Biological variation of hematology tests based on the 1999-2002 National Health and Nutrition Examination Survey.

OBJECTIVE: Biological variation consists of between-person (BP) and within-person (WP) variation. Estimates of WP coefficients of variation (CVw) and BP coefficients of variation (CVg) for hematology laboratory tests were estimated from the 1999-2002 National Health and Nutrition Examination Survey (NHANES). METHODS: NHANES is a survey of the civilian noninstitutionalized U.S. population that uses a stratified, multistage probability design. Between- and within-person variations were estimated for 18 hematology tests. For WP variation, a nonrandom sample was obtained with a median of 17 days between two test measurements. Between-person variation was estimated from the WP sample and additional participants were matched for age group, gender, and race and ethnicity to the WP sample. RESULTS: The BP and WP variations were estimated on as many as 2496 and 852 sample participants, respectively. Mean corpuscular hemoglobin concentration had the lowest CVg (2.25% for men and 2.40% for women), and mean corpuscular volume had the lowest CVw (0.31% for men and 0.37% for women). The index of individuality (CVw/CVg) ranged from 0.06 for mean corpuscular volume for men and women to 0.62 for segmented neutrophil number for men, and 0.55 for segmented neutrophil percent for women. Women had higher CVw compared with men for hematocrit, hemoglobin, mean corpuscular volume, red blood cell count, and red blood cell distribution width. Several hematology tests' CVw also differed by age group, including mean corpuscular volume; eosinophil, lymphocyte and segmented neutrophil percent; monocyte and segmented neutrophil number; white blood cell count; and red blood cell distribution width.

Multiplex RT Q-PCR assay for simultaneous quantification of three viruses used for validation of virus clearance by biopharmaceutical production.

Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log(10) clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6log(10) dynamic range with a limit of detection (LOD) of approximately 1 genome copy/microL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies.

Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion.

Methods that allow the accurate and reliable detection of ultra-low molecular levels of proteins using techniques such as quantitative immuno-PCR (qIPCR) have demonstrated numerous technical difficulties. Protein detection methods lose specificity when the protein target is immersed within a matrix of thousands of molecules having wide ranges of concentrations. In addition, sensitivities are limited because of high background signals. To validate the performance of an immunomagnetic bead qIPCR method designed to remove the 'matrix' effect for HIV-1 p24 antigen detection, regression analyses were performed using samples from patients infected with HIV-1 diluted to approximately 100-1000, 10-100, 1-10, and 0.1-1.0 HIV-1 p24 Ag molecules/reaction. The number of HIV-1 p24 Ag molecules was derived from quantified HIV-1 RNA determinations. The modified immunomagnetic qIPCR bead assay demonstrated a limit of quantification of 10-100 HIV-1 p24 molecules per reaction, with an average correlation coefficient of 0.948+/-0.028 over a 4-log dynamic range. This method detects less than one HIV-1 virion (a limit of detection unreported previously for HIV-1), and thus, has the potential to identify HIV-1 infection and monitor the dynamics of the disease course earlier than nucleic acid methods. The immunomagnetic qIPCR bead assay is a simple and inexpensive method for ultra-low protein detection of infectious agents, toxins, and cancer markers at a level unrecognized previously using any enzymatic or molecular method.

Immuno-polymerase chain reaction as a unique molecular tool for detection of infectious agents.

Theoretically, the immuno-polymerase chain reaction (IPCR) method is the most sensitive technique for the detection of proteins and gains its uniqueness through the exponential amplification of a signal-generating nucleic acid intermediate attached to a protein target. This method is similar to PCR for the detection of nucleic acid targets, and has now been shown to offer the ability to detect infectious agents where nucleic acids are not present. Although the technical development of IPCR has taken a torturous path down a winding avenue of encouraging advances, the method remains rarely utilized by the scientific community and completely unused as a clinical diagnostic test approved by a national accrediting agency. Although the use of real-time instrumentation has enhanced the performance of IPCR to higher levels of statistical accuracy and reproducibility, as compared with the conventional method, its application remains limited by the high standards required for clinical diagnoses of infectious diseases. This review summarizes experimental data published to date describing the utilization of the IPCR method as it relates to the detection and diagnosis of human infectious disease, and examines the progressive development of this method, as well as the factors impeding its universal application as a clinical diagnostic tool. With further standardization and validation, the IPCR method has the potential to become the most analytically sensitive method available for the detection of target proteins of infectious diseases.

Applications of real-time immuno-polymerase chain reaction (rt-IPCR) for the rapid diagnoses of viral antigens and pathologic proteins.

One of the major challenges of performing the IPCR has been to establish a robust, sensitive, and specific method which is easily adapted and highly standardized for routine use in a clinical laboratory. Presently, the performance of IPCR typically involves elaborate and multiple, time-consuming steps prone to high variation in reagents and technical application. Further advances in the technology and instrumentation used for the signal detection of IPCR has resulted in the development of real-time IPCR (rt-IPCR). Rt-IPCR is still relatively undeveloped in comparison to the use of both real-time PCR and IPCR as evidenced by the low number (eight citations) of publications in the scientific literature. However, increased use of rt-IPCR has shown that the method displays improved statistical validation of accuracy over IPCR. Inter-assay error is typically 5-10% vs 15-20% for IPCR. The primary advantage of using rt-IPCR in place of IPCR is the immediate interpretation of positive data (quantification of proteins) as the PCR reaction proceeds. This aspect is key to real-time diagnosis and has great importance for specific emergency situations (i.e., biological and environmental contaminations of toxins in biothreat situations), as well as cases where specific tumor/viral antigens and pathologic proteins may be present in body tissues in extremely low concentrations and rapid, early diagnosis is important for immediate palliative treatment. This review summarizes all of the experimental data published to date utilizing the rt-IPCR method for various analytes (vascular endothelial growth factor, mumps Ag, rViscumin, various IgG, gliadin, HIV-1 p24 Ag, Rotavirus VP6, pathologic and recombinant prion, and prostate specific Ag) and describes the molecular scaffold formats, solid formats, instrument detection systems, and probes/primers or fluorescent dyes used in these assays. With further standardization and validation, rt-IPCR has the potential to become the most analytically sensitive method available for the detection of proteins.

Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR.

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.

Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen.

Presently, the assay that attains maximal sensitivity and dynamic range of HIV-1 viral copy number (50 copies per milliliter) is nucleic acid amplification of HIV RNA in plasma. Enzyme-linked immunosorbent assay (ELISA) methods for quantification of HIV-1 p24 antigen have been relatively insensitive. In this report, we show data that indicate real-time immuno-polymerase chain reaction (IPCR), a combination of the ELISA and PCR techniques, is more sensitive for HIV-1 p24 antigen detection than other currently reported methods. When derived from an IPCR standard curve, a dose response was observed from patient samples with known viral loads diluted within a 3-log range (1.68-6,514 viral RNA copies per milliliter). IPCR detected 42% (22/52) of patient samples that had fewer than 50 viral RNA copies per milliliter by reverse transcriptase-PCR. IPCR shows the potential to become the most analytically sensitive test available for determination of HIV-1 viral load by the detection of HIV-1 p24 antigen.

Real-time PCR provides improved detection and titer determination of bacteriophage.

The plaque assay is the traditional method for the quantification of bacteriophage, particularly for lambda cloning vectors. Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this "gold standard" assay may be inaccurate due to the subjective interpretation of the results. The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations. QPCR, with an improved primer set specific for lambda phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification. Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample. When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs. one log, respectively). Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences.