Clinical Chemistry Specific Proteins Presentation

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1 Clinical Chemistry Specific Proteins Presentation November 2004 AEROSET and c8000 are registered trademarks of Abbott Laboratories. All other trademarks, brands, trade names and product names are the property of their respective companies Clinical Chemistry Specific Proteins Presentation, version 1 1

2 Agenda Basic Information Sample Handling Reagent Handling Calibration Quality Control Reaction Methodology Interfering Substances Precision Method Comparison Assay Specific Information Troubleshooting Tips Questions & Answers Clinical Chemistry Specific Proteins Presentation, version 1 2

3 Basic Information Clinical Chemistry Specific Proteins Presentation, version 1 3

4 Specific Protein Assay Highlights Apolipoprotein A1 Lipid transport found in HDL and chylomicrons Apolipoprotein B Lipid transport - found in Non-HDL lipoproteins Complement C3 Increased with acute phase reactions, AMI, cancer, pregnancy, viral hepatitis & diabetes Complement C4 Increased in some malignancies and acute phase reactions Haptoglobin Binds Hemoglobin Immunoglobulin A Serum and body secretion Immunoglobulin Immunoglobulin G Secondary immune response Found in blood, crosses placenta Immunoglobulin M Primary immune response Prealbumin T3, T4, and Vitamin A transport Indicator of protein malnutrition Transferrin Iron Transport Clinical Chemistry Specific Proteins Presentation, version 1 4

5 Clinical Implications Assay Decreased Levels Increased Levels Apolipoprotein A1 Atherosclerosis Low risk of coronary disease Apolipoprotein B Severe hepatic dysfunction Atherosclerosis, hyperlipidemias Complement (C3) AutoImmune disease, chronic Inflammatory Disease hepatitis, lupus Complement (C4) Autoimmune disease, chronic Acute inflammatory process hepatitis, acute glomerular nephritis Haptoglobin Hemolytic anemia, sickle cell anemia, liver disease Acute and chronic inflammatory disease Immunoglobulin (IgA) Immune deficiency states, non-iga myelomas IgA myelomea, chronic cirrhosis, chronic liver disease Immunoglobulin (IgG) Immunoglobulin (IgM) Prealbumin Immune deficiency states, non-igg Myelomas Immune deficiency states, non-igm myelomas Malnutrition, liver disease, acute Inflammation IgG myeloma, chronic infections, Liver disease Waldenstrom s macroglobulinemia, chronic infections, liver disease Hodgkin s disease, corticosteroid therapy Transferrin Inflammation, chronic hepatitis Iron deficiency, acute hepatitis, pregnancy Clinical Chemistry Specific Proteins Presentation, version 1 5

6 Ordering Information Clinical Chemistry Specific Proteins Presentation, version 1 6

7 Reagents Reagent List Number Kit configuration Tests per Kit Apolipoprotein A1 9D92-20 R1: 3 x 21 ml R2: 3 x 9 ml 243 Apolipoprotein B 9D93-20 R1: 3 x 21 ml 243 R2: 3 x 9 ml Complement C3 9D96-20 R1: 3 x 20 ml 279 R2: 3 x 8 ml Complement C4 9D97-20 R1: 3 x 20 ml 279 R2: 3 x 8 ml Haptoglobin 9D91-20 R1: 3 x 17 ml 230 R2: 3 x 7 ml Clinical Chemistry Specific Proteins Presentation, version 1 7

8 Reagents Reagent List Number Kit configuration Tests per Kit Immunoglobulin A 9D98-20 R1: 4 x 20 ml R2: 4 x 8 ml 373 Immunoglobulin G 9D99-20 R1: 4 x 20 ml R2: 4 x 20 ml 388 Immunoglobulin M 1E01-20 R1: 4 x 20 ml R2: 4 x 8 ml 373 Prealbumin 1E02-20 R1: 3 x 18 ml 242 R2: 3 x 6 ml Transferrin 1E04-20 R1: 5 x 20 ml 391 R2: 5 x 9 ml Clinical Chemistry Specific Proteins Presentation, version 1 8

9 Calibrators Calibrator ApoA1/ApoB List Number 6E54-02 Prealbumin 6E57-02 Specific Protein Multiconstituent 1E Clinical Chemistry Specific Proteins Presentation, version 1 9

10 Sample Handling Clinical Chemistry Specific Proteins Presentation, version 1 10

11 Sample Handling Serum or Plasma* in glass or plastic tubes *Prealbumin is serum only Serum with or without gel barrier Plasma without a gel barrier Acceptable anticoagulants: Lithium Heparin Ammonium Heparin Sodium Heparin EDTA Clinical Chemistry Specific Proteins Presentation, version 1 11

12 Sample Handling Assay Temperature Maximum Storage Apolipoprotein A1 2 to 8 C -20 C Apolipoprotein B 2 to 8 C -20 C -70 C Complement C3 2 to 8 C -20 C Complement C4 2 to 8 C Haptoglobin 2 to 8 C -20 C Immunoglobulin A (IgA) 2 to 8 C Immunoglobulin G (IgG) -20 C Prealbumin Transferrin Immunoglobulin M (IgM) 2 to 8 C -20 C 3 days 2 months 3 days 2 months 1 year* 8 days 8 days 2 days 1 week 2 weeks 3 days 6 months 5 days 6 months 1 * Thaw overnight at 2-8 C Clinical Chemistry Specific Proteins Presentation, version 1 12

13 Reagent Handling Clinical Chemistry Specific Proteins Presentation, version 1 13

14 Reagent Handling Reagent is a liquid, ready-to-use, two-reagent kit. The unopened reagents are stable until the expiration date when stored at 2-8 C. Do not mix fresh reagent with in use reagent. Do not mix materials from different kit lot numbers Mix gently, do not shake or foaming may occur. Due to small reagent volumes, do not use a transfer pipette to remove bubbles. Reagent onboard Stability IgA: 28 days IgG: 23 days ApoA, ApoB, C3, C4, Haptoglobin, IgM, Prealbumin, Transferrin: 57 days Clinical Chemistry Specific Proteins Presentation, version 1 14

15 Reagent Handling R1 and R2 both 20 ml bottles If running all 10 specific protein assays:requires twenty 20 ml reagent cartridge adapters List Number 09D22-11, 10 adapters Same list number for AEROSET and c Clinical Chemistry Specific Proteins Presentation, version 1 15

16 Reagent handling c8000 R2 reagent carousel requires one large cartridge segment, B, C, D (L/N 04J31-01) to hold 20 ml round bottle reagent adapters. Two large cartridge segments are required to hold all 10 Specific Protein R2 reagents at the same time Clinical Chemistry Specific Proteins Presentation, version 1 16

17 c8000 Base configuration D C A B R2 C D B A R1 R1 Segment A 12 large B positions B Segment B 12 Blarge positions Segment C 12 large B positions Segment D 20 large positions R2 Segment A 14 Small positions Segment B 9 large positions Segment C 14 small positions Segment D 14 small positions Clinical Chemistry Specific Proteins Presentation, version 1 17

18 Calibration Clinical Chemistry Specific Proteins Presentation, version 1 18

19 Calibration IgA now uses 1:25 autodilution of highest calibrator (SP5) to create the lowest non-zero calibrator (C1) Must edit linear high with highest calibrator value for all specific protein assays upon initial use and with each calibrator lot change Clinical Chemistry Specific Proteins Presentation, version 1 19

20 Calibration Calibration Stability ApoB: 38 days IgA: 25 days IgG: 23 days ApoA1, C3, C4, Haptoglobin, IgM, Prealbumin, Transferrin: 57 Days Calibration Method ApoA, ApoB, Prealbumin: Linear C3, C4, Haptoglobin, IgA, IgG, IgM, Transferrin: Spline Clinical Chemistry Specific Proteins Presentation, version 1 20

21 Calibration Preparation of Apo A1/Apo B calibrators Reconstitute with 1.0 ml Type II water Allow to stand for 10 minutes Dissolve by swirling don t shake Prepare the four calibrator dilutions using the reconstituted calibrator and diluent. Reconstituted calibrator is stable for 2 weeks when stored capped at 2 to 8 C Diluted calibrators are stable for 1 day when stored capped at 2 to 8 C Clinical Chemistry Specific Proteins Presentation, version 1 21

22 Apolipoprotein A1 Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 22

23 Apolipoprotein B Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 23

24 Complement C3 Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 24

25 Complement C4 Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 25

26 Haptoglobin Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 26

27 Immunoglobulin A Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 27

28 Immunoglobulin G Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 28

29 Immunoglobulin M Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 29

30 Prealbumin Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 30

31 Transferrin Calibration AEROSET Conventional Units c8000 SI Units Clinical Chemistry Specific Proteins Presentation, version 1 31

32 Quality Control Clinical Chemistry Specific Proteins Presentation, version 1 32

33 Quality Control Abbott control product is not available, however current AEROSET peer group information is available with BioRad Immunology Plus. BioRad peer value information can be found on WWCS Webpage US: BIO-RAD ROW: contact your local BioRad Representative Clinical Chemistry Specific Proteins Presentation, version 1 33

34 Quality Control BioRad Control Name Liquichek Immunoassay Lyphochek Immunoassay Plus Assays ApoA, ApoB, C3, C4, Hapt,IgA, IgG, IgM, PAlb, TRF IgA, IgG, IgM Catalog # 591, 592, 593 (6 x 1 ml) 594, 595, 596 (6 x 3 ml) 370 (Trilevel, 12 x 5 ml) 371, 372, 373 (12 x 5 ml) Liquichek Immunoassay Plus IgA, IgG, IgM 360 (Trilevel, 12 x 5 ml) 361, 362, 363 (12 x 5 ml) Clinical Chemistry Specific Proteins Presentation, version 1 34

35 Reaction Methodology Clinical Chemistry Specific Proteins Presentation, version 1 35

36 Basic Principles in Turbidimetry and Nephelometry When a soluble antigen is mixed in suitable proportions with the corresponding antibody a precipitate is formed. This reaction was described in quantitative terms in the pioneering work by Heidelberger & Kendall in In a now classical experiment, they showed that when increasing amounts of an antigen are added to a number of test tubes containing a constant quantity of the corresponding antibody, and the amount of precipitate was analyzed, a precipitin curve as shown was obtained. Quantitative Immunoprecipitin Curve Ab Excess Zone Ag Excess Zone Immunoprecipitate Y Y Y Y Y Y Y Y Y Equivalence Zone Y Y Y Y Y Y Y Ag Concentration Heidelberger Curve Clinical Chemistry Specific Proteins Presentation, version 1 36

37 Three zones: Basic Principles in Turbidimetry and Nephelometry 1. The Antibody Excess Zone The amount of precipitate increases as more antigen is added. The supernatant still contains free antibody. 2. The Equivalence Zone Maximum precipitation occurs. The supernatant contains neither free antigen nor free antibody at the peak of the curve. 3. The Antigen Excess Zone Due to high antigen concentration, the formation of small soluble immune complexes is favored rather than real precipitate. When insufficient amounts of antibody are available to bind with antigen, an erroneously low result can occur. The supernatant contains free antigen Clinical Chemistry Specific Proteins Presentation, version 1 37

38 Reaction Methodology Nephelometry (IN) Measures the intensity of scattered light (that which is reflected at various angles). Turbidimetry (IT) Measures the intensity of light transmission (incident light beam passing through the cuvette). Turbidimetry and Nephelometry Both are based on optical detection systems that measure the concentration of very small particles suspended in a liquid. Dilute antigen solutions are mixed with a solution of corresponding antibody to form immune complexes. Immune complexes cause solution turbidity Clinical Chemistry Specific Proteins Presentation, version 1 38

39 Nephelometry or Turbidimetry? "These two instrument approaches basically differ only in the nature of the optics. The basic principle is to measure that portion of an incident light beam which passes through the cuvette - Turbidimetry. Light beam reflected at various angles is Nephelometry. Both approaches have advanced greatly in the last decade and the instrument and computer controlled packages associated with the optical systems have produced devices which perform extremely well. "It is not possible to say which is better." Each manufacturer has sought to deliver an instrument which is better than its competition, but in reality the comparisons are informative only when the kits themselves are examined." 1 1Ritchie, Robert MD. Serum Proteins in Clinical Medicine, Vol. 1, Foundation of Blood Research. Scarborough, Maine p Clinical Chemistry Specific Proteins Presentation, version 1 39

40 Assay Reaction Sequence Methodology Immunoturbidimetric Equation: Analyte + PEG/buffer (R1) + Antibody to analyte (R2) (serum or plasma) Turbidity due to analyte: antibody complex Clinical Chemistry Specific Proteins Presentation, version 1 40

41 Reaction Check Prozone:Antigen excess. Extremely high immunoglobulin samples result in formation of soluble complexes instead of an insoluble complex leading to incorrect results within the reportable range of the assay. Reaction check: used with IgA and IgM assays to evaluate the reaction for prozone Clinical Chemistry Specific Proteins Presentation, version 1 41

42 Prozone (Antigen Excess) Prozone (Antigen Excess) New IgA and IgM parameters will use a 1:5 sample dilution and the RCD flag as the standard configuration to reduce the likelihood of prozone. Auto Rerun/Retest is used to rerun LL or < samples undiluted. IgM High Sample Neat vs 1:5 Absorbance Data absorbance Sample 1 neat hi cal lo cal sample 1 1:5 read point Clinical Chemistry Specific Proteins Presentation, version 1 42

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44 Prozone (Antigen Excess) Precision of 1:5 dilution IgA and IgM c8000 IgA Neat Cntl Total CV Cntl Total CV c8000 IgM Neat IgA Diluted IgM Diluted AEROSET IgA Diluted AEROSET IgM Diluted % CV Goal = 4.1 % CV Goal = Clinical Chemistry Specific Proteins Presentation, version 1 44

45 IgA and IgG sensitivity improvements IgA sensitivity was improved by using a 1:25 auto dilution of Cal 5 to create a new low calibrator between zero and Cal Clinical Chemistry Specific Proteins Presentation, version 1 45

46 IgA and IgG sensitivity improvements IgG sensitivity was improved by auto retest of samples < 320 mg/dl with 3X sample volume. IgG % Recovery vs Target Observed % of Target IgG mg/dl 2.7 ul Svol 8.1 ul Svol Clinical Chemistry Specific Proteins Presentation, version 1 46

47 Percent Extrapolation (%EXT Flag) (AEROSET only) %EXT flagging is based on the absorbance value (or absorbance change) of the highest calibrator. The absorbance value (or absorbance change) is defined as the difference between the absorbance (or absorbance change) of the highest calibrator and the reagent blank (absorbance or absorbance change). Determines the numerical value beyond which results will be flagged if they exceed that percentage of the absorbance or rate of the highest calibrator. The value for this parameter is 1% for all specific protein assays Clinical Chemistry Specific Proteins Presentation, version 1 47

48 Percent Extrapolation (%EXT Flag) (AEROSET only) Clinical Chemistry Specific Proteins Presentation, version 1 48

49 Interfering Substances Clinical Chemistry Specific Proteins Presentation, version 1 49

50 Apolipoprotein A1 Interfering Substances Interfering Concentration Target % of Target Substances Conc Bilirubin 15 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 500 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 50

51 Apolipoprotein B - Interfering Substances Interfering Concentration Target % of Target Substances Conc Bilirubin 30 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 51

52 Complement C3- Interfering Substances Medical Decision Level 1 Medical Decision Level 2 Interfering Concentration Target % of Target Target Conc % of Target Substances Conc Bilirubin 30 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 52

53 Complement C4- Interfering Substances Medical Decision Level 1 Medical Decision Level 2 Interfering Substances Concentration Target Conc % of Target Target Conc % of Target Bilirubin 30 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 53

54 Haptoglobin - Interfering Substances Interfering Concentration Target % of Target Substances Conc Bilirubin 30 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 54

55 IgA - Interfering Substances Medical Decision Level 1 Medical Decision Level 2 Interfering Concentration Target % of Target Target Conc % of Target Substances Conc Bilirubin 30 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 55

56 IgG - Interfering Substances Medical Decision Level 1 Medical Decision Level 2 Interfering Concentration Target % of Target Target Conc % of Target Substances Conc Bilirubin 30 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 56

57 IgM - Interfering Substances Medical Decision Level 1 Medical Decision Level 2 Interfering Concentration Target % of Concentration Target % of Target Substances Conc Target Conc Bilirubin 30 mg/dl mg/dl mg/dl mg/dl Hemoglobin 250 mg/dl mg/dl mg/dl mg/dl Human Trig 750 mg/dl mg/dl mg/dl mg/dl Intralipid 1000 mg/dl mg/dl mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 57

58 Prealbumin - Interfering Substances Interfering Concentration Target % of Target Substances Conc Bilirubin 30 mg/dl mg/dl Hemoglobin 500 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 58

59 Transferrin - Interfering Substances Medical Decision Level 1 Medical Decision Level 2 Interfering Substances Concentration Target Conc % of Target Target Conc % of Target Bilirubin 30 mg/dl mg/dl Hemoglobin 1000 mg/dl mg/dl Human Trig 750 mg/dl mg/dl Intralipid 1000 mg/dl mg/dl Clinical Chemistry Specific Proteins Presentation, version 1 59

60 Precision Clinical Chemistry Specific Proteins Presentation, version 1 60

61 Apolipoprotein A1- Precision (< 4.9 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Apolipoprotein B - Precision (< 6.5 % Total CV) Level N Mean Within Within Run Total Total mg/dl Run SD %CV SD %CV Level Level Level Clinical Chemistry Specific Proteins Presentation, version 1 61

62 Complement C3 - Precision (< 3.9 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Complement C4 - Precision (< 4.5 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Clinical Chemistry Specific Proteins Presentation, version 1 62

63 Haptoglobin - Precision (< 6.0 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Immunoglobulin A - Precision (< 4.1 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Clinical Chemistry Specific Proteins Presentation, version 1 63

64 Immunoglobulin G - Precision (< 3.4 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Immunoglobulin M - Precision (< 4.4 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Clinical Chemistry Specific Proteins Presentation, version 1 64

65 Prealbumin - Precision (< 5.5 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Transferrin - Precision (< 5.0 % Total CV) Level N Mean mg/dl Within Run SD Within Run %CV Total SD Total %CV Level Level Level Clinical Chemistry Specific Proteins Presentation, version 1 65

66 Method Comparison Clinical Chemistry Specific Proteins Presentation, version 1 66

67 Method Comparison Apolipoprotein A1 Apolipoprotein B AEROSET vs. Hitachi c8000 vs. AEROSET AEROSET vs. Hitachi c8000 vs. AEROSET N Y-Intercept Corr Coef Slope Mean % Bias Range (mg/dl) 26.9 to to to to Clinical Chemistry Specific Proteins Presentation, version 1 67

68 Method Comparison Complement C3 Complement C4 AEROSET vs. Hitachi c8000 vs. AEROSET AEROSET vs. Hitachi c8000 vs. AEROSET N Y-Intercept Corr Coef Slope Mean % Bias Range (mg/dl) 27.8 to to to to Clinical Chemistry Specific Proteins Presentation, version 1 68

69 Method Comparison Haptoglobin Immunoglobulin A AEROSET vs. Hitachi c8000 vs. AEROSET AEROSET vs. Hitachi c8000 vs. AEROSET N Y-Intercept Corr Coef Slope Mean % Bias Range (mg/dl) 1.8 to to to to Clinical Chemistry Specific Proteins Presentation, version 1 69

70 Method Comparison Immunoglobulin G Immunoglobulin M AEROSET vs. Hitachi c8000 vs. AEROSET AEROSET vs. Hitachi c8000 vs. AEROSET N Y-Intercept Corr Coef Slope Mean % Bias Range (mg/dl) to to to to Clinical Chemistry Specific Proteins Presentation, version 1 70

71 Method Comparison Prealbumin Transferrin AEROSET vs. Hitachi c8000 vs. AEROSET AEROSET vs. Hitachi c8000 vs. AEROSET N Y-Intercept Corr Coef Slope Mean % Bias Range (mg/dl) 4.3 to to to to Clinical Chemistry Specific Proteins Presentation, version 1 71

72 Assay Specific Information Clinical Chemistry Specific Proteins Presentation, version 1 72

73 Apolipoprotein A1 Assay Method: Immunoturbidimetric Reagent List #: 9D92-20 Calibraton: Method: Linear Apo A1/Apo B Calibrator: 6E54-02 Assay Stability onboard Stability: 57 Days (1368 hours) Calibration Stability: 57 Days (1368 Hours) Assay Volumes Specimen: 2 ul R1 Reagent: 200 ul R2 Reagent: 67 ul Reportable range: 16 mg/dl (0.16 g/l) up to highest calibrator Limit of Quantitation: < 3 mg/dl (0.03 g/l) Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA Precision: < 4.9 % Total CV New Information 1:2 dilution protocol added Calibration interval extended to 57 days LH field contains lowest possible value for high calibrator Clinical Chemistry Specific Proteins Presentation, version 1 73

74 Apolipoprotein B Assay Method: Immunoturbidimetric Reagent List#: 9D93-20 Calibraton: Method: Linear Apo A1/Apo B Calibrator: 6E54-02 Assay Stability Onboard Stability: 57 Days (1368 hours) Calibration Stability: 38 Days (912 Hours) Assay Volumes Specimen: 2 ul R1 Reagent: 200 ul R2 Reagent: 67 ul Reportable range: 11 mg/dl (0.11 g/l) up to highest calibrator Limit of Quantitation: < 3 mg/dl (0.03 g/l) Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA Precision: < 6.5 % Total CV New Information 1:4 Dil 1 protocol added LH field contains lowest possible value for high calibrator Clinical Chemistry Specific Proteins Presentation, version 1 74

75 Complement C3 Assay Method: Immunoturbidimetric Reagent List#: 9D96-20 Calibraton: Method: Spline Specific Protein MCC: 1E78-02 Assay Stability Onboard Stability: 57 Days (1368 hours) Calibration Stability: 57 Days (1368 Hours) Assay Volumes Specimen: 3 ul R1 Reagent: 167 ul R2 Reagent: 47 ul Reportable range: 11 mg/dl (0.11 g/l) up to highest calibrator Limit of Quantitation: < 5 mg/dl (0.05 g/l) Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA Precision: < 3.9 % Total CV New Information Reference ranges adjusted to reflect recent literature Calibration interval extended to 57 days LH field contains lowest possible value for high calibrator Clinical Chemistry Specific Proteins Presentation, version 1 75

76 Complement C4 Assay Method: Immunoturbidimetric Reagent List#: 9D97-20 Calibraton: Method: Spline Specific Protein MCC: 1E78-02 Assay Stability onboard Stability: 57 Days (1368 hours) Calibration Stability: 57 Days (1368 Hours) Assay Volumes Specimen: 3 ul R1 Reagent: 167 ul R2 Reagent: 47 ul Reportable range: 2.9 mg/dl (0.029 g/l) up to highest calibrator Limit of Quantitation: < 1.0 mg/dl (0.01 g/l) Precision: < 4.5 % Total CV Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA New Information Reference ranges adjusted to reflect recent literature LH field contains lowest possible value for high calibrator Calibration interval extended to 57 days Clinical Chemistry Specific Proteins Presentation, version 1 76

77 Haptoglobin Assay Method: Immunoturbidimetric Reagent List#: 9D91-20 Calibraton: Method: Spline Specific Protein MCC: 1E78-02 Assay Stability onboard Stability: 57 Days (1368 hours) Calibration Stability: 57 Days (1368 Hours) Assay Volumes Specimen: 2 ul R1 Reagent: 167 ul R2 Reagent: 47 ul Reportable range: 8 mg/dl (0.08 g/l) up to highest calibrator Limit of Quantitation: < 4 mg/dl (0.04 g/l) Precision: < 6.0 % Total CV Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA New Information 1:4 Dil 2 protocol added Cal interval extended to 57 days LH field contains lowest possible value for high calibrator Clinical Chemistry Specific Proteins Presentation, version 1 77

78 Immunoglobulin A (IgA) Assay Method: Immunoturbidimetric Reagent List#: 9D98-20 Calibraton: Method: Spline Specific Protein MCC: 1E78-02 Assay Stability onboard Stability: 28 Days (672 hours) Calibration Stability: 25 Days (600 Hours) Assay Volumes Specimen: 20 ul Standard dilution: 1:5 R1 Reagent: 167 ul R2 Reagent: 47 ul Reportable range: 5 mg/dl (0.05 g/l) up to highest calibrator Limit of Quantitation: < 3 mg/dl (0.03 g/l) Precision: < 4.1 % Total CV Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA New Information No longer need to run in panel with IgA_D and IgA_R Samples are run at a 1:5 dilution initially (sample volume has increased from 6.5 ul to 20 ul) Added rerun rule for LL to rerun with Dil 1 Reaction check parameters added to detect prozone High calibrator is auto-diluted for C1 LH field contains lowest possible value for high calibrator LL field changed from 31 to 5 mg/dl Added 1:10 Dil 2 parameters Clinical Chemistry Specific Proteins Presentation, version 1 78

79 Immunoglobulin G (IgG) Assay Method: Immunoturbidimetric Reagent List#: 9D99-20 Calibraton: Method: Spline Specific Protein MCC: 1E78-02 Assay Stability onboard Stability: 23 Days (552 hours) Calibration Stability: 23 Days (552 hours) Assay Volumes Specimen: 2.7 ul R1 Reagent: 160 ul R2 Reagent: 160 ul Reportable range: 109 mg/dl (1.09 g/l) up to highest calibrator Limit of Quantitation: < 61 mg/dl (0.61 g/l) Precision: < 3.4 % Total CV Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA New Information LL changed from 194 mg/dl (1.94 g/l) to 320 mg/dl (3.20 g/l). Dil 1 protocol added to use 3x the standard sample volume extending the lower limit of reportable range to 109 mg/dl (1.09 g/l) Rerun rule added to use Dil 1 for samples < 320 mg/dl Reference ranges adjusted to reflect recent literature Clinical Chemistry Specific Proteins Presentation, version 1 79

80 Immunoglobulin M (IgM) Assay Method: Immunoturbidimetric Reagent List#: 1E01-20 Calibraton: Method: Spline Specific Protein MCC: 1E78-02 Assay Stability onboard Stability: 57 Days (1368 hours) Calibration Stability: 57 Days (1368 Hours) Assay Volumes Specimen: 20 ul Standard dilution: 1:5 R1 Reagent: 167 ul R2 Reagent: 47 ul Reportable range: 5 mg/dl (0.05 g/l) up to highest calibrator Limit of Quantitation: < 2 mg/dl (0.02 g/l) Precision: < 4.4 % Total CV Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA New Information Reaction check range changed Linear low changed from 14 mg/dl (0.14 g/l) to 5 mg/dl (.05 g/l) Samples are run at a 1:5 dilution initially (sample volume has increased from 3.0 ul to 20 ul) Dil 1 protocol runs samples undiluted Added rerun rule to use Dil 1 for LL samples Calibration interval extended to 57 days Clinical Chemistry Specific Proteins Presentation, version 1 80

81 Prealbumin Assay Method: Immunoturbidimetric Reagent List#: 1E02-20 Calibraton: Method: Linear Prealbumin Calibrator: 6E57-02 Assay Stability onboard Stability: 57 Days (1368 hours) Calibration Stability: 57 Days (1368 Hours) Assay Volumes Specimen: 3 ul R1 Reagent: 167 ul R2 Reagent: 33 ul Reportable range: 3 mg/dl (0.03 g/l) up to highest calibrator Limit of Quantitation: < 1 mg/dl (0.01 g/l) Precision: < 5.5 % Total CV Acceptable specimen: serum only New Information Calibration interval extended to 57 days Clinical Chemistry Specific Proteins Presentation, version 1 81

82 Transferrin Assay Method: Immunoturbidimetric Reagent List#: 1E04-20 Calibraton: Method: Spline Specific Protein MCC: 1E78-02 Assay Stability onboard Stability: 57 Days (1368 hours) Calibration Stability: 57 Days (1368 Hours) Assay Volumes Specimen: 2 ul R1 Reagent: 200 ul R2 Reagent: 67 ul Reportable range: 19 mg/dl (0.19 g/l) up to highest calibrator Limit of Quantitation: < 9 mg/dl (0.09 g/l) Precision: < 5.0 % Total CV Acceptable specimen: plasma or serum Anticoagulants Tested: Lithium heparin, ammonium heparin, sodium heparin, EDTA New Information Reference ranges adjusted to reflect recent literature Calibration interval extended to 57 days Clinical Chemistry Specific Proteins Presentation, version 1 82

83 Troubleshooting Tips Clinical Chemistry Specific Proteins Presentation, version 1 83

84 Troubleshooting Tips IgG Imprecision R1 Reagent: 160 ul, R2 Reagent: 160 ul, large volume in cuvettes Check: Mixers R2 sample or reagent probe, syringes, wash, mixer, tubing Clinical Chemistry Specific Proteins Presentation, version 1 84

85 Troubleshooting Tips IgM or Prealbumin Imprecision Any assay using 340nm as the primary wavelength is more sensitive to lamp age. IgM and PAlb are the only specific protein assays using this wavelength (340/700) View the reaction graph, primary wavelength only. Normal lamp function: Flat line between reads 2 and 16 and a smooth line bending from vertical to horizontal between reads 17 and 33 Lamp degradation: Progress curve not smooth (exhibits an erratic saw-tooth appearance) Clinical Chemistry Specific Proteins Presentation, version 1 85

86 Troubleshooting Tips All Specific Protein assays Active ingredient is in R2 component of 2 reagent assay systems R2 dispense and mixing drives the reaction For precision concerns, verify R2 mixer alignment and function, R2 reagent probe dispense and position, and R2 reagent syringe seal tips and o-ring Clinical Chemistry Specific Proteins Presentation, version 1 86

87 Troubleshooting Tips IgA and IgM Saline (0.85% or 0.90% sodium chloride) is used as the diluent for the standard dilution When diluent is not onboard: AEROSET assay button will have black text indicating illegal assay and it will not run until diluent is loaded. c8000 Error code 0218, Unable to process test, no Processing Modules available Clinical Chemistry Specific Proteins Presentation, version 1 87

88 Troubleshooting Tips Barcode errors Round bottles may rotate slightly in adapter when the reagent carousel rotates, moving the bar code label and affecting the ability of the bar code scanner to read the bar code label. Turn the bottle to allow the bar coded scanner to read the reagent bar code label Insert the bottle from the bottom of the adapter so the label is not scraped. Bottles may fit tightly in some adapters. Frequent installation and removal of bottles may damage barcode. If the adaptor is not seated correctly the bottle may tilt resulting in barcode errors Clinical Chemistry Specific Proteins Presentation, version 1 88

89 Questions and Answers Clinical Chemistry Specific Proteins Presentation, version 1 89

90 Questions and Answers Why are linearity claims only up to the highest calibrator? For non-linear assays (C3, C4, Hapt, IgA, IgG, IgM and TRF) accuracy by recovery cannot be guaranteed beyond the highest calibrator. AEROSET uses %EXT to prevent extrapolation and c8000 does not print results above the highest calibrator for non-linear assays. For linear assays (ApoA, ApoB and PAlb) accuracy above the highest calibrator is dependant on calibrator concentrations which vary from lot to lot. AEROSET uses %EXT and c8000 uses the LH parameter to prevent extrapolation. Even though R&D testing supports linearity beyond the high calibrator, linearity was defined as up to the high calibrator. Data from a large cohort of samples indicate very few samples will exceed the high calibrator concentration Clinical Chemistry Specific Proteins Presentation, version 1 90

91 Questions and Answers Why are the calibration modes for ApoA, ApoB, and PAlb linear if the calibration curves are not straight? Although the graphs appear non-linear for these assays, the calibrations approximate a linear response closely enough to insure accurate results across the calibration range. Therefore the calibration modes have been defined as linear for each of these assays Clinical Chemistry Specific Proteins Presentation, version 1 91

92 Questions and Answers When the AEROSET launched the Specific Protein assays, only two levels of controls were run for precision. Now, why are three levels of controls shown for precision? Commercial 3 level controls were used for the c8000 precision testing. The level 3 control used for c8000 testing is approximately the same concentration as the level 2 control used in the original AEROSET testing. The revised package inserts now contain a combination of AEROSET and/or c8000 data. The data was selected based on the highest Total %CV for each control level independent of the instrument it was tested on Clinical Chemistry Specific Proteins Presentation, version 1 92

93 Questions and Answers When a manual dilution is performed for the IgA and IgM assays, what value do I enter for the sample dilution? Enter the manual dilution factor only. The analyzer will automatically adjust for the use of a standard sample dilution. Example, a manual 1:4 dilution is prepared and tested using the standard sample dilution (1:5). Enter 4 for the manual dilution and the analyzer will automatically multiply the measured concentration by 20 (manual dilution X standard sample dilution) to determine the result Clinical Chemistry Specific Proteins Presentation, version 1 93

94 Questions and Answers Why are the linear low and LOQ not the same? LOQ is the lowest analyte concentration at which the CV=20%. Some Specific Protein assays become increasingly inaccurate as analyte concentrations drop below the lowest calibrator and approach LOQ. For this reason the linear low limit for these assays is based on accuracy by recovery studies rather than LOQ studies Clinical Chemistry Specific Proteins Presentation, version 1 94

95 Questions and Answers When a LH (AEROSET ) or > (c8000 ) error code is observed when running the IgA or IgM assay, what should the customer do next? The sample needs to be diluted and rerun. An autodilution can be performed using Dil 2 (AEROSET ) or 1:10 (c8000 ). c8000 Operations Manual does not direct to dilute and rerun. P Clinical Chemistry Specific Proteins Presentation, version 1 95

96 Questions and Answers When a LL (AEROSET ) or < (c8000 ) error code is observed when running the IgA or IgM assay, what should the customer do next? The sample needs to be rerun undiluted. An autodilution can be performed using Dil 1 (AEROSET ) or Undiluted (c8000 ). c8000 Operations Manual does not direct to dilute and rerun. P Clinical Chemistry Specific Proteins Presentation, version 1 96

97 Questions and Answers Why would a LH (AEROSET ) or > (c8000 ) flag be observed when a result is less than the highest calibrator? Operator failed to edit the LH field to equal the high calibrator concentration for a new lot of calibrators. Both the calibrator concentrations and the linear high field must be edited as specified in the calibrator value sheet Clinical Chemistry Specific Proteins Presentation, version 1 97

98 Questions and Answers Why are the following c8000 results observed for the IgM assay? (highest calibrator is 330 mg/dl(3.30 g/l) Std (1:5) = >330 mg/dl. Repeat Manual 1:5 dilution =1856 mg/dl Spline assays, when run using the Standard (1:5) sample dilution on the c8000, will not correct the result for the dilution factor. The analyzer should have multiplied the LH field by the dilution factor and printed the result for the Std (1:5) as > 1650 mg/dl. This will be corrected in a future version of the software Clinical Chemistry Specific Proteins Presentation, version 1 98

99 Any Questions?????? End of Document Clinical Chemistry Specific Proteins Presentation, version 1 99