V07063-part Diff technology Haemoglobin molecule RBC Ammonium salts Fe 2+ RBC Fe Lysis of RBC Fe 2+ Haemoglobin estimation
V part Diff technology Haemoglobin molecule RBC Fe 2+ RBC Fe Change of conformity
V07063-part Diff technology Haemoglobin molecule Methemoglobin-complex Stable coloumetric complex – directly proportional to Hb Absorbance of solution is measured against standard Fe 3+ Fe 2+ O2O2 3. Oxidation Fe 2+
DC detection method Particle counting DC - direct current - impedance principle - volumetric measurement WBC count and 3-part differential RBC count PLT count V07063-part Diff technology
DC Detection Method V07063-part Diff technology external electrode internal electrode aperture vacuum U = R x I Impulse
Impedance Principle External Electrode Internal Electrode Aperture V = R x C V = Voltage C = Current R = Resistance
Impedance Principle V = R x C V = Voltage C = Current R = Resistance External Electrode Internal Electrode Aperture
Problems- recirculation and coincidence A B C pulse A pulse B pulse C aperture cells
V07063-part Diff technology Samples are passing through the centre of the aperture with sheath flow solution for RBC & PLT Hydrodynamic Focusing Recirculation and coincidence are prevented Enhanced linearity & accuracy
V07063-part Diff technology time pulse height From pulse to histogram: pulse diagram DC Detection Method
V07063-part Diff technology Histogram Cumulative Distribution Curve cells DC Detection Method
V part Diff technology fl fl Erythrocyte (RBC) Histogram RBC detection: between 25 and 250 fL Distribution curves are separated by flexible discriminators: RL & RU RL RU RBC PLT
V07063-part Diff technology fl fl The histogram curve should start and end at the base line within the discriminators RL RU RBC PLT Erythrocyte (RBC) Histogram
V07063-part Diff technology fl 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 RL RU RBC PLT Example: RL flag message 100% 20% Abnormal Erythrocyte (RBC) Histogram
V07063-part Diff technology 2-6 fl12-30 fl fixed at 12 fl PL PU PLT RBC 100% 20% PLT detection: between 2 and 30 fL Fixed discriminator at 12 fL Platelet (Plt) Histogram
V07063-part Diff technology 2-6 fl12-30 fl PL PU PLT RBC 100% 20% 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 Example: abnormal PLT curve PU message Abnormal Platelet (Plt) Histogram
Lysing reaction to the WBCs Structure of WBS Mitochondria Nucleus Nucleolus Cell membrane Ribosome Cytoplasm Lysing reaction on the WBC Leukocyte (WBC) Histogram
Before lysing reaction Neutrophile Basophile Eosinophile Monocyte Lymphocyte Cell size in µm Lysing reaction and WBC After lysing reaction Lymphocyte Monocyten Basophile Eosinophile Neutrophile Cell volume in fl Lymphocyte Monocyte Basophile Eosinophile Neutrophile Leukocyte (WBC) Histogram
V07063-part Diff technology 2-6 fl12-30 fl fixed at 12 fl WL WU 100% 20% 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 T1 T2 Leukocyte (WBC) Histogram
V07063-part Diff technology ~30 fl -300 fl WL WU 100% 20% 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 T1 T2 Example: abnormal WBC curve WL message in case of Lyse resistant RBC Abnormal Leukocyte (WBC) Histogram
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. Beckman Coulter
CONDUCTIVITY MEASUREMENT Cell exposed to RF, the RF energy penetrates into cell and reveal information about its size and internal structure.
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. 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 Eosinophils Neutrophils Monocytes LUC Lymphocytes + Basophils Perox Activity Volume
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. ADVIA TECHNOLOGY
Fluorescence flow cytometry- (light scatter and fluorescent dyes)
49 Differential- FSc vs SSc (baso channel) SFL vs SSc (diff channel)
ACAS / Centroids SSC SFL Ghost Neut + Ba Mono Lymph Eo 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.
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 Ghost Neut + Ba Mono Lymph Eo 1. calculated centroids Mahalanobis- Distance
Diff scattergram: IG positive vs IG negative IG MASTER
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 /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
IPF – Immature Platelet Fraction Immature PLT are identified by its increase in fluorescence (more RNA), FSC is also higher. IPF
Peripheral smear examination still required!!
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
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!!