Why should you use automation? High volume: The Core Laboratory may report well over 450,000 CBCs and 200,000 differentials a year Many parameters measured Wide range of clinical applications
Automated techniques of blood countingSemi-automated instruments Require some steps, as dilution of blood samples Often measure only a small number of variablesFully automated instruments Require only that an appropriate blood sample is presented to the instrument. They can measure 8-20 variables including some new parameters which do not have any equivalent in manual methods.
Disadvantages of manual cell counting Cell identification errors in manual counting: – mostly associated with distinguishing lymphocytes from monocytes; bands from segmented forms and abnormal cells (variant lymphocytes from blasts) – lymphocytes overestimated, monocytes underestimated Slide cell distribution error: – increased cell concentration along edges, also bigger cells found there i.e. monocytes, eosinophils, and neutrophils Statistical sampling error
Advantages of automated cell counters They have a high level of precision for cell counting and cell sizing greatly superior to that of the manual technology The results are generally accurate Aberrant results consequent on unusual characteristics of blood are “flagged” for subsequent review
Advantages of automated cell counters Objective (no inter-observer variability) No slide distribution error Eliminate statistical variations associated with manual count based on high number of cells counted Many parameters not available from a manual count, e.g. MCV, and RDW More efficient and cost effective than manual method: – Some cell counters can process 120-150 samples per hour – CAP assumes 11 minutes for a manual cell count
Automated cell counters can provide CBC: WBC, RBC, Hgb, Hct, PLT, RBC indices WBC Differential: 5 “normal” white cell types RDW, PDW, MPV Reticulocyte count Nucleated red cell count All automated cell counters are screening devices. Abnormalities must be verified by a blood film, staining and scanning by an expert observer
Review process Whenever the automated cell counter flags a specimen, the technologist (or an automated system) has to • Retrieve the tube • Make a slide • Stain the slide • Review the slide • Either release the results from the cell counter (“scan”) or perform a manual differential
Importance of the review rate If it takes a technologist 10 minutes to prepare, label, stain, review, count, and release results from a slide, and If a technologist costs $ 25.00 per hour; it will cost $ 4.00 to make a slide. If the laboratory’s review rate changes by 1% (= 2,000 differentials a year), you pay or save almost $ 10,000
Initial set up of instrument Check instrument for visual damage Check for any loose parts or connections Make sure all computer boards are properly sealed Check the socket to verify proper voltage outlet Plug power cord into (voltage stabilizer) electrical supply Confirm the correct voltage on instrument Main power supply Photometric voltage Permit instrument to stabilize/equilibrate Let all components reach proper temperature Set in any parameters that may be required Ranges and temperatures
Calibration Calibration fine tunes your haematology analyzer and provides the most accurate results possible. In the normal process of tracking data for an extended period of time, your laboratory can make a specific decision to recalibrate a given parameter. Never adjust to a specific value for an individual sample. For best performance, calibrate all the CBC parameters. The WBC differential is calibrated at the factory. They do not require calibration in the laboratory.
CalibrationYou should calibrate your instrument: At installation After the replacement of any component that involves dilution characteristics or the primary measurements (such as the apertures) When advised to do so by your service representative
Daily maintenance Daily cleaning Background counts Electronic checks Compare open and closed mode sampling (using a normal patient sample) Run controls
Ensure the Instrument is Functioning ProperlyCheck the reagent containers for: Sufficient quantity Not beyond expiration date No precipitates, turbidity, particulate matter, or unusual colour Proper connections between the instrument and the reagent containers
Ensure the Instrument is Functioning ProperlyCheck the waste container for: Sufficient capacity Proper connectionsPerform daily startupIn addition to verifying daily startup results, verifyacceptable: Reproducibility Carryover Control Results
Quality control Purpose of Quality Control (QC) Assures proper functionality of instrumentation Means of assuring accuracy of unknowns Monitoring the Integrity of the Calibration - When controls begin to show evidence of unusual trends - When controls exceed the manufacturer’s defined acceptable limits
Data review A review of instrument data, such as background, control, and blood sample results, is helpful in detecting problems. Sometimes a questionable blood sample result is the only symptom of subtle reagent or pneumatic problems.
Principles of automated cell counters Impedance (conductivity) system (Coulter) Optical system (H*1) Both impedance and optical Selective lysis (e.g. lysis of red cells and counting of white cells) Special stains
Electrical impedance method The “Coulter Principle” Cell counting and sizing is based on the detection and measurement of changes in electrical impedance (resistance) produced by a particle as it passes through a small aperture Particles such as blood cells are nonconductive but are suspended in an electrically conductive diluent As a dilute suspension of cells is drawn through the aperture, the passage of each individual cell momentarily increases the impedance (resistance) of the electrical path between two electrodes that are located on each side of the aperture
Diagram illustrating the Coulter Principle A stream of cells in suspension passes through a small aperture across which an electrical current is applied. Each cell that passes alters the electrical impedance and can thus be counted and sized.
Histograms of Coulter S Plus IV Histograms showing the size distribution of white cells, red cells and platelets. Sizing is based on impedance technology.
Optical method Laser light is used • A diluted blood specimen passes in a steady stream through which a beam of laser light is focused • As each cell passes through the sensing zone of the flow cell, it scatters the focused light • Scattered light is detected by a photodetector and converted to an electric impulse • The number of impulses generated is directly proportional to the number of cells passing through the sensing zone in a specific period of time
Optical method The application of light scatter means that as a single cell passes across a laser light beam, the light will be reflected and scattered. The patterns of scatter are measured at various angles. Scattered light provides information about cell structure, shape, and reflectivity. These characteristics can be used to differentiate the various types of blood cells and to produce scatter plots with a five-part differential
Light scattering Cells counted as passed through focused beam of light (LASER) Sum of diffraction(bending around corners), refraction (bending due to change in speed) and reflection (light rays turned back by obstruction) Multi angle polarized scatter separation (M.A.P.S.S) 0° : indicator of cell size 10° : indicator of cell structure and complexity 90° polarized: indicates nuclear lobularity 90° depolarized: differentiates eosinophils
Types of Haematology Analyzers Smaller instruments: Measure WBCs, RBCs, Hgb, Hct, MCV, MCH, MCHC, and PLTs) Advanced cell counters: Add: – Red cell morphology information, RDW – Mean platelet volume – Leukocyte differential
Trends Current trends include attempts to incorporate as many analysis parameters as possible into one instrument platform, in order to minimize the need to run a single sample on multiple instruments (e.g CD4 counts, smear preparation) Such instruments are being incorporated into highly automated combined chemistry/haematology laboratories, where samples are automatically sorted, aliquoted, and brought to the appropriate instrument by a robotic track system
Hb concentration• Hb is measured automatically by a modification of the manual (HiCN) method.• To reduce toxicity of HiCN some systems replace it by a non- toxic material Na- lauryl sulphate.
Haemoglobin measurement Sample is diluted with Cyanmethemoglobin reagent • Potassium ferricyanide in the reagent converts the hemoglobin iron from the ferrous state (Fe++) to the ferric state (Fe+++) to form methemoglobin, which then combines with potassium cyanide to form the stable cyanmethemoglobin A photodetector reads color intensity at 546 nm Optical density of the solution is proportional to the concentration of haemoglobin
RBC countThe RBCs are counted automatically by two methods Aperture impedance: where cells are counted as they pass in a stream through an aperture. Or by light scattering technologyThe precision of an electronic counting for RBCs is much better than the manual count, and it is available in a fraction of time.This made the use of RBC indices of more clinical relevance.
Reliability of electronic counters They are precise but care should be taken so that they are also accurate. Some problems which could be faced: Two cells passing through the orifice at the same time, counted as one cell. RBC agglutination(clump of cells) Counting bubbles or other particles as cells.
PCV and red cell indices Pulse height analysis allows either the PCV or the MCV to be determined. MCV=PCV/RBC count MCH= Hb/RBC count MCHC= Hb/PCV MCH & MCHC are derived parameters.
Red cell distribution width (RDW) Automated instruments produce volume distribution histograms which allow the presence of more than one population of cells to be appreciated. Most instruments produce a quantitative measurement of variation in cell volume, an equivalent of the microscopic assessment of the degree of anisocytosis. This is known as the RDW.
Total WBC count The total WBC count is determined in whole blood in which RBCs have been lysed. Fully automated multichannel instruments perform WBC counting by Impedance Light scattering Or both.
Automated differential count Automated differential counters which are available now generally use flow cytometry incorporated into a full blood counter rather than being standard alone differential counters Automated counters provide a three-part or five- to seven- part differential count.
Differential cell counting 3-part differential usually cont Granulocytes or large cells Lymphocytes or small cells Monocytes(mononuclear cells) or (middle cells) 5-part classify cells to Neutrophils Eosinophils Basophils Lymphocytes Monocytes
Differential cell countingA sixth category designated “large unstained cells” include cells larger than normal and lack the peroxidase activity this include Atypical lymphocytes Various other abnormal cellsOther counters identify 7 categories including Large immature cells (composed of blasts and immature granulocytes) Atypical lymphocytes (including blast cells)
Differential cell countingAnalysis may be dependant on: Volume of the cell Other physical characteristics of the cells Sometimes the activity of cellular enzymes such as peroxidase Technologies used Light scattering and absorbance Impedance measurementAutomated differential counters employing flow cytometry classify far more cells than is possible with a manual differential count.
Accuracy in blood cell counting The accuracy of automated counters is less impressive than their precision. In general automated differential counters are favourable to the manual in 2 conditions Examination of normal blood samples Flagging of abnormal samples
Platelet count Platelets can be counted in whole blood using the same technologies of electrical or electro-optical detection as are employed for RBCs. Other parameters include MPV PDW Plateletcrit = MPV x platelet count.
Reticulocyte countAn automated reticulocyte count can be performed using the fact that various fluoro-chromes combine with the RNA of the reticulocytes. Fluorescent cells can then be enumerated using a flowcytometer.An automated reticulocyte counter also permits the assessment of reticulocyte maturity since the more immature reticulocytes have more RNAfluoresce more strongly than the mature ones normally found in peripheral blood.
The Sysmex XE-2100• Uses both impedance and flow cytometry• Throughput: 150 samples/hour• Provides 32 parameters, including reticulocyte and NRBC counts• Uses an optical fluorescent platelet count when the impedance count may be unreliable• Unique features: – Can enumerate, not just flag for, immature granulocytes (metamyelocytes, myelocytes, promyelocytes) – Immature platelet fraction
The Sysmex XE-2100 Flow Cytometry (forward light scatter, side light scatter, side fluorescence) for: WBC differential, NRBC, reticulocytes, optical platelets Radio frequency (RF) and direct current (DC) resistance for: Immature granulocytes
HEMOGLOBIN MEASUREMENT• Red cells are lysed, hemoglobin is converted intosodium lauryl sulfate (SLS)-methemoglobin:– Short reaction time• Absorbance at 555 nm is measured, and concentrationof hemoglobin is calculated– Good correlation with reference method
RED CELL AND PLATELET COUNTS Red cells and platelets are both counted by the electric impedance method. In samples with large platelets or small RBCs or RBC fragments, platelet counts by the light scattering method are used instead.
WBC/BASO CHANNEL• RBCs are lysed,degranulation ofbasophils isselectivelysuppressed• Forward and sidescatter are used toobtain a WBC and basophilcount
4-DIFFERENTIAL CHANNEL• Red cells are lysed,DNA and RNA ofWBCs are stainedwith fluorescent dye• Side fluorescenceand side scatter areused to obtain afour-part differential
IMMATURE GRANULOCYTE COUNT Includes promyelocytes, metamyelocytes, myelocytes, but NOT bands or blasts A lysing reagent causes disruption of mature WBC membranes, while immature myeloid cells with low membrane lipid content remain intact.
IMI CHANNEL Red cells are lysed, a lysing agent which disrupts the cell membranes of MATURE WBCs only is used. • Analysis by radiofrequency and direct current impedance detection
IMMATURE GRANULOCYTE COUNT• Correlation coefficient with visual count: 0.81• Prediction of infection:– At 90% specificity, sensitivity of 35 - 40%– At 90% sensitivity, specificity of 20%– Better predictor of sepsis than WBC; comparable to ANC
ABNORMAL CELLS IN THE DIFF AND THE IMI CHANNEL