Gentian Canine CRP Immunoassay Application Note For ABX .
v02-Jan2014Gentian Canine CRP ImmunoassayApplication Note for ABX Pentra* 400Intended UseMaterial Storage and StabilityThe canine CRP immunoassay on Horiba Medical's ABX Pentra400 is an in vitro diagnostic test for quantitative determinationof canine CRP in dog serum and plasma. The measurement ofcanine CRP is used in the diagnosis and treatment ofinflammatory diseases in dogs [1,2,3,4,5].All materials provided for the Gentian canine CRP test must bestored at 2-8 C. The expiry date is printed on the labels. Usingan Abbott Architect c4000, the on board stability of the Gentiancanine CRP reagents was found to be at least eight weeks.Sample MaterialMeasuring rangeThe recommended measuring range of the Gentian canine CRPimmunoassay on ABX Pentra 400 is 15 - 300 mg/L, with asecurity zone up to 700 mg/L.Normal ValuesHealthy dogs have CRP concentrations 15 mg/L with theGentian canine CRP method on ABX Pentra 400. An exactreference range cannot be determined as CRP concentrations inhealthy dogs are below the LoQ of the Gentian canine CRPassay.Recommended sample material is canine serum, canineheparinized plasma or canine EDTA plasma. Analyze thesamples as fresh as possible, and mix them well in advance.Sample stability testing showed that canine CRP (in serum) wasstable for 14 days at 4-22 C [6]. The samples can be shippedwithout any special cooling and must then be analyzed within14 days after shipment. Samples have been tested and shownto withstand up to four freeze and thaw cycles [6].Assay ProcedureAssay PrincipleClinical Decision LimitsThe diagnostic specificity of canine CRP can be enhancedwithout seriously impairing diagnostic sensitivity by using a cutoff limit somewhere above the normal range [2]. Eachlaboratory should establish its own cut-off.Assay ReagentsApplication Parameter SetupMaterials Provided by GentianGentian Canine CRP Reagent Kit R1 Reaction buffer (45 ml) R2 Immunoparticles (10,5 ml)Gentian Canine CRP Calibrator Kit(6 levels, 0,5 ml per level)Gentian Canine CRP Control Kit Control low (0,5 ml) Control high (0,5 ml)All materials are ready for use.*Not available for individual sale.The canine serum or plasma sample is mixed with canine CRPimmunoparticles. Canine CRP from the sample and theimmunoparticles' anti-canine CRP aggregate. The complexparticles created absorb light, and turbidimetric measurementsof absorption are related to canine CRP concentration viainterpolation on an established standard calibration curve.Results are automatically calculated by Pentra 400.REF 1501REF 1507*REF 1514*REF 1551REF 1519REF 1520*REF 1521*The application must be installed with the instrument settingsprovided for the Gentian canine CRP method. For instructionson how to install a new application, consult the instrumentmanual.Reagent PreparationThe reagents provided are ready for use. Mix the reagentsgently before placing them into the assigned reagents positions.The bottles provided can be used directly on ABX Pentra 400.Calibration Curve EstablishmentCalibrator StandardizationGentian canine CRP calibrator values are established on thebasis of internal canine CRP reference material. No internationalstandard is available for canine CRP.Calibration StabilityUse the Gentian canine CRP calibrator kit to establish acalibration curve as described in the instrument manual. Arecalibration must be performed when a new calibrator lotand/or a new reagent kit lot is to be used. The assignedconcentration values of the calibrators are lot dependent. Therelevant values are stated in the Analytical Value Sheetprovided with the calibrator kit.The stability of the calibration curve has not been tested on ABXPentra 400. Recalibration every 4th week is generallyrecommended.*Registered trademark of Horiba Medical.Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.1
QC ControlsThe Gentian canine CRP control samples should be assayedevery day the method is in use, to validate the calibration curve.Each control has an assigned concentration value range thatmust be met before measuring regular samples. The relevantconcentration ranges are given in the Analytical Value Sheetprovided with the control kit. If the measured concentrationsare outside the valid range, repeat the control measurements.Recalibrate if necessary. If the calibration cannot be performedwithout error, or valid control values cannot be reproduced,contact the local distributor for support.Measuring Patient SamplesWhen a satisfactory calibration curve has been established andthe control values are within the valid concentration range,canine serum or plasma samples may be measured. Check thatminimum sample volume is present in sample cups/tubes andassay the samples according to the instructions given in theinstrument manual.ResultsThe sample concentration of canine CRP is calculatedautomatically by the analyzer and presented in mg/L.Lower Quantification LimitThe lower quantification limit (LoQ) of canine CRP is defined asthe lowest sample concentration that can be measured with atotal error (TE) 29,6 % [7]. Total error is calculated from thepooled standard deviation of the sample's measuredconcentration and the bias between the sample's mean andtheoretical concentration value. A sample of 7,7 mg/L had atotal error of 18 % with the Gentian canine CRP method. TheLoQ of the Gentian canine CRP immunoassay on ABX Pentra 400is set to 8 mg/L.LinearityDilution of a high canine CRP serum was performed, and theconcentration of the resulting samples was measured. Recoveryfrom expected concentrations was calculated. Based oninterpolation, the assay is considered satisfactory linear in therange 10 - 300 mg/L.100 %80 %60 %40 %20 %10 %5%2,5 9,7103,0105,1105,299,9108,3107,9Mean (mg/L)Linear fity 0,99x 2,8302R² 0,999210000100200300400Expected canine CRP concentration (mg/L)ImprecisionFour samples (A-D) were assayed over eight days on an ABXPentra 400 analyzer. Four other samples (E-H) were assayed onanother Pentra 400 instrument. Each sample was measured induplicate, twice a day.SampleIDABCDPerformance CharacteristicsABX Pentra 400DilutionfactorMeasured canine CRP concentration (mg/L)Linearity ABX Pentra 400Mean(mg/L)25,463,8121,1257,8Within runCV (%)7,432,081,340,76Betweenrun CV (%)011,651,560,41Betweenday CV ,57G103,90,850,881,79H272,10,681,831,051: According to statistical convention and EP5-A2, vol 24, No 25,the CV is set to zero, if a negative value is returned.InterferenceCanine CRP samples of about 30 mg/L were spiked with 10 g/Lhemoglobin, 10 g/L Intralipid or 800 mg/L bilirubin. The sampleswere compared to corresponding control samples. None of thespiked samples demonstrated clinically significant interference– defined as more than 10 % difference between test sampleand control sample.Hemoglobin (10 g/L)Intralipid (10 g/L)Bilirubin (800 L)29,128,230,4Recovery(%)100,5101,2105,8Security ZoneSamples with a canine CRP concentration up to 700 mg/L returna value above 300 mg/L (upper limit of linearity range) and canbe sent to automatic diluted rerun.2Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.TotalCV (%)8,103,983,322,1815,75,382,162,22
Analytical 6Recovery(%)99,7103,499,4Method Comparison: Gentian vs Randox400Gentian - Mean (mg/L)The analytical recovery of the canine CRP assay was determinedby adding fixed amounts of canine CRP to samples andcalculating the recovery from theoretical concentrations.300200IdentityPassing & Bablok (I) fit(7.79 0.91x)10000Instrument VariationA total of 32 samples was assayed on both ABX Pentra 400,Abbott Architect c4000 and Beckman Coulter AU400 with theGentian canine CRP immunoassay. A statistical analysis of themeasured instrument variation was performed.Instrument Variation – Pentra 400 vs. Architect c4000Bland Altman biasBland Altman 95 % limits of agreement200300400Randox - Mean (mg/L)Shipping DamagePlease notify your distributor if the product received isdamaged.1,4 %-8,2 % to 5,3 %Passing Bablok slope0,96Passing Bablok intercept (mg/L)2,36Symbols KeyLot numberTemperature limitationsInstrument Variation – Pentra 400 vs. AU400Bland Altman biasBland Altman 95 % limits of agreement100-3,6 %Expiration date-4,1 % to 11,2 %Passing Bablok slope1,01Passing Bablok intercept (mg/L)2,06Consult instructions for useManufacturerMethod ComparisonCatalogue numberThe Gentian canine CRP test was compared to the Randoxhuman CRP immunoassay, used for canine CRP applications(with canine CRP calibrators). A set of 45 serum samples wasassayed on both methods at the Swedish University ofAgriculture Sciences Animal Hospital, using an Abbott Architectc4000. Results were subjected to statistical analysis.Method ComparisonN 45Manufacturer DistributorGentian AS,Bjornasveien 5N-1509 Moss, NorwayTEL: 47 99 33 99 05FAX: 47 69 24 09 62http://www.gentian.noscil animal care company GmbHDina-Weissmann-Allee 668519 Viernheim, DeutschlandTel: 49 (0) 6204 7890 0Fax: 49 (0) 6204 7890 200E-Mail: info-de@scilvet.comBland Altman bias-3,0 %Bland Altman limits of agreement-19,4 to 13,4 %Passing Bablok slope0,91ReferencesPassing Bablok intercept (mg/L)7,79[1] Ceron et al. Vet Clin Pathol. 2005; 34: 85-99[2] Kjelgaard-Hansen; PhD Thesis. 2004[3] Kjelgaard-Hansen et al. Vet Clin Pathol. 2003; 32: 81-87[4] Yamamoto et al. Vet Res Comun. 1993; 17: 259-266[5] Eckersall et al. Vet J. 2010; 185 (1): 23-27[6] Hillström et al. Vet Clin Pathol. 2014; in press.[7] Kjelgaard-Hansen et al. Comp Clin Path. 2003; 12: 69-743Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
Instrument Settings Canine CRP ABX Pentra 400General ParametersCharacteristics:Sample type:Serum / PlasmaNumber of reagents:2Reagent short name:C-CRP*Reagent number:999*On board stability:---Reagent:CassetteAutomatic Rerun:Post dilution:---Post concentration:---Decimal positions:1Results:Manual patient validationPre-dilutionDiluent Name: Factor:---Incubation time (cycles):---Linearity:Correlation:Low limit:High limit:Slope:Intercept:15.0300.01.000.00Delta Check:Delta check validity:Absolute variation:Relative variation:--------*Defined by user.4Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
Reference Check (mg/L):Man / Default:Women:Child:Low:---------High:---------Man / un Range (mg/L):Calibration ParametersPre-dilution:Type:Calibrator diluent:------Factor 1Factor 2Factor 3Factor 4------------Factor 5Factor 6Factor 7Factor 8------------Checks:Reagent limit absorbance checkReagent range low:---Reagent range high:---Reagent blank limit absorbance checkBlank range low limit:---Blank range high limit:---Control RequiredControl used 1:---Control used 2:---Control used 3:---5Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
Validity Backup:Backup time frame without calibration requiredInterval:30Time unit: DayCalibration:Calibration mode:Logit / Log5Level:6Calibration factor:---Run(s):2 Dev rep. (%):10.0Dev c. (%):---Calibrator used:C-CRP*Validity: On requestTime validityInterval:---Time unit:---Factor Calibration:Low limit check:---High limit check:---Relative variation check:---Analysis ParametersCleaner:Cleaner solution:H2OBeforeAfter*Defined by user.6Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
Wavelength (nm):Primary wavelength:600Secondary wavelength:---Blank: Reagent blankDiluent:H2OAnalysis Sequence:Cycle:Reagent needle:Volume (µL):Sample needle:Volume (µL):H2O volume ------------------------------Mixing Speed:80Test Name: C-CRP*Channel: 999* Modified on: N/AEnableCode: C-CRP*Local Code: C-CRP*Calculation ParametersCorrelation Factor:Slope:1.000Intercept: 0.000Sample Limit Check:Sample limit (Δ OD):---Sample limit cycle:---7Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
Reaction Direction: Reaction direction checkReaction direction:IncreaseAntigen Excess Check: Antigen excess limit (%):---Antigen excess point:---Definition Step A:Calculation type:End pointFirst reading (cycle):21Last reading (cycle):42Reaction Limit Check:Reaction limit absorbance:---Cycle:---OD Deviation Check:Linear regressionr2 thresholdFirst pointSDLast pointFirst point thresholdSDFactorDefinition Step B (N/A)Calculation type:End pointFirst reading (cycle):---Last reading (cycle):---Reaction Limit CheckReaction limit absorbance:---Cycle:---8Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
OD Deviation Check:Linear regressionr2 thresholdFirst pointSDLast pointFirst point thresholdSDFactorDefinition Step C (N/A)Calculation type:End pointFirst reading (cycle):---Last reading (cycle):---Reaction Limit CheckReaction limit absorbance:---Cycle:---OD Deviation CheckLinear regressionr2 thresholdFirst pointSDLast pointFirst point thresholdSDFactorDefinition Step D (N/A)Calculation type:End pointFirst reading (cycle):---Last reading (cycle):---Reaction Limit CheckReaction limit absorbance:---Cycle:---9Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
OD Deviation Check:Linear regressionFirst pointr2 thresholdSDLast pointFirst point thresholdSDFactorFormulaUnit ParametersUnit:Conversion Factor:mg/L1.0000------------Unit:Conversion Factor:mg/L1.0000------------10Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical.
Application Note for Gentian Canine CRP Immunoassay on Pentra 400, Horiba Medical. Analytical Recovery The analytical recovery of the canine CRP assay was determined by adding fixed amounts of canine CRP to samples and calculating the recovery from theoretical concentrations. Sample ID Expected concentration (mg/L) Identity Measured
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Student perspectives of CRP 25 Teacher perspectives of CRP 27 CRP and school leadership 31 Visibility of CRP in Australia 35 CRP across the Australian Curriculum 36 Section 3: Culturally responsive pedagogy: Challenges 43 Conceptual confusion or distortion 43 Super-diversity 44 Validation 44 Essentialism and stereotyping 46
ELISA to AlphaLISA This guide presents the simple conversion of an ELISA or other immunoassay to an AlphaLISA immunoassay. AlphaLISA is a highly sensitive, no-wash alternative to your existing immunoassay. In the AlphaLISA assay, a biotinylated antibody and an antibody-conjugated AlphaLISA Acceptor bead are used to capture the target analyte.
Canine Handlers shall not use their canine partners for off-duty work assignments unless authorized by the chief of police or his designee. The use of the canine shall be governed by the department’s policy. Canine Handler shall limit the use of their canine partner within this written policy and within the department’s policy. III.
A comprehensive literature search yielded 13 articles that studied HRQoL differences before and after a CRP following an MI. These studies were analyzed by CRP length; time between MI and CRP start; CRP components, type, and intensity; and effect on HRQoL. Findings indicated that CRPs do seem to positively influence HRQoL following an MI,
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