General ChemistryImmunodiagnosticsCentrifugationDisease ManagementHematologyHemostasisLab AutomationData ManagementFlowCytometryPrimary CareACVDifferential Technologyand Case Studies.Bulletin 9151Beckman Coulter ACVDifferential TechnologyTABLE OF CONTENTS DIFFERENTIAL TECHNOLOGY .................... 1Introduction ............................ 3Differential Methodology: Sample Preparation........... 3Differential Methodology: Sample Flow and Technology ...... 3Flow Cell ........................... 3Dual Focused Flow ...................... 3Focused Flow Impedance ................... 4Differential Methodology: Parameter Derivation .......... 5Signal Processing....................... 5DiffPlot Characteristics ..................... 6Lymphocytes ......................... 6Monocytes .......................... 6Neutrophils .......................... 6Eosinphils .......................... 6WBC/ BASO Methodology .................... 7DIFFERENTIAL REAGENT SYSTEM OVERVIEW ............ 7DIFFERENTIAL ANALYTICAL/ MECHANICAL SYSTEM OVERVIEW.... 8Sequential Dilution System.................... 8DIFF Bath ............................ 9Optical Bath........................... 9WBC/ BASO Bath......................... 10Differential Calculation...................... 10DIFFERENTIAL FLAGGING OVERVIEW ................ 11Immature Granulocytes ..................... 11Band Cells ........................... 11Blasts.............................. 11DiffPlot Flags .......................... 12CASE STUDIES........................... 16TABLETable 1. Differential Measurement Technologies ........... 3FIGURES Figure 1. Flow Cell ......................... 4Figure 2. Signal Processing..................... 5Figure 3. Correlation of Signals to DiffPlot Populations ........ 6Figure 4. WBC/ BASO Histogram .................. 7Figure 5. Sequential Dilution System................. 9Figure 6. Diff Sample Flow ..................... 9Figure 7. Optical Bench....................... 10Figure 8. WBC/ BASO Bath Assembly ................ 10Figure 9. DiffPlot Normal and Abnormal Cell Populations ....... 11Figure 10. DiffPlot Flags ...................... 12Figure 11. DiffPlot Flags ...................... 13Figure 12. DiffPlot Flags ...................... 14Beckman Coulter ACVDifferential Technology2INTRODUCTION The ACT 5diff instrument is a fully automated hematology analyzer providinga 5-part white cell differential. The 5-part white cell differential is determinedsimultaneously by the differential (DIFF) and white cell/ basophil (WBC/ BASO)methodologies. The differential methodology utilizes the technologies andprinciples of absorbance cytochemistry and focused ow impedance. The WBC/ BASO methodology utilizes differential lysis, Coulter Principle(impedance) and differential thresholds to provide a total white cell count and basophil percentage. These two methodologies, listed in Table 1,provide a robust, low cost and efcient 5-part white cell differential withinterpretive agging for abnormal samples. Differential Methodology:Whole blood is mixed in the DIFF bath with ACT 5diff FIX reagent incubated Sample Preparation and then stabilized with diluent. This reaction lyses the red cells and stains the primary granules of monocytes, neutrophils and specic granules ofeosinophils at different intensities. This reaction also preserves theleukocytes in their native size. The lymphocytes, monocytes, neutrophils and eosinophils each have unique nuclear and morphologic structure, and hence absorb light differently. The differential staining and uniquecellular structure provide unique light absorbance characteristics. Theabsorbance cytochemistry method is based on the well-known referencemethods of Sheenan et al* and Kass.***Sheenan, H.L., Storey, G.W. (1947) An improved method of staining leukocytegranules with Sudan Black B. J ournal of Pathology and Bacteriology, 59, 336.**Kass L. (1981) Staining of granulocyte cells by Chlorazol Black E. American J ournal of Clinical Pathology, 76, 810-812.Table 1. Differential Measurement TechnologiesFluid DifferentialDynamics Technology Measurement OutputDual Focused Absorbance Light AbsorbanceLymphocyteFlow Aperture cytochemistry and of Cytochemically MonocyteVolume (ACV stained cells NeutrophilTechnology)Volume vs. CountEosinophilVolume Aperture Differential LysisVolume vs. Count Basophilwith CoulterWhite Cell CountPrincipleDifferential Methodology: Sample Flow and TechnologyFlow Cell In the ow cell, sequential analyses for cell volume by the Coulter Principleand light absorbance are performed. The ow cell incorporates a 60 maperture for cellular volume analysis and an optical window of 42 m forlight absorbance.Dual Focused Flow Dual Focused Flow (DFF) technology is a process by which individual cells or particles are focused in a stream of diluent (hydrodynamic focusing). Cellsare aligned and pass through the ow cell in a singular fashion.Dual focused ow uses the sheath uid to surround and force cellssuspended in diluent to pass in a single le through the center of the owcell. The rst sheath ow focuses the sample through the impedanceaperture. The second sheath ow then focuses the cells as they exit theaperture onto the optical window. The use of hydrodynamic focusing in theACT 5diff instrument ow cell enables accurate and rapid cell-by-cellmeasurements on a large number of individual cells.Dual Focused Flow delivers sample, cell by cell, for the analysis of cellvolume and cellular content in one ow cell. The detection of an impedancepulse and a light absorbance pulse from a single cell as it passes throughthe ow cell within 200 microseconds, prevents the counting of artifactssuch as bubbles or static.Beckman Coulter ACVDifferential Technology4Figure 1. Flow CellImpedanceLight AbsorbanceFocused Flow Impedance Focused ow impedance, using the Coulter Principle, is the technology thatprovides cell by cell volume information for each cell as it passes throughthe aperture. Each time a cell passes through the aperture a change inelectrical resistance is measured. The change in resistance is proportionalto the volume of the cell. The ACT 5diff analyzer utilizes dual focused ow in conjunction withcytochemical staining, light absorbance and Coulter Principle to providecellular information for the complete WBC differential.Differential Methodology:Each stained cell as it passes through the ow cell is exposed to the light Parameter Derivation source and a measurement is taken. As the cell passes through the opticalwindow, light is scattered in all directions, but only the forward light scatteris detected by the sensors. The system measures the amount of light lostdue to diffraction and absorbance as compared to full transmission when nocell is present. The signals collected are converted into voltage pulses and are processed.The size and shape of the voltage pulses are equivalent to the unique nuclear and morphologic structure of the cells being analyzed. The light absorbance analyzes each cell for cellular content (granularity, nuclear content) after cytochemical staining. These simultaneous measurements provide the information for the white cell sub populations, lymphocytes,monocytes, neutrophils, eosinophils. Signal Processing The signals from the impedance aperture and from the absorbance ow cell are correlated by a window of time. The optical pulse must be detectedwithin a time window of the impedance pulse, otherwise the signal isdiscarded.The output signals from the focused ow impedance and the lightabsorbance measurements are combined to dene the white cell differential population clusters. This combined technology provides an efcient androbust technology for the white cell differential.Signal processing is displayed in Figure 2. Parameter DiffPlot developmentis displayed in Figure 3.Beckman Coulter ACVDifferential Technology6DiffPlot CharacteristicsLymphocytes Lymphocytes typically being small with regular shape are smaller in volumeand lower in absorbance than the other cells and are positioned in the lowerpart of the DiffPlot. Normal Lymphocyte populations typically have a homogeneous, gaussian volume distribution.Neutrophils Neutrophils, having cytoplasmic granules and segmented nuclei, will absorb light depending on their morphological complexity. A hypersegmented neutrophil will give a higher optical response whenFigure 3. Correlation of Signals to DiffPlot PopulationsFigure 2. Signal ProcessingVolumeAbsorbanceAbsorbanceMonoNeutLymphDebrisEosVolumeInci dent Li ght(Tungsten Lamp)Fl owcel l DetectorNo Cel lNo Absorbance / ScatterBase Li ne Si gnalSmal lCel lLow Absorbance / ScatterLow Si gnalLarge Granul ar Cel lHi gh Absorbance / ScatterModerate Si gnalLarge Stai ned Cel lVery Hi gh Absorbance / ScatterLarge Si gnalcompared to a young neutrophil population. The higher the complexity of the cell the further to the right they appear in the DiffPlot.Monocytes Monocytes are typically large cells with kidney shaped nucleus and agranular cytoplasm. These cells will thus neither scatter nor absorb largeamounts of light and thus are positioned to the low end of the absorbanceaxis. However due to their size are positioned high on the volume axis.Very large monocytes will be found in the IMM (Immature cell) region.Eosinophils The staining of the primary and specic granules of the eosinophilsdifferentiate them from the monocytes and neutrophils. The lymphocytesremain unstained.WBC/BASO Methodology Whole blood is mixed in the WBC/ BASO bath with ACT 5diff WBC Lysereagent and stabilized at 35C. This reaction lyses the red cells andspecically differentiates between basophils and other leukocytes. Theresistance of the basophils to the acidic lytic reagent is the basis for thedifferentiation of the basophils from the other leukocytes. The sample is thenpulled with a constant vacuum through an aperture. As each cell passesthrough the aperture, it generates a pulse proportional to its cellular volume.This cellular data is then displayed on the WBC/ BASO histogram. The totalleukocyte count and the basophil percentage are determined via the CoulterPrinciple and specic thresholds. Figure 4 displays the WBC/ BASOhistogram and differential thresholds. Figure 4. WBC/ BASO HistogramBA1 BA2 BA3BASOBeckman Coulter ACVDifferential Technology8DIFFERENTIAL REAGENTThe ACT 5diff system utilizes cytochemical staining and differential lysis andSYSTEM OVERVIEW the technologies of absorbance cytochemistry and the Coulter Principle toprovide the complete ve-part differential. These analyses are performedsimultaneously on the ACT 5diff system.ACT5diff Fixreagent contains a lysing agent and the vital stain,Chlorazole Black, which during the reaction time stains the granules of themonocytes, neutrophils and eosinophils blue and preserves the leukocytes intheir native size. ACT 5diff Fix is an alcoholic reagent, buffered to a pH of6.9, which gently lyses all red cells. This reaction provides the dilution usedto differentiate the leukocyte subpopulations using the absorbancecytochemistry technology. ACT5diff WBC Lysereagent is an acidic solution with a pH of 2.4 thatlyses the red cells and specically differentiates between the basophils andother leukocytes. This reaction provides the dilution for the total leukocytecount and basophil percentage analyses, which are obtained by using theCoulter Principle and using specic volume thresholds.ACT5diff Diluentreagent is used to: dilute the whole blood samples,stabilize cell membranes for accurate enumeration and sizing, provide theconductive medium, provide the sheath medium for focused ow and rinsesinstrument components after analysis. DIFFERENTIAL ANALYTICAL / MECHANICAL SYSTEM OVERVIEW Sequential Dilution System The leukocyte differential reactions require mixing and delivery of sample tothe analytical hardware. The initial delivery and mixing reactions are achieved by the Sequential Dilution System (SDS) and mixing within the DIFF andWBC/ BASO baths. This system accurately and repeatedly delivers specicvolumes of sample to the baths for the specic lytic or staining reactions.Figure 5 displays the specic reaction baths and dilutions. Initial aspiration of whole blood occurs at the sampling probe. The SDSsystem delivers specic volumes of blood to the respective baths (DIFF orWBC/ BASO) where the sampling needle aligns perfectly with the reagentinputs. Here, the preheated reagents mix homogeneously with the wholeblood sample. The reagent cleans the sampling needle internally andexternally. Each dilution is then mixed, incubated and stabilized at 35C.Once the reactions have taken place and the dilutions are ready for analysis,they need to be transported to the analytical areas.DIFF Bath For the DIFF bath sample, the stabilized sample is transported to themeasuring bath in the optical bench. This is accomplished using the syringeassembly for the DIFF bath. The DIFF syringe feeds the DIFF dilution to theoptical bath sensor while at the same time, two additional syringes providediluent sheath ow to the ow cell. Once the sensor detects the sample, the system begins differential measurements. Figure 6 displays the DIFFsample ow.Figure 5. Sequential Dilution SystemRinse First Dilution DIFF RBS/ Plt WBC/ BABath Bath Bath Bath BathBeckman Coulter ACVDifferential Technology10Optical Bench Light absorbance measurement is accomplished within the optical bench(see Figure 7). A tungsten halogen lamp provides the light source that isconcentrated and focused through the ow cell. As each cell passes throughthe ow cell, a sensor detects light that is scattered forward. The opticalmeasurement is derived as a function of the amount of light lost due todefraction and absorbance, as compared to full transmission when no cell ispresent. This data is then processed and displayed as the white cell subpop-ulations of lymphocytes, monocytes, neutrophils and eosinophils. Figure 7. Optical BenchFigure 6. DIFF Sample FlowWaste OutSheath 1Sheath 2Sample Flowfor AnalysisDiff BathWhole Blood Sample,FIX Reagent and DiluentSample Flowto Fill DiffSyringeSheath Stream 1 Sampl e Stream Sheath Stream 2El ectrodesSampl e Inj ectorWasteApertureLi ghtSourceEl ectrodesDetectorOpti calWi ndowWBC/BASO Bath The WBC/ BASO solution is mixed, stabilized and counted in the WBC/BASO bath. The WBC/ BASO bath receives the whole blood sample and lyticreagent. The sample is then pulled with a constant vacuum through an 80 m aperture. Each cell as it passes through the aperture generates a pulse proportional to its cellular volume. This cellular data is then displayedon the WBC/ BASO histogram. The basophils are differentiated from the restof the white cells through differential lysis and histogram thresholds. Thebasophil count and percentage are derived from this analysis. TheWBC/ BASO bath and electrode assembly are displayed below in Figure 8.Differential Calculation The results obtained from the DIFF analysis in the ow cell and the WBC/BASO analysis in the WBC/ BASO bath provide the information for thecomplete 5-part differential results. The WBC/ BASO results provide thebasophil percentage and total white cell count. The DIFF results provide thelymphocyte, monocyte, neutrophil and eosinophil results. To achievedifferential results equaling 100%, the neutrophil, lymphocyte and monocyteresults are corrected for the results obtained for the basophil analysis. DIFFERENTIAL FLAGGINGDifferential ags are generated whenever the white cell subpopulations OVERVIEW exceed the population statistics and interfere with separation thresholdareas of other subpopulations. The abnormal cell types and their locationson the WBC differential DiffPlot are described in Figure 9. Figures 10, 11and 12 describe the DiffPlot ags and the cell types associated with theseags.Figure 8. WBC/ BASO Bath AssemblyElectrodesBath WallWBC/ BASOBathBeckman Coulter ACVDifferential Technology12Immature Granulocytes Immature granulocytes are detected by their larger volume and by the presence of granules that increase the intensity of scattered light. Band Cells Band cells are typically larger or of similar size to the neutrophils but due totheir low level of cellular complexity they absorb less light. This places theband cells in the region between the Neutrophils and the Monocytes.Blasts Blast cells are generally larger than monocytes and may have similarabsorbance. Large blasts are included in the IMM cell area. Atypical Lymphocytes Small blasts will be found between the normal lymphocyte and monocytepopulations. Large lymphocytes, reactive lymphoid forms, stimulatedlymphocytes and blast cells are found in the ATL region.NRBCs Platelet Aggregates The presence of platelet aggregates is indicated by a distribution patternthat moves from the DiffPlot origin into the lymphocyte region. Nucleatedred cells having very small nuclei will be situated in the debris and smalllymphocyte regions.Figure 9. DiffPlot Normal and Abnormal Cell PopulationsBlasts,Promyelocytes,MonocytesLymphsDebrisNRBC's,Lyse Resistant RBC,Plt AggregatesAbnormalLymph NeutrophilsEosinophilsBandsAtyp.Lymphs,BlastsMyelocytes,Metamyelocytes,Imm. NeutrophilsImm.Eos,A granularEos,AtypicalNeutro-philsABSORBANCEVOLUMEFigure 10. DiffPlot FlagsDB Debris Platelet aggregates RBCs resistant to lysis (stroma) NRBCsSL Small Lymphocytes Small Lymphocytes Plt Aggregates NRBCs, RBCs resistant to lysis NE, LY, MO, EO, ATL & IMM aggedSL1 Small Lymph 1 Plt Aggregates NRBCs, RBCs resistant to lysis Small abnormal lymphs NE, LY, MO, EO, ATL, IMM%& #aggedNL Neut/ Lymph Small Neut w/ o granules Lymps with segmented nucleus Neuts with weak membranes NE, LY %& #aggedDiffPlot FlagsSL /SL1DBMonoAbsorbanceVolumeNeutLymphEosNLBeckman Coulter ACVDifferential Technology14DiffPlot FlagsFigure 11. DiffPlot FlagsMN Mono/ Neut Monos with hyperbasophilic granules Immature neuts with nonsegmented nucleus ATL, IMM %& #agged MO, NE %& #inherited ( . . . . )UM Upper Mono Large Monos Myelocytes, Promyelocytes Large Blasts NE, MO, IMM %& #aggedLN Lower Neut Fragile or aged neutrophils Plt Aggs, RBCs resistant to lysis All WBC Diff parameters aggedUN Upper Neut Large neutrophils Imm Grans (Metamyelocytes, myelocytes, promyelocytes) NE, IMM %& #aggedNE Neut/ Eos Immature Eos, Agranular Eos Atypical Neutrophils IMM %& #agged NE, EO %& #inhibited ( . . . . )UM UNNEMNLNMonoAbsorbanceVolumeNeutDebrisLymphEosFigure 12. DiffPlot FlagsATL Large Lymphs Reactive lymphs Plasma cells BlastsIMM Immature cells Large Monocytes Promyelocytes, Myelocytes,Metamyelocytes BlastsATLIMMAbsorbanceVolumeDebrisMonoNeutLymphEosRange (1) Range (1)WBC 5.5L 103/L 6.0/ 11.0 NE 53.8 % 50.0 / 80.0 FLAGSRBC 5.03 106/L 4.00 / 6.20 LY 37.4 % 25.0 / 50.0HGB 15.2 g/dL 11.0 / 18.8 MO 6.5 % 2.0/ 10.0HCT 44.5 % 35.0 / 55.0 EO 1.6 % 0.0/ 5.0MCV 89.0 fL 80.0 / 100.0 BA 0.7 % 0.0/ 5.0MCH 30.3 pg 26.0 / 34.0MCHC 34.2 g/dL 31.0 / 35.0RDW 17.7 % 10.0 / 20.0 NE# 2.77 103/L 2.0/ 8.0LY# 1.92 103/L 1.0/ 5.0PLT 169 103/L 150/ 400 MO# 0.33 103/L 0.1/ 1.0MPV 9.1 fL 6.0/ 10.0 EO# 0.08 103/L 0.0/ 0.4BA# 0.04 103/L 0.0/ 0.2VOLABSRBCWBC/BASOPLTNORMAL PATIENTBeckman Coulter ACT5diff Analyzer Case StudyHistoryA normal patient.CommentaryThe ACT 5diff differential correlates well with the manual differential. The differential DiffPlot displays the whitecell populations with each cell type welldened with no abnormalities. The redcell and platelet histograms areunremarkable with normal CBCparameter results.Manual Differential MorphologyNeutrophils 54%Bands 1%Lymphocytes 39%Monocytes 5%Eosinophils 1%Beckman Coulter ACT5diff Analyzer and Manual Differential Results16Beckman Coulter ACT5diff Analyzer and Manual Differential ResultsHistoryA 4-year-old femalepatient admitted to the emergency room with fever andvomiting. Enlarged lymph nodes noted on examination.CommentaryThe ACT 5diff differential correlateswell with the manual differential. Thelymph region of the DiffPlot demonstrates anincreased density of lymphocytes, even though the whitecell count is low. The MCHC issomewhat elevated and is believed to be related to the recent onset ofvomiting and fever. Otherwise thedifferential results and the histogramsappear unremarkable.Range (1) Range(1)WBC 3.6 L 103/L 6.0/ 11.0 NE 31.3 LL % 50.0 / 80.0 FLAGSRBC 5.14 106/L 4.00 / 6.20 LY 58.1 HH % 25.0 / 50.0HGB 13.8 g/dL 11.0 / 18.8 MO 6.2 % 2.0/ 10.0HCT 41.4 % 35.0 / 55.0 EO 4.0 % 0.0/ 5.0MCV 81.0 fL 80.0 / 100.0 BA 0.4 % 0.0/ 5.0MCH 26.8 pg 26.0 / 34.0MCHC 33.3 g/dL 31.0 / 35.0RDW 15.1 % 10.0 / 20.0 NE# 1.13 103/L 2.0/ 8.0 LymphocytosisLY# 2.10 103/L 1.0/ 5.0 NeutropeniaPLT 188 103/L 150/ 400 MO# 0.22 103/L 0.1/ 1.0MPV 8.7 fL 6.0/ 10.0 EO# 0.14 103/L 0.0/ 0.4BA# 0.01 103/L 0.0/ 0.2VOLABSRBCWBC/BASOPLTLYMPHOCYTOSISManual Differential MorphologyNeutrophils 32%Lymphocytes 60%Monocytes 5%Eosinophils 1%Basophils 1%Beckman Coulter ACT5diff Analyzer Case StudyRange (1) Range (1)WBC 4.5L 103/L 6.0/ 11.0 NE 41.5 LL% 50.0 / 80.0 FLAGSRBC 2.42 L 106/L 4.00 / 6.20 LY 22.6 L % 25.0 / 50.0 WBC: IMMHGB 8.5L g/dL 11.0 / 18.8 MO 12.7 HH % 2.0/ 10.0 RBC:HCT 24.8 L % 35.0 / 55.0 EO 21.9 HH % 0.0/ 5.0MCV 102H fL 80.0 / 100.0 BA 1.3 % 0.0/ 5.0MCH 35.0 H pg 26.0 / 34.0MCHC 34.1 g/dL 31.0 / 35.0RDW 12.8 % 10.0 / 20.0 NE# 1.87 L 103/L 2.0/ 8.0 NeutropeniaLY# 1.02 103/L 1.0/ 5.0 EosinophiliaPLT 312 103/L 150/ 400 MO# 0.57 103/L 0.1/ 1.0 MonocytosisMPV 7.8 fL 6.0/ 10.0 EO# 0.99HH 103/L 0.0/ 0.4 MacrocytesBA# 0.06 103/L 0.0/ 0.2VOLABSRBCWBC/BASOPLTEOSINOPHILIAHistoryA 38-year-old female patient with admitting diagnosis: Renal failure.CommentaryThe ACT 5diff differential DiffPlotdemonstrates an increased number ofcells in the eosinophil region. The WBCIMM ag signies the presence of anabnormal number of cells in the uppermonocyte, upper neutrophil and Channel127 regions of the DiffPlot. The IMMag indicates that a white cell countgreater than 0.4 103/ L or 2.5%can befound in this area. The MCV is elevatedand is represented very well on the redcell histogram. The platelet histogram is unremarkable.Manual Differential MorphologyNeutrophils 47% Macrocytosis +2Bands 1%Lymphocytes 19%Monocytes 6%Eosinophils 26%Basophils 1%Beckman Coulter ACT5diff Analyzer and Manual Differential Results18Beckman Coulter ACT5diff Analyzer and Manual Differential ResultsHistoryA 64-year-old female admitted withadvanced degenerative bone disease.CommentaryThe ACT 5diff DiffPlot displays apopulation of monocytes extending intothe upper monocyte and neutrophilregions, generating the IMM ag. Thecells associated with the IMM aginclude large monocytes and hyper-basophilic monocytes. The manual differential results agree with theautomated differential results and thewhite cell morphology correlate with theIMM ag. The *WBC ag indicates whitecell count interference with possiblyplatelet aggregates or nucleated redcells. This ag is generated from theWBC/ BASO histogram. Red cell andplatelet morphology are unremarkable.The red cell count, hemoglobin andhematocrit results appear decreased.Range (1) Range (1)WBC 15.8 HH 103/L 6.0/ 11.0 NE 64.1 % 50.0 / 80.0 FLAGSRBC 3.41 L 106/L 4.00 / 6.20 LY 11.1 LL % 25.0 / 50.0 WBC:*WBC, IMMHGB 11.0g/dL 11.0 / 18.8 MO 22.2 HH % 2.0/ 10.0 RBC:HCT 31.3 L % 35.0 / 55.0 EO 1.1 % 0.0/ 5.0 PLT:MCV 92 fL 80.0 / 100.0 BA 1.5 % 0.0/ 5.0MCH 32.1 pg 26.0 / 34.0MCHC 35.0 g/dL 31.0 / 35.0RDW 12.2 % 10.0 / 20.0 NE# 10.1 HH 103/L 2.0/ 8.0 LeukocytosisLY# 1.75 103/L 1.0/ 5.0 LymphopeniaPLT 190 103/L 150/ 400 MO# 3.51 HH 103/L 0.1/ 1.0 NeutrophiliaMPV 8.2 fL 6.0/ 10.0 EO# 0.17 103/L 0.0/ 0.4 NRBCsBA# 0.24 H 103/L 0.0/ 0.2 MonocytosisPlateletAggregatesVOLABSRBCWBC/BASOPLTMONOCYTOSISManual Differential MorphologyNeutrophils 65%Bands 5%Lymphocytes 13%Monocytes 17%Monocytes withincreasedgranulation and numerousvacuoles.Beckman Coulter ACT5diff Analyzer Case StudyRange (1) Range (1)WBC 16.3 HH 103/L 6.0/ 11.0 NE 91.5 HH % 50.0 / 80.0 FLAGSRBC 3.11 L 106/L 4.00 / 6.20 LY 3.0LL % 25.0 / 50.0 WBC:*WBC, IMMHGB 10.4 L g/dL 11.0 / 18.8 MO 2.5 % 2.0/ 10.0 RBC:HCT 30.1 L % 35.0 / 55.0 EO 2.0 % 0.0/ 5.0 PLT:MCV 97 fL 80.0 / 100.0 BA 1.0 % 0.0/ 5.0MCH 33.3 pg 26.0 / 34.0MCHC 34.5 g/dL 31.0 / 35.0RDW 13.3 % 10.0 / 20.0 NE# 14.9 HH 103/L 2.0/ 8.0 LeukocytosisLY# 0.49 LL 103/L 1.0/ 5.0 NeutrophiliaPLT 82 L 103/L 150/ 400 MO# 0.41 103/L 0.1/ 1.0 LymphopeniaMPV 8.7 fL 6.0/ 10.0 EO# 0.33 103/L 0.0/ 0.4 MyelemiaBA# 0.16 103/L 0.0/ 0.2 Immature CellsPlateletAggregatesVOLABSRBCWBC/BASOPLTLEFT SHIFTHistoryA 48-year-old female admitted with respiratory failure.CommentaryThe ACT 5diff differential results agreewith the ndings from the manualdifferential. The automated differentialresults (91.5%neutrophils) correlatewell with the thirty-three percent (33%)bands and sixty percent (60%)neutrophils reported in the manualdifferential. The DiffPlot demonstratesan increased intensity of cells in theneutrophil and upper neutrophil regions.This population of cells generates theIMM ag. The cells associated with this ag include myleocytes,metamyleocytes, promyleocytes, largeneutrophils and blasts. The *WBC agindicates white cell count interferencewith possibly platelet aggregates ornucleated red cells. Red cell counthemoglobin and platelet values aredecreased.Manual Differential MorphologyNeutrophils 60%Bands 33%Lymphocytes 7%NRBC 1%Beckman Coulter ACT5diff Analyzer and Manual Differential Results20Beckman Coulter ACT5diff Analyzer and Manual Differential ResultsHistoryA 70-year-old female admitted in respiratory failure.CommentaryThe ACT 5diff differential results agreewith the ndings from the manualdifferential. The automated differentialresults (86.2%neutrophils) correlatewell with the ninety-two percent (92%)neutrophils (bands and segs) reported inthe manual differential. The DiffPlot displays a small population of cells inthe lower lymph region, possibly plateletaggregates. The instrument generatesthe microcytosis interpretive ag basedon the customer denable limits and theMCV of 72 fL.Range (1) Range (1)WBC 17.8 HH 103/L 6.0/ 11.0 NE 86.2 HH % 50.0 / 80.0 FLAGSRBC 5.13 106/L 4.00 / 6.20 LY 4.8LL % 25.0 / 50.0 WBC:*WBCHGB 12.9 g/dL 11.0 / 18.8 MO 7.3 % 2.0/ 10.0 RBC:HCT 37.1 % 35.0 / 55.0 EO 1.0 % 0.0/ 5.0 PLT:MCV 72 LL fL 80.0 / 100.0 BA 0.7 % 0.0 / 5.0MCH 25.1 L pg 26.0 / 34.0MCHC 34.8 g/dL 31.0 / 35.0RDW 16.9 % 10.0 / 20.0 NE# 15.3 HH 103/L 2.0/ 8.0 LeukocytosisLY# 0.85 LL 103/L 1.0/ 5.0 LymphopeniaPLT 440 H 103/L 150/ 400 MO# 1.30 HH 103/L 0.1/ 1.0 NeutrophiliaMPV 7.9 fL 6.0 10.0 EO# 0.18 103/L 0.0/ 0.4 MonocytosisBA# 0.12 103/L 0.0/ 0.2 NRBCMicrocytesPlatelet aggregatesVOLABSRBCWBC/BASOPLTGRANULOCYTOSIS, MICROCYTOSISManual Differential MorphologyNeutrophils 83%Bands 9%Lymphocytes 4%Monocytes 4%Plateletsincreased,markedmicrocytosis.Beckman Coulter ACT5diff Analyzer Case StudyRange (1) Range (1)WBC 8.6 103/L 6.0/ 11.0 NE 6.1 RLL % 50.0 / 80.0 FLAGSRBC 3.99 L 106/L 4.00 / 6.20 LY 58.0 HH % 25.0 / 50.0 WBC: UM,ATL,IMMHGB 11.1g/dL 11.0 / 18.8 MO 33.6 RHH % 2.0/ 10.0 RBC:HCT 33.9 L % 35.0 / 55.0 EO 0.5 % 0.0/ 5.0 PLT: SCHMCV 85 fL 80.0 / 100.0 BA 1.8 % 0.0/ 5.0MCH 27.9 pg 26.0 / 34.0MCHC 32.9 g/dL 31.0 / 35.0RDW 13.9 % 10.0 / 20.0 NE# 0.53 RLL 103/L 2.0/ 8.0 LymphocytosisLY# 5.01 H 103/L 1.0/ 5.0 NeutropeniaPLT 18 RLL 103/L 150/ 400 MO# 2.90 RHH 103/L 0.1/ 1.0 Immature CellsMPV 8.4 R fL 6.0/ 10.0 EO# 0.04 103/L 0.0/ 0.4 Atypical Lymp,BA# 0.16 103/L 0.0/ 0.2 MonocytosisPlt interpretationimpossibleVOLABSRBCWBC/BASOPLTACUTE MYELOID LEUKEMIAHistoryA 78-year-old female admitted withAcute Myelogenous Leukemia.CommentaryThe ACT 5diff DiffPlot demonstrates an intense population of cells beginningin the lymphocyte region extendingthrough the lymphocytes, atypical lymphocytes, monocyte and uppermonocyte regions. The lack of separation within this cellular populationgenerates the WBC ags including UM,ATL, IMM and diff parameter R ags.The cells associated with these agsinclude large monocytes, myelocytes,promyleocytes and blasts, which is inagreement with the manual differentialresults. The platelet histogram displaysmarked interference from schistocytes,generating the platelet interpretationimpossible ag. Reassessment of theresults listed by a reference method is required. Reference method plateletresults were ve.Manual Differential MorphologyNeutrophils 14%Bands 11%Metamyelocytes 2%Blasts 7%Lymphocytes 47%Atypical Lymphs 5%Monocytes 14%Beckman Coulter ACT5diff Analyzer and Manual Differential Results22Numerousschistocytes, +2anisocytosis,plateletsdecreased andnumeroussmudge cells.Beckman Coulter ACT5diff Analyzer and Manual Differential ResultsHistoryA 27-year-old female presented to theemergency room with shortness ofbreath and severe joint and abdominalpain. Patient has a known history of Hb S, Sickle-Cell disease. Current diagnosis: Hb S, Sickle-Cell disease, crisis phase.CommentaryThe ACT 5diff differential DiffPlotdemonstrates a continuous populationof cells beginning in the lowerlymphocyte region. The lack ofseparation within this cellular populationgenerates the SL and SL1 ags. Thecells associated with these ags includesmall lymphocytes, nucleated red cellsand red cells resistant to cell lysis.Additionally, on the WBC/ BASOhistogram a population of cells appearsto the left of the lower threshold. Thispopulation of cells generates the *WBCag and differential R ags. These agsare in agreement with the manual differential results. The corrected whitecell count is 4.2x103/ L. The red cellhistogram displays a markedly widedistribution in agreement with the meancell volume and presence of targetcells. The platelet histogram isunremarkable.Range (1) Range (1)WBC 5.0L 103/L 6.0/ 11.0 NE 33.9 RLL % 50.0 / 80.0 FLAGSRBC 2.39 LL 106/L 4.00 / 6.20 LY 59.4 RHH % 25.0 / 50.0 WBC:*WBC,SL,SL1HGB 8.3LL g/dL 11.0 / 18.8 MO 2.2R % 2.0/ 10.0 RBC:HCT 23.8 LL % 35.0 / 55.0 EO 3.8R % 0.0/ 5.0 PLT:MCV 100H fL 80.0 / 100.0 BA 0.7 % 0.0/ 5.0MCH 34.8 H pg 26.0 / 34.0MCHC 34.9 g/dL 31.0 / 35.0RDW 17.8 % 10.0 / 20.0 NE# 1.69 RL 103/L 2.0/ 8.0 LymphocytosisLY# 2.96 R 103/L 1.0/ 5.0 NeutropeniaPLT 257 103/L 150/ 400 MO# 0.11 R 103/L 0.1/ 1.0 NRBCsMPV 9.7 fL 6.0/ 10.0 EO# 0.19 R 103/L 0.0/ 0.4 AnemiaBA# 0.03 103/L 0.0/ 0.2VOLABSRBCWBC/BASOPLTHB S, SICKEL-CELL CRISISManual Differential MorphologyNeutrophils 26%Lymphocytes 72%Eosinophils 1%Basophils 1%NRBC 18/ 100 WBCNumerous sicklecells, +3 targetcells, +1 aniso-cytosis, rarelarge platelet.Eastern Europe, Middle East, North Africa: Switzerland, Nyon(41) 22 994 0707. Australia, Gladesville(61) 2 9844 6000. Canada, Mississauga(1) 905 819 1234. China, Beijing(86) 10 6515 6028. Hong Kong(852) 2814 7431, 2814 0481. France, Villepinte(33) 1 49 90 90 00. Germany, Krefeld(49) 2151 33 35. Italy, Milan(39) 02 953921. Japan, Tokyo(81) 3 5404 8424. Latin America(1) (305) 380 4709. Mexico, Mexico City(525) 575 6805. Netherlands, Mijdrecht(31) 297 230630. Singapore(65) 6339 3633. South Africa/Sub-Saharan Africa, Johannesburg(27) 11 805 2014. Sweden, Bromma(46) 8 564 85 900. Switzerland, Nyon0800 850 810. Taiwan, Taipei (886) 2 2378 3456. Turkey, Istanbul (90) 216 309 1900. UK, High Wycombe(44) 01494 441181. USA, Brea, CA(1) 800 352 3433, (1) 714 993 5321. www.beckmancoulter.com Copyright 2002 Beckman Coulter, Inc. PRINTED IN U.S.A.SI M P L I F Y A U T OM AT E I N N OVAT E