Validation of reagents and mixes in haematology. Tine Venken

Transcription

1 Validation of reagents and mixes in haematology Tine Venken Molecular Biology and Cytometry Course 23-24/03/2017

2 Overview ISO 15189:2012 Validation of reagents Validation of mixes Daily practice Conclusions 2

3 International Standard ISO 15189: Reagents and consumables Acceptance testing Each new formulation of examination kits with changes in reagents or procedure, or a new lot or shipment, shall be verified for performance before use in examinations. Consumables that can affect the quality of examinations shall be verified for performance before use in examinations.

4 ISO 15189: Reagents and consumables Acceptance testing Each new formulation of examination kits with changes in reagents or procedure, or a new lot or shipment, shall be verified for performance before use in examinations Consumables that can affect the quality of examinations shall be verified for performance before use in examinations Patient risk Broader interpretation Each new formulation of examination kits with changes in reagents or procedure, or a new lot or shipment, or consumables that can affect the quality of examinations, shall be verified for performance before reporting the results 4

5 Validation of reagents Antibody-fluorochrome combination Titration Validation/verification of antibody-fluorochrome combinations and mixes

6 Ab-fluorochrome combination Specific MoAbs with high sensitivity Bright fluorochromes to detect weak antigens Spectral overlap MoAbs titration Standardisation/harmonisation Literature Company Price 6

7 Ab-fluorochrome combination Lymphoid Screening Tube Euroflow Euroflow: LST - Lymphoid Screening Tube FITC PE PerpCP-Cy5.5 PE-Cy7 APC APC-H7 Pacific Blue Pacific Orange CD8 and SmIgλ CD56 and SmIgκ CD5 CD19 and TCRγδ SmCD3 CD38 CD20 and CD4 CD45 ZNA: LST FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 FL9 FL10 FITC FITC PE PE ECD ECD PC5 PC7 APC APC APC- AF700 APC- AF750 APCH7 V450 PB KO CD8 Lambda CD56 Kappa CD34 CD117 CD5 CD19 CD3 CD71 CD10 CD14 CD38 CD20 CD4 CD45 7

8 Reagents Titration Evaluation by intensity Signal to noise ratio (S/N) Separation parameter (SP) # events Fluorescence intensity 8

9 Reagents Titration Evaluation by intensity MFI positive population # events Fluorescence intensity Evaluation by intensity MFI CD3+CD4+ CD3+CD4-1 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/1280 1/12800 MoAb Titration (1/x) 9

10 Reagents Titration Signal to noise ratio # events Fluorescence intensity Signal to noise ratio S/N CD3+CD /2 1/4 1/8 1/16 1/32 1/64 1/128 1/1280 1/12800 MoAb Titratie (1/x) 10

11 Reagents Titration Separation parameter (SP) # events = ² ² Fluorescence intensity SP 4.00 SP CD3+CD /2 1/4 1/8 1/16 1/32 1/64 1/128 1/1280 1/12800 MoAb Titratie (1/x) Optimal titration: 1/16 11

12 Reagents Validation of Abfluorochrome combinations EQC reports SKML Leukemie en lymfoomtypering concordance with reports Efficacy relative to positive (and negative) populations SKML data Commercial control material Specific routine sample with known expression patterns e.g. CD34 in a sample with immature cells Definition of positive and negative internal control e.g. CD19+ = CD3-/CD20+ B-lymphocytes 12

13 Reagents ZNA Leukemia & lymphoma typing 20 tubes 89 Ab-fluorochrome combinations Tube FL9 FL10 FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 Screening LST16 mix 20 & & λ 56 & κ 34 & & & 38 B-cell SB mix κ λ B-cell ERIC mix b B-cell IGH mix FMC7 45 IgA IgD IgM 81 B-cell HCL mix LAIR-1 11c 19 IgG 25 B-cell CLPD2 mix b Blasts AL mix DR & Paraprotein PCD mix cykappa cylambda Mastocytosis MAST DR T-cell T DR a T-cell ST TCRgd NK-cell NK NK-cell SNK c 19 AL/MDS MDS1 DR b 10 AL/MDS MDS2 DR e 14 AL/MDS MDS3 DR AL/MDS AL2 DR AL/MDS AL67 DR a AL/MDS ALOT cyt 3 45 cytmpo cyt79a AL/MDS ALBT DR 45 nutdt

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15 Reagents Example 1 CD15-APC (tube AL2) SKML : #SKML Fluorochrome Marker +/+ -/- +/- -/+ FL6 APC CD Discordancy: SKML Feedback Experts Aberrant population: 58% CD13+CD14-CD15+ (32%)CD19-CD33+CD34-CD36+CD45+CD56-CD64+CD117+HLADR+CyCD3-MPO+ Conclusion: AML 15

16 Reagents Example 1 CD15-APC (tube AL2) Discordancy: SKML Report CD15: heterogeneous (#61 labs) 27 labs: negative 27: positive with variable percentage 4 100% (mean 20%) 7: not included in report 16

17 Reagents Example 1 CD15-APC (tube AL2) Discordancy: SKML Discussion In general, the complete weak CD45/SSC low population was used to describe the aberrant population. In this casus it was difficult to distinguish between the aberrant and the myeloid population heterogeneous results 17

18 Reagents Example 1 CD15-APC (tube AL2) Efficacy SKML2015.2: CD15+ granulocytes Sample : CD15+ blasts 18

19 Reagents Example 2 CD41-CD61 Tube FL9 FL10 FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 AL67 DR a History: all samples negative Extra tube: PLT Tube FL9 FL10 FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 AL a NoPWNoLNoW RBC RBC RBC PLT PLT PLT 19

20 Validation of mixes Validation/verification of antibody-fluorochrome combinations and mixes Determining expiration date 20

21 Mixes Validation of antibodyfluorochrome mixes Lymphoid Screening Tube: LST FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 FL9 FL10 Fluorochrome FITC FITC PE PE ECD ECD PC5 PC7 APC APC APC- AF700 APC- AF750 APCH7 V450 PB KO Marker CD8 Lambda CD56 Kappa CD34 CD117 CD5 CD19 CD3 CD71 CD10 CD14 CD38 CD20 CD4 CD45 Titration 1/ Company A A B A C C C C B B C C B B B C 21

22 Mixes Validation of LST16 mix Goal: Detection of aberrant populations 516 samples Diagnostic (D) - follow up (FU) Bone marrow (BM), peripheral blood (PB), glands, other Result LST16 vs tube FacsCanto II: positive vs negative 22

23 Mixes Validation of LST16 mix Scores Negative: no aberrant population Positive: aberrant population Concordant: aberrant population, in accordance with tube FacsCanto II Discordant: Aberrant population, not in accordance with tube FacsCanto II No aberrant population, although expected Difficult to interprete (NI) Not discordant: no aberrant population, in accordance with tube FacsCanto II 23

24 Mixes Validation of LST16 mix Results Discordant Type # % Number Concordant Not discordant Discordant Negative Positive NI Total (7.8%) PB 2/ CD117+CD34- blasts CD38+ plasmacells BM 12/ CD117+CD34- blasts CD38+ plasmacells Glands 23/ Interpretation/discrimantion of CD3/CD19 and kappa/lambda Titration CD10 en CD5 insufficient for correct characterization of the aberrant population Other 0/

25 Mixes Validation of LST16 mix Discussion >90% aberrant populations detected Type Subtype Number Lowest detected % MBCN CLL/SLL MBL CD HCL FL MCL B-NHL MTCN nvt Aberrant populations of 1 à 2% can be detected 25

26 Mixes Validation of LST16 mix Conclusion LST16 is diagnostic and analytically specific and sensitive Sample type D/FU Clinical indication Tube PB/BM Blasts LST16 + AL Paraprotein LST16 + PCD Other LST16 Glands SB Other LST16 26

27 Mixes Expiration date ZNA: 10 mixes Tube FL9 FL10 FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 Screening LST16 mix 20 & & λ 56 & κ 34 & & & 38 B-cell SB mix κ λ B-cell ERIC mix b B-cell IGH mix FMC7 45 IgA IgD IgM 81 B-cell HCL mix LAIR-1 11c 19 IgG 25 B-cell CLPD2 mix b Blasts AL mix DR & Paraprotein PCD mix cykappa cylambda Lympho subsets Lympho subsets LST7 mix & LST5 mix Performance verification when prepared 27

28 Mixes Expiration date Mix fiche info on datasheet PCD After 1 After 2 month months Date preparation mix 15-Mar Jul-16 Date sample 26-Apr Sep-16 Difference (#days) Sample ID Fluorochrome Marker Mix fiche info OK/NOK OK/NOK FITC CD38 Monocytes HD HD PE CD56 CD19- NK-lymphocytes OK OK PCP5.5 CD138 Plasmacells, not expired! Positive Positive PE7 CD19 CD20+ B-lymphocytes LD LD V450 CD20 CD19+ B-lymphocytes OK OK V500 CD45 Leucocytes OK OK Expiration date OK OK All Ab mixes meet the criteria described on the mix sheet, even after 2 months 28

29 Daily practice Performance verification of new lot/shipment

30 ISO 15189: Reagents and consumables Acceptance testing Each new formulation of examination kits with changes in reagents or procedure, or a new lot or shipment, shall be verified for performance before use in examinations Consumables that can affect the quality of examinations shall be verified for performance before use in examinations Patient risk Broader interpretation Each new formulation of examination kits with changes in reagents or procedure, or a new lot or shipment, or consumables that can affect the quality of examinations, shall be verified for performance before reporting the results 30

31 Performance verification of new lot/shipment Risk analysis acceptance criteria Ongoing verification Only allowed if sufficient patient material available to repeat the test, if an antibody (mix) does not to meet the acceptance criteria (after the analysis, but before reporting the results to the clinic) Verification/acceptance well documented before reporting the results 31

32 Performance verification Situations A: Abs seperately pipetted in a fixed combination and concentration B: Batch of a mix of Abs in a fixed combination and concentration (home made/company) C: Abs added to a known mix D: Abs against Ags, not present on normal samples 32

33 Performance verification Efficacy relative to positive (and negative) populations Commercial control material Acceptance criteria defined on worksheet Acceptance in URT Westgard rules Levey Jennings chart Specific routine sample with known expression patterns e.g. CD34 in a sample with immature cells Definition of positive and negative internal control e.g. CD19+ = CD3-/CD20+ B-lymphocytes 33

34 Performance verification Acceptance Only positive results Negative results Ongoing validation Abs not expired Critical/non critical reagents 34

35 Performance verification Commercial control material (B) 35

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37 Performance verification Commercial control material (B) 37

38 Performance verification Specific routine sample with known expression patterns (B, C) Sample : 0.4% plasmacells: Positive: cyt lambda, CD138 HD: CD38, CD56 Negative: CD19 38

39 Performance verification Sample : 0.4% plasmacells: Positive: cyt lambda, CD138 HD: CD38, CD56 Negative: CD19 39

40 Performance verification Routine sample (A, D) 40

41 Performance verification Mix fiche info CD45, CD4, HLADR Immature cells CD34, CD117 cfr. other tubes PLT: CD41, CD61, CD235a 41

42 Conclusions

43 Conclusions Broader interpretation Each new formulation of examination kits with changes in reagents or procedure, or a new lot or shipment, or consumables that can affect the quality of examinations, shall be verified for performance before reporting the results Acceptance criteria IQC Critical/non critical Timing Appropriate (routine) samples Control material NVC: 43

44 Thank you for your attention!

45 Validation of reagents and mixes in haematology Tine Venken Molecular Biology and Cytometry Course 23-24/03/2017