CONCURRENT 10: THE COMPLETE BLOOD COUNT AND BEYOND: QUALITY ISSUES AND REFERENCE METHODS

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1 CONCURRENT 10: THE COMPLETE BLOOD COUNT AND BEYOND: QUALITY ISSUES AND REFERENCE METHODS Saturday May 14 th :30 3:00 PM ( hrs) International Society for Laboratory Hematology (ISLH)

2 CONCURRENT 10: THE COMPLETE BLOOD COUNT AND BEYOND: QUALITY ISSUES AND REFERENCE METHODS CHAIRS: Dr. Piero Cappelletti. SIPMEL, Pordenone, Italy Dr. Albert Huisman, UMC Utrecht, Utrecht, Netherlands International Society for Laboratory Hematology (ISLH)

3 Verification and Quality Control of automated Hematology Analyzers Dr. Albert Huisman, UMC Utrecht, Utrecht, Netherlands International Society for Laboratory Hematology (ISLH)

4 Verification and Quality Control of automated Hematology Analyzers Dr. Albert Huisman, UMC Utrecht, Utrecht, Netherlands International Society for Laboratory Hematology (ISLH)

5 Disclosure information: Dr Albert Huisman: Nothing to disclose The UMC Utrecht, department of Clinical Chemistry and Hematology has received funding for contract research from: Abbott Diagnostics Beckman-Coulter Diagnostic Grifols Mechatronics

6 Verification and quality control of automated Hematology Analyzers Automated Cell Analysis (Complete Blood Count (CBC))» Including 5 part Leukocyte differential count, reticulocyte count and new parameters (Reticulocyte Hemoglobin content Reticulated Platelets / Immature Platelet Fraction, ) Validation of a new analyzer Verification of a new analyzer ISO CLSI, ICSH, others Samples Precision Accuracy and comparability Sensitivity and specificity Reference intervals Quality control Daily / internal External Quality Assesment (EQA/Proficiency testing)

7 Why? Why bother about verification and quality control of a highly automated analyzer?

8 Because: CBC results form the starting point of numerous diagnostic schedules, treatments and interventions, for example: Erytrocyte / Platelet transfusions Work up of anemia (iron deficiency/ thalassemia / ) Hematological malignancies General work-up in various disease states Etctetera

9 Because: The ISO standard [Medical laboratories Particular requirements for quality and competence] requires a verification process

10 Advantages of Automated Cell Analysers: Excellent analyticalperformance Closed-tube analysis No inter-observer variability No slide distribution error Eliminate statistical variations Potential of reflex testing Availability of extra parameters e.g. MCV, RDW, %rp, More efficient (> 100 analyses/hour) and cost effective than manual method.

11 Automated cell counters: Complete Blood Count (CBC): Hemoglobin (Hb) concentration, RBC count & RBC indices (MCV, MCH, MCHC), WBC count, PLT count WBC differential count (5 normal WBC s) RDW, MPV Reticulocyte count Nucleated RBC count (NRBC s). Flagging, etc etc New parameters: Advanced RBC parameters: % microcytic RBC s, Reticulocyte Hemoglobin content,.. Reticulated Platelets / Immature Platelets.

12 Further advances in automation Multiple in line analysers Built-in Slide-Maker-Stainer Auto-validation in LIS Automated reflex/morphology etc. Including digital morphology or flow-cytometry solutions All Full blood analysis in line (ESR, HbA1c,..) Total Laboratory Automation Digital Morphology

13 Validation of a new analyzer: goal Provision of objective evidence that a hematologyanalyzer fulfills specified requirements (where the specified requirements are adequate for intended use). Validation is primarilya manufacturersresponsibilityto ensure that design goals are met and performance claims are stated (including safety and effectiveness). Validation study (manufacturer): collect data to support a regulatory submission and the registration of a hematology analyzer.

14 Validation of a new analyzer: goal Provision of objective evidence that a hematologyanalyzer fulfills specified requirements (where the specified requirements are adequate for intended use). Validation is primarilya manufacturersresponsibilityto ensure that design goals are met and performance claims are stated (including safety and effectiveness). Validation study (manufacturer): collect data to support a regulatory submission and the registration of a hematology analyzer.

15 Validation of a new analyzer (manufacturer) Objectives of a validation study: Generate data to assess safety and clinical efficacy from a medical perspective Develop performance information for labelingand marketing purposes Validate appropriate operationalperformance characteristics of the hematologyanalyzer in a typical end-user setting Develop data that are used to support submissionof the product for approval or clearance by regulatory bodies Source: CLSI H26A2

16 Validation of a new analyzer (manufacturer) Validation performance specifications: Limit of blank (LoB, background) Carryover Imprecision (reproducibility), short term and long-term Analytical measuring interval (AMI) (linearity) Lower limit of detection (LLoD) and lower limit of quantitation (LLoQ) Comparibility (correlation) Interferences Frequency and type of data invalidations Source: CLSI H26A2

17 Validation of a new analyzer (manufacturer) Performance specifications: current reference methods*: Selective microscopy for WBC differential RBC count and WBC count (impedance) Selective microhematocrit (PCV) for hematocrit (HCT) Hemoglobin by hemiglobincyanidemethod Selective PLT monoclonalantibody (contemporary reference method) Reticulocytesbyflow cytometry * Standardized and independent of manufacturer Source: CLSI H26A2

18 Validation of a new analyzer (manufacturer) Performance specifications: current reference methods: When no reference method is available: usuallycomparison with previous generation instrument Source: CLSI H26A2

19 Validation of a new analyzer (manufacturer) Currentreference methods, problems, need for improvement: Current reference methods are more or less outdated e.g.: microscopic 400 cell WBC differential count: elaborative and imprecise Huisman et al. Clin Lab Med 2015

20 Validation of a new analyzer (manufacturer) Currentreference methods, problems, need for improvement: Current reference methods are more or less outdated e.g.: microscopic 400 cell WBC differential count: elaborative and imprecise No reference methods available for clinically relevant parameters*: MCV Extended RBC parameters Reticulocyte Hbcontent MPV Reticulated platelets/immature platelet fraction. * Clinically relevant parameters are not standardized! This may lead to differences between different Hematology analyzers: Differences in clinical interpretation (confusion for clinicians) May prevent wider use Invalid aggregation of data Huisman et al. Clin Lab Med 2015

21 Validation of a new analyzer (manufacturer) Currentreference methods, problems, need for improvement: Current reference methods are more or less outdated e.g.: microscopic 400 cell WBC differential count: elaborative and imprecise No reference methods available for clinically relevant parameters: MCV Extended RBC parameters Reticulocyte Hbcontent MPV Reticulated platelets/immature platelet fraction. Urgent need for improvement, role for professional societies Huisman et al. Clin Lab Med 2015

22 Verification

23 Instrument Verification by the end user laboratory The end user should asses whether the manufacturers claims on performance of the specific instrument also applyto the intended use criteria set by the laboratory: Verification Huisman et al. Clin Lab Med 2015

24 Instrument Verification by the end user laboratory The verification process includes performance analysis of: Accuracy Precision Reportable range of test results and reference intervals (normal ranges) Background (limit of blank) Carryover (sample) Lower limits of detection Quantitation Clinically reportable intervals (CRIs) Huisman et al. Clin Lab Med 2015

25 Instrument Verification by the end user laboratory According to ISO a certain level of verification of any new (hematology) analyzer has to be done (preferably) accordingto a professional standard. ISO also stimulates laboratories to take patient risk factors into consideration to meet these standards There are currenly 2 internationaldocuments available for verification of a hematologyanalyzer: Consensus documents with recommendations (not based on strong evidence but rather on expert opinion ). Huisman et al. Clin Lab Med 2015

26 Instrument Verification by the end user laboratory According to ISO a certain level of verification of any new (hematology) analyzer has to be done (preferably) accordingto a professional standard. ISO also stimulates laboratories to take patient risk factors into consideration to meet these standards There are currenly 2 internationaldocuments available for verification of a hematologyanalyzer: Clinical Laboratory Standards Institute (CLSI) (standard H26A2) published in 2010 International Council on Standardisation in Hematology (ICSH) guideline published in Huisman et al. Clin Lab Med 2015

27 Consensus documents with recommendations CLSI ICSH (published in the International Journal of Laboratory Hematology, the official ISLH journal

28 Instrument Verification by the end user laboratory Verification CLSI The laboratory (enduser) should follow the procedures of manufacturer validation, but the verification may be abbreviated; the goal is to verify that the manufacturer s stated performance is correct ICSH Verification is conformation of the evaluation performed by the manufacturer or published in the literature; the verification may be abbreviated (ie, focused to meet specific requirements at the test site) Precision/imprecision (within-run reproducibility, closeness of agreement between test results Should be performed with normal samples and samples at medical decision levels available in clinical laboratories. No specifications for number of measurements and reporting Single run of 10 measurements on the same sample (all reported parameters), 3 levels (normal, abnormal low, and abnormal high, around clinical decision points). Reported as SD and %CV

29 Instrument Verification by the end user laboratory Verification CLSI The laboratory (enduser) should follow the procedures of manufacturer validation, but the verification may be abbreviated; the goal is to verify that the manufacturer s stated performance is correct ICSH Verification is conformation of the evaluation performed by the manufacturer or published in the literature; the verification may be abbreviated (ie, focused to meet specific requirements at the test site) Precision/imprecision (within-run reproducibility, closeness of agreement between test results Should be performed with normal samples and samples at medical decision levels available in clinical laboratories. No specifications for number of measurements and reporting Single run of 10 measurements on the same sample (all reported parameters), 3 levels (normal, abnormal low, and abnormal high, around clinical decision points). Reported as SD and %CV

30 Instrument Verification by the end user laboratory Precision (between batch, longterm Comparability (comparison between evaluation HA and current HA) CLSI Should be performed with normal samples and samples at medical decision levels available in clinical laboratories. No specifications for time period and number of measurements Should be performed but not specified, if different modes are available, whether an extensive mode-to mode comparability should be performed ICSH Single sample, repeated daily, for a period of 20 30d. Three levels (all parameters, abnormal low and abnormal high, around clinical decision points). Fixed blood (control material) may be required At least samples, measured on both HA with samples with various disorders and with interfering substances

31 Instrument Verification by the end user laboratory Precision (between batch, longterm Comparability (comparison between evaluation HA and current HA) CLSI Should be performed with normal samples and samples at medical decision levels available in clinical laboratories. No specifications for time period and number of measurements Should be performed but not specified, if different modes are available, whether an extensive mode-to mode comparability should be performed ICSH Single sample, repeated daily, for a period of 20 30d. Three levels (all parameters, abnormal low and abnormal high, around clinical decision points). Fixed blood (control material) may be required At least samples, measured on both HA with samples with various disorders and with interfering substances

32 Instrument Verification by the end user laboratory Accuracy (closeness of agreement between measurement and true value) Reference intervals CLSI Should be performed; not otherwise specified Must be established or verified for all reportable parameters ICSH Depending on availability of reference method, often not applicable; in practice often compared with current HA Should be calculatedfor all components of the CBC; at least 120 samples from apparently healthy individuals (60 male, 60 female)

33 Instrument Verification by the end user laboratory Accuracy (closeness of agreement between measurement and true value) Reference intervals CLSI Should be performed; not otherwise specified Must be established or verified for all reportable parameters ICSH Depending on availability of reference method, often not applicable; in practice often compared with current HA Should be calculatedfor all components of the CBC; at least 120 samples from apparently healthy individuals (60 male, 60 female)

34 Instrument Verification by the end user laboratory Samples: Beware of pre-analytical variables! Time is one of the most important variables due to time dependent alterations of cells (volume and morphology) Usually anonymous surplus material Broad range of underlyingpathology(garantee that all cells are recognized ) Results should encompass the entire analyticalrange (very low (e.g. extreme trombocytopenia) to very high levels (e.g. extreme leucocytosis / CML). Age and gender (pediatricsamples?) All types of tubes that may enter the laboratory

35 Instrument Verification by the end user laboratory Huisman et al Clin Lab Med 2015

36 Instrument Verification by the end user laboratory: Precision Accuracy is the proximity of a measurement result to the true value and mainly dependent on systematic error (the term bias should be avoided); precision is the reproducibility of the measurements and mainly dependent on random error. Huisman et al IJKH 2016

37 Precision The desired precision of a CBC parameter is dependenton the biological variation of this parameter Work of George Cembrowski et al (Clin Lab Med 2015; IJLH 2016).

38 Cembrowski et al IJLH 2016

39 Precision: In order to generate (clinically) relevant results the analytical precision (%CV reproducibility) of a CBC result should be less than half and preferableless than ¼ of the biological variation. State of the art possibilities of current generation of HA

40 Instrument Verification by the end user laboratory

41 Instrument Verification by the end user laboratory De analytical %CV does add to the total %CV

42 Instrument Verification by the end user laboratory Room for improvement: Reticulocytes WBC WBC differential

43 Instrument Verification by the end user laboratory Carry-over, is it important? Background, is it important?

44 Instrument Verification by the end user laboratory Carry-over, is it important? Example: carry over 1%, Sample 1: platelet count 1000 x 10 9 /L Sample 2: platelet count 10 x 10exp 9 /L (subsequent sample) A carry-over of 1% will result in a falsely increased platelet count of 20 x 10 9 /L (100% increase)

45 Instrument Verification by the end user laboratory Background, is it important? A background (for example in the WBC channel) can lead to false positive results in CSF samples

46 Instrument Verification by the end user laboratory Reference: Tracy George et al.

47 Quality Control Internal Quality Control Performed on a daily basis Electronic built in quality control flags Are there mechanical / electronical problems? Control materials often provided by manufacturer Usually 3-control levels (low-/medium-/high- level) ~Wide range Often manipulated blood 1 versus multiple times/day depending on stability of analyser Comparison between different analysers possible Expensive 3.5 SD (Cembrowski et al)

48 Quality Control Internal Quality Control Statistical QC based on generated results Moving Average Built in software or LIS Large workload required Always Available Cheap

49 Quality Control External Quality Control / Proficiency testing Several times/year How do you perform in comparison with other labs and other types of equipment? ¹Calibrator

50 Acknowledgements: Jolande Vis Sue Ellen Verbrugge

51 Thanks!

52 Contact: Dr Albert Huisman University Medical Center Utrecht Department of Clinical Chemistry and Haematology G Heidelberglaan CX Utrecht Netherlands

53 Thank you for being with us!

54 See you at ISLH 2017 in Honolulu, Hawai ISLH 2017

55 See you at ISLH 2017 in Honolulu, Hawai ISLH 2017 May