Validation of a new automated irradiation system for off-line ECP

Extracorporeal photopheresis (ECP) is a cell therapy originally employed for cutaneous T cell lymphoma and later for GvHD, solid organ rejection and other immunological diseases demonstrating an excellent safety profile. Mononuclear cell (MNCs) apoptosis triggered by UV-A light irradiation in the presence of 8-methoxypsoralene has a key role in priming the cells, ultimately leading to immunomodulation. We report preliminary data about an evaluation of the new automated irradiator device LUMILIGHT (Pelham Crescent srl) for off-line ECP. Fifteen MNCs samples collected by apheresis from 15 adult patients undergoing ECP at our Center were cultured immediately after irradiation along with untreated samples and evaluated at 24, 48 and 72 h timepoints for T cell apoptosis and viability by flow cytometry with Annexin V and Propide Iodidum staining. Post irradiation Hematocrit (HCT), calculated by the device, was compared with that of the automated cell counter. Bacterial contamination was also tested. In irradiated samples after 24–48 and 72 h, the average total apoptosis was 47 %, 70 % and 82 %, respectively, showing a significant difference from untreated samples; residual viable lymphocytes at 72 h were, on average, 18 %. The greatest initiation of apoptosis occurred from 48 h of irradiation onwards. Average early apoptosis of irradiated samples decreased over time (26 %, 17 % and 10 % at 24, 48 and 72 h, respectively). HCT measured by LUMILIGHT was over-estimated, possibly due to the low pre irradiation red blood cell contamination. Bacterial tests resulted negative. Our study showed the LUMILIGHT device to be a valid instrument for MNCs irradiation with good handling and no major technical problems as well as no adverse events in the patients. Our data need to be confirmed in larger studies.     

Validation of a new automated immunoassay for measurement of intact osteocalcin

Bone turnover is assessed indirectly by measurement of biochemical markers of bone turnover. Osteocalcin, a 49-amino-acid protein is a major noncollagenous protein of bone matrix, synthesized by osteoblasts and odontoblasts. Various assays exist for assessment of osteocalcin and concentrations in the same serum or plasma sample may vary enormously. The used antibodies may recognize intact osteocalcin and/or circulating fragments of osteocalcin. We here describe and validate a new automated immunoassay system for measurement of intact osteocalcin (DPC IMMULITE assay) using monoclonal antibodies (mouse) against the C-terminus of osteocalcin (AA 44-49). For detection polyclonal antibodies (goat) directed against the N-terminus (AA 1-17) conjugated with alkaline phosphatase are used. While different laboratory assays show marked clinical discordance, we evaluated our results comparatively to an established IRMA method (Nichols). We observed a highly significant correlation between both assays (r = 0.9352, p < 0.0001, n = 286) for healthy persons and also for patient samples (osteoporosis, diabetes type 1, rheumatoid arthritis). Very low inter- and intraassay covariance as well as highly significant linearity (analytical recovery near 100%) tested by serial dilutions were demonstrated for the DPC IMMULITE intact osteocalcin assay. We conclude that the IMMULITE assay is a useful method for assessment of intact osteocalcin giving valuable results in comparison to an established non-automated assay.     

Validation of a new automated method for analysis of gated-SPECT images

We recently presented a new method for quantification of CArdiac FUnction--denoted CAFU--as the first step in the development of an automated method for integrated interpretation of gated myocardial perfusion single photon emission computed tomography (SPECT) images. The aim of this study was to validate CAFU in the assessment of global and regional function of the left ventricle. Quantitative gated-SPECT (QGS), the most widely used software package for quantification of gated-SPECT images, was used as reference method for the measurements of ejection fraction (EF) and ventricular volumes, and visual analysis by an experienced physician was used as reference method for the measurements of regional wall motion and thickening. Two different groups of consecutive patients referred for myocardial perfusion scintigraphy were studied. Global function was evaluated in 316 patients and regional function in 49 other patients. The studies were performed using a 2-day stress/rest 99 m-Tc-sestamibi protocol. A good correlation was found between EF values from QGS and CAFU (EF CAFU = 0.84 EF QGS + 13, r = 0.94), but CAFU values were on average 4 EF points higher than QGS values. With CAFU the segments with normal thickening according to the physician showed significantly higher thickening values (in all parts of the myocardium) compared to the segments classified as having abnormal thickening. In conclusion, this study demonstrates that CAFU can be used to quantify global and regional function in gated-SPECT images. This is an important step in our development of an automated method for integrated interpretation of gated-SPECT myocardial perfusion scintigraphy studies.     

Validation of an immunoradiometric assay to measure plasma levels of active renin

An immunoradiometric assay (IRMA) for active renin in human plasma was analytically and clinically validated. Analytical validation established 1) precision, 2) recovery, 3) linearity, 4) cross-reactivity, 5) sample stability, and 6) the validity and specificity of the 125I-labeled anti-renin monoclonal in the Diagnostics Pasteur immunoradiometric renin kit. Clinical validation included 1) establishing normal reference range for renin, 2) comparing plasma renin activity (PRA) results to immunoreactive renin levels in subjects on Upjohn research protocols, and 3) comparing the renin responsiveness of sodium replete subjects to that of sodium deplete subjects prior to, during, and after infusion with Upjohn renin inhibitory peptide, ditekiren. This study was undertaken to demonstrate the research validity of an assay tool for the differentiation of enzymatically active renin from inactive renin or a form of prorenin.     

Validation of an automated immunoturbidimetric assay for measurement of plasma D-dimer.

The performance characteristics and diagnostic accuracy of a new rapid automated quantitative immunoturbidimetric D-dimer assay, Auto-Dimer (Biopool, Ume?, Sweden) were evaluated in a population of 135 outpatients with suspected deep-vein thrombosis of a lower limb. Enzyme-linked immunosorbent assay was used as the reference method. The Auto-Dimer assay showed good reproducibility. The correlation between Auto-Dimer and enzyme-linked immunosorbent assay was high (r = 0.91, p < 0.05) and agreement in classifying patients above or below the cut-off was good (kappa coefficient 0.74, 95% CI 0.62-0.86). At a cut-off of 340 microg/l the sensitivity and specificity of Auto-Dimer were high (100% and 61%, respectively). These data show that Auto-Dimer is a reliable screening test for exclusion of deep-vein thrombosis. The assay could be included in prospective patient management studies in order to obtain further information on its clinical usefulness.     

Evaluation of a new fully automated one-step C-peptide chemiluminescence assay (LIAISON C-Peptid)

The determination of C-peptide, a 31 amino acid fragment of proinsulin which is a byproduct of insulin formation, is used as a marker for insulin secretion. Clinically, the determinations are performed to detect autonomous insulinoma, factitious hypoglycemia, and in general to assess the function of beta-cells in patients with diabetes mellitus. The analysis is frequently performed by radioimmunoassays (RIA), which have several disadvantages, for instance the use of radioactivity and time and resource requirements. We performed an evaluation of a new fully automated chemiluminescence assay (LIAISON C-Peptid, Byk-Sangtec) at two clinical sites, in Germany and Italy, with regard to imprecision and clinical relevance of the obtained data, and the correlation with a standard RIA method and another chemiluminescence test. The new assay showed a good correlation with the RIA (r = 0.950) and the chemiluminescence assay (r = 0.967). The intra-assay variability and inter-assay variability was 3.5% and 8.7% in Germany, and 2.4% and 9.6% in Italy. The clinical evaluation of samples derived from 19 oral glucose tolerance tests, 13 insulin suppression tests, and 2 insulin secretion stimulation tests revealed a clinical specificity of 100%, i.e. all cases resulted in the same clinical diagnosis with all tests. With regard to the practical performance of the assays, the new chemiluminescence test, as a single-step fully automated method, offered the advantage of being a non-radioactive, less complex and much faster method than the RIA and also had timely advantages over the comparative chemiluminescence test. In general, the new LIAISON chemiluminescence assay compared favorably with the RIA and comparative chemiluminescence test and offers an attractive alternative for C-peptide analysis.     

Analytical and clinical evaluation of a new heart-type fatty acid-binding protein automated assay.

BACKGROUND: The accurate and rapid recognition of myocardial injury in patients presenting in the emergency department (ED) with chest pain continues to be a clinical challenge. Heart-type fatty acid-binding protein (H-FABP) appears to be one of the best candidates among the new early cardiac markers studied. METHODS: We evaluated the analytical characteristics of a new quantitative and fully automated H-FABP assay (Randox Laboratories Ltd., Crumlin, UK) and compared its clinical performance with respect to the myoglobin (Myo) assay (Dade Behring, Milan, Italy). A precision study was carried out by testing three levels of quality control (QC) material and two in-house pool (P) samples. To test the accuracy of H-FABP determinations in plasma (lithium-heparin) samples, H-FABP concentrations measured in a set of matched sera and plasma samples were compared. A total of 77 non-consecutive patients (51 males and 26 females; 62+/-16 years) who presented to the ED with chest pain suggesting myocardial ischemia were enrolled. The patients were classified into two groups (acute myocardial infarction, n=22; non-acute myocardial infarction, n=55) on the basis of the discharge diagnosis. RESULTS: The between-day imprecision for three levels of control material and serum pool samples was 6.26%-8.04% (range 2.32-44.03 microg/L) and 9.03%-12.63% (range 11.85-65.13 microg/L), respectively. In the serum vs. plasma study, bias was +0.178 (95% CI -0.033 to +0.389). The best cut-off and the associated diagnostic efficacy were 95 microg/L and 89.47% for Myo and 5.09 microg/L and 98.70% for H-FABP, respectively. CONCLUSIONS: H-FABP determination in patients with ischemic symptoms may be a more reliable early indication of cardiac damage than myoglobin.     

Validation of an automated intact N-terminal propeptide of type I procollagen (PINP) assay

Objectives

N-terminal propeptide of type I procollagen assay (PINP) reflects the rate of type I collagen synthesis

Design and methods

Different sera were fractioned by gel filtration and analyzed with intact and total PINP assays. The sizes of the antigens were determined by western blotting. The thermal stability was tested at + 37 °C, + 4 °C and room temperature (RT).

Results

Automated intact PINP assay hardly measured monomeric form. In haemodialysis patients intact and total PINP assays gave significantly different results. The monomeric PINP antigen in serum was larger than the trimeric PINP antigen. PINP were thermally stable at least 7 days at + 4 °C and at RT but the results of both assays were decreased similarly at + 37 °C.

Conclusions

The IDS-iSYS intact PINP assay is precise and sensitive. It seems that monomeric form is not derived from the thermal instability of the trimers but acts as a confounding factor.     

Validation of an automated latex particle–enhanced immunoturbidimetric von Willebrand factor activity assay

Summary. Background: Laboratory diagnosis of von Willebrand disease (VWD) requires accurate measurement of plasma von Willebrand factor (VWF) activity. Objectives: To evaluate laboratory characteristics, diagnostic accuracy and testing utilities of an automated latex particle–enhanced immunoturbidimetric VWF assay (VWF:Lx) based on a monoclonal antibody recognizing the VWF‐platelet glycoprotein (GP) Ib binding domain. Methods: Laboratory characteristics including lower detection limit, linearity, precision, sample stability, and method comparison between VWF:Lx and VWF ristocetin cofactor activity by platelet aggregometry (VWF:RCo) were examined. To assess VWF:Lx diagnostic accuracy, 492 patient plasma samples, including 40 previously characterized VWD patient samples, were tested for VWF antigen (VWF:Ag) and VWF:RCo by either aggregometry or flow cytometry, and VWF:Lx with supplemental VWF multimer analysis when indicated. Based on results of VWF:Ag, VWF:RCo and VWF multimer analysis, and available clinical information, samples were categorized as: normal; VWD types 1, 2A/B, 2M, or severe 1 vs. 2M; or acquired VWF abnormalities (AVWA) due to subtle loss of highest molecular weight multimers. Results: VWF:Lx had excellent laboratory characteristics and linear correlation with VWF:RCo (R² = 0.93). VWF:Lx accurately classified virtually all normal and VWD patient samples. Compared with VWF:RCo, VWF:Lx had superior sensitivity and specificity for distinguishing severe type 1 vs. 2M VWD and identifying AVWA. A proposed screening panel comprising VWF:Ag and VWF:Lx had 100% and 83% sensitivity for detecting VWD and AVWA, respectively. Conclusions: VWF:Lx has excellent laboratory characteristics and diagnostic accuracy compared with VWF:RCo, and can be used as part of an initial VWD screening panel and as a supplementary test.     

Evaluation of a sex hormone-binding globulin automated chemiluminescent assay

Abstract

Background. Sex hormone-binding globulin (SHBG) is the main transport protein of sex steroids. This study evaluated the analytical performance of the recently developed Access SHBG assay (Beckman Coulter) and compared it with other commercial methods for the determination of serum SHBG. Clinical validation was also performed. Methods. Analytical performance including within-run and between-run imprecision was assessed for Access SHBG assay on the automated Beckman UniCel DxI800 analyzer. Linearity was assessed using five dilutions of the serum samples. For methods comparison, SHBG levels were determined also with Immulite 2000 analyzer (Siemens Healthcare) using clinical serum samples (n = 104). For clinical validation 135 specimens from healthy subjects, pregnant women, hypothyroid and hyperthyroid patients were analyzed. Results. Total coefficients of variation were < 5.5%. Linearity test showed > 90% recovery for all samples and for all dilution rates. Comparison analysis (Bland-Altman difference analysis and Passing-Bablock regression) showed an acceptable agreement between selected methods. SHBG values measured by Access SHBG assay in different groups of subjects were in agreement with other clinical evidence. Conclusions. Automated Access SHBG assay appears to be a reliable and easy to perform assay, as necessary for application in routine diagnostics.     

Validation of automated ultrasound-CT registration of vertebrae

Purpose

Image-guided spine surgery requires registration of the patient anatomy and preoperative computed tomography (CT) images. A technique for intraoperative ultrasound image registration to preoperative CT scans was developed and tested. Validation of the ultrasound-CT registration technique was performed using porcine cadavers.

Methods

An ultrasound-CT registration technique was evaluated using 18 thoracic and lumbar vertebrae of 3 porcine cadavers with 10 different sweep patterns for ultrasound acquisition. For each sweep pattern at each vertebra, 100 randomly simulated initial misalignments were introduced. Each misalignment was registered. The resulting registration transformations were compared to gold standard registrations based on implanted fiducials to assess accuracy and robustness of the technique.

Results

The orthogonal-sweep acquisition was found to perform best and yielded a registration accuracy of 1.65 mm across all vertebrae on all porcine cadavers, where 82.5% of the registrations resulted in target registration errors below the 2 mm threshold recommended by a joint report from the experts in the field. In addition, we found that registration accuracy varies by the sweep pattern and vertebral level, but neighboring vertebrae tend to result in statistically similar accuracy. Ultrasound-CT registration took an average of 2.5 min to run, and the total registration time per vertebra (also including time for ultrasound acquisition and reconstruction) is approximately 8 min.

Conclusions

A previously described ultrasound-CT registration technique yields clinically acceptable accuracy and robustness on multiple vertebrae across multiple porcine cadavers. The total registration time is shorter than that of surface point-based manual registration.

     

Analytical and clinical performance of a new,automated assay panel for the diagnosis of antiphospholipid syndrome

Summary. Background: Anticardiolipin (aCL) and anti‐β₂glycoprotein I (aβ₂GPI) antibodies are part of the criteria for antiphospholipid syndrome (APS). Therefore they are widely measured and about 30 commercial kits are available. Objectives: To investigate the analytical and clinical performances of four fully automated, chemiluminescent assays: HemosIL AcuStar aCL IgG, HemosIL AcuStar aCL IgM, HemosIL AcuStar aβ₂GPI IgG, and HemosIL AcuStar aβ₂GPI IgM. Methods: Cut‐off values were assessed by testing 250 blood donors. Total coefficients of variation (CV) were determined with six plasma pools and two controls ranging from 4.3 to 2694 U mL^?1 depending on the assay. Samples from 218 well‐characterized patients and 103 controls were measured in three laboratories to determine inter‐laboratory variation. The results of the 321 samples were compared with three commercial assays (REAADS, INOVA and VARELISA). Results: Cut‐off values were assigned to 20 arbitrary units for all the tests. Total CV ranged from 4.3 to 11.2%. No interference of hemoglobin, bilirubin, triglycerides, heparins and rheumatoid factor was observed. Inter‐laboratory variability was low and no sample changed status. Overall status agreement between HemosIL assays and the comparator kits ranged from 82 to 96%. Sensitivity, specificity, agreement when predicting APS and the odds ratios when predicting a thrombotic or obstetric event gave comparable results between HemosIL AcuStar and the three other assays. Conclusions: Our study demonstrates that the fully automated HemosIL AcuStar aPL assay panel showed similar performances to the three commercial ELISAs commonly used by various laboratories to detect antiphospholipid antibodies.     

Assessment of iron status with a new fully automated assay for transferrin receptor in human serum.

Serum transferrin receptor is considered as a reliable marker of iron status particularly when iron deficiency is associated with chronic disorders such as inflammation, infection or malignancy. The present study aims to illustrate the performances of a new fully automated assay using immunonephelometry. The intra and between-assay precision was found to be very good (CVs < 4%). In healthy subjects there was no statistically significant difference between men and women. With a cut-off of 1.76 mg/l for diagnosing iron deficiency either alone or combined with anemia of chronic diseases, the sensitivity and specificity were respectively 82% and 96.8%. Unlike conventional biochemical and hematological tests, soluble transferrin receptor was unaffected by confounding pathologies. In genetic hemochromatosis the concentration of soluble transferrin receptor was mostly decreased due to the regulatory effect of iron intracellular level. Our study confirms the reliability of soluble transferrin receptor for the assessment of iron status. It is now possible to assay soluble transferrin receptor, ferritin and transferrin on the same apparatus within 15 minutes.