Automated cell counters- THE BASICs Pankhi Dutta MD DM (haematopath) Consultant haematopathologist SevenHills Hospital, Mumbai.

Introduction Automated cell counter-backbone of the haemat lab Wallace Coulter in 1956 – impedance method Various technologies today More accurate, more precise reports at a faster rate Basic CBC + newer parameters Inherent technological limitations

Basic parameters – 3 part counter NO. 4 DATUM: 9/10/95 15:11 MODE: VOLLBLUT WBC 5,8 x 103/µl RBC 4,84 x106/µl HGB 13,7 g/dl HCT 42,0 % MCV 86,8 fl MCH 28,3 pg MCHC 32,6 g/dl PLT 257 x103/µl LYMPH% 31,2 % MXD% 6,8 % NEUT% 62,0 % LYMPH# 1,8 x103/µl MXD# 0,4 x103/µl NEUT# 3,6 x103/µl 250 RBC RDW-SD 40,0 fl 40 PLT PDW 13,1 fl MPV 10,4 fl P-LCR 28,1 % WBC 300 Haemoglobin RBC, WBC, PLT count Red cell indices RDW 3-part differential Histograms

3-part differential analyser Two chambers Hb + WBCs RBCs + PLTs Vacuum Blood cell DC supply Registor (constant current) Internal electrode External electrode Aperture Transducer chamber Blood cell suspension

Haemoglobin estimation 1. Lysis of RBC Haemoglobin molecule Fe2+   Fe2+ Fe2+   Fe2+ Ammonium salts     Fe2+ Fe2+ Fe2+ Fe2+ RBC RBC 3-part Diff technology V0706

2. Change of conformity Haemoglobin molecule RBC RBC       Fe2+ 3-part Diff technology V0706

3. Oxidation Haemoglobin molecule O2       Methemoglobin-complex Fe3+ Fe2+   Fe2+ Fe2+   Fe2+ Fe2+ Fe2+ Methemoglobin-complex Stable coloumetric complex – directly proportional to Hb Absorbance of solution is measured against standard 3-part Diff technology V0706

DC detection method Particle counting DC - direct current - impedance principle - volumetric measurement WBC count and 3-part differential RBC count PLT count 3-part Diff technology V0706

DC Detection Method aperture vacuum internal external electrode U = R x I Impulse 3-part Diff technology V0706

Impedance Principle External Electrode Internal Electrode Aperture V = Voltage C = Current R = Resistance V = R x C XT-Series Product file Chapter V. Principles and Technologies Date: 23.07.2002, TTO – page 10

Impedance Principle Aperture External Electrode Internal Electrode V = Voltage C = Current R = Resistance V = R x C XT-Series Product file Chapter V. Principles and Technologies Date: 23.07.2002, TTO – page 11

Problems- recirculation and coincidence pulse A pulse B pulse C aperture cells

Hydrodynamic Focusing for RBC & PLT Samples are passing through the centre of the aperture with sheath flow solution  Recirculation and coincidence are prevented  Enhanced linearity & accuracy 3-part Diff technology V0706

DC Detection Method From pulse to histogram: pulse diagram time 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pulse height 3-part Diff technology V0706

DC Detection Method 30 20 10 Cumulative Distribution Curve Histogram 1 4 5 6 7 8 9 10 11 12 13 14 30 cells 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 10 Cumulative Distribution Curve 1 2 3 4 5 6 7 8 9 10 11 12 13 14 4 1 1 2 3 4 5 4 3 2 1 Histogram 3-part Diff technology V0706

NO. 4 DATUM: 9/10/95 15:11 MODE: VOLLBLUT WBC 5,8 x 103/µl RBC 4,84 x106/µl HGB 13,7 g/dl HCT 42,0 % MCV 86,8 fl MCH 28,3 pg MCHC 32,6 g/dl PLT 257 x103/µl LYMPH% 31,2 % MXD% 6,8 % NEUT% 62,0 % LYMPH# 1,8 x103/µl MXD# 0,4 x103/µl NEUT# 3,6 x103/µl 250 RBC RDW-SD 40,0 fl 40 PLT PDW 13,1 fl MPV 10,4 fl P-LCR 28,1 % WBC 300

Erythrocyte (RBC) Histogram RL RU PLT RBC 25-75 fl 200-250 fl RBC detection: between 25 and 250 fL Distribution curves are separated by flexible discriminators: RL & RU 3-part Diff technology V0706

Erythrocyte (RBC) Histogram RL RU PLT RBC 25-75 fl 200-250 fl The histogram curve should start and end at the base line within the discriminators 3-part Diff technology V0706

Abnormal Erythrocyte (RBC) Histogram RL RU 100% Example: RL flag message PLT RBC 20% 25-75 fl 200-250 fl In case of abnormal histogram curves the flag messages: RL; RU or MP are generated and results must be checked RL : Abnormal height at lower discriminator RU : Abnormal height at upper discriminator MP : (Multi Peak) RBC Anisocytosis 3-part Diff technology V0706

Platelet (Plt) Histogram PU 100% PLT RBC 20% fixed at 12 fl 2-6 fl 12-30 fl PLT detection: between 2 and 30 fL Fixed discriminator at 12 fL 3-part Diff technology V0706

Abnormal Platelet (Plt) Histogram PU Example: abnormal PLT curve PU message 100% PLT RBC 20% 2-6 fl 12-30 fl In case of abnormal histogram curves the flag messages: PL; PU or MP are generated and results must be checked PL : Abnormal height at Lower discriminator PU : Abnormal height at Upper discriminator MP : (Multi Peak) Platelet Anisocytosis 3-part Diff technology V0706

Leukocyte (WBC) Histogram Lysing reaction to the WBCs Structure of WBS Lysing reaction on the WBC Mitochondria Nucleus Nucleolus Cell membrane Ribosome Cytoplasm

Leukocyte (WBC) Histogram Lysing reaction and WBC Cell size in µm Before lysing reaction Neutrophile Basophile Eosinophile Monocyte Lymphocyte 10 - 15 9 - 14 11 - 16 12 - 20 7 - 12 0 2 4 6 8 10 12 14 16 18 20 22 After lysing reaction 0 50 100 150 200 250 300 Lymphocyte Monocyten Basophile Eosinophile Neutrophile 30 - 80 60 - 120 70 - 130 80 - 140 120 - 250 Cell volume in fl Lymphocyte Monocyte Basophile Eosinophile Neutrophile

Leukocyte (WBC) Histogram WL WU T1 T2 100% 20% fixed at 12 fl 2-6 fl 12-30 fl WBC detection: between 30 and 300 fL Leukocytes are separated in 3 parts: lymphocytes, mixed cells (mono, eo, baso) and neutrophils by discriminators: T1, T2 3-part Diff technology V0706

Abnormal Leukocyte (WBC) Histogram WL WU T1 T2 Example: abnormal WBC curve WL message in case of Lyse resistant RBC 100% 20% ~30 fl -300 fl The histogram curve should start within the lower and upper discriminator at the base line Abnormal curves are flagged with WL, WU, T1, T2, F1, F2  results must be checked 3-part Diff technology V0706

about 3-part differential counters QUESTIONS? about 3-part differential counters

5-part differential counters Various technologies :- Fluorescence flowcytometry Volume Conductivity Scatter Peroxidase staining

Beckman Coulter VOLUME MEASUREMENT VCS utilises the Coulter Principle of counting and sizing to measure the volume of the cell by using Direct Current (DC) across the two electrode in a flow cell. VOLUME: As opposed to using 0 light loss to estimate cell size, VCS utilises the Coulter Principle of (DC) Impedance to physically measure the volume that the entire cell displaces in an isotonic diluent. This method accurately sizes all cell types regardless of their orientation in the light path.

CONDUCTIVITY MEASUREMENT Cell exposed to RF, the RF energy penetrates into cell and reveal information about its size and internal structure. CONDUCTIVITY: Alternating current in the radio frequency (RF) range short circuits the bipolar lipid layer of a cell’s membrane, allowing the energy to penetrate the cell. This powerful probe is used to collect information about the internal structure of the cell, including chemical composition and nuclear volume. OPACITY: By correcting the Conductivity signal so that it is no longer influenced by cell size, we obtain a measurement that is related only to the internal structure of the cell. This allows VCS Technology to separate cells of similar size, but different internal composition. It also allows the instrument to calculate the Nuclear/Cytoplasmic ratio - a feature useful in distinguishing variant Lymphocytes from normal Lymphs. Opacity is a transformation of data derived from the ratio of RF/DC components obtained during data acquisition. It is calculated for every cell measurement and has the effect of removing the size component yielding a measurement which is more related to the internal conductivity of the cell.This parameter is plotted on the X axis of DF2 plot. Opacity  RF/DC

SCATTER MEASUREMENT As cells are pass in single stream (flow cell) they are struck by laser strike which gets scattered. The light scatter at angles between 10 and 70 deg is used by VCS instruments. SCATTER: When a cell is struck by the coherent light of a LASER beam, the scattered light spreads out in all directions. Using a proprietary new detector, median angle light scatter signals are collected to obtain information about cellular granularity, nuclear lobularity and cell surface structure. ROTATED LIGHT SCATTER (RLS): Using a method similar to that used in Opacity, Coulter eliminates the size component of the light scatter signals. In so doing, we were able to determine the optimum angle of scatter for each cell type and design a scatter detector that covered this range (10 to 70). This allows VCS Technology to accurately separate what would normally be mixed cell types (such as Neutrophils and Eosinophils) into distinct clusters without mathematical manipulation. It also enhances the separation between the non granular cell types. Rotated Light scatter  Log10 (LS) / DC The scattered light gives information about cell surface and granularity

3D Data Analysis Lymphs Monos Basos NRBCs Eos Neuts

ADVIA TECHNOLOGY WBC and Differential Peroxidase Channel Stain Cells With Peroxidase :Eosinophils- Strong Staining :Neutrophils- Medium Staining :Monocytes- Weak Staining :Lymphocytes and Basophils- No Staining :Large Unstained Cells (LUC) No staining Also Measure Cell Size Using Low Angle ScatterPlot 2D Scattergram To Give 4 Part Differential Volume Monocytes Neutrophils LUC Lymphocytes + Basophils Eosinophils Perox Activity 45

ADVIA TECHNOLOGY The ADVIA WBC differential is calculated from a 3 step process. Cells are stained by peroxidase reagent and analyzed for size and peroxidase stain intensity. Cell specific lysis reagents are used to separate basophils from all other white cells. Basos are subtracted from the lymph/baso cluster in the perox channel to calculate the lymphs.

Sysmex X-class analyzers-Fluorescence flow cytometry Jaldeep Bhansali 47

Fluorescence flow cytometry- (light scatter and fluorescent dyes)

Differential- FSc vs SSc (baso channel) Differential scattergrams SFL vs SSc (diff channel) FSc vs SSc (baso channel) Differential scattergrams Two scattergrams are used for the WBC differential 1. Diff Channel for the measurement of lymphocytes, monocytes, neutrophils, and eosinophils. The cell populations are separated by the side scatter based upon the internal structure of the cells and side fluorescence based upon the RNA and DNA content of the cells. The neutrophile population contains also the basophile which are counted in the WBC/BASO channel 2. WBC/BASO channel for the measurement of the total white blood cells and the basophils. The cells are separated by internal structure (side scatter) and cell size (forward scatter)

ACAS / Centroids 1. The first centroids are provided: The starting position of centroids has been determined from thousands of samples. These values are stored in the instrument and are used as the starting position for cluster analysis. SSC SFL Mono Lymph Ghost Neut + Ba Eo XT-Series Product file Chapter V. Principles and Technologies Date: 23.07.2002, TTO – page 50

ACAS / Mahalanobis Distance 2. Cluster analysis of scattergram If a cell is detected, distances between this signal and the given centroids are calculated (Mahalanobis distance). This distance reveals to which given cell population the signal belongs. SSC SFL 1. calculated centroids Mahalanobis- Distance Mono Lymph Ghost Neut + Ba Eo XT-Series Product file Chapter V. Principles and Technologies Date: 23.07.2002, TTO – page 51

Differential fluorescent staining- Immature granulocytes(Sysmex) Jaldeep Bhansali 52

IG MASTER Diff scattergram: IG positive vs IG negative

Reticulocyte parameters (RET channel) Separate channel Polymethine dye stains N.A. in WBCs, nRBCs , retics & platelets. Size vs fluorescence Retic count

Retic channel Reticulocyte count Reticulocyte fractions (LFR, MRF, HRF) Immature reticulocyte fraction Ret-He (reticulocyte Hb content) Platelet –O (fluorescent platelets) Fragmented red cells (FRC) An example of efficient multitasking!!

New haematological parameter can predict iron deficiency where classical serum tests fail Reticulocyte Haemoglobin Equivalent--- Ret-He CHr- Siemens (FDA approved)

Ret-He Ret-He - Hb content equivalent of reticulocytes Units of “pg”(normal range- 28 – 35 pg) Monitors state of iron supply during the course of erythropoiesis, provides information on availability of functional iron. Can classify hypochromic anemia, classical vs functional ID, helps to select optimal therapy & to monitor response to EPO and iron treatment.

Spurious platelet counts- Siegenthaler & Spertini, NEJM May 2006 48 yr old M with severe burns Automated CBC- Hct- 37%, MCV-91fl, RDW- 15.8%, WBC-7,400/ul, PLT count- 274000/ul PS – microspherocytes and spherocytes Manual platelet count- 85,000/ul

Overcoming the problems with impedance counting Manual method- haemocytometer with phase contrast (Time consuming, laborious, operator dependency is more) Flowcytometric method using RBC/PLT ratio (anti CD41, anti CD61) Am J Clin Path 2001 (Expensive, requires a flowcytometer and experience with FCM)

Optical (fluorescent) platelets (good correlation with reference methods) Sysmex fluorescent PLT-O results are unmatched by ordinary platelet technologies of other analyzers Jaldeep Bhansali 60

Fluorescent PLT (platelet-O) Giant PLT Microcytic RBC

PLT abn. Distribution giant thrombocytes

IPF – Immature Platelet Fraction Immature PLT are identified by its increase in fluorescence (more RNA), FSC is also higher.

Peripheral smear examination still required!!

Thrombocytopenia

Platelet clump(EDTA induced)

Platelet count after collection in citrate !!

Criteria for smear review Smear review- increases manual work, reduces TAT & productivity Need to reduce smear review rate without risk of missing anything significant Different labs – different criteria Criteria depend upon patient population, type of analyser in use, etc.

Consensus rules for smear review International consensus group for hematology review – ISLH, 2002 ( Dr. Berend Houwen) Laid down rules for action following automated CBC including smear review Rules tested in 15 labs (13,298 samples) Data analysed, rules refined, 43 rules laid down Guidelines for individual laboratories

Review : Criteria for automated CBC & WBC diff analysis

Examples of some consensus rules (Lab Haematol, 2005) Rule no Parameter Primary And/or Action 1 neonate 1st sample Slide review 10 MCV <75fl or >105fl Specimen <24hrs old 15 RDW >22 1st time 16 No WBC diff/incomplete Manual diff & slide review 7 Platelet <100 or >1000

Hierarchial blood film evaluation Examiner Differentiation Analyser flagged normal ? Abnormal RBCs ? Atypical mononuclear WBCs Common RBC abnormality Granulocyte left shift Atyipcal (variant) lymphocytes normoblasts Routine technician Sr. technician ? Blasts ? Organisms Myelocytes Plasma cells Dohle bodies Targets Auer rods Physician Diagnostic cells Report Report Report Report

QUIZ-

Fragments ?

Eosinophilia

Iron Deficiency Anemia

Photo of slide

Lymphocytosis

Summary Automated cell counters-backbone of the diagnostic laboratory Fast, accurate, precise Impedance and various other technologies All have various limitations Accurate information on the technologies helpful to recognize problematic areas Maintenance, calibration, QC procedures Slides still need to be reviewed (criteria) Finally………

IT IS THE MAN BEHIND THE MACHINE WHO MATTERS MOST!! THANK YOU!!