6 Unified Fluids Circuit (UFC) The UFC assembly uses Unifluidics technology.The UFC block is made up of eight acrylic plates. Machined within these plates are the pathways for the fluids and air flow, valves, and four reaction chambers. An additional chamber is mounted on the outside surface of the UFC block.The reagent pump assembly, mounted to the bottom of the UFC block, is also acrylic.
8 Dividing the SampleThe Sample Shear Valve divides the sample into 5 aliquots for the different types of tests. The reagents and sample segments are delivered to their respective reaction chambers for mixing and aspiration.Side View of UFC
10 The HGB Method ADVIA 120 HGB contains: - Potassium cyanide, 20 mmol/L - Dimethyllaurylamine oxide, 2.0%Reaction:- Red blood cells are lysed to release hemoglobin.- The heme iron in the hemoglobin is oxidized from the ferrous to the ferric state,and then it is combined with cyanide in the ADVIA 120 HGB reagent to formthe reaction product.CYANISATIONHEMOGLOBIN + HGB reagent METHEMOGLOBIN CYANIDE HGBFe++Fe+++Fe+++.CN
11 The HGB Method HGB reaction chamber Lamp assembly UFC Filter + PhotodiodeHGB reaction chamberLampassemblyOptical readings are obtained colorimetrically at 546 nm.
12 The HGB Method 1. ADVIA 120 Sheath/Rinse reading from previous cycle 2. Draining and refilling with the reaction solution3. Reaction solution readings (Sample Mean)4. Draining and refilling with the ADVIA 120 Sheath/Rinse5. ADVIA 120 Sheath/Rinse readings (Baseline Mean)seconds
13 Calculating reported parameters The HGB MethodCalculating reported parametersHGB Hemoglobin (directly measured)MCH (HGB ÷ RBC) x (Mean Corpuscular Hemoglobin)MCHC (HGB ÷ [RBC x MCV]) x (Mean Corpuscular Hemoglobin Concentration)
14 FLOWCELL TECHNOLOGY Sample stream Sheath stream Shuttle chamber ADVIA 120 SHEATH encases the sample streamas the two fluids pass through the flowcell. Lightdetects the cells as they pass through the light path.
15 The RBC Method ADVIA 120 RBC/PLT contains: - Sodium dodecyl sulfate, mmol/L- Disodium EDTA dihydrate, 4.03 mmol/L- Tetrasodium EDTA dihydrate, 3.36 mmol/L- Sodium chloride, 109.3 mmol/L- Glutaraldehyde, 0.11%- BufferReaction:- ADVIA 120 RBC/PLT reagent contains sodium dodecyl sulfate (SDS) andglutaraldehyde that causes sphering of the red blood cells and platelets.When red cells and platelets are isovolumetrically sphered, shape is eliminatedas a variability factor.- RBCs and platelets are fixed
16 The RBC Method No matter what your shape or size .... We can make you a SPHERE
17 The RBC Method Low angle scatter 2o - 3o (Volume) High angle scatter 5o - 15o (HGB concentration)
18 The RBC Method Laserdiode Sample stream Beamsplitter Dark stop Mirror Referentie signaalLaserdiode Sample stream Beamsplitter Dark stop MirrorAbsorption Low-angle High-angledetector scatter scatterdetector detectorFront view of the dark stop
19 The RBC Method The RBC Scatter cytogram is the graphical representation of two light-scatter measurements:the high-angle light scatter (5° to 15°) is plotted alongthe x axis, and the low-angle light scatter (2° to 3°) isplotted along the y axis.1. Low-angle light scatter (2° to 3°)2. High-angle light scatter (5° to 15°)3. Mie map containing RBCs4. Platelets detected in RBC method
20 The RBC Method The Volume/Hemoglobin Concentration (V/HC) cytogram is a linear version of the RBC mapthat appears on the RBC cytogram.On the V/HC cytogram, hemoglobin concentration isplotted along the x axis and cell volume is plotted alongthe y axis. Only red blood cells appear on this cytogram.fL volume markerfL volume markerg/dL HC markerg/dL HC marker
24 The RBC Method The RBC Volume histogram represents the distribution of red blood cells by cell volume.The histogram has a range from 0 fL to 200 fL.Normal samples have a bell-curve shapeddistribution with a mode channel between60 fL and 120 fL.The mean corpuscular volume (MCV) andthe red cell distribution width (RDW) aredetermined from this histogram.MCV is the mean of the of RBC Volumehistogram.RDW is the coefficient of variation of thepopulation.
25 The RBC Method The RBC hemoglobin concentration (RBC HC) histogram represents the distribution of red bloodcells by cellular hemoglobin concentration.The histogram has a range from 0 g/dL to 50 g/dL.Normal samples have a bell-curve shapedHgb concentration distribution with a meanchannel between 28 g/dL and 41 g/dL.The cell hemoglobin concentration mean (CHCM)and the hemoglobin distribution width (HDW) areobtained from this histogram.CHCM is the mean of the RBC HC histogram.HDW is the standard deviation of the RBC HChistogram.
26 The RBC Method The RBC CH (cellular hemoglobin) histogram represents the distribution of red blood cells bythe amount of hemoglobin present in each cellindependent of cell volume.The histogram has a range from 0 picograms to100 picograms.Cellular Hemoglobin Content (CH) is the mean ofthe RBC CH histogram.Cell hemoglobin distribution width (CHDW)is the standard deviation of the RBC CH histogram.
27 Calculating reported parameters The RBC MethodCalculating reported parametersRBC Number of Red Cells (directly measured) (Red Blood cel Count)MCV Mean of RBC Volume histogram (Mean Corpuscular Volume)HCT (RBC x MCV) ÷ (Hematocrit)MCH (HGB ÷ RBC) x (Mean Corpuscular Hemoglobin)MCHC (HGB ÷ [RBC x MCV]) x (Mean Corpuscular Hemoglobin Concentration)
28 Calculating reported parameters The RBC MethodCalculating reported parametersCHCM Mean of RBC HC histogram (Corpuscular Hemoglobin Concentration Mean)CH Mean of RBC CH histogram (Corpuscular Hemoglobin content)RDW x (SD of RBC Volume histogram ÷ MCV) (Red cell volume Distribution Width)HDW SD of RBC HC histogram (Hemoglobin concentration Distribution Width)
29 Calculating reported parameters The RBC MethodCalculating reported parameters%MICRO Percent of red blood cells smaller than 60 fL%MACRO Percent of red blood cells larger than120 fL%HYPO Percent of red blood cells with less than 28 g/dL HGB%HYPER Percent of red blood cells with more than 41 g/dL HGBThe three severity levels are: +, ++ or +++ and are customized by Bayer for each customer sitebased on the technologists severity levels on the manual differentials
30 Area of Platelet Analysis The Platelet MethodThe 2-Dimensional platelet analysis (2D-PLT method)is based on the integrated analysis of red blood celland platelet measurements.Area of Platelet Analysis
31 Refractive Index = Platelet Content The Platelet MethodUsing the Mie theory of light scattering for homogeneousspheres , the low-angle and high-angle light scatter signalsfor each cell are transformed into volume and refractive index values.The PLT Scatter cytogram is the graphical representationof two light-scatter measurements(5° to 15°), scatter is plotted on the x axis(2° to 3°), scatter is plotted on the y axisVolume = SizeRefractive Index = Platelet Content
32 The Platelet Method PLATELET CYTOGRAM 2 1 Platelets 2 Large platelets 345PLATELET CYTOGRAM1 Platelets2 Large platelets3 Red blood cells4 RBC fragments5 RBC ghosts
33 The Platelet MethodThe 2D-PLT VOL histogram shows the distribution of cells by volume. Volume data are obtained from the integrated analysis.The histogram has a range from 0 fL to 60 fL.
34 Calculating reported parameters The Platelet MethodCalculating reported parametersPLT PLT Count x RBC Cal Factor x PLT Cal Factor (Platelet count)MPV Mean of 2D-PLT Vol histogram (Mean Platelet Volume)Large LPLT Platelets with volumes greater than 20 fL (Large Platelets)
35 The three severity levels are: +, ++ or +++ The Platelet MethodMorphology FlagsThe three severity levels are: +, ++ or +++LPLT The percentage of large platelets (%LPLT) is greater than (Large Platelets) % of the platelet countRBCF The presence of RBC fragments is suspected. This flag is (RBC Fragments) triggered if the number of events in the RBC Fragment area of the PLT Scatter cytogram is greater than 100,000 cells/ulRBCG The presence of RBC ghosts is suspected. This flag occurs if (RBC Ghosts) the number of events in the RBC Ghost area of the PLT Scatter cytogram is greater than 100,000 cells/ul
36 The Retic Method ADVIA 120 autoRETIC contains: - Oxazine 750, 11.4 mg/L- Buffer- N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, mmol/LReaction:The ADVIA 120 autoRETIC reagent contains a zwitterionic detergent (surfactant)that isovolumetrically spheres the red cells.It also contains a cationic dye, Oxazine 750, that stains cells according to theirRNA content.
38 The Retic MethodLaserdiode Sample stream Beamsplitter Dark stop MirrorReferentie signaalAbsorption Low-angle High-angledetector scatter scatterdetector detectorFront view of the dark stop
39 The Retic MethodThe RETIC Scatter ABS cytogram is the graphical representation of the absorption and light-scatter measurements:absorption (cell maturation) is plotted along the x axislight scatter (cell size) is plotted along the y axis.1 RTC Platelet threshold2 RTC Coincidence threshold3 RTC threshold4 Low/Medium RTC threshold5 Medium/High RTC thresholdA Mature RBCsB Low absorption reticsC Medium absorption reticsD High absorption reticsE PlateletsF Coincidence events
40 The Retic Method The RETIC Volume histogram represents the overlaid distributions of mature RBCs and reticulocytes by cell size only.The histogram has a range from 0 fL to 200 fL..The RETIC hemoglobin concentration (RETIC HC) histogram represents the overlaid distributions of matureRBCs and reticulocytes by cellular hemoglobin concentration only.The histogram has a range from 0 g/dL to 50 g/dL.Mature RBC population (red)Reticulocyte population (blue)
41 Reticulocyte population (blue) The Retic MethodThe RETIC cellular hemoglobin (RETIC CH) histogram represents the overlaid distributions of mature RBCs and reticulocytes by the actual weight or mass of hemoglobin present in each cell.The histogram has a range from 0 pg to 100 pg.Mature RBC population (red)Reticulocyte population (blue)
42 Calculating reported parameters The Retic MethodCalculating reported parameters%RETIC x (RETIC Count) x % Retic Cal Factor (%Reticulocytes)#RETIC RBC x (%Retic ÷ 100) (#Reticulocytes)MCVr Mean of the RETIC Volume histogram for the reticulocyte (Mean Cell Volume population reticulocytes)CHr Mean of the RETIC CH histogram for the reticulocyte (Cellular Hemoglobin population content reticulocytes)CHCMr Mean of the Retic HC histogram for the reticulocyte population (Cell Hemoglobin Concentration Mean reticulocytes)
43 Calculating reported parameters The Retic MethodCalculating reported parametersIRF-H x (#HRetic ÷ RETIC Count) (Immature Reticulocytes Fraction High)IRF-M+H x ([#HRetic + #MRetic] ÷ RETIC Count) (Immature Reticulocytes Fraction Medium + High)These Parameters Not FDA Cleared For Reporting - Investigational Use Only
45 The Perox Method ADVIA 120 PEROX 1 contains: - Sodium dodecyl sulfate, 0.36 mmol/L- Sorbitol, 620 mmol/L- Sodium chloride, 8.35 mmol/L- Formaldehyde, 5.5%- BRIJ-35, mmol/L- BufferReaction:- Surfactants (sodium dodecyl sulfate and Brij-35) in combination with thermal stresslyse the red blood cells.- Formaldehyde fixes the white blood cells.
46 The Perox Method ADVIA 120 PEROX 2 contains: - 4-Chloro-1-naphthol, 44.8 mmol/L- Diethylene glycol, 99.2%ADVIA 120 PEROX 3 contains:- Stabilizer- Hydrogen peroxide, 0.3%Reaction:- The 4-Chloro-1-naphthol in ADVIA 120 PEROX 2 serves as a substrate that enables thehydrogen peroxide in ADVIA 120 PEROX 3 to form a dark precipitate at sites of peroxidaseactivity in the granules of white blood cells as described by the following equation:cellular peroxidaseH2O2 + 4-chloro-1-naphthol dark precipitate within the cells
47 The Perox Method If you have the granules - we have the stain I’m meltingPEROXSTAINBoy,your granuleslook great !But youstill lookpaleIf you have the granules - we have the stain
48 Number of neutrophil granules The Perox MethodNumber of neutrophil granulesBone marrowBloodPromyelocytesMyelocytesMetamyelocytes# granulesBand cellsMature PMNBlastsCell maturation
49 The Perox MethodCytochemical classification according to peroxidase activityCel type PeroxidaseMyeloblasts -, sometimes ½+ (especially micromyeloblasts)Promyelocytes 3+Myelocytes 3+Metamyelocytes 3+Band cellsNeutrophils 2+Eosinophils 4+Basophils ½-1+ (stay unstained in the ADVIA 120)Lymphoblasts -Prolymphocytes -Lymphocytes -Atypical lymphocytes -Monoblasts -Promonocytes ½-1+Monocytes 1+Plasma cells -Nucleated red blood cells -
51 The Perox Method Scatter signal to measure the volume of the cells Absorption signal for peroxidase activity measurementCells with medium peroxidase activity absorbs less lightthan cells with high peroxidase activity
52 The Perox MethodThe PEROX cytogram is divided into 100 counting channels on each axis. The cells absorb light proportional to the amount of peroxidase stain present, and this is represented on the x axis. Cells scatter light proportional to their size, and this is represented on the y axis.When the light scatter and absorption data are plotted, distinct populations or clusters are formed. Cluster analysis identifies each population based on its position, area, and density, and then the number of cells in each population is processed. The lines that separate the different cell populations are calculated by the software on a sample-by-sample basis.Light scatter = Cell SizeAbsorbed light = Peroxidase Activity1 Noise2 Nucleated Red Blood Cells3 Platelet Clumps4 Lymphocytes and Basophils5 Large Unstained Cells6 Monocytes7 Neutrophils8 Eosinophils
54 Calculating reported parameters The Perox MethodCalculating reported parametersWBCP RawWBC x (PeroxCalFactor) (White Blood cell Count Perox)%NEUT ([100 x Neutrophil Count] + %HPX) ÷ PHA Cells (%Neutrophils)#NEUT (%NEUT ÷ 100) x WBC (#Neutrophils)%LYMPH ([100 x Lymphocyte Count] ÷ PHA Cells) - %BASO (%Lymphocytes)#LYMPH (%LYMPH ÷ 100) x WBC (#Lymphocytes)%MONO (100 x Monocyte Count) ÷ PHA Cells (%Monocytes)#MONO (%MONO ÷ 100) x WBC (#Monocytes)
55 Calculating reported parameters The Perox MethodCalculating reported parameters%EOS (100 x Eosinophil Count) ÷ PHA Cells (%Eosinophils)#EOS (%EOS ÷ 100) x WBC (#Eosinophils)%LUC (100 x LUC Count) ÷ PHA Cells (%Large Unstained Cells))#LUC (%LUC ÷ 100) x WBC (#Large Unstained Cells)
56 The three severity levels are: +, ++ or +++ The Perox MethodMorphology FlagsThe three severity levels are: +, ++ or +++ATYP The presence of atypical lymphocytes is suspected (Atypical Lymphocytes)IG The presence of immature granulocytes is suspected (Immature Granulocytes)MPO Sample is a weak peroxidase stainer. (Myeloperoxidase deficiency)NRBC The presence of nucleated red blood cells is suspected. (Nucleated Red Blood Cells)PLT-CLM Presence of clumped platelets is suspected (Platelet Clumps)
57 The Baso Method ADVIA 120 BASO contains: - Hydrochloric acid, 9.00 mmol/L- Phthalic acid, mmol/L- Preservative- SurfactantReaction:- The ADVIA 120 BASO reagent contains phthalic acid and a surfactant which lysesthe red cells, platelets, and the cytoplasm of all white cell types except basophils.
59 The Baso Method Laserdiode Sample stream Beamsplitter Dark stop Mirror Referentie signaalAbsorption Low-angle High-angledetector scatter scatterdetector detectorFront view of the dark stop
60 Nuclear Configuration The Baso MethodWhen the high-angle light scatter (nuclear configuration) is plotted on the x axis, and the low-angle light scatter (cell size) is plotted on the y axis, distinct populations or clusters are formed. Cluster analysis identifies each population based on its position, area, and density, and then counts the number of cells/nuclei in each population.The BASO cytograms is representative of a patient specimen.1 Noise2 Blast cell nuclei3 Mononuclear WBCs (Monocyte and Lymphocyte nuclei)4 Basophils5 Baso Suspect6 Saturation7 Polymorphonuclear WBCs (Neutrophil and Eosinophilnuclei)Cell SizeNuclear Configuration
62 Calculating reported parameters The Baso MethodCalculating reported parametersWBCB RawWBC x (BasoCalFactor) (White Blood cell Count Baso)%BASO x (BASO Count ÷ BASO PHA Cells ) (%Basophils)#BASO (%BASO ÷ 100) x WBCB (#Basophils)%BLAST 100 x (Blasts ÷ BASO PHA Cells ) (%Blasts)%MN x (MN ÷ BASO PHA Cells ) (%Mononuclear cells)%PMN x (PMN ÷ BASO PHA Cells ) (%Polymorphonuclear cells)%BASO Suspect 100 x (BASO Suspect ÷ BASO PHA Cells ) (%BASO Suspect)
63 The three severity levels are: +, ++ or +++ The Baso MethodMorphology FlagsThe three severity levels are: +, ++ or +++BLASTS The presence of blasts is suspected (Blasts)LS The presence of nonsegmented neutrophils (bands) is suspected (Left Shift)