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Validation procedures for cell analyzers

Validation procedures for cell analyzers. Dr Archana Vazifdar Dept. of Hemato-Pathology, Super Religare Laboratories Limited, Mumbai. Principles of automation. Impedance – count and size cells by change in resistance produced as they are suspended in an electrically conductive medium

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Validation procedures for cell analyzers

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  1. Validation procedures for cell analyzers Dr Archana Vazifdar Dept. of Hemato-Pathology, Super Religare Laboratories Limited, Mumbai

  2. Principles of automation • Impedance – count and size cells by change in resistance produced as they are suspended in an electrically conductive medium • Optical scatter- measures scatter properties of cells by laser light • Single angle/ Multi-angle scatter

  3. RBC & Platelets measured in one channel • RBC volume > 30-36 fl • Platelet volume 2-20 fl • Hb & WBC measured in second channel • DLC in third channel

  4. Interpretation of data

  5. Normocytic Normochromic • RBC count • Spurious increase: • Giant PLT • High WBC counts (>50) • Spurious decrease: • Cold /warm agglutinins • Very small RBC • Cryoglobulins

  6. ADVIA 120 COULTER • Platelet count • Spurious increase: • RBC/ WBC fragments • Cryoglobulins • Lipids • Spurious decrease: • Platelet clumps • Giant platelets CELL-DYN

  7. WBC (FCM) Impedance- VCS Baso,mono, eos, blasts neutro lympho Normal WBC histogram Normal WBC scatterplot

  8. Optical scatter: ADVIA120 DLC by Peroxidase method • Spurious increase • PLT clumps & large platelets • Nucleated red cells • Resistant RBC’s • Spurious decrease: • Clotted sample • Fragile cells- CLL • Lymphoid aggregates- UTI, B- cell NHL, CMML • Storage associated degeneration

  9. Flags • A signal to the operator that the analyzed sample may have a significant abnormality/ does not meet acceptance criteria/ cannot be displayed • Cause of errors: • Analyzer • Sample • Random run error

  10. RBC flags Suspect flags • N’rbc, R’rbc, Micro RBC, RBC fragments, • interfere with WBC & platelet counts • H & h errors • short sample, aged sample Definitive flags • Anemia, anisocytosis, microcytosis, macrocytosis, poikilocytosis • Erythrocytosis

  11. Hb 8.5 RBC 3.2 FLAG: Anemia, Microcytosis, anisocytosis • Left shift of curve: • Microcytosis • Iron Deficiency Anemia • βthalassemia trait • Anemia of chronic diseases

  12. ACTION: • RBC indices • Mentzer’s index (MCV/RBC)= 18.3 • MI ≤ 13- BTT, ≥ 13- IDA Conclusion: s/o Iron Deficiency Anemia Advise Iron studies

  13. Hb 6.4 PLT 140 MPV 7.9 PCT .148 PDW 15 • Flags: • N’rbc, Micro RBC/ RBC fragments • Giant plt • Thrombocytopenia Lt of curve not touching baseline: Noise Schistocytes &/ extremely small rbc Giant platelets

  14. Action: • RBC Indices- MCV, RDW • PLT Histogram- MPV & PDW • Review PS- RBC morphology • -PLT count (100) Conclusion: RBC count falsely ↓ Platelets falsely ↑ (mask t’penia) Hemolytic anemia

  15. Hb- 8.6, MCH- 26.5, MCHC- 32.2 Flags: Dimorphic RBC population, anisocytosis Bimodal peak: Dimorphic RBC population Transfused cells Combined deficiency Therapeutic response in IDA Action: Review PS to identify cause

  16. 50/ F, Hb-8.9, MCV-73, MCH- 25.6, RDW-26.8 Blood transfusion

  17. 45/F, Severe pallor Hb-5.1, MCV-96.7, MCH- 29.6, MCHC-31.4, RDW-24.5 TLC/Plt-Normal S. Fe- 25 TIBC- 144 S. Fe saturtn- 20.8 S. B12- 158 Dual/Combined deficiency

  18. H&H Spurious ↑MCHC: Flags: H&H error, N’rbc, dimorphic reds Anemia, macrocytosis, anisocytosis • Sample related problems- turbidity-↑ Hb • Lipemia/ TPN • Cryoglobulins • Autoagglutination • Hemolysis (in-vitro/vivo) • Spurious ↓ Hct • Clotted sample Right portion of curve extended: RBC agglutination N’rbcs Leukocytosis

  19. Action: Review PS: L/F agglutination vs n’rbc’s After warming in H2O bath @ 37ºC for 15 mins corrected Conclusion: False ↓ RBC, Hct, False ↑ MCV, MCH & MCHC Cold agglutinin disease

  20. Causes of H&H mismatch: • partial sample aspiration/ improper mixing • Hb/ MCV measurement error/ very low • High WBC counts (interfere with Hb measurment) • Cold agglutinins Short sample (microtainer) Repeat collection

  21. PlateletsSmallest guys largest culprits!! • As platelet counts fall, reliability of analyzer decreases. • Conventional methods are unable to provide consistently accurate results in lower range • Clinicians using thresholds of 5-10 X 109/l must be aware of the limitations in precision and accuracy of cell counters Linearity : 10–1,000 X 109/l

  22. Common platelets flags • PLT Clumps • ↓Plt counts • Interferences with WBC Results (↑WBC counts) • Giant platelets • Small platelets • PIC/POC delta- difference > 20,000 • Thrombocytopenia- true/false

  23. Flags: Small platelets Increased small sized particles: Noise, debris, lipids, bacteria, fungi ? Wiskott Aldrich syndrome Debris/ noise Conclusion: Falsely elevated platelet counts

  24. Flags: Giant platelets, platelet clumps Cellular interference Action: Review PS for platelet count Non fitted curve with increase in large cells: Large platelets, clumps Conclusion: Falsely ↑RBC count Falsely ↑WBC count Falsely ↓ Plt count, ↑MPV Giant platelets

  25. 45/M • PIC/POC delta • Excessive noise included in impedance count • Debris, bacteria, fungi • Plt clumps • Giant plt

  26. WBC Flags IG, Band, Blasts Aty ly, Variant ly MPO, non viable WBC N’RBC, rst RBC Plt clump Outside Reportable Range Leukocytosis, monocytosis, basophilia, eosinophilia Unable to Find Clear Separation between WBC subpopulations

  27. Shoulder on the left of curve: N’rbc Lyse resistant RBC Platelet clumps/ Giant platelets Fibrin Impedance noise

  28. CML Flags: IG, Blasts, eosinophilia,monocytosis, lymphopenia Leukocytosis Thrombocytosis Anemia

  29. Flags: Aty lymphocyte, Variant lymphocyte Non-viable wbc Leukocytosis T’penia Acute Leukemia

  30. 38/F, k/c/o DM Plt 100 Flag: leukocytosis, n’rbc, dimorphic reds Conclusion: 21 nrbc’s/100 wbc- corr WBC= 17.35 DM in sepsis with liver abscess

  31. Newer Aspects: VCS-Neutrophil population data • VCS: • Quantitative • Operator independent • Routinely available • Inexpensive • INCREASE MEAN NEUTROPHIL VOLUME (MNV) • DECREASE MEAN NEUTROPHIL SCATTER (MNS) – left shift • Lacking leukocytosis or neutrophilia Suggestive of acute bacterial sepsis

  32. Automated malaria detection • “Gold standard” - thick & thin smear • Need for rapid, sensitive & cost-effective screening technique • Hemazoin pigment • Activation of neutrophils & monocytes • Increase volume heterogeneity (anisocytosis) of monocytes & lymphocytes, detected by VCS • ‘Positional parameters’, used as objective criteria for detecting presence of plasmodium Clin. Lab. Haem., 26, 367–372 Automated detection of malaria

  33. Plasmodium falciparum Normal Monocytes Reactive LY Parasitized RBC shoulder Vol SD lymphocyte X SD Monocyte / 100 > 3.7 Am J Clin Pathol 2006;126:691-698 Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY

  34. Specificity is 94% and sensitivity 98% • PPV is 70% and NPV 99.7%. • A flag indicating potential presence of malaria is a valuable diagnostic method for detection of malaria and may become a routine parameter in it’s diagnosis

  35. Reticulocyte Indices • most promising from a clinical viewpoint are the CHr and the MCVr. • CHr: • directly reflects hemoglobin synthesis in marrow, & measures iron availability. • ↓ IDA & BTT (independent of iron stores) • MCVr: ↑rapidly following iron therapy • ↓ with the development of iron-deficiency • ↓ in macrocytosis after therapy with B12 &/or folic acid • Available in very few analyzers, not standardized

  36. Case 1 38/M, No history available

  37. Result after treatment in H20 bath @ 37 ̊C Cold agglutinin disease

  38. Case 2 27/M, Hb 7, MCV 94, MCH 32, MCHC 35.7, RDW 14.6, Plt 158 Flags: Blasts, IG, n’rbc, rbc fragments, giant platelets

  39. 50 nrbc’s/100 WBC Spherocytes + Giant platelets Conclusion: Severe hemolysis following Primaquine ingestion in G6PD deficiency

  40. Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding. All other parameters WNL, ? ITP Flags: n’rbc, micro rbc/ rbc fragments

  41. Action: Change anticoagulant to Sodium Citrate Platelet count- 243 Conclusion EDTA dependant pseudothrombocytopenia (EDP)

  42. EDP EDTA dependant pseudothrombocytopenia (EDP): • Hypothesis- antigen-binding site in the GPIIb/IIIa complex , normally hidden/cryptic, is modified by or exposed only in presence of EDTA • In-vitro phenomena • Associated with autoimmune/ neoplastic pathology, but also seen in healthy individuals • Abnormal plt from CMPD, more prone to clumping by EDTA • Alternate anticoagulants; 10% trisodium citrate/ ACD

  43. Case 4: 15/M, Fever

  44. Malaria discriminant factor= 6.3 Conclusion: Plasmodium falciparum , PI 15% Thrombocytopenia

  45. THANK YOU Archana Vazifdar, M.D. SRL RELIGARE LTD.

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