Whole Blood Specimens Research Articles

Multicenter Comparison of Different Real-Time PCR Assays for Quantitative Detection of Epstein-Barr Virus

Quantification of Epstein-Barr virus (EBV) in peripheral blood is important for the diagnosis and management of serious EBV diseases, including posttransplant lymphoproliferative disorder. A variety of PCR-based methods are currently in use; however, there is little information on their comparability. This study assessed the relative performance of different quantitative assays. A multicenter comparative study was performed at eight sites using three panels consisting of serial dilutions of quantified EBV DNA and extracts from a total of 19 whole-blood specimens. Samples were distributed and tested blindly. Instrumentation, probe chemistries, amplification targets, and other test-related aspects varied considerably between laboratories. Each laboratory's calibration curve indicated strong evidence of a consistent log-linear relationship between viral load and cycle threshold, suggesting that intralaboratory tracking of a given patient would yield similar relative quantitative trends among the participating test sites. There was strong concordance among laboratories with respect to qualitative test results; however, marked quantitative discordance was seen. For most samples, the across-laboratory interquartile range of the reported viral load (in copies/microl) was roughly 0.6 log-units, and for one sample the overall range was approximately 4.2 log-units. While intralaboratory tracking of patients may yield similar results, these data indicate a need for caution when attempting to compare clinical results obtained at different institutions and suggest the potential value to be gained by more standardized testing methodology.

Value of measuring subpopulations of T and B lymphocytes in patients with musculoskeletal tumours

Aims of our study were to assess the clinical value of measuring subpopulations of T and B lymphocytes in patients with musculoskeletal tumours as an immunodiagnostic procedure in the primary diagnosis of tumours. In this prospective study, blood samples were obtained from 145 patients aged 04-98 years with musculoskeletal tumours. CD3, CD4, CD8, Helper/Suppressor ratio and CD19 subpopulations of lymphocytes were measured in specimens of whole blood in EDTA, upon the principle of Ortho Cytoron Absolute flow cytometry. Histological tumour diagnosis was obtained by the histopathology investigation of biopsy sample and lymphocytes values allocated accordingly. Out of 145 patients, osteomyelitis was diagnosed in 15 (10.34%). Median values of subpopulations of lymphocytes were: CD3 2456, CD4 1479, CD8 929, Helper/Suppressor ratio 1.8 and CD19 560 and all were raised above normal values in patients with osteomyelitis. Results were also calculated for osteosarcomas, Ewing sarcomas, giant cell tumours, chondrosarcomas and other tumours. These causes had median values within normal reference levels. To confidently out rule or confirm diagnosis of osteomyelitis, we measured the cut off point values and they were: CD3 2420, CD4 1400, CD8 873 and CD19 550. The main clinical use of measuring subpopulations of lymphocytes is in establishing the correct diagnosis between suspected osteomyelitis and other musculoskeletal tumours. Levels of measured subpopulations of lymphocytes above the presented cut off values are important and accurate confirming factor for the clinical diagnosis of suspected osteomyelitis. II.

Direct comparison of enzyme measurements from dried blood and leukocytes from male and female Fabry disease patients

Anderson-Fabry disease is an X-linked disorder that is caused by deficiency of the lysosomal enzyme alpha-galactosidase A. Symptoms include chronic progressive painful small-fibre neuropathy, cornea verticillata, renal failure and heart disease. Interestingly, female heterozygous patients may also show severe symptoms. After clinical suspicion, usually the determination of alpha-galactosidase activity in leukocytes is requested first. Alternatively, an enzymatic assay using dried blood specimens has been described. Dried blood samples require less material and are substantially more stable (several months at room temperature) than whole-blood specimens. To validate the new method and to asses its usefulness for diagnosis of female patients, enzyme activities of alpha-galactosidase, beta-galactosidase and beta-glucuronidase from 78 known Fabry patients were compared (29 males, 47 females) between both materials. In summary, the determination of alpha-galactosidase activity using dried blood and leukocytes as well as the ratio of alpha-galactosidase to beta-glucuronidase in dried blood can improve the diagnostic specificity in cases of female patients who are difficult to identify when only leukocyte enzyme activities are considered.

Multicentric evaluation of a new commercial cytomegalovirus real-time PCR quantitation assay

Automated real-time PCR systems have become the most common method in the quantitation of viral load during cytomegalovirus (CMV) infection in immuno-compromised patients. In order to evaluate a new commercially available CMV real-time PCR assay (CMV R-gene™, Argene, France), a pp65 antigenemia assay and four different “in-house” real-time PCR assays were compared to the CMV R-gene™ for the detection and the quantitation of CMV load in 506 specimens of whole blood from transplant patients in four French hospital laboratories. The CMV R-gene™ was more sensitive than the pp65 antigenemia: there were 18% antigenemia-negative versus CMV R-gene™-positive samples. A significant correlation was found between DNA quantitation by CMV R-gene™ and the number of positive cells detected by the pp65 antigenemia test (Spearman's rank test, r = 0.63, p < 0.0001). A CMV DNA load equivalent to 50 pp65-positive cells/200 000 polymorphonuclear leukocytes was 5.26 log 10 copies/mL of whole blood. When the CMV R-gene™ kit was compared to the four other “in-house” real-time PCR assays, there were few discordant results (6.7% total for the four laboratories), all detected with a weak positive CMV DNA viral load. Spearman's coefficients showed a good ( r = 0.82 for laboratory 1, r = 0.66 for laboratory 3) to excellent ( r = 0.99 for laboratory 2, r = 0.94 for laboratory 4) correlation between CMV R-gene™ and the four real-time “in-house” PCR assays. However, the results of CMV DNA viral load generated by CMV R-gene™ test were constantly higher than those generated by three out of four “in-house” PCR assays. This mean variation in CMV DNA viral load measured by CMV R-gene™ and “in-house” PCRs was of 0.77 log 10, 0.04 log 10, 0.77 log 10 and 0.97 log 10, for laboratories 1, 2, 3 and 4, respectively. We concluded that there was variability between results of different real-time PCR assays for CMV DNA quantitation. This observation emphasized the need of a standardised commercial assay to allow an “inter-laboratory” comparison of results. Our study showed that CMV R-gene™ is an accurate, efficient, reliable and versatile tool for rapid diagnosis and monitoring of CMV disease in transplantation recipients.

Simultaneous Quantification of Epstein-Barr Virus, Cytomegalovirus, and Human Herpesvirus 6 DNA in Samples from Transplant Recipients by Multiplex Real-Time PCR Assay

We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.

Investigation of False Positive Results with an Oral Fluid Rapid HIV-1/2 Antibody Test

BackgroundIn March 2004, the OraQuick® rapid HIV antibody test became the first rapid HIV test approved by the US Food and Drug Administration for use on oral fluid specimens. Test results are available in 20 minutes, and the oral fluid test is non-invasive. From August 2004–June 2005, we investigated a sudden increase in false-positive results occurring in a performance study of OraQuick® oral-fluid rapid HIV tests in Minnesota.Methodology/Principal FindingsIn a field investigation, we reviewed performance study data on oral-fluid and whole-blood OraQuick® rapid HIV test device lots and expiration dates and assessed test performance and interpretation with oral-fluid and whole-blood specimens by operators who reported false-positive results. We used multivariate logistic regression to evaluate client demographic and risk characteristics associated with false-positive results. Next, we conducted an incidence study of false-positive OraQuick rapid HIV tests in nine US cities and tested both oral-fluid and finger-stick whole-blood specimens from clients; reactive tests were confirmed with Western blot. Sixteen (4.1%) false-positive oral-fluid results occurred in the performance study from April 15, 2004 through August 31, 2004 with unexpired devices from six test lots among 388 HIV-uninfected clients (specificity, 95.9%; 95% CI: 93.4–97.6). Three test operators who had reported false-positive results performed and interpreted the test according to package-insert instructions. In multivariate analysis, only older age was significantly associated with false-positive results (adjusted odds ratio = 4.5, 95% CI: 1.2–25.7). In the incidence study, all valid oral-fluid and whole-blood results from 2,268 clients were concordant and no false-positive results occurred (100% specificity).Conclusions/SignificanceThe field investigation did not identify a cause for the increase in false-positive oral-fluid results, and the incidence study detected no false-positive results. The findings suggest this was an isolated cluster; the test's overall performance was as specified by the manufacturer.

Optimized Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. This unit describes a technique to measure ZAP-70 protein expression in whole-blood specimens from CLL samples. The protocols presented include an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP-70 protein with significantly improved signal-to-noise ratio, an optimized combination of antibodies-fluorophores to maximize ZAP-70 expression levels, standardized methodology for instrument setup, including compensation, to improve inter- and intra-laboratory reproducibility, and a method to index ZAP-70 protein expression levels to internal positive and negative cell populations. Residual normal T and B cells function as internal positive and negative controls. These are used to index ZAP-70 protein expression levels in the CLL population.

Molecular Typing of Novel Rickettsia rickettsii Isolates from Arizona

Seven isolates of Rickettsia rickettsii were obtained from a skin biopsy, two whole-blood specimens, and from Rhipicephalus sanguineus ticks from eastern Arizona. Molecular typing of seven isolates of R. rickettsii and DNA samples from two other Rh. sanguineus ticks infected with R. rickettsii was conducted by PCR and DNA sequencing of rompA and 12 variable-number tandem repeat regions (VNTRs). All DNA specimens from Arizona were identical to each other and to reference human and Dermacentor andersoni isolates of R. rickettsii from Montana in their rOmpA gene sequences and 10 VNTRs. Two of the twelve VNTRs had differences in the number of repeat sequences in isolates from Arizona compared to those from Montana, thus conferring the novelty of the Rh. sanguineus-associated R. rickettsii.

Anatomical mapping of human herpesvirus reservoirs of infection

Following primary infection, all eight human herpesviruses persist lifelong in the human host. However, a mapping of all anatomic sites of human herpesvirus persistence is lacking. Fresh tissue specimens representing approximately 40 major anatomic sites from eight autopsies were screened using a recently developed real-time PCR method for detection of all eight human herpesviruses. Patients with evidence of active herpesvirus infection (herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), herpesvirus 6 (HHV-6), herpesvirus 7 (HHV-7), and herpesvirus 8 (HHV-8)) at the time of death were excluded to avoid detection of widely disseminated infection. Despite this precaution, widespread HSV-1 positivity (with blood positivity) was detected in one case—an elderly male who died of cardiac arrest. In a middle-aged male with HIV-AIDS, HSV-1 was found in neural and pharyngeal tissues, skin, cartilage, bone, and urinary bladder, whereas in two other cases, HSV-1 was restricted to neural tissues. HSV-2 was detected in a single site, the anus, in the male with HIV-AIDS. VZV was detected only twice, once in the adrenal gland and once in the small intestine. CMV was detected in three cases, most commonly in nasal mucosa, trachea, thyroid, intestine, and liver. EBV was detected in all eight cases, especially in nasal mucosa, tonsil, spleen, lymph node, tongue, and intestine, but in only two of six whole-blood specimens. HHV-6, like EBV, was detected in all eight cases, most commonly in salivary glands, thyroid, stomach, intestines, liver, and pancreas. HHV-7, like EBV and HHV-6, was detected in all eight cases, most commonly in salivary glands, tonsil, lymph nodes, and bone marrow. HHV-8 was detected in only two sites (both lymph nodes) from two cases. Herpesviruses were detected in three of six whole-blood specimens, including HSV-1, EBV, HHV-6, and HHV-7. These results represent the most comprehensive mapping of herpesvirus tissue distribution in humans reported to date.

Correction of factitious hyperkalemia in hemolyzed specimens

Background Hemolysis in pediatric specimens is common due to difficult blood draws and small-bore intravenous catheters. Values of serum K + become falsely elevated secondary to release of intracellular contents. If a reliable correction factor existed for this factitious elevation, repeat K + measurements might be avoided. Objective The aim of the study was to establish a correction factor for factitiously elevated K +, using free plasma hemoglobin (p-Hgb) as a measure of in vitro hemolysis. Methods Twenty whole-blood specimens drawn from healthy adults via a 23-gauge needle were divided into 4 aliquots: (1) no manipulation, (2) mechanical hemolysis via a 27-gauge needle, (3) addition of potassium acetate (KAc), and (4) addition of KAc and mechanical hemolysis. KAc was added to mimic potentially significant hyperkalemia. All specimens had standard K + and p-Hgb measurements performed. Results Nonhemolyzed and hemolyzed K + ranged from 3.2 to 8.1 mEq/L and 3.5 to 10.0 mEq/L, respectively. A linear relationship existed between the change in K + and p-Hgb from the nonhemolyzed to hemolyzed specimens. A correction factor for K + of 0.00319 (95% confidence interval, 0.00290-0.00349) × p-Hgb was obtained. Conclusions A reliable correction factor for factitious hyperkalemia in a clinically relevant range exists. By example, using the above correction factor, one can predict that the delta K + in a specimen with 500 mg/dL of p-Hgb will be 1.6 mEq/L (range, 1.5-1.7). We suggest that when the lower bound of the predicted delta K + results in a corrected value within the reference range, a second blood draw is unnecessary.

Detection and Quantification of Human Immunodeficiency Virus Type 1 p24 Antigen in Dried Whole Blood and Plasma on Filter Paper Stored under Various Conditions

The quantification of human immunodeficiency virus type 1 (HIV-1) by an assay measuring heat-dissociated (HD) p24 antigen (Ag) in specimens of whole blood and plasma stored on filter paper, and of plasma stored in tubes, was compared to HIV-1 RNA plasma levels determined by real-time reverse transcription (RT)-PCR. The stability of p24 Ag on filter paper under conditions simulating specimen transport was also evaluated. The HD p24 Ag in both plasma and whole-blood specimens stored on filter paper correlated with plasma HIV-1 RNA levels (Spearman rank rho = 0.74 [P < 0.0001] and rho = 0.56 [P = 0.0001], respectively). The sensitivity of the HD p24 Ag assay was similar when plasma and whole blood on filter paper were contrasted to the real-time RT-PCR assay (80% versus 82.5% and 78.6% versus 83.3%, respectively). However, while the specificity of the HD p24 Ag assay of plasma on filter paper was 100%, the specificity was diminished in whole-blood specimens. The storage of specimens on filter paper for 2 weeks at 37 degrees C, 24 degrees C, or 0 degrees C did not alter the detection or quantification of HD p24 Ag. These results suggest that transport and storage of plasma on filter paper and quantification of HD p24 Ag may be a reliable method for HIV-1 load monitoring.

Detection of Mycoplasma pneumoniae in serum specimens from patients with mycoplasma pneumonia by PCR

There are few data on detection of Mycoplasma pneumoniae from blood, serum or plasma, and systematic studies on this diagnostic approach in community-acquired pneumonia (CAP) are scarce. Compared to testing respiratory specimens, this approach has the advantages that it is less dependent on proper specimen collection, serum is easily stored and handled, and the pathogen is detected in a primary sterile site, where colonization can be ruled out. In this study, acute-phase serum specimens from 29 patients of Vienna University Hospital (treated between 11/1994 and 6/2004; female: 14, male: 15; median age: 31 years, range: 15–66 years) with CAP and serologically verified M. pneumoniae infection, who had not received anti-mycoplasma therapy prior to serum collection, were tested for M. pneumoniae by conventional PCR and real-time PCR. Conventional PCR yielded negative results for all specimens, but real-time PCR detected M. pneumoniae in 15/29 patient sera (52%). These findings indicate that M. pneumoniae is present in the bloodstream of a substantial proportion of patients with mycoplasma pneumonia. Despite the possible adherence of M. pneumoniae to human erythrocytes, the pathogen can be detected from serum, if a method with enhanced sensitivity is applied. However, the negative predictive value of PCR from serum with regard to etiological diagnosis is low. With regard to the potential clinical benefit of blood-based PCR diagnosis of mycoplasma pneumonia the diagnostic accuracy of this approach using either serum or whole-blood specimens should be addressed by large-scale studies.

Evaluation of Glucose-6-Phosphate Dehydrogenase Stability in Blood Samples under Different Collection and Storage Conditions

Fujimoto et al. (1) reported that the stability of galactose 1-phosphate in dried blood spots for neonatal screening was adversely affected by humidity and temperature, especially when low-value samples are evaluated. We extend these findings to glucose-6-phosphate dehydrogenase (G-6-PD; EC 1.1.1.49) activity, deficiency of which is by far the most common genetic disease worldwide (2) and accounts for more than one-half of the cases of severe jaundice among male newborns in Greece, China, and in Sephardic Jewish groups (3). Tests for G-6-PD deficiency are thus included in newborn screening programs in some regions. We collected whole-blood specimens from …

Preclinical and Clinical Performance of the Efoora Test, a Rapid Test for Detection of Human Immunodeficiency Virus-Specific Antibodies

Barriers to effective diagnostic testing for human immunodeficiency virus type 1 (HIV-1) infection can be reduced with simple, reliable, and rapid detection methods. Our objective was to determine the accuracy, sensitivity, and specificity of a new rapid, lateral-flow immunochromatographic HIV-1 antibody detection device. Preclinical studies were performed using seroconversion, cross-reaction, and interference panels, archived clinical specimens, and fresh whole blood. In a multicenter, prospective clinical trial, a four-sample matrix of capillary (fingerstick) whole-blood specimens and venous whole blood, plasma, and serum was tested for HIV-1 antibodies with the Efoora HIV rapid test (Efoora Inc., Buffalo Grove, IL) and compared with an enzyme immunoassay (EIA) (Abbott Laboratories) licensed by the Food and Drug Administration. Western blot and nucleic acid test supplemental assays were employed to adjudicate discordant samples. Preclinical testing of seroconversion panels showed that antibodies were often detected earlier by the rapid test than by a reference EIA. No significant interference or cross-reactions were observed. Testing of 4,984 archived specimens yielded a sensitivity of 99.2% and a specificity of 99.7%. A prospective multicenter clinical study with 2,954 adult volunteers demonstrated sensitivity and specificity for the Efoora HIV rapid test of 99.8% (95% confidence interval [CI], 99.3 and 99.98%) and 99.0% (95% CI, 98.5 and 99.4%), respectively. Reactive rapid HIV-1 antibody detection was confirmed in 99.6% of those with a known HIV infection (n = 939), 5.2% of those in the high-risk group (n = 1,003), and 0.1% of those in the low-risk group (n = 1,012). For 21 (0.71%) patients, there was discordance between the results of the rapid test and the confirmatory EIA/Western blot tests. We conclude that the Efoora HIV rapid test is a simple, rapid assay for detection of HIV-1 antibodies, with high sensitivity and specificity compared to a standardized HIV-1 EIA.

Early Prediction of Hepatitis C Virus (HCV) Infection Relapse in Nonresponders to Primary Interferon Therapy by means of HCV RNA Whole-Blood Analysis

Routine analysis of serum and/or plasma specimens for hepatitis C virus (HCV) RNA does not always correctly reflect the response to antiviral therapy. Analysis of whole-blood specimens for detection of viral RNA should provide more-accurate prognostic information. Whole-blood, serum, and plasma specimens (268 sample sets) were obtained from 56 patients who did not respond to initial interferon (IFN)- alpha 2b monotherapy (5 MU every 2 days for 3 months). Specimens were analyzed for HCV RNA by 4 different types of reverse-transcriptase polymerase chain reaction (RT-PCR) (Cobas Amplicor HCV-2.0 [Roche], LightCycler real-time PCR [Roche], and 2 in-house RT-PCRs) to determine whether specimen type can predict the rate of virologic response to high-dose treatment with IFN (10 MU every 2 days) and ribavirin (1-1.2 g/day). Of the 56 patients who provided specimens, serum and plasma obtained from 18 tested negative for HCV RNA at the end of treatment, indicating a complete virologic response. In contrast, analysis of whole-blood specimens obtained at the same time revealed the presence of viral RNA in 12 of these 18 patients. All 12 subjects had relapse of HCV in serum and plasma: 11 relapsed a median of 4 weeks after the end of treatment, and 1 relapsed 20 weeks after the end of treatment. None of these 12 patients--all of whom consistently had whole-blood specimens that tested positive and plasma and serum specimens that tested negative for HCV RNA up to 20 weeks before the end of treatment--showed a sustained virologic response (P=.0002). Results of whole-blood tests for detection of HCV RNA were highly predictive of viral relapse (positive predictive value, 100%) and thus may be useful tools for monitoring and tailoring IFN/ribavirin therapy. Testing of only serum or plasma specimens underestimates the true circulating HCV load and leads to an overestimation of antiviral response rates.

Rapid and simple mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.

FLOW CYTOMETRIC EVALUATION OF LEUKOCYTE FUNCTION IN RAT WHOLE BLOOD

The aim of this study was to establish a standard flow cytometric method to measure the phagocytic function of and intracellular hydrogen peroxide (H2O2) production by rat leukocytes. Thirty-six adult, male Sprague-Dawley rats were included in this study. Whole-blood specimens from the inferior vena cava were collected in a heparinized tube and ethylenediaminetetraacetic acid (EDTA) anticoagulated tube. The phagocytic function of and intracellular H2O2 generation by leukocytes were measured with FACS Vantage trade mark flow cytometer (Becton Dickinson, San Jose, CA), using fluorescent microspheres and dihydrorhodamine-123 as probes, respectively. Several conditions were optimized in this study, including anticoagulants (heparin and EDTA), fluorescent probes (0.75- and 1.72-microm-diameter microspheres), incubation time, and concentration of the chemicals used in the experiment. Neutrophils, monocytes, and lymphocytes could be clearly defined and separated in whole blood by flow cytometry and tested for phagocytosis and intracellular H2O2 generation without the need for further purification and handling of the cells. Intracellular H2O2 production by and phagocytic function of neutrophils and monocytes were inhibited in EDTA-anticoagulated blood compared with heparin- anticoagulated blood (P < 0.01). Neutrophils showed similar phagocytic function to 0.75- and 1.72-microm microspheres, but monocytes showed weak phagocytic activity to 1.72-microm beads compared with 0.75-microm beads (P < 0.01). In conclusion, a flow cytometric method to measure the phagocytic function of and intracellular H2O2 production by rat leukocytes has been developed. Quantitative flow cytometric analysis of rat leukocyte function is convenient and feasible and provides a reliable and rapid assay to assess phagocytosis and intracellular H2O2 production by rat neutrophils and monocytes.

Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients.

Quantitative monitoring of human cytomegalovirus (HCMV) infection is helpful in determining appropriate antiviral management of transplant recipients. Quantitative PCR technologies have demonstrated accuracy in measuring systemic HCMV loads. A total of 298 consecutive whole-blood specimens submitted to the Clinical Virology Laboratory at Vanderbilt University Medical Center from 15 February to 31 October 1999 were included in the study. In addition to a qualitative colorimetric microtiter plate PCR assay (MTP-PCR) and a semiquantitative pp65 antigenemia assay, each specimen was measured for HCMV loads by a quantitative PCR assay performed on an ABI PRISM 7700 Sequence Detection System (TaqMan). Compared to results of the MTP-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 70.5, 97.5, 87.8, and 92.8% for the antigenemia assay and were 96.7, 92.0, 75.6, and 99.1% for the TaqMan assay, respectively. There was a high correlation between antigenemia values and HCMV loads as determined by the TaqMan (r = 0.989; P < 0.001). Antigenemia values of 0, 1 to 10, 11 to 100, 101 to 1,000, and over 1,000 positive cells per 2 x 10(5) leukocytes corresponded to median HCMV loads measured by TaqMan of 125, 1,593, 5,713, 16,825, and 5,425,000 copies/ml, respectively. Corresponding to antigenemia values of 1 to 2, 10, and 50 positive cells per 2 x 10(5) leukocytes, HCMV viral loads of 1,000, 4,000, and 10,000 copies/ml are proposed as cutoff points for initiating antiviral therapy in patient groups with high, intermediate, and low risk of CMV diseases.

Reference limits for urine/blood ratios of ethanol in two successive voids from drinking drivers.

Specimens of venous whole blood and two successive urinary voids were collected from 450 individuals apprehended for driving under the influence of alcohol in Sweden. The first specimen of urine (UAC-1) was obtained as soon as possible after arrest, and the second void (UAC-2) was collected about 60 min later (mean 66 min, range 30-130). A specimen of venous blood was drawn approximately 30 min after the first urine sample was collected. Ethanol was determined in blood and urine by headspace gas chromatography, a method with high analytical precision (coefficient of variation approximately 1%). The mean UAC for the first void was 2.60 g/L (range 0.21-5.35) compared with 2.40 g/L (range 0.16-5.50) in the second void. The mean concentration of alcohol in venous blood (BAC) was 1.97 g/L (range 0.08-4.57). The concentrations of ethanol in the two voids of urine were highly correlated (r = 0.97, residual standard deviation [SD] 0.22 g/L). The UAC and BAC results were also highly correlated; r = 0.958 (residual SD 0.28 g/L) for the first void and r = 0.978 (residual SD 0.21 g/L) for the second void. The concentration of ethanol in the first void (UAC-1) was higher than the second void (UAC-2) in 383 (87%) instances, decreasing by 0.23 g/L/h on average. In 57 instances (13%), UAC-1 was less or equal to UAC-2 with a mean increase of 0.19 g/L. When BAC exceeded 0.5 g/L (N = 429), the mean UAC-1/BAC ratio was 1.345 with 95% reference limits of 0.968 and 1.72, which agreed well with median (2.5th and 97.5th percentiles) of 1.325 (0.938 and 1.79). For the second void, the mean UAC-2/BAC ratio was 1.221 with 95% reference limits of 0.988 and 1.45 and with a median (2.5th and 97.5th percentiles) of 1.226 (0.997 and 1.46). These reference limits are appropriate to use when a person's venous BAC needs to be estimated with reasonable scientific certainty from the concentration determined in specimens of urine.

Interference of hematocrit in the tacrolimus II microparticle enzyme immunoassay.

The authors studied the effect of hematocrit, bilirubin, and alkaline phosphatase on microparticle enzyme immunoassay for tacrolimus II (MEIA II) using specimens of whole blood obtained from 33 patients undergoing cyclosporine treatment. Tacrolimus was added to these samples at a final concentration of 7.5 microg/L and 15 microg/L. Both coefficients of variation were over 20% (21% at 7.5 macrog/L of tacrolimus and 22% at 15 microg/L of tacrolimus). No correlation was found between bilirubin and tacrolimus concentrations or between alkaline phosphatase and tacrolimus concentrations. On the other hand, negative correlations were found between hematocrit values and tacrolimus concentrations (r2 = 0.47; P < 0.0001 at 7.5 microg/L tacrolimus, r2 = 0.54; P < 0.0001 at 15 microg/L tacrolimus). Negative correlations were also found between hematocrit and the tacrolimus concentration using normal human red blood cells diluted with physiological saline solution (r2 = 0.93; P < 0.0001 at 7.5 microg/L tacrolimus, r2 = 0.91; P < 0.0001 at 15 microg/L tacrolimus). The results showed that the hematocrit interferes with the MEIA II for tacrolimus, and the magnitude of the interference is clinically significant. Beyond the normal range of hematocrit values, caution should be exercised in interpreting results as one may need to compensate for the levels of tacrolimus.