Automated Cell Counter And Quality Control

Automated Cell Counter AndQuality ControlSpeaker :- Dr Saikat MandalModerator :-Dr Supriyo Roy Choudhury

World Of Automation

History Antonie van Leeuwenhoek Invented Microscope

The world of cells was colorless until Paul Ehrlich stained blood cells

Wallace Coulter :First automated analyzer for counting and sizing cells and presented it in 1956

Automation In HematologyCell counts(Automated hematology analyzers)Diagnosis of hemoglobinopathies(HPLC)Immunophenotyping(Flow cytometry)Coagulation(Coagulometers)

Automated Hematology AnalyzersHemogram-Backbone of any lab evaluation

As a routine investigationIncluding anaemia, polycythemia, infection, inflammation, allergy, drug toxicity, malignancy, bleeding tendency etc

Aim-to study RBC, WBC series and platelets

Advantages

Speed with efficient handling of large number of samplesAccuracy and precision in quantitative blood testsAbility to perform multiple tests on a single platformSignificant reduction of labor requirementsInvaluable for accurate determination of red cell indices

Disadvantages

Flagging of a laboratory test result demands labour intensive manual examination of a blood smearComments on red cell morphology cannot be generatedPlatelet Clumps are counted as single. so low count.Erroneously increased or decreased results due to interfering factorsExpensive with high running costs

Types of countersSemi automated : Some steps carried out manually like dilution of blood Measures only a few parameters

Fully automated: Require only anticoagulated blood samples. Measures multiple parameters

Components of a cell counter 3 Basic componentsHydraulics: Includes aspirating unit, dispencers,diluters,mixing chambers, aperture baths & hemoglobinometerPneumatics : Vacuums & pressure for operating valvesElectronics : Analyzer &computing circuit

Principles of WorkingElectrical ImpedanceOptical Light Scatter FluorescenceLight absorptionElectrical conductivity

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Electrical ImpedanceFirst introduced by Wallace CoulterBlood cells are poor conductor of electricity2 chambers filled with a conductive buffered electrolyte solutionSeparated by a small apertureDC current between two electrodes

Electrical ImpedanceDiluant displacement causes potential differenceVoltage pulse displayed on an osciloscopeNo. of impulse = No. of cellsHeight = vol. of cellsFreq dist curve & size dist histogramsRequisite High dilution

Optical Light ScatterEach cell flows in a single line through a flow cellA laser device focussed On striking on cells scattering in different directionsSensor capture & multipliesForward angle light scatter (FALS)-Cell SizeSide scatter(SS)-Granularity

Other MethodsPeroxide based counters: MPO is used to count neutrophils.Lymphocytes not stained

Fluroscence based: Retic and platelet count.Immature pltlets detected best

Immunological based: Accurate platelet count using CD41/CD61 antibodies

INTERPRETATION

3 Part Analyzer

5 Part Analyzer

IP Messages Interpretive messages Assist the laboratory in screening for abnormal samples that may need verificationSeen at the bottom end of hemogram

Indicators that may appear after the data @ : Data is outside the linearity limit* : Data is doubtful+ or :Data is outside the reference limits.---- : Data doesnt appear due to analysis error or abnormal sample++++ : Data exceeds display limit.

Discrimination Thresholds WBC Discriminator WBC LOWER discriminator-the optimum position in 30 - 60 fL .WBC is calculated from the particle counts more than this LOWER discriminator.

RBC Discriminator RBC LOWER discriminator- optimum position in 25 -75 fL and UPPER discriminator, 200 - 250 fL,. RBC is calculated from the particle counts between this LOWER discriminator and UPPER discriminator.

PLATELET Discriminator PLT LOWER discriminator, the optimum position in 2 6 fL and and UPPER discriminator- 12 - 30 fL,

HistogramsThese are the graphical presentation of numerical datas of different cell populations in a cell counter.On the X-axis is the cell size and on the Y-axis is the number of cells.Used to determine: The average size Distribution of size Detect subpopulation

Flagging SystemWhenever any significant abnormalities of any cell present , signalled by certain asteriks on the report.Every instrument has its own flagging system.

Parameters Measured Directly MeasuredDerived From Histograms Calculated1.RBC Count 2.WBC Count3.Platlet count4.Hemoglobin5.Reticulocyte Count1.MCV2.RDW3.DLC4.PDW1.Hematorit ( MCV/RBC Count)2.MCH (Hb/RBC Count)3.MCHC (Hb/Hct)

RBC HistogramGaussian(Bell Shaped)curvePeak ideally within 80-100 fl2 flexible discriminator LD (25-75fl) UD(200-250fl)

RU-Flag NormoblastsCold agglutininsALL- L1

RL- Flag

Large plateletsFragmented RBCsPlatelet aggregation

MP-FlagBlood transfusionDimorphic anaemiaTreated IDA

Red Cell Distribution WidthRDW-SD- 20% height on y axis.Normal-35-45 fl

RDW-CV- SD/MCV X 100Normal- 11.5-14.5 %

WBC HISTOGRAM

LD(30-60fl)Flexible UD at 300 fl2 Troughs T1(78-114fl) T2(