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

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

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 RBC-Plt- separated by Volume & refractive index based counting/sorting MCV does not exceed 150–160 fl and, as there is no particle above that size in the blood stream in health or in disease.

Interpretation of data 3 steps for performing a CBC- interpretation of numeric, graphic & flag data, Delta checks, Review of peripheral smear

Normocytic Normochromic RBC count Spurious increase: Giant PLT High WBC counts (>50) Spurious decrease: Cold /warm agglutinins Very small RBC Cryoglobulins As cell passes through an orifice, the change in resistance produced is measured as a single puls No. of pulses= no. of cells Size of pulse= volume of cell All info is then depicted on a histogram RBC histo- X axis, volume, Y axis, No. of RBC, MCV- mean of distribution curve

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

WBC (FCM) Impedance- VCS Baso,mono, eos, blasts neutro lympho The traditional microscopic method based on the count of 100 cells has 3 types of error: statistical error, distributional error owing to unequal distribution of cells in the smear, and error in identifying cells related to the subjective interpretation of the examiner. Newer analyzers analyze thousands of cells per sample and can produce morphologic and quantitative flags, which have significantly reduced error and allow reliable absolute counts at low and high concentrations. WBC analyzed by fcm, using impedance (VCS), Optical scatter (cytochem) VCS- Simultaneous measurement of volume, conductivity and scattered laser light, CELLS R ANALYZED IN NEAR- NATIVE STATE. x-axis represents laser light scatter, the y-axis volume and the z-axis conductivity Normal WBC histogram Normal WBC scatterplot

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 EDC include immature or atypical cells such as blasts, IGs, atypical lymphocytes, hematopoietic progenitor cells (HPCs), and NRBCs.70 The principal aims of the EDC are to further reduce the need for microscopic revision, to obtain more precise and accurate counts when elevated basophil/ monocyte counts are produced, they must be examined with caution because they can be artifacts due to the presence of abnormal cells such as blasts, plasma cells, and lymphoma cells.

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

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

Anemia, Microcytosis, anisocytosis Hb 8.5 RBC 3.2 FLAG: Anemia, Microcytosis, anisocytosis Left shift of curve: Microcytosis Iron Deficiency Anemia β thalassemia trait Anemia of chronic diseases IDA & ACD- NORMAL RDW

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

N’rbc, Micro RBC/ RBC fragments Giant plt Thrombocytopenia Hb 6.4 PLT 140 MPV 7.9 PCT .148 PDW 15 Flags: N’rbc, Micro RBC/ RBC fragments Giant plt Thrombocytopenia FRBCs are identified only on the basis of size and hemoglobin content, independent of their shape Lt of curve not touching baseline: Noise Schistocytes &/ extremely small rbc Giant platelets

Hemolytic anemia 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

Dimorphic RBC population, anisocytosis 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 NN anemias: Hypoplastic, malignancy, Sideroblastic (low retic) Acute bld loss, Sev hemolysis, AIHA, G6PD, Sickle, Action: Review PS to identify cause

50/ F, Hb-8.9, MCV-73, MCH- 25.6, RDW-26.8 Blood transfusion Volume/Hemoglobin Concentration (V/HC) cytogram hemoglobin concentration (chromia) is plotted along the x axis and cell volume/size is plotted along the y axis, Nine zone grid, central zone NN Blood transfusion

Hb-5.1, MCV-96.7, MCH- 29.6, MCHC-31.4, RDW-24.5 TLC/Plt-Normal 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

H&H error, N’rbc, dimorphic reds Anemia, macrocytosis, anisocytosis 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

Review PS: L/F agglutination vs n’rbc’s Action: Review PS: L/F agglutination vs n’rbc’s After warming in H2O bath @ 37ºC for 15 mins corrected spuriously low RBC counts and to abnormally high MCV (each small RBC clump is considered as one single particle Haematocrit (RBC · MCV) is erroneous and spuriously low, contrasting with Hb that is measured after RBC lysis and is unaffected by agglutinins. As a rule, the MCHC is spurious, usually >36 g/dl. Conclusion: False ↓ RBC, Hct, False ↑ MCV, MCH & MCHC Cold agglutinin disease

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

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 109/l must be aware of the limitations in precision and accuracy of cell counters Linearity : 10–1,000 X 109/l Thresholds for use of prophylactic plt tx has reduced frm 20 × 103/µL to 10 × 103/µL (20 × 109/L to 10 × 109/L). Other Authors have suggested that in patients without fever or bleeding, there may be even lower values. However, the utilization with confidence of these new thresholds requires knowledge of the limitations in precision and accuracy of the analyzers at these count levels.

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

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

Falsely ↓ Plt count, ↑MPV 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

45/M PIC/POC delta Excessive noise included in impedance count Debris, bacteria, fungi Plt clumps Giant plt CAUSE- Plt clumps, giant plt, ….?

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

Shoulder on the left of curve: N’rbc Lyse resistant RBC Platelet clumps/ Giant platelets Fibrin Impedance noise N’RBC: neonatal hemolytic disease, in premature neonates and neonates affected by hypoxia in the perinatal period. adults: thalassemic syndromes, myeloproliferative diseases (myelofibrosis), bone marrow metastases of solid tumors, extramedullary hematopoiesis, and all of the conditions of hematopoietic stress (eg, septicemia, massive hemorrhage, and severe hypoxia) Resistant RBC’s: physiological (neonates) pathological (abnormal hb, liver disease, uraemia, chemotherapy) extended lysis mode

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

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

38/F, k/c/o DM Plt 100 Flag: leukocytosis, n’rbc, dimorphic reds it is useful not only to identify the presence of NRBCs, but also to estimate the NRBC count: 1.to evaluate the efficacy of transfusion therapy, as with thalassemic syndromes in which it is advisable to maintain an NRBC concentration of less than 5/100 WBCs 2. persistence of NRBCs in the peripheral blood of subjects undergoing stem cell transplantation has been shown to be a poor prognostic factor 3. hospitalized patients after general or cardiothoracic surgery or with other nonhematologic disease, the mortality rate was 21.1% for patients with NRBCs, whereas it was 1.2% for patients without Flag: leukocytosis, n’rbc, dimorphic reds Conclusion: 21 nrbc’s/100 wbc- corr WBC= 17.35 DM in sepsis with liver abscess

VCS-Neutrophil population data 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

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

Vol SD lymphocyte X SD Monocyte / 100 > 3.7 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

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

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 CHr- reduction indicates iron-deficient erythropoiesis, even in conditions in which traditional biochemical markers such as ferritin and transferrin are inadequate (eg, in cases of inflammation or anemia from chronic disease), and, on the other hand, it is useful for monitoring early response to intravenous iron therapy because it increases significantly after only 48 hours. exceptions are heterozygotes for β-thalassemia whose CHr is always reduced independent of iron stores.

Case 1 38/M, No history available

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

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

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

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

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

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

Case 4: 15/M, Fever

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

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