ISLH Laboratory Hematology 7:69-74 © 2001 Carden Jennings Publishing Co., Ltd. Official Publication Performance Evaluation of the ABX Micros CRP: The First Instrument Reporting a Complete Blood Count, 3-Part Leukocyte Differential, and C-Reactive Protein Quantitation BRUCE H. DAVIS, NANCY C. BIGELOW Maine Medical Center Research Institute, Scarborough, Maine significant carryover. Correlation of CRP with the Roche COBAS Integra was excellent with a slope of approximately 0.98, no significant bias, and an r 2 value of >0.99. No interference was seen in the analysis of any of the samples from patients with clinical conditions associated with interfering substances. Samples held at either 25°C or 4°C were stable for 48 hours. The CRP sensitivity was greater than the manufacturer’s claim of 0.2 mg/dL. The ABX Micros CRP demonstrated performance suitable for clinical practice. The ease of use, small sample volume (18µL), and rapid turnaround time (<5 minutes for CBC and CRP) are features that will make this instrument useful in point-ofcare testing areas including both adult and pediatric intensive care units and in office laboratories, particularly those of pediatricians, rheumatologists, and infectious disease specialists. Lab. Hematol. 2001;7:69-74. ABSTRACT ABX Diagnostics has developed the ABX Micros CRP analyzer, the first instrument to provide both a complete blood count (CBC) with a 3-part differential and a quantitative C-reactive protein (CRP) determination. The CRP measurement is based on a whole-blood immunoturbidimetric method using latex beads coated with antiCRP–specific–monoclonal antibody. The evaluation reported here of the linearity, sensitivity, precision, carryover, and clinical performance of the ABX Micros CRP was performed prior to release of the instrument in the United States. Precision of CRP analysis was assessed by replicate determinations of K3EDTA-anticoagulated whole-blood samples with CRP values in the range of <0.4 mg/dL to 10 mg/dL. Linearity was evaluated by assaying pooled normal samples spiked with CRP control material. The stability of CRP was studied in blood samples held at both room temperature and refrigerated conditions over a 72-hour period. Correlation studies were performed by analyzing 100 blood samples on the ABX Micros CRP and comparing the results with those obtained using the Roche COBAS Integra CRP assay, which is based on latex nephelometry. Within-run and between-run precision of both CBC and CRP values had a coefficient of variation (CV) of <10%. Excellent linearity was observed and there was no KEY WORDS: Inflammation · Hematology instrumentation · Point-of-care testing · Laboratory automation · Acute phase reactant INTRODUCTION One of the recent trends in laboratory medicine is the merging of testing in the traditionally separate disciplines of hematology and chemistry. This merger has created the unfortunate term, chematology, and has been driven by the desire to save money. One expensive, still experimental, approach to achieving that goal has been to put hematology and chemistry instruments on the robotic track, forcing Correspondence and reprint requests: Bruce H. Davis, MD, Maine Medical Center Research Institute, 81 Research Drive Scarborough, ME 04074 (e-mail: [email protected]). Received December 1, 2000; accepted December 4, 2000 69 70 B.H. Davis and N.C. Bigelow data generated with this system correlates with that of the FDA-approved Roche COBAS Integra CRP assay, which is based on latex nephelometry. MATERIALS AND METHODS Stability Five samples from healthy patients were divided into 2 aliquots each, 1 of which was placed at room temperature and the other at 4°C. At baseline (immediately after blood collection) and at 24, 48, and 72 hours post-collection, each aliquot was carefully mixed and tested in duplicate using the ABX Micros CRP. Results are shown in Table 2 and are expressed as percentage deviation from the baseline measurement. FIGURE 1. MICROS CRP instrument from ABX DIAGNOSTICS (Montpellier, France) provides a CBC with a 3part leukocyte differential and C-reactive protein values on whole-blood samples. technical specialists to become cross-trained generalists and on-site computer and mechanical repair experts. An alternative approach is to focus on the point-of-care arena and create specialized instruments that are focused on specific clinical needs. One such arena is the outpatient or specialized inpatient setting in which patients with suspected or documented inflammatory diseases such as infections and rheumatoid diseases are evaluated. ABX Diagnostics (Montpellier, France) has developed an instrument that provides both a complete blood count (CBC) with a 3-part leukocyte differential and a quantitative C-reactive protein (CRP) measurement, a combination of testing that is tailored to this setting. The ABX Micros CRP (Figure 1) is a fully automated hematology analyzer identical to the ADVIA 60 (Bayer Diagnostics, Tarrytown, NY), an FDA-approved hematology analyzer that measures various CBC parameters, including 3part differential leukocyte counts, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell counts and distribution width, platelet count, and a mean platelet volume and distribution width. ABX has modified the Micros to create the ABX Micros CRP by integrating another assay using turbidimetric measurements with monoclonal antibody–coated latex beads to measure CRP in whole blood. CRP is an acute-phase reactant that provides diagnostic information similar to the classic erythrocyte sedimentation rate (ESR) and is an indicator of tissue injury or disease activity in inflammatory processes [1-7]. The purpose of this study was to evaluate the performance characteristics of the ABX Micros CRP and to demonstrate the extent to which Precision A blood sample from a healthy subject was used to estimate the within-run precision for the normal range using replicate testing (>10 times). Serial analyses of low-control (LC) and high-control (HC) specimens (ABX Diagnostics) were performed to determine between-run and between-day precision. All normal and abnormal blood specimens were analyzed in duplicate using paired-precision analysis to assess day-to-day intra-assay precision. Linearity ABO- and Rh-matched blood samples with high and low CRP levels were mixed in the following proportions and assayed in duplicate: 100%:0%, 90%:10%, 80%:20%, 65%:35%, 50%:50%, 35%:65%, 20%:80%, 5%:95%, 0%:100%. In addition, known amounts of CRP control material were added to 0.5 mL aliquots of a normal blood sample in increments of 0.2 mg and run in duplicate. Carryover The ABX Micros CRP carryover was evaluated by assaying the LC specimens in triplicate (LC1-3), followed by triplicate testing of the HC specimens (HC1-3), then rerunning the LC in triplicate again (LC4-6). It is assumed that carryover TABLE 1. Carryover for the ABX Micros CRP Assay* Sample Number 1 Number 2 Number 3 Mean ± SD UL/CI Carryover (%)† Low Control High Control Low Control 0.57 0.60 0.59 0.59 ± 0.01 0.603955 0.13 4.46 4.68 4.65 4.60 ± 0.10 0.61 0.57 0.59 0.59 ± 0.02 *SD indicates standard deviation; UL, upper limit of 95% confidence interval; CI, confidence interval. †(0.61 – 0.603955) ÷ 4.60 100. Performance Evaluation of the ABX Micros CRP 71 by medical chart review using the presence of infection or other inflammatory disease as the clinical indicator. Investigation of Clinical Conditions Associated With Interfering Substances Samples were obtained from patients with conditions known to be associated with hyperbilirubinemia, hyperhemoglobinemia, hypergammaglobulinemia, and hyperlipidemia, and evaluated with the ABX Micros CRP; the results were evaluated using paired precision and clinical correlation. This evaluation is not meant to be an exhaustive study, but does provide information about the testing of patients with clinical conditions associated with substances known to interfere with other CRP methods. Statistical Analysis Linearity and procedural correlation data were analyzed using linear regression analysis, and comparative data on patient and control specimens were evaluated using analysis of variance methods when appropriate. All statistical procedures were performed using Statistica for Windows (StatSoft, Tulsa, OK) and Excel (Microsoft, Redmond, WA). RESULTS FIGURE 2. Linearity of C-reactive protein (CRP) measurements on ABX Micros CRP. has not occurred if the LC4 value lies within the 95% confidence interval (CI) defined by the LC1-3 replicates. If the LC4 value was greater than the upper limit of the 95% CI (UL), then the following formula was used to calculate carryover: (LC4 – UL/CI) ÷ (HC1-3 ÷ 3), where LC4 is the first low control replicate following the high control testing, UL/CI is the upper limit of the 95% CI, and HC1-3 ÷ 3 is the average of the triplicate high control values. Procedural Correlation Twenty-five samples from healthy patients and 75 samples from patients with diseases in which CRP levels are likely to be elevated were assayed in duplicate with the ABX Micros CRP. These values were compared with the value obtained with the Roche COBAS Integra CRP assay according to institution-approved guidelines and Institutional Review Board (IRB) approval. All samples were stored at room temperature and tested within 12 hours of collection, except for the stability evaluation phase of the study (described above). When samples were assayed on multiple instruments, not more than 1 hour elapsed between the beginning of testing with the first instrument and the beginning of testing with the second. Sensitivity and specificity for clinical utility was determined Assay Precision A blood specimen from a healthy subject was assayed 10 times in succession and the mean level of CRP was found to be 0.39 ± 0.02 mg/dL, with a coefficient of variation (CV) of 6.0%. The within-run precision derived from paired duplicate analyses of CRP on all patient samples and healthy donors was a mean CV of 17.6% ± 37.3% (range 0% to 200%, N = 100). When those samples that had CRP levels below the linearity limit determined by this study (0.05 mg/dL) were excluded, low imprecision was demonstrated with a mean CV of 6.5% ± 11.7% (range 0% to 72.7%, n = 86). The between-run precision estimates were obtained by assaying the LC and HC specimens 4 times at approximately 2-hour intervals every day for 17 days. CVs ranged from 1.4% to 11.6% (mean, 4.1% ± 2.8%) for the LC with an overall mean of 0.56 ± 0.03 mg/dL, and 0.5% to 3.1% (mean, 1.8% ± 0.7%) for the HC with an overall mean of 4.25 ± 0.14 mg/dL. Between-day precision studies for the LC and HC specimens produced CVs of 5.4% and 3.3%, respectively. Assay Linearity The linearity of the ABX Micros CRP was initially assessed using duplicate assays of dilutions of ABO- and Rh-matched blood samples with high and low CRP levels mixed in various proportions and showed a loss of linearity in undiluted specimens with CRP levels between 7 mg/dL and 10 mg/dL (Figure 2). Specimens with CRP values above 10 mg/dL were diluted prior to analysis according to the product manual and showed good linearity. Linearity between the normal range 72 B.H. Davis and N.C. Bigelow for the samples with the lowest CRP levels, with increases of 5% to 10% at 48 hours in samples stored at both ambient and low temperatures, in contrast to decreases of 1% to 3% for those with elevated CRP levels (Table 2). At 72 hours post-collection, deviations from baseline were more apparent, but were still less than 11% for both normal and abnormal samples irrespective of storage temperature. FIGURE 3. Intermethod correlation between ABX Micros CRP and Roche Integra CRP (latex) of 100 samples. CRP indicates C-reactive protein. and approximately 7 mg/dL were studied further by adding increasing volumes of 5 µL to 230 µL of a sample with a high CRP level (21.3 mg/dL) to 0.5 mL aliquots of a normal specimen and showed excellent linearity (Figure 2). Procedural Correlation Samples were collected from 25 healthy subjects and from 75 subjects with known disease (44% female), of which 42 were older than 4 years (mean age, 35.3 years) and 58 were younger than 1 year. Samples were assayed in duplicate using the ABX Micros CRP and Roche COBAS Integra. The correlation coefficient between the 2 methods was 0.975 for all samples tested (Figure 3), with the ABX Micros CRP method exhibiting a greater deviation from regression in the 7 to 10 mg/dL range, in which whole-blood samples were not diluted, in accordance with the manufacturer’s original instructions. When only the 25 normal samples were tested, the 2 methods were almost perfectly correlated (r 2 = 0.996, Figure 4). Investigation of Potentially Interfering Substances During the course of the evaluation, 2 samples were found to have potentially interfering substances for a CRP assay. One sample with hyperhemoglobinemia secondary to an active malaria infestation gave clinically relevant duplicate values of 6.61 mg/dL and 6.92 mg/dL. A second sample was from a patient with hyperlipidemia (cholesterol, >1000 mg/dL) and a hypergammaglobulinemia (>3 g/dL), but no clinical evidence of active inflammation. The ABX Micros CRP gave appropriately normal CRP values for the sample with both hyperlipidemia and hypergammaglobulinemia (0.18 mg/dL and 0.19 mg/dL). Both samples demonstrated a CV with paired precision comparable to samples without these potentially compounding substances. These findings show that although users should be aware of the potential for interfering substances to cause erroneous results, the presence of hyperlipidemia, hypergammaglobulinemia, and hyperhemoglobinemia are not absolute contraindications for using the ABX Micros CRP. DISCUSSION The data presented in this study demonstrate that withinrun, between-run, and between-day precision for the ABX Micros CRP assay is quite acceptable, with the best precision Sample Carryover As shown in Table 1, the CRP value for the first LC sample following triplicate testing of the 3 HC samples was slightly above the upper limit of the 95% CI for the 3 low level controls initially tested (0.610 versus 0.604) resulting in a carryover estimate of 0.13%. Sample Stability Five normal and 5 abnormal blood specimens were tested immediately after collection and following storage at both ambient (approximately 25°C) and low temperatures (approximately 5°C) at 24, 48, and 72 hours. As expected, the percentage deviation from baseline was somewhat greater FIGURE 4. Intermethod correlation between ABX Micros CRP and Roche Integra CRP (latex) of 25 healthy donor samples show excellent agreement. CRP indicates C-reactive protein. Performance Evaluation of the ABX Micros CRP TABLE 2. Stability of Normal and Abnormal Samples Assayed on ABX Micros CRP (n = 10)* Time (h) Variable Normal sample RT Mean Deviation Percentage deviation Abnormal sample RT Mean Deviation Percentage deviation Normal sample at 5°C Mean Deviation Percentage deviation Abnormal sample at 5°C Mean Deviation Percentage deviation 0 24 48 72 0.131 0.138 0.007 5.3 0.139 0.008 6.1 0.142 0.011 8.4 4.054 3.981 0.073 –1.8 4.002 0.052 –1.3 3.730 0.324 –8.0 0.131 0.144 0.013 9.9 0.139 0.008 6.1 0.117 0.014 –10.7 4.054 3.935 0.119 –2.9 3.995 0.059 –1.5 3.816 0.238 –5.9 *Mean CRP values expressed as mg/dL. Deviation is the deviation of the sample mean from baseline. Percentage deviation is the mean deviation expressed as a percentage. RT indicates room temperature. obtained for the low control (0.56 mg/dL) tested 4 times per day for 17 days (CV 1.4 to 11.6%). This CRP assay is linear for anticoagulated blood without dilution up to a CRP value of 7 mg/dL. Although the ABX Micros CRP was originally designed for CRP concentrations up to 10 mg/dL in undiluted blood, this study demonstrated a slight loss of linearity in the range of 7 to 10 mg/dL. Thus, our findings indicate that samples with CRP values > 7 mg/dL on initial analysis with the ABX Micros CRP should be diluted and reanalyzed to obtain accurate results. When this protocol was followed, we observed results from the ABX Micros CRP assay to be highly correlated with those of the Roche COBAS Integra CRP. Carryover was found to be negligible and samples stored for up to 48 hours at either room temperature or under refrigeration were shown to be stable for the ABX Micros CRP assay. Finally, we observed no interference in the CRP measurements in a limited sampling of patients with rheumatoid factor, hyperhemoglobinemia, hyperlipidemia, or hypergammaglobulinemia, conditions associated with interference problems with other CRP methods [8]. Thus our findings support the claim that the ABX Micros CRP provides reproducible, linear measurement of CRP in the range of 0.05 to 7.0 mg/dL using undiluted, EDTAanticoagulated whole-blood samples and >7.0 mg/dL using diluted samples. Furthermore, the ease of operation, small sample volume requirement, and reporting of both CBC and CRP results in less than 5 minutes are features that make this a suitable instrument for point-of-care testing. 73 An elevated CRP level is indicative of inflammatory disease activity and is preferred to the ESR as a disease marker. But the simpler nature of the ESR procedure means it is more available and that results are reported out more frequently than the immunologic CRP method. If it is available, the CRP assay is typically run in batches, and results are reported once daily (or even less frequently), precluding its use in realtime patient management. Yet it is clear that the CRP has clinical utility in the management of neonatal sepsis [9-14], suspected appendicitis in the acute care setting [1,2,15], and more recently, in cardiac injury and stroke [16-20]. Such clinical conditions typically involve patients in either intensive care settings or emergency care, where the use of point-of-care testing has been found to improve patient care and reduce health care costs. It is clear that instruments such as the ABX Micros CRP should provide a solution to a real clinical need. The CRP measurement with the ABX Micros CRP was shown to have a lower limit of linearity down to 0.05 mg/dL, which is not sufficient to be defined as a high-sensitivity CRP method as employed in many of the studies demonstrating CRP as a risk factor in cardiovascular diseases. However, the ABX Micros CRP appears to be sufficiently sensitive to identify not only the elevated CRP values that occur in neonatal sepsis, but also those in patients with subtler abnormalities that have been found in studies using high-sensitivity CRP methods which define prognostic risk at threshold values in the range of 0.2 to 0.4 mg/dL [16-18,20]. Therefore, aside from the intended use of the ABX Micros CRP in the physician office setting of rheumatologist, pediatrician, or internist, this instrument may find utility in point-of-care settings such as neonatal intensive care units, emergency care units, or outpatient cardiac care settings. The clinical use of the CRP is generally well defined as a marker in the patient with infection or sepsis, although its use may be challenged by newer diagnostic tests of responses to infections, such as procalcitonin, interleukin-6, and neutrophil CD64 measurements [11,21-27]. But at least for now, the advantage of combining CBC and CRP determinations on a single instrument platform, such as the ABX Micros CRP, should be a valuable diagnostic tool for reliable point-of-care testing of individuals with proven or suspected inflammatory diseases. The combination of CBC parameters with other laboratory determinations may be a new trend in laboratory hematology. ACKNOWLEDGMENTS This work was supported by ABX Diagnostics, Irvine, CA, and was performed as part of the clinical evaluation for the FDA 510(k) submission of the ABX Micros CRP. Dr. Davis and Ms. Bigelow are paid consultants to ABX Diagnostics, Montpellier, France. This work was presented in part at the annual ISLH meeting in Banff, Canada, in April 2000 and the American 74 B.H. Davis and N.C. Bigelow Society of Hematology meeting in San Francisco, California, in December 2000. REFERENCES 1. Clyne B, Olshaker J. The C-reactive protein. J Emerg Med. 1999;17:1019-1025. 2. Gronroos J, Gronroos P. 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