Specimen Dilution Research Articles

Specimen Dilution Improves Sensitivity of the Amplified Mycobacterium tuberculosis Direct Test for Smear Microscopy-Positive Respiratory Specimens

Specimen dilution has been proposed as a strategy to minimize amplified Mycobacterium tuberculosis direct (MTD) test inhibition (N. Pollock, J. Westerling, and A. Sloutsky, Am. J. Clin. Pathol. 126:142-147, 2006; A. Sloutsky, L. L. Han, and B. G. Werner, J. Clin. Microbiol. 42:1547-1551, 2004). We evaluated the impact of respiratory specimen dilution on MTD test accuracy in a public health laboratory. The difference in MTD test sensitivity between the dilution and conventional methods was 15.9% (P = 0.001) for smear microscopy-positive specimens and -3.6% (P = 0.38) for smear microscopy-negative specimens.

Improved COBAS TaqMan Hepatitis C Virus Test (Version 2.0) for Use with the High Pure System: Enhanced Genotype Inclusivity and Performance Characteristics in a Multisite Study

We have evaluated the COBAS TaqMan hepatitis C virus (HCV) test (version 2.0) for use with the High Pure system (HCVHPS V2), a new, revised real-time reverse transcription-PCR assay developed to improve the genotype quantitation of version 1.0 (HCVHPS V1). Revisions were made in the wash buffer and in the reverse transcription temperature. The genotype inclusivity of HCVHPS V2 was evaluated at three different sites, using HCVHPS V2, HCVHPS V1, and the COBAS AMPLICOR HCV MONITOR test (version 2.0) (CAHCM). The fully automated COBAS Ampliprep/COBAS TaqMan HCV test was also used in one of the participating laboratories. The mean differences in HCV RNA values between HCVHPS V2 and CAHCM and between HCVHPS V2 and HCVHPS V1 ranged from -0.21 to 0.13 log and from 0.24 to 1.27 log, respectively, with >0.5-log differences for genotypes 2, 3, 4, and 5. With a NIBSC panel of HCV genotypes 1 through 6, the measured HCVHPS V2 values were within 0.25 log of the nominal values for all 6 genotypes. When serial dilutions of genotype-specific clinical HCV specimens were tested, the assay showed a limit of detection between 10 and 20 IU/ml and a linear range of 25 IU/ml to 3.91 x 10(8) IU/ml. Clinical and analytical specificities of 100% were demonstrated with 100 HCV-seronegative specimens as well as with 12 non-HCV members of Flaviviridae and 22 additional microorganisms. These data indicate that HCVHPS V2 is a robust and accurate test for the quantitation of all six HCV genotypes and useful in monitoring viral load in all clinical HCV specimens.

CURRENT–VOLTAGE PROPERTIES OF SELF-ASSEMBLY MONOLAYERS ON AU(111) SUBSTRATE USING SCANNING TUNNELING MICROSCOPY

This study observes the morphology of self-assembled organic thin film that is formed at room temperature using a Scanning Tunneling Microscopy (STM) and examines its electrical properties using a Scanning Tunneling Spectroscopy (STS). The specimen used in this experiment that has a functional group for both sides and will be controlled to make self-assembled onto the Au (111) substrate. After the dilution of the specimen by 1 mM/ml and self-assembled onto the Au (111) substrate. Using a STM, the images of organic thin film can be observed. In addition, the electrical properties of organic thin film can also be examined by the junction structure of STM–organic thin film– Au (111) substrate by using a STM tip. As a result, we measured current–voltage (I–V) curve using STS, I–V curve also clearly shows several current peaks between the negative bias region (-1.42 V) and the positive bias region (1.30 V), respectively.

Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR

Background Molecular diagnosis of Norovirus infection can be a complex multistep process, which requires significant user intervention and expertise, and is not amenable to automation without extensive validation and optimization. Objectives To develop a real-time multiplexed RT-PCR assay with automated sample preparation that requires only a single-step and a single-tube for reverse transcription, amplification, and detection while exceeding the sensitivity of conventional PCR for broad-spectrum Norovirus detection. Study design Limit of detection was assessed against dilutions of clinical specimens. Fifty archived extractions were used to compare TaqMan sensitivity with either a separate RT using random primers or a single-step RT-PCR. The sensitivity of the novel assay was compared with conventional RT-PCR using 100 specimens from gastroenteritis cases. Results Automated extraction reduced RNA recovery by 0.5 logs compared to manual extraction but was more effective at removing PCR inhibitors from stool specimens. The optimized single-step real-time RT-PCR demonstrated no reduction in sensitivity. Together, the sensitivity of the novel assay was 19% higher than manual extraction with conventional RT-PCR. Conclusions A semi-automated and simplified molecular diagnostic protocol for the rapid detection of Norovirus has been achieved. PCR inhibitors are present in human fecal specimens and cause a significant problem for Norovirus detection by RT-PCR.

Real-Time PCR Assay for Detection and Quantification of Hepatitis B Virus Genotypes A to G

The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response. The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques. In this study, we describe the development, evaluation, and validation of a novel real-time PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens. A computer algorithm was used to design degenerate real-time PCR primers and probes based upon a large number (n = 340) of full-length genomic sequences including HBV genotypes A to H from Europe, Africa, Asia, and North and South America. Genotype performance was tested and confirmed using 59 genotype A to G specimens from two commercially available worldwide genotype panels. This assay has a dynamic range of at least 8 log(10) without the need for specimen dilution, good clinical intra- and interassay precision, and excellent correlation with the Bayer Diagnostics VERSANT HBV DNA 3.0 (branched DNA) assay (r = 0.93). Probit analysis determined the 95% detection level was 56 IU/ml, corresponding to 11 copies per PCR well. The high sensitivity, wide linear range, good reproducibility, and genotype inclusivity, combined with a small sample volume requirement and low cost, make this novel quantitative HBV real-time PCR assay particularly well suited for application to large clinical and epidemiological studies.

Specimen Dilution Increases the Diagnostic Utility of the Gen-Probe Mycobacterium Tuberculosis Direct Test

The Mycobacterium Tuberculosis Direct (MTD) Test is widely used for detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. Based on high specificity, it is accepted that a positive MTD result, in proper clinical context, “rules in” a diagnosis of tuberculosis. Test utility for “ruling out” tuberculosis has been limited by lower sensitivity and negative predictive value (NPV), based in part on poorly defined reaction inhibitors in clinical specimens. After discovering that specimen dilution could decrease concentrations of reaction inhibitors without reaching the MTBC detection limit, the Massachusetts State Laboratory Institute in 2003 began routinely testing paired undiluted and diluted samples in parallel. We retrospectively analyzed results from 105 respiratory specimens (87 smear-positive, 18 smear-negative) tested consecutively over 23 months using this dilution protocol. MTD results for each sample pair were merged into a sum result (positive or negative) and compared with culture outcome as the “gold standard.” We found that the MTD sum result always matched the culture outcome, even for smear-negative specimens. Accordingly, the MTD using the dilution protocol had sensitivity, specificity, positive predictive value, and NPV of 100% vs culture.

Specimen Dilution Increases the Diagnostic Utility of the Gen-Probe Mycobacterium Tuberculosis Direct Test

The Mycobacterium Tuberculosis Direct (MTD) Test is widely used for detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. Based on high specificity, it is accepted that a positive MTD result, in proper clinical context, "rules in" a diagnosis of tuberculosis. Test utility for "ruling out" tuberculosis has been limited by lower sensitivity and negative predictive value (NPV), based in part on poorly defined reaction inhibitors in clinical specimens. After discovering that specimen dilution could decrease concentrations of reaction inhibitors without reaching the MTBC detection limit, the Massachusetts State Laboratory Institute in 2003 began routinely testing paired undiluted and diluted samples in parallel. We retrospectively analyzed results from 105 respiratory specimens (87 smear-positive, 18 smear-negative) tested consecutively over 23 months using this dilution protocol. MTD results for each sample pair were merged into a sum result (positive or negative) and compared with culture outcome as the "gold standard." We found that the MTD sum result always matched the culture outcome, even for smear-negative specimens. Accordingly, the MTD using the dilution protocol had sensitivity, specificity, positive predictive value, and NPV of 100% vs culture.

An investigation into the extent of possible dilution of specimens received for urinary drugs of abuse screening

Recent American and Swedish studies have shown an increase in "false" negative results when analysing dilute urine specimens for drugs of abuse. In the light of these studies, it was decided to perform a pilot study to determine the extent of possible specimen adulteration and dilution in a random batch of 50 urine specimens presented to this laboratory, using creatinine, osmolality, pH and relative density. It was found that 20% of the specimens were outside the pH range associated with the optimum working of Syva EMIT Drugs of Abuse in Urine (DAU) immunoassay screening techniques, and that if the National Institute on Drug Abuse (NIDA) recognized dilution cut-off of 1.8 mmol/L for urine creatinine concentration is applied, 84% of the specimens surveyed here would need to be repeated to ensure accurate results. Because of these findings, it is recommended that routine creatinine and pH estimations should be performed on all specimens submitted for urinary drugs of abuse screening, or at least when unexpectedly negative results are obtained.

Hyaluronidase as a Liquefying Agent for Chemical Analysis of Vitreous Fluid

Vitreous humor is a suitable specimen for postmortem clinical chemistry because the analytes remain relatively stable after death and they closely reflect blood levels immediately prior to death. The viscous nature of vitreous fluid, however, presents analytical problems including imprecision and inaccuracy. Various preanalytical treatments, such as boiling, high speed centrifugation, microfiltration and dilution have been used. These techniques are labor intensive and add to imprecision and inaccuracy. Because glycosaminoglycans contribute significantly to the viscosity of vitreous humor, we used hyaluronidase as a liquefying agent. We compared the results of analyses in 33 vitreous humor specimens after hyaluronidase treatment with the results after either no treatment or specimen dilution. Seventeen of the 33 specimens could not be analyzed without dilution. Even after dilution, several analytes still could not be measured. Hyaluronidase treatment negated the need for sample dilution and had no significant effect on the analyses.

Specific detection of human BK polyomavirus in urine samples of immunocompromised patients.

A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were positive when specimens were submitted because of clinical suspicion of BK virus infection.

Interpretation of GC–MS opiate results in the presence of pholcodine

Although the cross reactivity of pholcodine with opiate immunoassays has been well documented there is little published information the potential for pholcodine interference with chromatographic analyses. Wilson and Smith [Ann. Clin. Biochem. 36 (1999) 592] recently described the ‘misidentification’ of morphine in quality control specimens that had been spiked with pholcodine. This report describes a sensitive, rapid gas chromatography—mass spectrometry (GC–MS) method for the detection and quantitation of pholcodine and morphine, together with 6-monoacetylmorphine (6-MAM), codeine and dihydrocodeine in urine. This method was used to analyse urine specimens collected from volunteers given single and multiple doses of pholcodine to establish the significance this drug on the analytical results obtained when performing drug screening according to the proposed UK and EU legally defensible workplace drug testing guidelines. The maximum urinary free morphine concentration achieved following a single 10mg oral dose of pholcodine was 1.39mg/l at 2–4h post dose. Following multiple 10mg oral doses of pholcodine the maximum urinary free morphine concentration was determined as 0.4mg/l at 170h after the final dose was administered. This apparent anomaly in the morphine concentrations obtained following single and multiple pholcodine doses can be explained in part by differences in the concentration of the specimens, and may be overcome by applying a correction factor for specimen dilution using their creatinine concentration. The data from this study suggests that even following one single 10mg dose of pholcodine, free morphine concentrations greater than both the proposed UK workplace drug testing guidelines threshold of 0.3mg/l total morphine and the proposed European Union threshold of 0.2mg/l total morphine can be achieved. This highlights the need for caution when interpreting confirmatory opiate data, especially in medicolegal and clinical cases, and in cases where the use of pholcodine is suspected.

Causes of Unsatisfactory Performance in Proficiency Testing

Proficiency testing (PT) provides a measure of the effectiveness of laboratory quality assurance programs. Test reports are released from processes that the laboratory judges to be in conformance with quality specifications; an evaluation of unsatisfactory performance (UNSAT) by a PT provider is an unexpected outcome for the laboratory. An understanding of the root cause(s) of testing errors provides an opportunity for the continuous improvement of laboratory services. We used participant data from the New York State Department of Health PT program to characterize the quality of testing in the toxicology specialty. Outcomes from laboratory investigations into causes of UNSAT and information on quality control practices collected from all program participants were used to identify the root causes of error. Two classes of error were encountered: spurious test results caused by lapses in standard operating procedures and instrument malfunctions (300 per million assays) and common-cause analytic error (7000 per million assays or 0.7% rate of UNSAT). Causes of spurious results included inaccurate mathematical correction for specimen dilution, misinterpretation of instrument codes, and instrument sampling errors. Calibration drift was most frequently cited as the common-cause analytic error. Approximately one-half of the laboratories used an allowable error for the quality control of analytical systems that exceeded the threshold error specified by manufacturers for stable instrument performance. The causes of spurious results suggest the need for ongoing competency testing of analysts where analyst intervention is required in an otherwise automated process, and for continued diligence in mistake-proofing instrument design. The intrinsic quality of laboratory testing is unlikely to improve until the allowable error in quality control is consistent with manufacturer specifications for stable system performance.

Effect of Urine Specimen Dilution on the Performance of Two Commercial Systems in the Detection of Chlamydia trachomatis Infection in Men

Effect of Urine Specimen Dilution on the Performance of Two Commercial Systems in the Detection of Chlamydia trachomatis Infection in Men

Effect of urine specimen dilution on the performance of two commercial systems in the detection of Chlamydia trachomatis infection in men.

Effect of urine specimen dilution on the performance of two commercial systems in the detection of Chlamydia trachomatis infection in men.

Differential effects of serial hemodilution with hydroxyethyl starch, albumin, and 0.9% saline on whole blood coagulation

Study Objectives: To determine by thrombelastography assessed coagulation, the effects of progressive hemodilution with three intravascular volume expanders. Design: Prospective, controlled, whole blood, volumetric ex vivo hemodilution study. Setting: University of Pennsylvania Medical Center Operating Rooms. Patients: 60 ASA physical status I and II patients; phlebotomy prior to administration of IV fluids or medications. Interventions: Analysis of whole blood clotting determined by six thrombelastographic channels for control and five volumetric hemodilutions (11%, 25%, 33%, 50%, and 75%) with 0.9% saline, 5% albumin, and 6% hydroxyethyl starch (n = 20 for each diluent group). Measurements and Main Results: Thrombelastographic parameters R (minutes), angle α (°), MA (mm), and lysis (%) were measured and compared to the sample control for each dilution of the same specimen. There was no significant difference between control groups in any thrombelastographic variable (R, angle α, MA, or lysis). No changes were seen in any variable from any diluent at 11% hemodilution. Seventy-five percent hemodilution caused significantly hypocoagulable changes from control for all thrombelastographic parameters for all three diluents. Thrombelastographic indices differed significantly from controls at intermediate hemodilutions. Both colloids caused decreases in measured angle α and MA at lower hemodilution than did 0.9% saline. Albumin 5% caused significant hypocoagulable changes from control values at lower hemodilution than did either 0.9% saline or 6% hydroxyethyl starch for all thrombelastographic parameters. Saline 0.9% increased angle α significantly at 50% hemodilution. Abnormal lysis did not occur at any dilution. Differing ex vivo effects of three different intravascular fluids thrombelastography assessed coagulation are found. Conclusion: No differences were found after 11% hemodilution with any volume expanders. Hemodilution with up to 50% saline maintained thrombelastographic indices. Albumin produced early and profound hypocoagulable effects. Significant hypocoagulability occurred for all three diluents at 75% hemodilution. The study supports the use of albumin in patients at risk for thrombosis, and saline in patients with a need for normal hemostasis.

Evaluation of commercial kits for a rapid detection of Escherichia coli O157 in stool

To introduce rapid detection of E. coli O157 in the stool without culturing, nine different commercial kits for detection of E. coli O157 were compared. VIP, Reveal, Path Stik, Quix, Now EH and EZ coli were found to detect E. coli O157 greater than 10(6) CFU/ml. TECRA and Novapath could detect more than 10(5) CFU/ml. VIDAS reacted positive with more than 10(4) CFU/ml. TECRA, Novapath, Quix could detect 1:4, 1:4, 1:9 dilution of stool specimen spiked with E. coli O157 respectively, but others could not detect with 1:9 dilution of stool specimen. When stool specimens spiked with E. coli O157 were diluted ten times, centrifuged slightly and supernatants were tested, Path Stik, Quix and EZ coli could detect 10(7) CFU/ml. TECRA and Novapath could detect 10(6) CFU/ml, whereas VIDAS could detect 10(6) CFU/ml. Quix is therefore one of the most suitable kit for detection of E. coli O157 in the stool at the bedside, although some non-specific reactions were observed. Path Stik is also convenient kit at the bedside. VIDAS, although it requires special equipment, is the most sensitive kit for detection of E. coli O157 and is suitable for detection of low levels of E. coli O157 in the clinical laboratory.

A Rapid Assay to Screen for Drug‐Resistant Herpes Simplex Virus

A rapid assay was developed to screen for herpes simplex virus (HSV) isolates that are resistant to acyclovir and other antiviral agents. The assay is a modified plaque reduction assay (PRA) in which the number of plaques seen in the absence of acyclovir was compared with that seen in the presence of a single cutoff concentration of acyclovir (2 microg/mL). This assay utilizes a cell line that expresses beta-galactosidase only after infection with HSV. Since histochemically stained plaques are easily visualized, small plaques can be easily enumerated. This allows the assay to be performed on dilutions of untitered specimens in the small wells of a 24-well plate and allows the results to be read only 2 days after inoculation of the virus. The assay performed well compared with a standard PRA and should be a valuable tool in identifying drug-resistant HSV in a timely manner.

Methodologic considerations in the interpretation of postmortem carboxyhemoglobin concentrations

The interpretation of postmortem carboxyhemoglobin (COHb) concentrations can have major implications even when COHb is not the direct cause of death. Much litigation may hinge on the length of time an individual was alive during the fire. Therefore, the reliability of an analytical method to measure COHb, ranging from low to sub-lethal levels, is critical to the proper interpretation of results. This study used 40 blood specimens from fire deaths and 15 blood specimens from non-fire deaths to compare COHb levels obtained from a spectrophotometric method (IL 482 CO-Oximeter; CO-Ox) with a reference gas Chromatographic method (GC). Since spectrophotometric methods are influenced by the amount of total hemoglobin (Hb) present in the blood, multiple saline dilutions of specimens were performed to yield Hb as low as lg/dl. For GC COHb concentrations < 5% and Hb ≥ 4g/dl, the average and median ratios of CO-Ox COHb to GC COHb concentrations were 2.8 and 2.6 respectively. These ratios were 8.0 and 6.8 respectively when Hb < 4g/dl. At GC COHb levels ranging from 5 to 40% and Hb ≥ 4g/dl, the average and median ratios were 1.6 and 1.0 respectively. These ratios were correspondingly 2.1 and 1.9 when Hb < 4 g/dl. These data clearly indicate COHb can be influenced by the analytical methods used.

Coagulation abnormalities in patients with Gaucher's disease: Effect of therapy

Various coagulation defects have been associated with Gaucher's disease, including factor IX deficiency and acquired von Willebrand's disease (VWD). We performed repeated coagulation assays in 9 patients with Gaucher's disease over a period of 2 years. The prothrombin time (PT) and fibrinogen levels were normal in 8 of 9 patients, while the partial thromboplastin time (PTT) was abnormal in 5 of 9; all mixing PTT tests showed correction. Factor IX was normal repeatedly in the 7 of 7 patients tested. In contrast, factor XI was decreased in 3 of 9 patients assayed. Anticardiolipin (ACL) IgM was normal in all patients. ACL IgG was highly variable; levels were abnormal at least once in 6 of 8 patients, but were also normal at least once in 7 of 8 patients. Factor VIII was also quite variable: levels were decreased at least once in 4 of 9 patients, and normal at least once in 8 of 9 patients. Von Willebrand factor antigen (VWF Ag) studies were normal in 7 of 8 patients, but VWF activity was decreased at least once in 4 of 8 patients. In some patients, these problems could be overcome by specimen dilution. In ony 1 patient was VWF Ag decreased; this patient had a factor VIIIC level of 13% , and VWF activity of 18.7%. Coagulation assays performed before and after alglucerase administration failed to demonstrate any significant improvement in these assays, and neither was there a consistent improvement over the duration of therapy. We suggest that previously reported decreases in factor IX and VWF may be secondary to the interfering presence of increased cerebroside levels. Caution must be used in the interpretation of clotting assays in the patient with Gaucher's disease.

Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay.

To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR.