.SymbolRs#Ch.Gene (GeneID)MutationPeptide changeRestriction Enz.HPA1rs591817ITGB3 (3690)196 T>CLeu33ProMspIHPA2rs606517GP1BA (2811)524 C>TMet145ThrSfaNIHPA3rs591117ITGA2B (3674)2622 T>GIle843SerFokITable 2:Tunisian cohort collection took place from January to December 2009. This table summarizes the number and genotypes of patients enrolled and questionnaires collectedSexMaleFemaleTotal (%)TOTAL455196 (100)SCD Patients403575 (78)Healthy Controls51621 (22)Hemoglobin Genotype of SCD patientsSS141529 (38.6)Sβ010919 (25.3)Sβ+112 (2.6)SC112 (2.6)SO101 (1.3)unknown13922 (29.3)Total (%)403575 (100)Questionnaires filled282654No questionnaires111021 Disclosures:No relevant conflicts of interest to declare.

Low prevalence of JAK2 V617F mutation in patients with first unprovoked venous thromboembolism

Low prevalence of <i>JAK2</i> V617F mutation in patients with first unprovoked venous thromboembolism

Low-Intensity Exercise Exerts Beneficial Effects on Plasma Lipids via PPARγ

An important mechanism by which physical activity reduces the risk of cardiovascular disease is through regulating plasma lipids. We investigated whether low-intensity exercise modulates lipid metabolism and the transcription factors peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) responsible for controlling reverse cholesterol transport (RCT). Thirty-four sedentary adults, mean age 45.6 +/- 11.1 yr, participated in an 8-wk low-intensity exercise program consisting of walking 10,000 steps, three times a week. Subjects were randomly allocated to either an exercise group or a sedentary control group, and serum lipid or lipoprotein concentrations were determined. Compared with controls, there was a significant decrease in total cholesterol (preexercise, 5.73 +/- 1.39 mmol x L; postexercise, 5.32 +/- 1.28 mmol x L) and a significant increase in HDL (preexercise, 1.46 +/- 0.47 mmol x L; postexercise, 1.56 +/- 0.50 mmol x L) after the exercise program. There was a significant increase in serum oxidized LDL (oxLDL) concentrations in the exercise group before and after exercise (0 wk, 554 +/- 107 ng x mL; 4 wk, 698 +/- 134 ng x mL; 8 wk, 588 +/- 145 ng x mL). A significant increase in leukocyte mRNA expression for PPARgamma (4 wk, 1.8 +/- 0.9-fold; 8 wk, 4.3 +/- 1.9-fold) was observed, which was reinforced by increased PPARgamma DNA-binding activity postexercise (preexercise, 0.22 +/- 0.09 OD units; postexercise, 1.13 +/- 0.29 OD units). A significant increase in gene expression was observed for the oxLDL scavenger receptor CD36 (4 wk, 3.8 +/- 0.6-fold; 8 wk, 2.7 +/- 0.5-fold) and LXRalpha (8 wk, 3.5 +/- 0.8-fold). Two LXRalpha-regulated genes involved in RCT, namely, ATP-binding cassette transporters A1 and GI (ABCA1 and ABCG1, respectively), were significantly up-regulated postexercise (8 wk: ABCA1, 3.46 +/- 0.56-fold; ABCG1, 3.06 +/- 0.47-fold). We propose that the net effect of these changes may be to increase oxLDL uptake, to stimulate RCT, and thus to promote clearance of proatherogenic lipids from the vasculature, ultimately contributing to the cardiovascular benefits of low-intensity aerobic exercise.

Effect of Anticoagulants on Plasma Nevirapine and Efavirenz Concentrations

Antiretroviral drug measurements as part of therapeutic drug monitoring are performed in several human immunodeficiency virus (HIV) clinics in developed as well as developing countries. Heparin is routinely used as an anticoagulant for plasma drug estimations. EDTA plasma for drug estimations, if found suitable, would have several practical advantages. In this study, plasma nevirapine (NVP) and efavirenz (EFV) levels were compared in paired blood collected in heparin and EDTA Vacutainer tubes (Greiner Bio-one, Austria). The study involved HIV-infected individuals who were receiving antiretroviral treatment at the Government Hospital of Thoracic Medicine, Tambaram, Chennai, India. Their treatment consisted of administration of NVP or EFV, along with lamivudine and stavudine/zidovudine. Blood was collected at the same time in heparin and EDTA Vacutainer tubes. Plasma concentrations of NVP and EFV were estimated, according to validated methods, by high-performance liquid chromatography (HPLC). The mean NVP concentrations in heparin and EDTA plasma were 8.79 and 8.59 microg/mL, respectively. This difference was not statistically significant (P = 0.203). In the case of EFV, the corresponding values were 3.03 and 2.78 microg/mL, respectively; the difference was significant (P < 0.05). However, the percent variation in EFV between both types of plasma was <10%. Blood collected in heparin or EDTA Vacutainer tubes could be used for plasma NVP determination.

A comparison of platelet parameters in EDTA‐ and citrate‐anticoagulated blood in dogs

Platelet aggregates are a common artifact in canine blood. Aggregates may affect the accuracy of platelet counts, with important consequences for patient care. The purpose of this study was to determine if platelet counts in dogs were more accurate if blood was collected into citrate instead of EDTA as an anticoagulant. Blood was collected from 50 dogs with neoplasia admitted to the oncology service at Cornell University. EDTA and citrate Vacutainer tubes were filled with blood in random order. Platelet counts and parameters (mean platelet volume [MPV], platelet distribution width [PDW], mean platelet component concentration [MPC], platelet component distribution width [PCDW], and automated platelet clump count [APCC]) were determined using an optical-based hematology analyzer (ADVIA 120). Blood smears from each anticoagulated sample were scored visually for platelet aggregates. The median platelet count was significantly lower (median decrease, 27 x 10(9)/L) in citrate-anticoagulated blood compared with EDTA-anticoagulated blood. This was attributed to platelet activation and aggregation: significantly more aggregates were seen in smears of citrate- than of EDTA-anticoagulated blood. Aggregates were typically small and not detected by the analyzer. Also, the MPV and MPC (or density) were significantly higher (median increase, 3 fL) and lower (median decrease, 33 g/L) in citrate-anticoagulated samples, respectively. Platelets aggregate, likely from activation, when blood from dogs with neoplasia is anticoagulated with citrate for hematology testing, resulting in lower platelet counts. Citrate also yields inaccurate results for MPV and MPC, likely because of inadequate sphering of platelets. Thus, we recommend that citrate not be used as an anticoagulant when accurate platelet counts are desired in dogs.

Accurate quantification of dimethylamine (DMA) in human plasma and serum by GC–MS and GC–tandem MS as pentafluorobenzamide derivative in the positive-ion chemical ionization mode

Dimethylamine (DMA) circulates in human blood and is excreted in the urine. Major precursor for endogenous DMA is asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is hydrolyzed to DMA and l-citrulline by dimethylarginine dimethylaminohydrolase (DDAH). In previous work, we reported a GC–MS method for the quantification of DMA in human urine. This method involves simultaneous derivatization of endogenous DMA and the internal standard (CD 3) 2NH by pentafluorobenzoyl chloride (PFBoylCl) and extraction of the pentafluorobenzamide derivatives by toluene. In the present work, we optimized this derivatization/extraction procedure for the quantitative determination of DMA in human plasma. Optimized experimental parameters included vortex time and concentration of PFBoylCl, carbonate and internal standard. The GC–MS method was thoroughly validated and applied to measure DMA concentrations in human plasma and serum samples. GC–MS quantification was performed by selected-ion monitoring of the protonated molecules at m/ z 240 for DMA and m/ z 246 for (CD 3) 2NH in the positive-ion chemical ionization mode. Circulating DMA concentration in healthy young women ( n = 18) was determined to be 1.43 ± 0.23 μM in serum, 1.73 ± 0.17 μM in lithium heparin plasma, and 9.84 ± 1.43 μM in EDTA plasma. DMA was identified as an abundant contaminant in EDTA vacutainer tubes (9.3 ± 1.9 nmol/monovette, n = 6). Serum and lithium heparin vacutainer tubes contained considerably smaller amounts of DMA (0.42 ± 0.01 and 0.95 ± 0.01 nmol/monovette, respectively, each n = 6). Serum is recommended as the most appropriate matrix for measuring DMA in human blood. The present GC–MS method should be useful for the determination of systemic and whole body DDAH activity by measuring circulating and excretory DMA in experimental and clinical studies.

Affordable flow cytometry for enumeration of absolute CD4+ T-lymphocytes to identify subtype C HIV-1 infected adults requiring antiretroviral therapy (ART) and monitoring response to ART in a resource-limited setting.

BackgroundThe World Health Organization (WHO)'s "3 × 5 program" has spurred efforts to place 3 million people on combination antiretroviral therapy (ART) for treatment of AIDS in resource-limited countries. Paradoxically, the cost of CD4+ T-lymphocyte count essential for decision-making to commence HIV positive adults on ART as well as for monitoring responses to ART remains unaffordable in most resource-limited countries. Thus, low-cost methods for enumerating CD4+ T-lymphocyte are urgently needed.ObjectiveTo evaluate Cyflow cytometry (Cyflow SL, Partec, Munster, Germany) for enumeration of absolute CD4+ T-lymphocyte in subtype C HIV-1 seropositive subjects using FACSCount (Becton and Dickinson, Immunocytometry Systems, San Jose, CA, USA) as the "predicate method".MethodsA total of 150 HIV-1 seropositive subjects were included in the evaluation exercise. Fifty-eight specimens were collected from pregnant HIV-1 seropositive women (subtype C drug resistance study). Twenty-seven specimens were collected from women and their spouses with AIDS followed in a Duke ART study to assess the immunologic and virologic responses to generic ART, comprising Stavudine, Lamivudine and Nevirapine (Stalanev, Varichem Labs, Harare, Zimbabwe). Sixty-five specimens were collected from AIDS patients enrolled in an ongoing Kaposi Sarcoma (KS) study to investigate impact of ART on KS progression. Enumeration of CD4+ T-lymphocytes using FACSCount is routinely conducted for all the three studies. The Medical Research Council of Zimbabwe and Medicines Control Authority of Zimbabwe approved the studies. Whole blood was collected in EDTA vacutainer tubes and aliquoted into two tubes (200 μL in each). CD4+ T-lymphocyte counts were enumerated using a Cyflow counter, in the Department of Immunology and a FACSCount in the Department of Obstetrics and Gynaecology within 6 hours of phlebotomy following manufacturers' instructions.ResultsUsing linear regression analysis, there was a very strong correlation (R = 0.991) between the overall CD4+ T-lymphocyte counts obtained by FACSCount and those obtained by Cyflow. When data analysis was stratified by study groups, there was a strong correlation between the FACSCount and Cyflow CD4+ T-lymphocyte counts from subjects in the three independent studies; Subtype C resistance (R2 = 0.987), Duke ART (R2 = 0.980) and KS (R2 = 0.994), Table 1.Using Bland-Altman plots, the overall, absolute CD4+ T lymphocytes obtained by the two methods were in excellent agreement (mean difference 1.21, 95% Confidence Interval {CI): -2.1 to 3.3). For the 0–250 CD4+ T-lymphocytes range, the CD4 counts obtained using FACSCount were also in good agreement with those obtained using Cyflow counter (mean difference = 2.6 cells/μL, 95% CI: -1.1 to 6.3). Similarly, in the 251–500 (mean difference 1.0, cells/μL, 95% CI: -3.7 to 5.6) and the 501–1200 (mean difference = 0.29 cells/μL, 95% CI: -8.1 to 8.7) CD4 T-lymphocytes range, good agreement was observed.ConclusionThe Cyflow counter is as accurate as the FACSCount in enumerating absolute CD4+ T-lymphocytes in the range 1–1200 cells/μL. Cyflow cytometry is relatively affordable, easy to use technology that is useful not only in identifying HIV seropositive individuals who require ART but also for monitoring immunologic responses to ART.

A modified method for preparing platelet-rich plasma: An experimental study

In this study, our aim was to prepare low-cost autogenous platelet-rich plasma (PRP) for use in minor bone grafting procedures. This was the preliminary study of an experimental study concerning augmentation of bony defects with a mixture of PRP and beta-tricalcium phosphate. Venous blood was collected from marginal ear veins of New Zealand White rabbits in EDTA (K3) vacutainer tubes. Blood samples were divided into 2 groups, and PRP was prepared through 2 methods, 1 of which is a variant of the other. The 2 PRP preparation methods described in this study were modified from Landesberg et al (J Oral Maxillofac Surg 58:297, 2000) after several trials, because the platelet enrichment percentages we obtained using their method were very low. Complete blood counts of venous blood samples, PRP, and platelet poor plasma (PPP) were made. Statistical analyses were performed using SPSS for Windows (SPSS, Chicago, IL). It took approximately 30 minutes to prepare PRP with both methods. Platelet counts in PRP were increased with respect to venous blood platelet counts. The differences between the experimental groups and genders were evaluated statistically with Mann-Whitney U tests (P < .05). Correlations between the groups and genders were also evaluated (P < .05). It was experimentally shown that PRP with high platelet counts can be prepared using this modified method without the need for costly autotransfusion systems.

Spanish population data for the 15 STRs loci included in Powerflex-16

Short tandem repeat (STR) loci are the most informative PCR-based genetic markers available to date for attempting to individualize biological material. The 16 STR loci: D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX, FGA, and the locus amelogenin can be amplified simultaneously using the the PowerPlex16 kit (Promega, Madison, WI, USA). This paper presents allele distribution data in the Spanish population. The data demonstrate that these loci can be useful for providing estimates of the frequency of a DNA profile in forensic identity testing and that a multiple locus profile is extremely rare in all the population. Whole blood was obtained in EDTA vacutainer tubes by venipuncture from unrelated individuals (N=323) residing in Andalucia (Spain). Extracted DNA samples were amplified at the 16 loci using the PowerPlex 16 kit (Promega). Samples were analyzed using the ABI Prismk 310 Genetic Analyzer (PE Biosystems, Foster City, CA) according to the manufacturer’s recommended protocol. All 15 loci are highly polymorphic in the Spanish sample population with the locus TPOX (66.8%) having the lowest observed heterozygosity, and the locus D18S51 (87.5%) displaying the highest heterozygosity. The most discriminating loci were D18S51 (PD=0.969) and PentaE (PD=0.968). The combined probability of exclusion for the 15 STR loci is 0.99999953. There was little evidence for departures from Hardy-Weinberg expectations (HWE) in this sample population. Based on the exact test, the locus that departed significantly from HWE was vWA ( p=0.0488). After employing the Bonferroni correction for the number of loci analyzed, these observations are not likely to be significant. An interclass correlation test analysis was performed to detect any correlations

Population data from Chile using PowerPlex-16

Short tandem repeat (STR) loci are routinely used for paternity and forensic identity testing. Furthermore, STR loci are useful components of national DNA databanks, such as those in the United Kingdom and the United States. The typing of STR loci is facilitated by the ability to amplify several loci simultaneously in a multiplex polymerase chain reaction (PCR). The 16 STR loci D3S1358, TH011, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX, FGA and the locus amelogenin can be amplified simultaneously using the PowerPlex16 kit (Promega, Madison, WI, USA). This paper presents allele distribution data in the general Chilean population. The data demonstrate that these loci can be useful for providing estimates of the frequency of a DNA profile in forensic identity testing and that a multiple locus profile is extremely rare in all the population. Whole blood was obtained in EDTA vacutainer tubes by venipuncture from unrelated individuals (N = 323) residing in Chile. Extracted DNA samples were amplified at the 16 loci using the PowerPlex16 kit (Promega). Samples were analyzed using the ABI Prismk 310 Genetic Analyzer (PE Biosystems, Foster City, CA) according to the manufacturer’s recommended protocol. All 15 loci are highly polymorphic in the Chilean sample population with the locus TPOX (67.3%) having the lowest observed heterozygosity, and the locus PentaE (92.9%) displaying the highest heterozygosity. The most discriminating loci were PentaE (PD= 0.983) and FGA (PD= 0.966). The combined probability of exclusion for the 15 STR loci is 0.99999961. There was little evidence for departures from Hardy–Weinberg expectations (HWE) in this sample population. Based on the exact test, the locus that departed significantly from HWE was D16S539 ( p = 0.002). After employing the Bonferroni correction for the number of loci analyzed, this observation is not likely to

Allele Frequencies for the 13 CODIS STR Loci in a Sample of Southern Croatians

Abstract Whole blood obtained by venipuncture was collected in EDTA vacutainer tubes from individuals attending Split Hospital, Split,Croatia. Blood was dried onto clean cotton cloth, sealed in individual plastic bags and transported to the United States for testing. Approximately 3 mm squares were extracted using Chelex (1). The DNA was not quantified. PCR amplification was performed using the AmpFlSTR Profiler Plus™ and Cofiler™ PCR amplification kits (PE-Biosystems, Foster City, CA) following the manufacturer's protocol using 2 µL of sample. The amplified products were separated and detected using the ABI Prism™ 377 DNA sequencer (PE-Biosystems, Foster City, CA). Allele frequencies and goodness of fit tests were performed using Excel spread sheets. Each locus (n = 13) was tested for the following: Hardy-Weinberg Global Chi-square goodness of fit using all genotypes with expected values greater than 1.0, all other genotypes were pooled to form a residual class (df = number of genotypes minus the number of alleles)( p1); T statistic testing θ = 0 recommended by NRC 2 (2)(p2); and the Chi-square comparing expected and observed total homozygosity and heterozygosity (df = 1)(p3). The Bonferroni corrected threshold was 0.0038. No loci exceeded the Bonferroni threshold; only one locus (D8S1179) was significant (p (0.05) for one of three tests of deviation from Hardy-Weinberg equilibrium due to an excess of heterozygotes and deficiency of homozygotes prior to the Bonferroni correction for multiple tests.

Han Chinese Population Data for Ten STR Loci in Changsha, China

Abstract Whole blood obtained by venipucture was collected in EDTA vacutainer tubes from unrelated individuals residing in Changsha, China. The DNA was extracted by phenol chloroform method (1). PCR amplification was performed using the AmpFlSTR Profiler Plus™ PCR amplification kit (PE-Biosystems, Foster City, CA) following the manufacturer's protocol (2). The amplified products were separated and detected using the ABI Prism™ 377 DNA sequencer (PE-Biosystems, Foster City, CA). The data were analyzed using The Promega Software, POWERSTATS.

Simultaneous extraction of hepatitis C virus (HCV), hepatitis B virus, and HIV-1 from plasma and detection of HCV RNA by a reverse transcriptase-polymerase chain reaction assay designed for screening pooled units of donated blood.

Testing for viral nucleic acids should reduce the residual risk of transmitting viral infections by transfusion of blood components. The AmpliScreen Hepatitis C (HCV) Test, Version 2.0, was designed for screening pools composed of samples from individual units of blood or plasma. An ultracentrifugation step during sample processing simultaneously extracts and concentrates HCV, HIV type 1, and hepatitis B virus particles from plasma. An HCV internal control RNA serves as an extraction and amplification control for each processed sample. Processed samples are amplified by reverse transcriptase polymerase chain reaction and detected by hybridization of the amplified products to HCV- and internal-control-specific oligonucleotide probes. Plasma samples containing known quantities of HCV were used to evaluate analytical sensitivity and precision. The analytical sensitivity of the test was 25 IU of HCV per mL of pooled plasma; all HCV genotypes were detected with similar efficiency. The test did not react with other blood-borne viruses. The within-run and total coefficients of variation were 1.3-13.0 percent and 1.7-22.0 percent, respectively, with low copy samples producing the more variable results. The test performed well using plasma collected in either EDTA, ACD, or PPT Vacutainer tubes. Plasma samples containing elevated levels of hemoglobin, albumin, triglycerides, or bilirubin did not interfere with the test. The test detected HCV RNA 23 to 32 days prior to seroconversion for four of the five seroconversion panels tested. The AmpliScreen HCV Test, Version 2.0 provides a reproducible and specific method for screening pooled blood units for HCV. Theoretically, this test has sufficient sensitivity to detect a single infected unit containing 2.4 x 10(3) IU of HCV per mL in a pool with 95 uninfected units and should reduce the window period by at least 20 to 30 days.

Determination of free acetaldehyde in total blood for investigating the effect of aspartate on metabolism of alcohol in mice

To explore the effect of sodium l-aspartate monohydrate (aspartate) as a NAD + regenerating agent for acetaldehyde in alcohol metabolism, a simple HPLC method has been developed for the measurement of free acetaldehyde in total mice blood digested with alcohol and aspartate. The blood samples were collected in EDTA Vacutainer tubes, and treated with 2,4-dinitrophenylhydrazine (DNP hydrazine) reagent in total blood. Acetaldehyde DNP hydrazone was extracted from total blood and analyzed by HPLC using an Ultrasphere ODS column. The compounds were separated using acetonitrile–water (50:50, v/v) as mobile phase and detected at 356 nm. The detection limit for acetaldehyde DNP hydrazone was 0.1 ppm. A blank determination was carried out for each analysis and subtracted from the results. The amount of acetaldehyde in blood has been determined as a function of time lapse after sole alcohol administration and aspartate ingestion followed by alcohol administration, respectively. This comparative analysis demonstrates that the ingestion of aspartate before the administration of alcohol dramatically decreases the aldehyde level in blood, and aspartate may be utilized as a prospective antagonist for acceleration of ethanol metabolism and prevention of acetaldehyde toxicity.

On the accurate measurement of serotonin in whole blood

Levels of serotonin (5-hydroxytryptamine; 5-HT) in whole blood reflect the levels in the platelets, but there are problems with instability of 5-HT in measurement of whole blood extracts. We therefore developed and validated an assay for whole blood 5-HT using HPLC with fluorometric detection. The procedure involved collecting blood in EDTA vacutainer tubes and aliquoting it with internal standard, alpha-methyl-5-HT, before freezing. The aliquots were thawed on ice and proteins precipitated with perchloric acid containing EDTA and ascorbic acid. The 5-HT was stable in fresh whole blood for 24 h after sampling when stored at room temperature, refrigerated, or ice-cooled. Measurements of the levels of 5-HT in blood collected in heparin, citrate or EDTA were similar, but in blood collected in ACD-buffer the levels were approximately 25% lower. Cell lysis by sonication decreased the 5-HT levels, but this was compensated for by the internal standard. Determinations of 5-HT by HPLC were in good agreement with those by gas chromatography - mass spectrometry. The intra-individual variability between days was 3.6% (n=6). However, single and repeated ingestion of bananas, which are rich in 5-HT, elevated 5-HT in whole blood, indicating dietary influences on platelet 5-HT.