EDTA Whole Blood Samples Research Articles

Research on the stability changes in expert consensus of the ACTH detection preprocessing scheme

Abstract Objectives Adrenocorticotropic hormone (ACTH) is extremely unstable and can easily degrade at room temperature. The experts agreed that the samples should be transported in an ice bath. If it cannot be detected immediately, the plasma should be separated and frozen, which is difficult to carry out in routine practice. This study was performed to explore the preanalytical factors that influence the stability of adrenocorticotrophic hormone (ACTH) measurements. Methods ACTH levels in 21 EDTA whole-blood samples were measured immediately after 0 h and then divided into three equal groups according to the corresponding values (low, L; median, M; and high, H). Next, three sample processing methods (including seven subtypes) were used: whole blood was uncentrifuged (named the A method), stored at 4 °C or 22 °C, centrifuged but not subjected to plasma removal (the B method), stored at 4 °C or 22 °C, and centrifuged with the plasma removed and stored (the C method) at 4 °C, 22 °C, and −20 °C. Each subtype contained three samples, namely, L, M, and H; these samples were retested using a Siemens XP2000 at different times. The change bias was calculated at 0 h. Results Compared to that at 0 h, there was no significant change in ACTH up to 24 h when the sample was stored at 4 °C or 22 °C with the B method (p>0.05), while it significantly changed (up or down >10 %) at 4 °C/24 h (bias is expressed as the mean±SEM; 13.37±21 %, p<0.05) and 22 °C/12 h (9.13±7.68 %, p<0.05) with the A method; and 4 °C/24 h (8.93±5.54 %, p<0.05), 22 °C/12 h (9.5±4.47 %, p<0.05) and −20 °C/3 h (12.03±4.8 %, p<0.05) with the C method. Conclusions After ACTH samples were centrifuged, the presence of plasma without removal did not affect the detection value, and the sample was stored at 4 °C for up to 24 h. There was a significant difference in the detection of ATCH when the sample was stored at −20 °C and thawed again (p<0.05).

B-306 Performance Evaluation of an Automated Assay for Measurement of Tacrolimus* on the ADVIA Centaur XP System

Abstract Background Tacrolimus (FK506) (PROGRAF) is a macrolide antibiotic of fungal origin (Streptomyces tsukubaensis). Tacrolimus is an inhibitor of calcineurin, a phosphatase that activates T-cell proliferation. At the cellular level, tacrolimus binds a family of binding proteins termed FK506-binding proteins (FKBPs). The correlation between steady-state trough concentration and area under the curve (AUC) provides reliable therapeutic monitoring for tacrolimus exposure at the trough level. The objective of this study was to determine the performance characteristics of the Tacrolimus assay on the ADVIA Centaur® XP System. Methods The ADVIA Centaur Tacrolimus assay is a competitive immunoassay using direct chemiluminescent technology. Free tacrolimus in the patient sample competes with the bound tacrolimus in the solid phase for binding with the acridinium ester-labeled anti-tacrolimus antibody. The amount of tacrolimus present in the patient sample has an inverse relationship to the amount of relative light units detected by the system. Results The ADVIA Centaur Tacrolimus assay demonstrated a mean %CV of 3.1% for intra-assay and 6.0% for total precision (across a sample range of 3.10 to 26.15 ng/mL). Close correlation to LC-MS/MS was demonstrated, with r, slope, and intercept of 0.957, 0.973, and 0.475, respectively. LoD and LoQ were 0.7 ng/mL and 1.9 ng/mL, respectively. Likewise, comparison to the Atellica IM assay resulted in r, slope, and intercept of 0.966, 1.09, and -0.508, respectively. Linearity was demonstrated across a range from LoQ to 30.0 ng/mL. Tacrolimus metabolite percent cross-reactivity ranged from 1.7 to 34.2%. A panel of potential interferants including the following were tested and found not to interfere (≤10%): cephalosporin to 100 µg/mL, erythromycin to 13.8 mg/dL, phenobarbital to 10 mg/dL, rapamycin to 5 µg/mL, sulfamethoxazole to 150 µg/mL, tobramycin to 3.3 mg/dL, trimethoprim to 4.2 mg/dL, bilirubin (conjugated) to 60 mg/dL, bilirubin (direct) to 60 mg/dL, and rheumatoid factor to 500 IU/mL. Conclusion The resulting data demonstrates that the ADVIA Centaur Tacrolimus assay is an accurate and precise method of quantifying tacrolimus concentration in EDTA whole-blood samples. *Assay under development by Randox Laboratories Ltd. for Siemens Healthineers. Not available for sale. Future availability cannot be guaranteed.

DETECTION OF VECTOR-BORNE INFECTIONS IN LIONS AND TIGERS AT TWO ZOOS IN TENNESSEE AND OKLAHOMA, USA.

Protozoal and bacterial vector-borne infections are frequently diagnosed in domestic felids. However, with the exception of Mycoplasma haemofelis and Cytauxzoon felis, their occurrence in managed nondomestic felids housed in the United States is largely unknown. Following a case in February 2020 of fulminant cytauxzoonosis in an African lion (Panthera leo), EDTA-whole blood samples were collected opportunistically from February 2020 through June 2020 from 34 adult tigers (Panthera tigris) and eight adult African lions from the same sanctuary in eastern Tennessee as well as 14 adult tigers from a zoo in southern Oklahoma. Samples were analyzed for Cytauxzoon felis, Bartonella spp., hemotropic Mycoplasma, Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp., and Hepatozoon spp. DNA by PCR amplification. All animals were asymptomatic at the time of collection. None of the Oklahoma animals were positive for vector-borne organisms, but these pathogens were detected in tigers at the Tennessee facility, including Cytauxzoon felis (11.8%), "Candidatus Mycoplasma haemominutum" (5.9%), and Ehrlichia ewingii (2.9%). During the study period, two animals developed clinical signs of cytauxzoonosis and were assessed for vector-borne infections as part of their diagnostic evaluation. This study documents the presence of tick-borne diseases in managed nondomestic felids in the southeastern United States and underscores that ectoparasite control measures should be practiced to minimize exposure of carnivores in managed care.

The role of cytomegalovirus infection in ischemic heart failure progression

Abstract Objective To study an association between cytomegalovirus infection (CMV) and molecular biomarkers (NT-proBNP, tumor necrosis factor (TNF-α), Interleukin-1β) and evaluate prognostic role of CMV infection in ischemic heart failure (HF) progression during the 12-month follow-up period. Methods A total of 104 patients (61.5% men, median age of 59 [53; 62.5] years) with stable coronary artery disease and baseline left ventricular ejection fraction (LVEF) of 43% [36; 57]% were enrolled in the study. At baseline evaluation HF patients were of New York Heart Association (NYHA) class I (7.7%), class II (61.0%), and class III (31.3%). Sixty five percent of patients had prior myocardial infarction and 70.2% received prior myocardial revascularization (coronary artery bypass graft/stent). Cytomegalovirus DNA concentrations in EDTA whole-blood samples were measured using a polymerase chain reaction baseline and at 12 months of follow-up period. Serum levels of NT-proBNP, Interleukin-1β, TNF-α were measured baseline using an enzyme immunoassay. Two-dimensional transthoracic echocardiography was performed at baseline and at the 12 months. Results At baseline, all patients were divided into 2 groups: group A comprised CMV seropositive patients (n=52); group B comprised CMV seronegative patients (n=52). Plasma concentration of cytomegalovirus DNA was 1709.4 [615; 3176] copies/mL. The values of cytomegalovirus DNA significantly correlated with NT-proBNP (r=0.781), TNF-α (r=0.799) and Interleukin-1β (r=0.756). Levels of NT-proBNP were higher (p=0.0001) in group A by 36.6% than in group 2 (559 [364; 756] vs. 354.5 [279; 545.5] pg/mL, respectively). Levels of TNF-α were also higher (p<0,001) by 35.3% (8.5 [6.5; 10.9] vs. 5.5 [4.1; 7.3] ng/mL, respectively) and levels of Interleukin-1β (p<0,001) by 17.6% (19.3 [15.8; 23.75] vs. 15.9 [13.15; 18.7] ng/mL, respectively) in group A than in group B. During the 12-month follow-up period in group A the rate of HF progression was 51.6% cases, and in group B 26.9% (p=0.009). Based on ROC-analysis, baseline plasma concentration of cytomegalovirus DNA ≥2020 copies/mL (AUC=0.798; specificity 67%, sensitivity 82%; p<0.001) were identified as a cut-off values predicting development of HF progression during the 12-month follow-up period. 12-month levels of cytomegalovirus DNA did not differ (p=0,678) in comparison to baseline ones and were 1737.9 [321; 3384] copies /mL. In group A LVEF significantly increased by 18.8% from 50.5 [36.5; 56.0] to 41.0 [35.0; 50.0]%, end-systolic dimension significantly increased by 7.3%, end-diastolic dimension by 9.6% (p<0,0001), while in group B these parameters did not change. Conclusion Our data suggest that values of cytomegalovirus DNA are associated with NT-proBNP, TNF-α, Interleukin-1β levels, and may be considered as non-invasive biomarker for prediction of ischemic heart failure progression during the 12-month follow-up period. Funding Acknowledgement Type of funding sources: None.

Evaluation of a commercial microbial enrichment kit used prior DNA extraction to improve the molecular detection of vector-borne pathogens from naturally infected dogs

Accurate detection of vector-borne pathogens (VBPs) is extremely important as the number of reported cases in humans and animals continues to rise in the US and abroad. Validated PCR assays are currently the cornerstone of molecular diagnostics and can achieve excellent analytical sensitivity and specificity. However, the detection of pathogens at low parasitemia still presents a challenge for VBP diagnosis, especially given the very low volume of specimens tested by molecular methods. The objective of this study is to determine if a commercially available microbial enrichment kit, used prior DNA extraction, is capable of expanding the overall microbial community and increasing detectable levels of VBPs in canine blood samples through host DNA depletion. This study used EDTA-whole blood samples from dogs naturally infected with varying parasitemia levels of either Anaplasma phagocytophilum, Babesia gibsoni, or Ehrlichia ewingii. For two VBPs, EDTA-blood samples were diluted to determine the effect of microbial concentration at low parasitemia. Paired EDTA-blood samples from each dog were subjected to traditional, automated DNA extraction with or without the microbial concentrating kit (MolYsis®) prior DNA extraction. Relative amounts of pathogen DNA in paired samples were determined by real-time PCR and Next-Generation Sequencing targeting conserved regions of 16S rRNA (for bacteria) and 18S rRNA (for protozoa). Results from the three molecular methods suggest that the microbial concentrating kit did not improve the detection of VBPs, although significantly reduced the presence of host DNA. Alternative methods for VBP enrichment in clinical samples prior to molecular testing should continue to be investigated, as it may significantly improve clinical sensitivity and reduce the number of false-negative results.

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method.

Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method—the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

Phenotypic Corrections of Ex Vivo Erythrocytes Derived from Chronic Hemolytic Anemia Patients Due to Krϋppel-like Factor 1 (KLF-1) Mutations By AG-348: A Novel Synthetic Pyruvate Kinase Activator

Introduction: Pyruvate kinase-red blood cell (PK-R) enzyme is an isoform of PK-liver (PK-L) expressed by tissue-specific promoter on the PKLR gene. The PK-R functions in the glycolytic pathway to generate pyruvate & adenosine triphosphate (ATP), the energy carrier molecule supporting 120-day red blood cells (RBCs) survival. Patients with PKLR mutations have PK deficiency (PKD) & defective RBCs leading to different degree of chronic hemolysis from non-transfusion to transfusion dependent. A novel synthetic AG-348 compound, an allosteric activator of PK, enhances PK-R activity in patients with PKD (Kung et al., 2017). Previously, our group described a novel form of severe hemolytic anemia due to mutations of the Krϋppel-like factor 1 (KLF1 disease) (Viprakasit et al., 2014). Besides deregulated globin gene switching with persistent embryonic globin expression, all KLF1 patients had low PK-R activity leading to hemolytic anemia & transfusion dependency. Decreased KLF1 expression in trans lead to phenotypes mimic that of PKD since the PKLR genes are regulated by the KLF1. At present, there is no specific treatment for KLF1 disease besides supportive care & RBCs transfusion. Objective: To determine ex vivo effects of AG-348 on PK-R activity, ATP production & potential adverse effects on RBC physiology (hemolysis, inducing reactive oxygen species (ROS) and apoptosis) in RBCs from KLF1 patients. Methods: EDTA-whole blood samples were collected from two non-regularly transfused KLF-1 patients. The genotypes of the PKLR and KLF1 genes were confirmed by Sanger sequencing. RBCs were isolated by cellulose column and prepared into a suspension; 1 x 108 RBCs/mL and subsequently incubated with various concentrations of AG-348 (Agios Co. Ltd.); 0.002 - 10 μM at 37⁰C overnight. The AG-348-treated RBCs were then harvested and determined for PK-R activity by coupled enzyme assay with lactate dehydrogenase and ATP production. Furthermore, we evaluated possible adverse consequences of this compound by evaluating hemolysis, intracellular ROS and apoptosis and numeration by flow cytometry. Results: Two adolescent patients: patient A & B had chronic anemia with baseline hemoglobin (Hb) ≤ 8 g/dL with high reticulocyte count (10 %) and high level of Hb F. The genotypes of KLF1 gene were identified in both subjects as compound heterozygotes of one null and one missense mutation (Table 1). Both patients had low PK-R activity but no PKLR gene mutation identified. Using an ex vivo assay, the PK-R activities of patients' RBCs significantly increased after incubation with AG-348 in a dose-dependent manner for 100% to 150% compared to baseline and 0.1% DMSO as control. Moreover, the ATP production, a direct product from RBC glycolytic pathway driven by PK catalysis also significantly increased in AG-348 incubated RBCs in a similar manner (Figure 1A and 1B). Nevertheless, there were no changes in normal RBC controls for both PK activity and ATP generation. Also, AG-348 did not adversely induce hemolysis in the KLF1-mutated RBCs as determined by Hb released (µg/mL) in the culture medium compared with 0.1% DMSO (Figure 1C). Similarly, intracellular ROS detected by mean fluorescent intensity (MFI) & apoptotic signals did not increase in normal and KLF1-mutated RBCs (data not shown). Conclusion: Our ex vivo study using RBCs derived from KLF-1 patients is the first investigation to suggest a potential clinical role of the AG-348 as a therapeutic intervention in this hereditary condition. Our results were consistent with the previous study that AG-348 enhanced PK-R activity & ATP production in RBCs from patients with classical PKD due to PKLR mutations (Kung et al., 2017) and currently this compound is under investigation in patients with non-transfusion dependent & transfusion dependent PKD (NCT03548220 and NCT03559699, respectively). For the safety profile, the AG-348 does not induce ROS and apoptosis in our ex vivo setting. Interestingly, our data showed that AG-348 might be effective for RBCs with lower PK activity and potentially improves RBCs survival, no matter the causes of PKD. Our study warrants further investigation to determine whether this PK correction could be translated into a clinical benefit in our KLF-1 patients. Finally, our finding suggests that the activity of AG-348 might include those with higher RBCs turnover & exhaust PK activity such as other hemolytic anemias especially thalassemia & hemoglobinopathy. Disclosures No relevant conflicts of interest to declare.

Molecular detection of Bartonella species and haemoplasmas in wild African buffalo (Syncerus caffer) in Mozambique, Africa

AbstractThe African buffalo (Syncerus caffer), a mammal species whose population is declining, can play a role as a reservoir or carrier of a wide number of arthropod-borne pathogens. Translocation procedures have been used as an alternative approach for species conservation. However, the veterinary aspects of this sort of procedures are extremely important to minimize the impact on animal health. In order to detectBartonellaand haemoplasmas, two important group of bacterial that have an impact in both human and animal health, EDTA whole-blood samples were screened for the presence of these bacterial pathogens by molecular techniques. As a result, a molecular occurrence of 4.1 and 15.4% forBartonellaspp. and haemoplasmas, respectively, was reported among 97 wild buffaloes sampled during a translocation procedure from Marromeu to Gorongosa Reserve, Mozambique. Additionally, phylogenetic analyses of the obtained sequences were conducted. At least, three bovine-associated pathogens, namelyB. bovis,M. wenyoniiand ‘CandidatusM. haemobos’, as well as a probably newBartonellagenotype/species were detected inS. caffer.Further studies are needed in order to determine whether these bacterial species may cause impact in buffaloes and other sympatric ruminant species living in the release site.

Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.

Molecular detection of hemotrophic mycoplasmas among domiciled and free-roaming cats in Campo Grande, state of Mato Grosso do Sul, Brazil

Hemoplasmas are bacteria living in feline red blood cells. Feline hemoplasmosis is frequently associated with old male cats that have access to the streets. This study aimed to detect the presence of hemoplasma speciess in domiciled and free-roaming cats in Campo Grande, state of Mato Grosso do Sul (MS), Brazil, using molecular techniques. Between January 2013 and April 2013, EDTA-whole blood samples were collected from 151 domestic cats (65 free-roaming and 86 domiciled cats). Samples were subjected to PCR assays targeting hemoplasmas 16S rRNA, followed by sequencing, BLAST analysis and phylogenetic analysis. Results show an occurrence of 36.4% for hemoplasmas. Twenty-three cats (15.2%) were positive for 'Candidatus Mycoplasma haemominutum', 17 (11.2%) for M. haemofelis and 15 (9.9%) for 'Candidatus M. turicensis', from PCR. Coinfection by two or three hemoplasmas was found in 25 cats (16.6%). No statistically significant difference between genders or between lifestyles was observed for the presence of hemoplasmas among the cats. Results show different hemoplasma species are present in cat population (Campo Grande, MS, Brazil). It is suggested that a differential diagnosis for feline hemoplasmosis should be made when cats show nonspecific clinical signs of disease with systemic manifestation.

Design and establishment of a biobank in a multicenter prospective cohort study of elderly patients with venous thromboembolism (SWITCO65+)

In the field of thrombosis and haemostasis, many preanalytical variables influence the results of coagulation assays and measures to limit potential results variations should be taken. To our knowledge, no paper describing the development and maintenance of a haemostasis biobank has been previously published. Our description of the biobank of the Swiss cohort of elderly patients with venous thromboembolism (SWITCO65+) is intended to facilitate the set-up of other biobanks in the field of thrombosis and haemostasis. SWITCO65+ is a multicentre cohort that prospectively enrolled consecutive patients aged ≥65 years with venous thromboembolism at nine Swiss hospitals from 09/2009 to 03/2012. Patients will be followed up until December 2013. The cohort includes a biobank with biological material from each participant taken at baseline and after 12 months of follow-up. Whole blood from all participants is assayed with a standard haematology panel, for which fresh samples are required. Two buffy coat vials, one PAXgene Blood RNA System tube and one EDTA-whole blood sample are also collected at baseline for RNA/DNA extraction. Blood samples are processed and vialed within 1 h of collection and transported in batches to a central laboratory where they are stored in ultra-low temperature archives. All analyses of the same type are performed in the same laboratory in batches. Using multiple core laboratories increased the speed of sample analyses and reduced storage time. After recruiting, processing and analyzing the blood of more than 1,000 patients, we determined that the adopted methods and technologies were fit-for-purpose and robust.

Establishment of a CYP2C19 Genotyping Assay for Clinical Use

Conversion of clopidogrel (Plavix) to its active metabolite is catalyzed largely by the P450 enzyme 2C19 (CYP2C19). Numerous allelic variants of CYP2C19 exist. The *1 allele is considered wild type, whereas the *2 and *3 alleles have no in vivo enzymatic activity. Conversely, the *17 allele has increased expression, resulting in increased clopidogrel activation. Poor metabolizers (*2/*2 and *2/*3 genotypes) experience higher rates of therapeutic failure. For this reason, we have validated a CYP2C19 genotyping assay for the *1, *2, *3, and *17 alleles. Genomic DNA extracted from 30 deidentified EDTA whole-blood samples from patients was analyzed at 2 independent facilities using specific TaqMan realtime polymerase chain reaction primers and probes. Concordant genotypes were generated on all samples tested. Of the 30 samples, 15 were CYP2C19*1/*1, 8 were CYP2C19*1/*17, 5 were CYP2C19*1/*2, and 2 were CYP2C19*2/*17. There were no CYP2C19*3 alleles or *2/*2 homozygous genotypes detected. This CYP2C19 genotyping assay is appropriate for clinical testing, demonstrating excellent interlaboratory concordance, enabling the selection of the most effective clopidogrel treatment regimen for patients undergoing percutaneous coronary intervention.

Molecular characterization of Hepatozoon sp. in cats from São Luís Island, Maranhão, Northeastern Brazil

Few molecular studies have been done concerning the molecular characterization of Hepatozoon species among domestic and wild felids. The present work aimed to characterize molecularly the presence of Hepatozoon sp. DNA in cat blood samples from São Luís Island, Maranhão state, Northeastern Brazil. EDTA-whole blood samples were collected from 200 domestic cats with outdoor and wood areas access from São Luís, Maranhão, Brazil. Each sample of extracted DNA was used as a template in PCR reactions aiming to amplify a partial sequence of 18S rRNA of Hepatozoon spp. We also performed sequence alignment to establish the identity of the parasite species infecting these animals using DNA sequences based on 18S rRNA. From 200 sampled cats, Hepatozoon DNA was only found in one animal (0.5%). The found Hepatozoon DNA showed 97% of identity with Hemobartonella felis isolates 1 and 2 from Spain. When analyzing the phylogenetic tree, the found Hepatozoon DNA was in the same clade than H. felis isolates. Our findings suggest that more than one species of Hepatozoon could infect felids in Brazil.

Molecular diagnosis of bacteriemia in patients with neutropenic febrile oncohematologic.

9033 Background: The rapid microbiologic diagnosis in the course of fever of unknown origin plays an important role for the appropriate treatment in neutropenic patients. Blood culture helps identify bacterial and fungal bacteriemia. However, its efficacy can be limited due to its low sensitivity when the patient is receiving antibiotics and to the long time required to identify the pathogen (24-72 hours). Septifast (Roche Molecular Systems) is a new molecular technique which detects in 6 hours the 25 most important bacterial and fungal species reported to cause bloodstream infections. The aim of the study was to compare Septifast with conventional blood cultures in blood samples from neutropenic febrile patients. Methods: Forty-five blood samples from 19 patients were prospectively tested. All of them presented fever with neutrophil count <0.5x109/L. The patients (pts) were receiving antibiotic therapy (piperaciline-tazobactam/imipenem (19pts), vancomicine (7pts), amikacine (3pts), aciclovir (2pts) and antifungal therapy (9pts). For each sample, two blood culture vials for aerobic/anaerobic cultures and an EDTA whole-blood samples for Septifast were drawn. Results: Thirty-two percent of patients presented a positive result. Eight of the 45 (17.7%) blood samples studied were positive by PCR assay (S.aureus: 2; P.aeruginosa: 3; E.coli: 1; Coagulase-negative Staphylococci: 2). Two blood samples were positive for P.aeruginosa and Coagulase-negative Staphylococci by both Septifastâ and blood culture. The entire negative PCR test results correlated with negative blood culture samples. The negative predictive value was 100% with a sensitivity of 100% and a specificity of 86% (Table). Conclusions: Septifast is an easy and sensitive method for detecting pathogens in severely immunocompromised patients, allowing results in a shorter time and with higher sensitivity than conventional blood cultures. Comparision results from Septifast test and conventional blood culture system. Blood culture Results Positive samples (%) Negative samples (%) Total Septifast Positive samples (%) 2 (4.4) 6 (13.3) 8 (17.7) Negative samples (%) 0 (0) 37 (82.2) 37 (82.2) Total (%) 2 (4.4) 43 (95.5) 45 (100)

Detection of Cytomegalovirus (CMV) DNA in EDTA Whole-Blood Samples: Evaluation of the Quantitative artus CMV LightCycler PCR Kit in Conjunction with Automated Sample Preparation

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.

Comparison of the Reintroduced MEIA® Assay With HPLC-MS/MS for the Determination of Whole-Blood Sirolimus From Transplant Recipients

Therapeutic monitoring with dosage individualization of sirolimus drug therapy is standard clinical practice for organ transplant recipients. For several years sirolimus monitoring has been restricted as a result of lack of an immunoassay. The recent reintroduction of the microparticle enzyme immunoassay (MEIA) for sirolimus on the IMx analyser has the potential to address this situation. This study, using patient samples, has compared the MEIA sirolimus method with an established HPLC-tandem mass spectrometry method (HPLC-MS/MS). An established HPLC-UV assay was used for independent cross-validation. For quality control materials (5, 11, 22 microg/L), the MEIA showed acceptable validation criteria based on intra- and inter-run precision (CV) and accuracy (bias) of <8% and <13%, respectively. The lower limit of quantitation was found to be approximately 3 microg/L. The performance of the immunoassay was compared with HPLC-MS/MS using EDTA whole-blood samples obtained from various types of organ transplant recipients (n = 116). The resultant Deming regression line was: MEIA =1.3 x HPLC-MS/MS + 1.3 (r = 0.967, S(y/x) = 1) with a mean bias of 49.2% +/- 23.1% (range, -2.4% to 128%; P<0.001). The reason for the large and variable bias was not explored in this study, but the sirolimus-metabolite cross-reactivity with the MEIA antibody could be a substantive contributing factor. Whereas the MEIA sirolimus method may be an adjunct to sirolimus dosage individualization in transplant recipients, users must consider the implications of the substantial and variable bias when interpreting results. In selected patients where difficult clinical issues arise, reference to a specific chromatographic method may be required.

CEDIA® Sirolimus Assay Compared With HPLC-MS/MS and HPLC-UV in Transplant Recipient Specimens

The role of the therapeutic drug monitoring laboratory in support of immunosuppressant drug therapy is well established, and the introduction of sirolimus (SRL) is a new direction in this field. The lack of an immunoassay for several years has restricted the availability of SRL assay services. The recent availability of a CEDIA SRL assay has the potential to improve this situation. The present communication has compared the CEDIA SRL method with 2 established chromatographic methods, HPLC-UV and HPLC-MS/MS. The CEDIA method, run on a Hitachi 917 analyzer, showed acceptable validation criteria with within-assay precision of 9.1% and 3.3%, and bias of 17.1% and 5.8%, at SRL concentrations of 5.0 microg/L and 20 microg/L, respectively. The corresponding between-run precision values were 11.5% and 3.3% and bias of 7.1% and 2.9% at 5.0 microg/L and 20 microg/L, respectively. The lower limit of quantification was found to be 3.0 microg/L. A series of 96 EDTA whole-blood samples predominantly from renal transplant recipients were assayed by the 3 methods for comparison. It was found that the CEDIA method showed a Deming regression line of CEDIA=1.20xHPLC-MS/MS-0.07 (r=0.934, SEE=.47), with a mean bias of 20.4%. Serial blood samples from 8 patients included in this evaluation showed that the CEDIA method reflected the clinical fluctuations in the chromatographic methods, albeit with the variable bias noted. The CEDIA method on the H917 analyzer is therefore a useful adjunct to SRL dosage individualization in renal transplant recipients.

Long-term stability of human immunodeficiency virus viral load and infectivity in whole blood.

We intended to evaluate the stability of human immunodeficiency virus (HIV) type 1 virions in whole blood and in culture medium. EDTA whole-blood samples taken from 12 patients were left at room temperature for up to 7 days, and aliquots of a laboratory virus stock spiked in EDTA, in heparinized or in citrated whole blood, with or without the addition of Triton X-100, or spiked in culture medium were left at room temperature for up to 120 days before plasma was separated and frozen at -80 degrees C. Viral load was measured for all frozen plasma samples using different viral load assays. p24 antigen and infectivity were also measured in the spiked samples. The patient whole-blood samples did not show any decrease in viral load during this 7-day period. The spiked samples decayed by not more than 1 log after 120 days (about 4 months), with the fastest decay in medium. Virus infectivity decayed very slowly from 20,000 units mL-1 to undetectable amounts after 56 days. These results indicate that HIV-1 virions in whole blood possess a long-term stability in terms of viral load, p24 antigen level and infectivity, which is not sufficiently recognized by laboratory and health care workers.

Thrombotic thrombocytopenic Purpura: Understanding a Disease No Longer Rare

Serial studies of plasma samples from patients during episodes of thrombotic thrombocytopenic purpura (TTP) have often shown either the presence of unusually large (UL) von Willebrand factor (vWf) multimers or, alternatively, absence of the largest plasma vWf forms. The presence of ULvWf multimers in TTP patient plasma may reflect impaired processing of the ULvWf forms released from endothelial cells. The disappearance of ULvWf and large vWf multimers in some TTP patient plasma samples during acute TTP episodes may be predominantly because these ULvWf forms, along with the largest vWf multimers, bind to platelets and cause aggregation. Serial flow cytometry studies of EDTA-whole blood samples from patients with initial episode, intermittent, and chronic relapsing types of TTP confirm that vWf is the likely aggregating agent, perhaps in association with fluid shear stress. The amount of vWf bound to single platelets has been found to be significantly increased during TTP relapses relative to remission periods in patients with all types of TTP. A substance in normal platelet-poor plasma and the cryoprecipitate-depleted fraction of normal plasma (cryosupernatant) is capable in vitro of reversibly reducing the size of ULvWf multimeric forms released by endothelial cells into the somewhat smaller vWf multimers ordinarily in circulation. This activity has characteristics of a limited disulfide bond reductase. The process of ULvWf breakdown may be made irreversible by the tandem proteolysis, catalyzed by a vWf metalloproteinase, of partially reduced vWf multimers. Several patients with chronic relapsing TTP have decreased or absent plasma vWf metalloproteinase activity, apparently on a congenital basis. Adult initial episode and intermittent TTP patients have been found to have vWf metalloproteinase activity inhibited by an autoantibody during, but not after, TTP epidsodes.

Thrombotic thrombocytopenic purpura: understanding a disease no longer rare.

Serial studies of plasma samples from patients during episodes of thrombotic thrombocytopenic purpura (TTP) have often shown either the presence of unusually large (UL) von Willebrand factor (vWf) multimers or, alternatively, absence of the largest plasma vWf forms. The presence of ULvWf multimers in TTP patient plasma may reflect impaired processing of the ULvWf forms released from endothelial cells. The disappearance of ULvWf and large vWf multimers in some TTP patient plasma samples during acute TTP episodes may be predominantly because these ULvWf forms, along with the largest vWf multimers, bind to platelets and cause aggregation. Serial flow cytometry studies of EDTA-whole blood samples from patients with initial episode, intermittent, and chronic relapsing types of TTP confirm that vWf is the likely aggregating agent, perhaps in association with fluid shear stress. The amount of vWf bound to single platelets has been found to be significantly increased during TTP relapses relative to remission periods in patients with all types of TTP. A substance in normal platelet-poor plasma and the cryoprecipitate-depleted fraction of normal plasma (cryosupernatant) is capable in vitro of reversibly reducing the size of ULvWf multimeric forms released by endothelial cells into the somewhat smaller vWf multimers ordinarily in circulation. This activity has characteristics of a limited disulfide bond reductase. The process of ULvWf breakdown may be made irreversible by the tandem proteolysis, catalyzed by a vWf metalloproteinase, of partially reduced vWf multimers. Several patients with chronic relapsing TTP have decreased or absent plasma vWf metalloproteinase activity, apparently on a congenital basis. Adult initial episode and intermittent TTP patients have been found to have vWf metalloproteinase activity inhibited by an autoantibody during, but not after, TTP epidsodes.