Internal quality control of a turbidimetric immunoassay for canine serum C-reactive protein based on pooled patient samples

BACKGROUND: Optimized internal quality control (IQC) procedures are important to ensure that only results without medically important errors are used for medical decision-making and to ensure that unnecessary rejection of valid analytical runs is avoided. Additionally, estimates of the analytical performance can be derived from IQC data. In the absence of available species-specific standards of a compound, the use of alternative control materials based on patient samples is a possibility, although investigations on the suitability of this approach are needed. OBJECTIVES: The objective of the study was to plan and implement a simple IQC procedure with control material based on pooled canine serum samples for a turbidimetric immunoassay (TIA) for the determination of human C-reactive protein (CRP) that recently was validated for the determination of canine serum CRP, and to assess the clinical analytical performance of the assay. METHODS: Proposed guidelines for the planning and implementation of IQC procedures were followed by using 2 control materials. Quality requirements of the assay were defined objectively by means of available data on biological variation, and goals for IQC performance were defined according to recommendations (probability of error detection [P(ed)] >.90 and of false rejection [P(fr)] <.05). Analytical performance was evaluated by means of medical decision charts. RESULTS: The control rule of 1(2.5s) (ie, rejection of the analytical run if at least 1 of 2 control materials deviates from the mean by more than 2.5 SD) fulfilled the criteria of predicted IQC performance (P(ed) =.94-1.00, P(fr) =.03). The IQC method was successfully implemented over a 14-week period. The observed coefficient of variation in the period of monitoring was 3.8% (low) and 2.9% (high), which equals excellent analytical performance. CONCLUSIONS: It was possible to plan and implement a simple IQC procedure for the CRP-TIA with control materials based on canine serum samples that fulfilled the criteria of high error detection and low false rejection of valid analytical runs. The assay showed excellent long-term analytical performance over a 14-week period.     

Evaluation of a point-of-care test for canine C-reactive protein

Background: In veterinary medicine, there is increasing interest in measuring C‐reactive protein (CRP) as a tool for diagnosis and monitoring of inflammatory diseases. Reported CRP concentrations for healthy dogs have ranged from 0 to 8.9 mg/L. Objectives: The aims of this study were to evaluate a canine‐specific point‐of‐care (POC) lateral flow immunoassay for qualitative CRP measurement in healthy and diseased dogs and to compare results with those obtained by a quantitative ELISA. Methods: Blood samples from 73 client‐owned dogs were available for testing: 16 healthy dogs and 57 dogs with a variety of infectious, inflammatory, or neoplastic diseases. CRP was measured in heparinized whole blood samples and serum with the TECOmedical Dog CRP‐visual POC test. A red line develops in the POC device if CRP is ≥5 mg/L, and results are scored as negative or positive. An ELISA validated previously for canine serum was used as the reference method. Results: For all dogs, serum CRP concentrations measured by the ELISA ranged from 0.1 to ≥350 mg/L (median=38 mg/L). Percentages of the CRP POC test results that agreed with the ELISA results were 98.6% for whole blood and 97.3% for serum samples. For serum samples, sensitivity of the POC test was 96.4% and specificity was 81.3%. For whole blood, sensitivity was 94.7% and specificity was 93.8%. Conclusions: The POC test had very good agreement with the ELISA test and had high sensitivity and specificity; therefore, it can be used as a qualitative test to screen for increases in CRP concentrations.     

Evaluation of a commercially available human serum amyloid A (SAA) turbidometric immunoassay for determination of equine SAA concentrations

The aim of the present study was to evaluate whether equine serum amyloid A (SAA) concentrations could be measured reliably with a turbidometric immunoassay (TIA) developed for use with human serum. Intra- and inter-assay imprecision were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by linearity under dilution. The assay was subsequently used for measuring SAA concentrations in clinically healthy horses, horses with inflammatory diseases, horses with non-inflammatory diseases, and in horses before and after castration. In pools with low, intermediate and high SAA concentrations, the intra-assay imprecisions were 24.4%, 1.6% and 2.1%, and the inter-assay imprecisions were 33.2%, 4.6% and 6.5%. Slight signs of inaccuracy were observed, but these inaccuracies were negligible when considering the large dynamic range of the SAA response. The assay was able to detect the expected difference in SAA levels in different groups of horses. It was also able to demonstrate the expected dynamic changes in SAA after castration. In conclusion, equine SAA concentrations can be measured reliably using the TIA designed for human SAA.     

Preliminary evaluation of a particle-enhanced turbidimetric immunoassay (PETIA) for the determination of serum cystatin C-like immunoreactivity in dogs

Abstract: Serum cystatin C often is used in humans as a rapid and more sensitive marker than serum creatinine for glomerular filtration rate. The purpose of the present study was to evaluate whether cystatin C-like immunoreactivity (CLI) could be measured reliably in canine serum and to investigate whether dogs with clinical renal insufficiency had higher CLI levels than did clinically healthy dogs and dogs with nonrenal diseases. A commercially available particle-enhanced turbidimetric immunoassay (PETIA) for human serum cystatin C was used to measure canine serum CLI in a linear and proportional manner, with a mean recovery of 104%± 7.5% and coefficients of variation of 1.7 to 9.6%. The assay was then applied to serum samples from 17 clinically healthy dogs, 12 dogs with nonrenal diseases, and 8 dogs with renal insufficiency. Serum CLI was significantly higher in dogs with renal insufficiency (median serum CLI = 5.01 mg/L) than in clinically healthy dogs and dogs with nonrenal diseases (median serum CLI = 1.06 mg/L and 1.62 mg/L, respectively). Thus, canine serum CLI could be reliably measured using a commercially available PETIA designed for human serum cystatin C, and dogs with clinical renal insufficiency had, as expected, significantly higher serum CLI levels.     

Canine C-reactive protein (CRP) does not share common antigenicity with human CRP

Differences in antigenicity between human and canine C-reactive proteins were investigated by Western blotting analysis. It was confirmed that several commercial anti-human CRP sera reacted with canine CRP. However, 34 anti-canine CRP sera prepared by immunization of rabbits and goats with canine CRP all reacted with canine CRP but not with human CRP in either immunoelectrophoresis or Western blotting.Immunization with human CRP produced a cross-reacting antibody that reacted with canine CRP. Conversely, immunization with canine CRP did not produce a cross-reacting antibody that reacted with human CRP. These findings may be interpreted as showing that, while canine and human CRPs do not share common antigenicity, they do contain structurally similar antigenic determinants.Abbreviations CRP C-reactive protein - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Evaluation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of C-reactive protein in canine serum

The purpose of this study was to evaluate a commercially available enzyme-linked immunosorbent assay for determination of canine serum C-reactive protein (CRP). The concentration of CRP could be determined accurately and the intra- and inter-assay coefficients of variation were in the range of 6.9-10.1 and 7.5-29.0%, respectively. This level of imprecision between runs is usually considered unacceptable for diagnostic purposes, but the overall results indicated that the assay was useful in differentiating dogs suffering from infections, from dogs suffering from various other diseases (neoplastic diseases, endocrine/metabolic disorders), and healthy dogs. The assay was also able to detect dynamic changes of CRP during development and after cessation of spontaneous occurring inflammatory stimuli in two clinical cases.     

Evaluation of an enzymatic immunoassay for free thyroxine determination in canine serum

Free thyroxine (FT4) and cholesterol were measured in 400 dogs with either suspected hypothyroidism or dermatological signs such that hypothyroidism needed to be ruled out. Hypothyroidism was diagnosed in 68 dogs from the history, physical examination and stated lower reference limit (<7 pmol/L) for FT4 in euthryoid dogs. Dogs with FT4 concentrations in the range 6–9 pmol/L were finally categorized as hypo- or euthyroid either on the basis of retesting after 2 months or on their clinical response to thyroid replacement therapy over at least 2 months.The enzyme immunoassay evaluated in this paper is considered to be of clinical value and offers many advantages compared with radioimmunoassays.Abbreviations FT4 free thyroxine fraction - MEIA microparticle enzyme immunoassay - RIA radioimmunoassays - T4 thyroxine - TBG thyroxine-binding globulin     

Serum concentration of some acute phase proteins in naturally occurring canine babesiosis: a preliminary study

BACKGROUND: Serum concentrations of acute phase proteins can provide valuable diagnostic information in the detection, prognosis, or monitoring of disease. Information available on the acute phase response in naturally occurring canine babesiosis is limited. OBJECTIVE: The purpose of this investigation was to retrospectively evaluate serum concentrations of haptoglobin, C-reactive protein, and ceruloplasmin in dogs naturally infected with Babesia canis. METHODS: Haptoglobin, C-reactive protein, and ceruloplasmin concentrations were measured in serum samples from dogs with uncomplicated (n = 6) and complicated (n = 1) babesiosis and compared with 6 healthy dogs. RESULTS: Serum C-reactive protein and ceruloplasmin concentrations were significantly higher in dogs with babesiosis; however, serum haptoglobin concentration was significantly lower compared with control dogs (P <.01). CONCLUSIONS: Results of this study suggest that acute phase protein concentrations could be beneficial in the diagnosis and determination of the severity of babesiosis in dogs.     

Analytical and Clinical Validation of a Time-resolved Immunofluorometric Assay (TR-IFMA) for Canine C-reactive Protein in Serum

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of C-reactive protein (CRP) in canine serum. CRP was isolated from canine acute-phase serum by affinity chromatography on agarose coupled with phosphorylethanolamine. This isolated dog CRP was used as standard to calibrate the assay. Intra-assay and inter-assay coefficients of variation were in the ranges 5.3–7.1% and 4.8–13.3%, respectively. Accuracy, evaluated by adding 2 and 10 μg/ml of CRP to serum samples, provided recoveries of 99.9% and 106.8%. High correlation was found between CRP measurements by TR-IFMA and a by commercial enzyme-linked immunosorbent assay (R² = 0.98). The limit of detection for the TR-IFMA method was 0.000067 μg/ml and the measurement of CRP in serial dilutions of acute-phase dog sera generated curves with the same slope as the one constructed with purified CRP. The TR-IFMA provides a precise, accurate and highly sensitive assay for CRP determination in dog samples. CRP levels in dogs with different diseases ranged between 10.2 and 210.7 μg/ml and were significantly higher than those observed in healthy dogs (< 7.1 μmg/ml).     

Serum C-reactive protein concentration as an indicator of remission status in dogs with multicentric lymphoma

BACKGROUND: The acute-phase protein C-reactive protein (CRP) is used as a diagnostic and prognostic marker in humans with various neoplasias, including non-Hodgkin's lymphoma. OBJECTIVE: To evaluate if CRP could be used to detect different remission states in dogs with lymphoma. ANIMALS: Twenty-two dogs with untreated multicentric lymphoma. METHODS: Prospective observational study. Blood samples were collected at the time of diagnosis, before each chemotherapy session, and at follow-up visits, resulting in 287 serum samples. RESULTS: Before therapy, a statistically significant majority of the dogs (P = .0019) had CRP concentrations above the reference range (68%, 15/22). After achieving complete remission 90% (18/20) of the dogs had CRP concentrations within the reference range, and the difference in values before and after treatment was statistically significant (P < .001). CRP concentrations of dogs in complete remission (median, 1.91; range, 0.2-103) were significantly different (P = .031) from those of dogs with partial remission (median, 2.48; range, 0-89), stable disease (median, 1.77; range, 1.03-42.65), or progressive disease (median, 8.7; range, 0-82.5). There was profound variation of CRP measurements within each dog. CONCLUSIONS: CRP is useful in determining complete remission status after treatment with cytotoxic drugs. However, the individual variation between dogs means CRP concentration is not sufficiently different in other remission states to permit its use in monitoring progression of the disease. Greater reliability in determining remission status might be achieved by combining CRP concentration with other serum markers.     

Validation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of cortisol in canine plasma samples

Samples were obtained from clinically normal dogs before and after ACTH stimulation and dexamethasone suppression tests. The test kit Enzymun-Test (Boehringer Mannheim) for determining cortisol concentrations in human plasma was used in connection with the analyser system Enzymun-Test (Boehringer Mannheim) System ES300 following the manufacturer's instructions. The intra-assay and inter-assay coefficients of variation were 1.28% and 5.64%, respectively. The mean recovery when assaying samples with a cortisol content of more than 100 nmol/L was 95.41%, but this percentage decreased in samples with lower cortisol levels. The sensitivity of the assay was 2.76 nmol/L. The results of the ACTH stimulation and dexamethasone suppression tests were similar to those published previously. The ELISA method evaluated allows a precise and sensitive determination of cortisol concentrations in canine plasma samples. The major drawback observed was the loss of accuracy at low cortisol concentrations. Since the assay tends then to report lower cortisol concentrations, the generally accepted concentration of 40 nmol/L may not be suitable as the cutoff value in dexamethasone suppression tests.     

Physiological levels of C-reactive protein in normal canine sera

This study was undertaken to investigate whether the level of C-reactive protein (CRP) in the serum of dogs undergoes physiological variation, using 10 normal Beagle dogs (5 males and 5 females), 1–2 years old, maintained in a healthy condition in a controlled environment. The CRP concentration in the sera collected seven times each day at intervals of approximately 3 h ranged from 0.8 to 16.4 µg/ml (mean 5.06±3.60) in one experiment and from 0.8 to 14.0 µg/ml (mean 4.50±2.80) in a second experiment. On examining the 24-h variations in the concentration of CRP in serum, neither consistent changes nor a definite pattern of circadian rhythm was detected. During 28 days observation, only very slight changes, which seemed attributable to analytical errors, were seen in any of the dogs, except one. The concentration of CRP in the serum during the 28 days ranged from 0.8 to 22.6 µg/ml (mean 3.65±1.40). The concentrations underwent no significant variations in individual dogs, but significant differences were found between the dogs (p<0.01).     

Analytical validation of commercially available methods for acute phase proteins quantification in pigs

The aim of this study was to validate commercially available methods for porcine haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and major acute phase protein (Pig-MAP) determinations. Intra and inter assay coefficients of variation (CVs) were lower than 20% in all cases with exception of inter assay CVs for CRP and Pig-MAP assays with samples of low acute phase proteins concentration, and for SAA assay at any acute phase proteins concentration. All methods showed good linearity and detection limits were low enough to detect APPs levels in healthy animals. Hp and SAA were very affected by haemolysis. Lipaemia influenced mainly on SAA determination. Over 15-fold increase was observed in CRP and SAA concentrations after artificially induced inflammation by a single subcutaneous dose of turpentine, whereas Hp and Pig-MAP increased less than 5-fold.     

Evaluation and comparison of two immunoturbidimetric assays for the heterologous determination of porcine serum C-reactive protein

The objective was first to evaluate the performance (intra- and inter-assay imprecision, inaccuracy, detection limit and overlap performance) of two different commercially available human C-reactive protein (CRP) immunoturbidimetric assays (TIA) applied to porcine serum CRP; and secondly to investigate inter-laboratory discrepancies and the influence of calibration materials when comparing results. The intra-laboratory performance seemed acceptable for clinical purposes for both assays with excellent overlap performance, acceptable detection limits, no significant inaccuracies and low imprecision. Although a significant discrepancy in the measured CRP levels was observed when comparing results, the methods agreed significantly on diagnostic classification, suggesting a low clinical significance. Both assays performed reliably for the heterologous determination of porcine serum CRP for clinical purposes. The results emphasise the need to establish species-specific standards to decrease inter-laboratory discrepancies.     

Quantitative capillary reversed passive latex agglutination test for C-reactive protein (CRP) in the dog

A capillary reversed passive latex agglutination test (capillary RPLA) was developed which allows quantification of serum C-reactive protein (CRP) within approximately 15 min. The logarithmic regression line (calibration curve) obtained after measuring each CRP concentration three times in twofold dilutions of a standard canine serum containing 222 g/ml of CRP was y=6.394+0.030x (r=0.995). Capillary RPLA permitted quantification of CRP in the range 6.9–222 g/ml. The coefficients of variation ranged from 10.28% to 12.40%. The recovery rates (percentage recovery) of CRP by capillary RPLA were within the range 87% to 106%. On measuring the CRP concentrations in sera from 78 dogs by capillary RPLA, single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), close correlations were demonstrated between SRID and capillary RPLA (y=7.250+1.109x, r=0.978), between SRID and ELISA (y=3.042+1.059x, r=0.967), and between capillary RPLA and ELISA (y=1.778+0.929x, r=0.962). Capillary RPLA may be considered useful as a routine biochemical technique for measurement of serum CRP concentration in the dog.Abbreviations CRP C-reactive protein - ELISA enzyme-linked immunosorbent assay - RPLA reversed passive latex agglutination test - SRID single radial immunodiffusion     

Evaluation of a Commercially Available Human Serum Amyloid A (SAA) Turbidimetric Immunoassay for Determination of Feline SAA Concentration

Serum amyloid A (SAA) is an acute-phase protein in cats likely to be useful for diagnosing and monitoring inflammatory diseases, especially if rapid, reliable and automated assays can be made available. A commercially available automated human SAA turbidimetric immunoassay (SAA-TIA) was evaluated for determination of SAA in cats. Intra-assay and inter-assay imprecisions were in the ranges 2.1–9.9% and 7.0–12.5%, respectively, and without significant inaccuracy. Eighty-eight cats were divided into groups according to (A) the presence or absence of an acute-phase response (APR) (n = 23 and 65, respectively) and (B) clinical diagnosis (clinically healthy cats, cats diagnosed with inflammatory/infectious diseases, endocrine/metabolic diseases, neoplastic diseases, and miscellaneous disorders (n=43, 13, 8, 4 and 20, respectively)). The observed SAA concentrations were, as expected, different for (A) cats with and without an APR and (B) cats with inflammatory/infectious diseases compared to other diagnostic groups, except neoplastic diseases. In conclusion, the SAA concentration in cats could be measured reliably using the commercially available TIA designed for measuring human SAA, which should facilitate implementation of the parameter for routine diagnostic purposes. Hansen, A.E., Schaap, M.K. and Kjelgaard-Hansen, M., 2006. Evaluation of a commercially available human serum amyloid A (SAA) turbidimetric immunoassay for determination of feline SAA concentration. Veterinary Research Communications, 30(8), 863–872