73. Oxidation Haemoglobin molecule O2 Methemoglobin-complex Fe3+Fe2+Fe2+Fe2+Fe2+Fe2+Fe2+Methemoglobin-complexStable coloumetric complex – directly proportional to HbAbsorbance of solution is measured against standard3-part Diff technologyV0706
8DC detection method Particle counting DC - direct current- impedance principle- volumetric measurementWBC count and 3-part differentialRBC countPLT count3-part Diff technologyV0706
9DC Detection Method aperture vacuum internal external electrode U = R x IImpulse3-part Diff technologyV0706
10Impedance Principle External Electrode Internal Electrode Aperture V = VoltageC = CurrentR = ResistanceV = R x CXT-Series Product fileChapter V. Principles and TechnologiesDate: , TTO – page 10
11Impedance Principle Aperture External Electrode Internal Electrode V = VoltageC = CurrentR = ResistanceV = R x CXT-Series Product fileChapter V. Principles and TechnologiesDate: , TTO – page 11
12Problems- recirculation and coincidence pulse A pulse B pulse Caperturecells
13Hydrodynamic Focusing for RBC & PLTSamples are passing through the centre of the aperture with sheath flow solution Recirculation and coincidence are prevented Enhanced linearity & accuracy3-part Diff technologyV0706
14DC Detection Method From pulse to histogram: pulse diagram time 1 2 3 4567891011121314pulse height3-part Diff technologyV0706
17Erythrocyte (RBC) Histogram RLRUPLTRBC25-75 flflRBC detection: between 25 and 250 fLDistribution curves are separated by flexible discriminators: RL & RU3-part Diff technologyV0706
18Erythrocyte (RBC) Histogram RLRUPLTRBC25-75 flflThe histogram curve should start and end at the base line within the discriminators3-part Diff technologyV0706
19Abnormal Erythrocyte (RBC) Histogram RLRU100%Example:RL flag messagePLTRBC20%25-75 flflIn case of abnormal histogram curves the flag messages: RL; RU or MP are generated and results must be checkedRL : Abnormal height at lower discriminatorRU : Abnormal height at upper discriminatorMP : (Multi Peak) RBC Anisocytosis3-part Diff technologyV0706
20Platelet (Plt) Histogram PU100%PLTRBC20%fixed at12 fl2-6 fl12-30 flPLT detection: between 2 and 30 fLFixed discriminator at 12 fL3-part Diff technologyV0706
21Abnormal Platelet (Plt) Histogram PUExample: abnormal PLT curve PU message100%PLTRBC20%2-6 fl12-30 flIn case of abnormal histogram curves the flag messages: PL; PU or MP are generated and results must be checkedPL : Abnormal height at Lower discriminatorPU : Abnormal height at Upper discriminatorMP : (Multi Peak) Platelet Anisocytosis3-part Diff technologyV0706
29Leukocyte (WBC) Histogram Lysing reaction to the WBCsStructure of WBSLysing reaction on the WBCMitochondriaNucleusNucleolusCell membraneRibosomeCytoplasm
30Leukocyte (WBC) Histogram Lysing reaction and WBCCell size in µmBefore lysing reactionNeutrophileBasophileEosinophileMonocyteLymphocyte9 - 147 - 12After lysing reactionLymphocyteMonocytenBasophileEosinophileNeutrophileCell volume in flLymphocyteMonocyteBasophileEosinophileNeutrophile
31Leukocyte (WBC) Histogram WLWUT1T2100%20%fixed at12 fl2-6 fl12-30 flWBC detection: between 30 and 300 fLLeukocytes are separated in 3 parts: lymphocytes, mixed cells (mono, eo, baso) and neutrophils by discriminators: T1, T23-part Diff technologyV0706
32Abnormal Leukocyte (WBC) Histogram WLWUT1T2Example: abnormal WBC curve WL message in case of Lyse resistant RBC100%20%~30 fl-300 flThe histogram curve should start within the lower and upper discriminator at the base lineAbnormal curves are flagged with WL, WU, T1, T2, F1, F2 results must be checked3-part Diff technologyV0706
405-part differential counters Various technologies :-Fluorescence flowcytometryVolume Conductivity ScatterPeroxidase staining
41Beckman 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.
42CONDUCTIVITY 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
43SCATTER MEASUREMENTAs 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) / DCThe scattered light gives information about cell surface and granularity
45ADVIA 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 stainingAlso Measure Cell Size Using Low Angle ScatterPlot 2DScattergram To Give 4 Part DifferentialVolumeMonocytesNeutrophilsLUCLymphocytes+BasophilsEosinophilsPerox Activity45
46ADVIA TECHNOLOGYThe 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.
48Fluorescence flow cytometry- (light scatter and fluorescent dyes)
49Differential- FSc vs SSc (baso channel) Differential scattergrams SFL vs SSc (diff channel)FSc vs SSc (baso channel)Differential scattergramsTwo scattergrams are used for the WBC differential1. 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 channel2. 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)
50ACAS / Centroids1. 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.SSCSFLMonoLymphGhostNeut + BaEoXT-Series Product fileChapter V. Principles and TechnologiesDate: , TTO – page 50
51ACAS / Mahalanobis Distance 2. Cluster analysis of scattergramIf 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.SSCSFL1. calculated centroidsMahalanobis-DistanceMonoLymphGhostNeut + BaEoXT-Series Product fileChapter V. Principles and TechnologiesDate: , TTO – page 51
53IG MASTER Diff scattergram: IG positive vs IG negative
54Reticulocyte parameters (RET channel) Separate channelPolymethine dye stains N.A. in WBCs, nRBCs , retics & platelets.Size vs fluorescenceRetic count
55Retic channel Reticulocyte count Reticulocyte fractions (LFR, MRF, HRF)Immature reticulocyte fractionRet-He (reticulocyte Hb content)Platelet –O (fluorescent platelets)Fragmented red cells (FRC)An example of efficient multitasking!!
56New haematological parameter can predict iron deficiency where classical serum tests fail Reticulocyte HaemoglobinEquivalent--- Ret-HeCHr- Siemens(FDA approved)
57Ret-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.
58Spurious platelet counts- Siegenthaler & Spertini, NEJM May 2006 48 yr old M with severe burnsAutomated CBC- Hct- 37%, MCV-91fl, RDW- 15.8%, WBC-7,400/ul, PLT count /ulPS – microspherocytes and spherocytesManual platelet count- 85,000/ul
59Overcoming 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)
60Optical (fluorescent) platelets (good correlation with reference methods) Sysmex fluorescent PLT-O results are unmatched by ordinary platelet technologies of other analyzersJaldeep Bhansali60
71Criteria for smear review Smear review- increases manual work, reduces TAT & productivityNeed to reduce smear review rate without risk of missing anything significantDifferent labs – different criteriaCriteria depend upon patient population, type of analyser in use, etc.
72Consensus 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 reviewRules tested in 15 labs (13,298 samples)Data analysed, rules refined, 43 rules laid downGuidelines for individual laboratories
73Review : Criteria for automated CBC & WBC diff analysis
74Examples of some consensus rules (Lab Haematol, 2005) Rule noParameterPrimaryAnd/orAction1neonate1st sampleSlide review10MCV<75fl or >105flSpecimen <24hrs old15RDW>221st time16No WBC diff/incompleteManual diff & slide review7Platelet<100 or >1000
88Summary Automated cell counters-backbone of the diagnostic laboratory Fast, accurate, preciseImpedance and various other technologiesAll have various limitationsAccurate information on the technologies helpful to recognize problematic areasMaintenance, calibration, QC proceduresSlides still need to be reviewed (criteria)Finally………
89IT IS THE MAN BEHIND THE MACHINE WHO MATTERS MOST!! THANK YOU!!