Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations

Presentation on theme: "Validation procedures for cell analyzers Dr Archana Vazifdar Dept. of Hemato-Pathology, Super Religare Laboratories Limited, Mumbai."— Presentation transcript:

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 > 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 CELL-DYN COULTER Platelet count Spurious increase: RBC/ WBC fragments Cryoglobulins Lipids Spurious decrease: Platelet clumps Giant platelets

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

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 FLAG: Anemia, Microcytosis, anisocytosis Hb 8.5 RBC 3.2 Left shift of curve: Microcytosis Iron Deficiency Anemia β thalassemia trait Anemia of chronic diseases

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

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

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

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

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

17 Dual/Combined deficiency 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 S. B

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

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

20 Short sample (microtainer) Repeat collection 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

21 Platelets Smallest 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 10 9 /l must be aware of the limitations in precision and accuracy of cell counters Linearity : 10–1,000 X 10 9 /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 Increased small sized particles: Noise, debris, lipids, bacteria, fungi ? Wiskott Aldrich syndrome Conclusion: Falsely elevated platelet counts Flags: Small platelets Debris/ noise

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

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

26 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 WBC Flags

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

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

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

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

31 V CS: Quantitative Operator independent Routinely available Inexpensive INCREASE MEAN NEUTROPHIL VOLUME (MNV) DECREASE MEAN NEUTROPHIL SCATTER (MNS) – left shift –Lacking leukocytosis or neutrophilia Newer Aspects: VCS-Neutrophil population data 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 Normal Plasmodium falciparum Monocytes Reactive LY Parasitized RBC Vol SD lymphocyte X SD Monocyte / 100 > 3.7 Am J Clin Pathol 2006;126: Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY shoulder

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 37 ̊C Cold agglutinin disease

38 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 Case 2

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

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

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

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

Similar presentations

Ads by Google