1、Principles and TechnologySYSMEX EUROPE GMBHPrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodThe PulseSample BeakerlFirst Dilution 1:500(WBC)lSecond Dilution 1:50.000(RBC)Sample BeakerDB-1Dilutions for Semiautomatic AnalyserPrinciples and TechnologySYSMEX EUROPE GMBH DC Detection Method
2、The PulseWBC Dilution+3 Drops Quicklyser for Lysing and HGBRBC DilutionDilutions for Semiautomatic AnalyserPrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodThe PulseExternal ElectrodeApertureInternal ElectrodePrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodTransducerVacu
3、umBlood cellsResistanceConstant Current(ca.100 V)Internal ElectrodeExternal ElectrodeApertureSample BeakerBlood SuspensionPrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodTransducerExternal ElectrodeApertureInternal ElectrodeVacuumPrinciples and TechnologySYSMEX EUROPE GMBH DC Detectio
4、n MethodExternalElectrodeApertureInternal ElectrodeU=R x IThe PulsePrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodU=R x IExternalElectrodeApertureInternalElectrodeThe PulsePrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodTime secPulse PictureVolume flPulses/VolumeBlood
5、suspensionTransducerStart-SensorStop-SensorManometerCounting Time(defined Volume)forerunafterrunAbsolute Counting IPrinciples and TechnologySYSMEX EUROPE GMBHTime secPulse PictureVolume flPulses/VolumeBlood suspensionTransducerStart-SensorStop-SensorManometerCounting Time(defined Volume)PrerunAfterr
6、un DC Detection MethodAbsolute Counting IPrinciples and TechnologySYSMEX EUROPE GMBHTime secPulse PictureVolume flPulses/VolumeBlood suspensionTransducerStart-SensorStop-SensorManometerCounting Time(defined Volume)PrerunAfterrun DC Detection MethodAbsolute Counting IPrinciples and TechnologySYSMEX E
7、UROPE GMBHTime secPulse PictureVolume flPulses/VolumeBlood suspensionTransducerStart-SensorStop-SensorManometerCounting Time(defined Volume)PrerunAfterrun DC Detection MethodAbsolute Counting IPrinciples and TechnologySYSMEX EUROPE GMBHTime secPulse PictureVolume flPulses/VolumeBlood suspensionTrans
8、ducerStart-SensorStop-SensorManometerCounting Time(defined Volume)PrerunAfterrrun DC Detection MethodAbsolute Counting IPrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodTransducerSampleStartStopDefined VolumeVacuumCounteroffanDisplaySum of Pulses/VolumeonPulse Processing digitalAbsolut
9、e CountingPrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodTransducerSampleStartStopDefined VolumeVacuumCounteroffanDisplaySum of Pulses/VolumeonPulse Processing digitalAbsolute Counting IISignal-PulsesDisturbing PulsesSignal-PulsesDiscriminatorlNot to be changed by User:Sample VolumeD
10、ilution RatioCounted VolumeAperture DiameterlAutomatically checked by the instrument:Counting TimeCondition of aperture during measurement(Noise-Control)lAdvantages:No calibration of countersAutomatic check of aperturePrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodSampleVacuumReferen
11、ce Sample for Instrument CalibrationPulses per Timeelectrical VoltageDigital-AnalogConverterAnalog-DigitalConverter(Digital-Voltmeter)Display:digitalVolt(=Target Value of Reference Sample)Adjustment of Voltage to Target Value of Reference SamplePulsprocessing analogSignal-PulsesDisturbingPulseSignal
12、-PulsesDiscriminatorPulsprocessing digitallMeasurement of Particlesper Time PeriodlElements of Uncertainty:Reference SampleState of the Aperture VacuumRelative CountingPrinciples and TechnologySYSMEX EUROPE GMBH DC Detection MethodlMeasurement of particlesin a defined volumelCalculation of cell-conc
13、entration with known Sample volume and defined Sample dilutionlno calibration of counting valueslMeasurement of particles per time periodlThe count is received indirectly through a reference-sample.It is taken for granted that the sample and the reference-sample have the same characteristics and giv
14、e the same result.lCalibration of counting values in necessaryAbsolute CountingRelative CountingComparison of Absolute and Relative CountingPrinciples and TechnologySYSMEX EUROPE GMBH12345678910 1112 13 14Pulse HeightVolume(Time)DC Detection MethodPrinciples and TechnologySYSMEX EUROPE GMBH123456789
15、1011121314Cumulative Size Distribution CurvePulse Picture1020301234567891011121314410 0012 345 43 21 DC Detection MethodHistogramPrinciples and TechnologySYSMEX EUROPE GMBH1234567891011121314Histogram410 0012 345 43 21Cumulative Size Distribution Curve1020301234567891011121314410 0012 345 43 21 DC D
16、etection MethodHistogramPrinciples and TechnologySYSMEX EUROPE GMBHErythrocyte Histogram25-75 fl200-250 fl DC Detection MethodHistogramPrinciples and TechnologySYSMEX EUROPE GMBHPlatelet Histogram2-6 fl12-30 flfixed at12 fl DC Detection MethodHistogramPrinciples and TechnologySYSMEX EUROPE GMBH DC D
17、etection MethodHistogramLeukocyte-Histogramfixed at300 fl30-60 flLDT1T2UDLymphocytesNeutrophilesMonocytesBasophilesEosinophilesPrinciples and TechnologySYSMEX EUROPE GMBH Operation SequencesHistogramSV2SV3SV1SV4NCNOCNONCCCNONCCNCDiluent Inlet(A)(B)Ball FoatOpen to the AirorPressure PumpWaste(Vacuum)
18、lAll solenoid valves are de-energized.lC(Common)and NO(Normally Open)solenoid valve ports are connected.lBall Float is in the down position.“READY”-StatusPrinciples and TechnologySYSMEX EUROPE GMBH Operation SequencesHistogramSV2SV3SV1SV4NCNOCNONCCCNONCCNCDiluent Inlet(A)(B)Ball FloatOpen to the Air
19、Waste(Vacuum)COUNT KEY onlSV1 and SV 4 are energized.lC and NC solenoid valve ports are connected.lDiluent is aspirated through SV4,SV2,Transducer,SV1 and SV3.lThe Ball Float in the manometer ispushed upward.lThe inner portion of transducer and manometer is automatically rinsed to minimize carry-ove
20、r effect.Principles and TechnologySYSMEX EUROPE GMBH Operation SequencesHistogramCountinglSV1 and SV4 are de-energized and SV2 is energized.lThe diluted sample is aspirated throughthe aperture and the Ball Float moves downward.lWhen the Ball Float reaches level A,the counter starts counting.lWhen th
21、e Ball Float reaches level B,the counting stops and SV2 is de-energized.lThe volume of aspirated sample is the same as the manometer-volume between points A and B.SV2SV3SV1SV4NCNOCNONCCCNONCCNCDiluent Inlet(A)(B)Open to the AirWaste(Vacuum)Principles and TechnologySYSMEX EUROPE GMBH Operation Sequen
22、cesHistogramSV2SV3SV1SV4NCNOCNONCCCNONCCNCDiluent Inlet(A)(B)Ball FloatPressure PumpWaste(Vacuum)lSV1 and SV3 are energized.lPressure Pump is activated.lPressure is applied to the aperture to remove the clog.lThe same function occurs if the FLUSH key is pressed.Clog removalPrinciples and TechnologyS
23、YSMEX EUROPE GMBH Operation SequencesHistogramSV2SV3SV1SV4NCNOCNONCCCNONCCNCDiluent Inlet(A)(B)Ball FloatOpen to the AirWaste(Vacuum)lWhen power switch is turned on,SV1 and SV4 are energized.lDiluent is aspirated through the transducer.lThe same function occurs if the FILL key is pressed.lThe diluen
24、t removes diluent and anyblood sample remaining in the system.Diluent FillPrinciples and TechnologySYSMEX EUROPE GMBHHaemoglobinHistogramlHaemoglobin is the dye at the RBC.lIt is a Fe-containing Protein.lHaemoglobin is exclusively produced by Erythroblasts in the bone marrow.lHemoglobin is an import
25、ant part of the Erythrocytes.lThere is a direct relation between Haematokrit,Erythrocyte-concentration and Haemoglobin.lThe main function of Haemoglobin in the body is to transport Oxygen and CO2.Principles and TechnologySYSMEX EUROPE GMBHHaemoglobinHistogramNewborn14-26 g/dlChildren10-26 g/dl Women
26、 12-16 g/dl Men 14-18 g/dl 02468101214161820NewbornBabiesChildrenWomenMenPatient LimitsPrinciples and TechnologySYSMEX EUROPE GMBHHaemoglobinHistogramlA special reagent is needed,containing Cyanidefor lysing RBClF-Series=QuicklyserSysmex KCN-Method Principles and TechnologySYSMEX EUROPE GMBHHaemoglo
27、binHistogramlSLS means SodiumlaurylsulfatelSLS ins not toxic(ingredient in a lot of soaps tooth-paste)lSysmex Sulfolyser is now used with the majority of instruments(K-,E-,NE-,SF-,SE-,XE-systems)lOnly Sysmex hematology systems have a stable KCN-free Haemoglobin-measurement as standard.Sysmex SLS-Met
28、hodPrinciples and TechnologySYSMEX EUROPE GMBHHaemoglobin-Measurementa ab bHeamoglobin moleculeHydrophobic areaHydrophilic areaSO3-Na+HaemoglobinPrinciples and TechnologySYSMEX EUROPE GMBHa abbHaemoglobin moleculeaHydrophobic areaHydrophilic areaSO3-Na+HaemoglobinCyanide-free SLS-methodPrinciples an
29、d TechnologySYSMEX EUROPE GMBHlHydrophobic area of SLS bind to GlobinlConformation changelOxidation:Fe2+-Fe3+lHydrophilic area of SLS bind to Fe3+-SLS-Hb.Fe 2+Fe 3+Fe 3+SLSHaemoglobinCyanide-free SLS-methodPrinciples and TechnologySYSMEX EUROPE GMBHlphotometric measurement at 540 nm(F-Series,K-800,K
30、-1000,K-4500)Lamp Lens FilterSampleCellpackFlowcellPhotosensorHaemoglobinCyanide-free SLS-methodPrinciples and TechnologySYSMEX EUROPE GMBHLampHaemoglobinLampLens FilterDiluentFlow CellPhoto SensorFirst step:BLANK MEASUREMANTPrinciples and TechnologySYSMEX EUROPE GMBHLampHaemoglobinLampLens FilterDi
31、luentFlow CellPhoto SensorSampleLampLens FilterSampleFlow CellPhoto SensorSecond step:MEASUREMENT WITH LYSED SAMPLEPrinciples and TechnologySYSMEX EUROPE GMBHLampHaemoglobinStartFlow Cell is rinsed with Diluent.Measurement and saving of Blank.Measurement of sample dilutionSample value-Blank=HB-value
32、Printing of result EndPrinciples and TechnologySYSMEX EUROPE GMBHHaematocritlHaematocrit is determined by comparing the total or cumulative volume of red blood cells to the volume of whole blood.lDetermined normally by centrifugation or by electric pulse measurementlHaematocrit is besides Haemoglobi
33、n an important parameter in anemia-diagnosis.HaematocritPrinciples and TechnologySYSMEX EUROPE GMBHHaematocrit00,10,20,30,40,50,6NewbornChildrenMenBabiesWomenNewborn0,51-0,65Children0,35-0,43 Women 0,38-0,48 Men 0,42-0,52 Patient LimitsPrinciples and TechnologySYSMEX EUROPE GMBHHaematocritlDetermini
34、ng the volume of corpuscles in the bloodlNormally equivalent to mass of erythrocytes.lBlood is drawn into special capillaries,which contain dried heparin.lAfter closing,capillaries are placed into a centrifuge.lIn the centrifuge blood is divided into its corpuscular(cells)and fluid(plasma)components
35、CentrifugationPrinciples and TechnologySYSMEX EUROPE GMBHHaematocrit-MeasurementHaematocritPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPrinciples and TechnologySYSMEX EUROPE GMBHTotal volume VTCentrifugationHaematocritPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugeCentr
36、ifugationHaematocritTotal volume VTPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugeVTVHct(%)=V/VT x100CentrifugationHaematocritTotal volume VTPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritCentrifugationlReading is done with a scale out of triangles.00,10,20,30,40,50,60,70,
37、80,91,0lThe auxiliary line through the borderbetween erythrocytes-and leukocytesgives the heamatocrit.lCV app.1%Principles and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height TransducerPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height TransducerStart-
38、SensorStop-Sensordefined volume VTPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height TransducerStart-SensorStop-Sensordefined volume VTPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height TransducerStart-SensorStop-Sensordefined volume VTPrin
39、ciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height TransducerStart-SensorStop-Sensordefined volume VTPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height TransducerStart-SensorStop-Sensordefined volume VTPrinciples and TechnologySYSMEX EUROPE GMB
40、HCentrifugationHaematocritPulse Height TransducerStart-SensorStop-Sensordefined volume VTPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height TransducerStart-SensorStop-Sensordefined volume VTPrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritPulse Height
41、 PhPulse DiagramPh=k x VEry VEry=1/k x Ph Ph Pulse hightk ConstantVEry Volume of 1 ErythrocytePrinciples and TechnologySYSMEX EUROPE GMBHCentrifugationHaematocritCumulative Pulse Hct(%)=V/VT x 100V=VEry Ph Pulse hightk ConstantVEry Volume of 1 ErythrocyteVTVPrinciples and TechnologySYSMEX EUROPE GMB
42、HTransducerPh=k x VEryVEry=Ph x 1/k Ph Pulse Heightk KonstantVEry Volume EryPhHct(%)=V/VT x 100V=VEry=1/k x PhHct(%)=VT 1/k x Ph x 100VVTCumulative Pulse Height DetectionK-SeriesHaematocritPrinciples and TechnologySYSMEX EUROPE GMBHErythrocyte IndicesCalculated(Parameters)Principles and TechnologySY
43、SMEX EUROPE GMBHErythrocyte-IndicesMean Corpuscular Volume(MCV)Hct(%)RBC(x 106/l)MCV(fl)=Mean Corpuscular Haemoglobin (MCH)Hb(g/dl)RBC(x 106/l)MCH(pg)=Mean Cellular Haemoglobin Concentration(MCHC)Hb(g/dl)Hct(%)MCHC(g/dl)=RBC-IndicesPrinciples and TechnologySYSMEX EUROPE GMBHBiological Variation-40-2
44、0020406080100of quantitative parameters of CBCDeviation fromMedian in%WBC103/l5,0190PLT103/l14Hgbg/dl4,5RBC x 106/l42HCT%90MCVfl34MCHCg/dl31MCHpgBiological VariationPrinciples and TechnologySYSMEX EUROPE GMBHSpecifications Example:SYSMEX K-4500SpecificationsPrinciples and TechnologySYSMEX EUROPE GMB
45、Hl18 Parameter:WBC,RBC,HBG,HCT,PLTMCV,MCH,MCHRDW-CV,RDW-SD,MPV,PDW,P-LCRSCR%(LYMPH%),MCR%(MONO/EO/BASO%),LCR%(NEUT%)SCC#(LYMPH#),MCC#(MONO/EO/BASO#),LCC#(NEUT#)l3 Histograms:WBC,RBC,PLTSpecificationsPrinciples and TechnologySYSMEX EUROPE GMBHReliable Measurement Principles forhighest accuracyWBC/RBC
46、/PLT DC Detection MethodHBGSLS-Haemoglobin MethodHCTCumulative Pulse Height DetectionWBC-DiffDC Detection MethodSpecificationsPrinciples and TechnologySYSMEX EUROPE GMBHThroughputl80 Samples/HourCycle Time with single Samplesl45 Seconds/SampleSpecificationsPrinciples and TechnologySYSMEX EUROPE GMBH
47、Sample VolumelClosed Mode(Sampler)200 llManual Open Mode100 llMCP-Mode200 llCapillary Mode 40 SpecificationsPrinciples and TechnologySYSMEX EUROPE GMBHSpecificationsParametersWBC,NEUT%,LYMPH%,EO/MONO/BASO%,NEUT#,LYMPH#,EO/MONO/BASO#,RBC,HKT,HGB,MCV,MCH,MCHC,RDW-SD,RDW-CV,PLT,PDW,MPV,P-LCRReagentsDil
48、uentCELLPACKLyse-ReagentSTROMATOLYSER-3WPhaemoglobin-Reagent SULFOLYSERCleaning Reagent(Detergent)CELLCLEANDiameter of AperturesWBC100 mRBC/HCT/PLT75 mThroughput80 Samples/Hour(appr.45 Seconds Cycle-Time/Sample)Principles-DC Detection Method(RBC,WBC,PLT)-SLS-Hemoglobin(HGB)-Cumulative Pulse Height D
49、etection-Calculation from RBC and HCT(MCV)-Calculation from RBC and HGB(MCH)-Calculation from HCT and HGB(MCHC)Sample VolumeSampler Mode200 lClosed Mode200 lManual Open Mode100 lMCP-Mode200 lCapillary Blood Mode 40 lData MemoryExternal data memory available(K-DPS)Principles and TechnologySYSMEX EURO
50、PE GMBH-PrecisionOne fresh normal whole blood sample was measured 10 times consecutively in Sampler Mode.From those results the Coefficient of Variation has been calculated.WBCCV 3,0%RBCCV 1,5%HGBCV 1,5%HCTCV 1,5%MCVCV 1,5%MCHCV 1,5%MCHCCV 1,5%PLT CV 4,0%AccuracyWBC 3,0%RBC 3,0%PLT 3,0%Linearity-WBC
