metodo, entrenamiento y principios del advia 120

ADVIAADVIA 120 120

TECHNOLOGYTECHNOLOGY

ADVIA 120

• Hematology Analyzer• Complete Blood Count (CBC)• White Blood Cell Differential (Diff)• Reticulocyte Analysis (Retic)

• Analysis on one aspiration of whole blood• 120 CBC/Diff samples per hour• Random Access Test Selectivity

Sample Handling

3 Modes of Aspiration

• Multiple tube size• Multiple tube types• Small sample volume (157uL)

Auto

Open Closed

Unified Fluidics Circuit

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.

Unified Fluids Circuit (UFC)

Dividing the Sample

The 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

ADVIAADVIA 120 120

METHODSMETHODS

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 form the reaction product.

HEMOGLOBIN + HGB reagent METHEMOGLOBIN CYANIDE HGB

CYANISATION

Fe++ Fe+++ Fe+++.CN

The HGB Method

ADVIA 120 HGB contains:

- Potassium cyanide, 20 mmol/L- Dimethyllaurylamine oxide, 2.0%

UFC Filter + Photodiode

HGB reaction chamber

Lampassembly

The HGB Method

Optical readings are obtained colorimetrically at 546 nm.

seconds

1. ADVIA 120 Sheath/Rinse reading from previous cycle

2. Draining and refilling with the reaction solution

3. Reaction solution readings (Sample Mean)

4. Draining and refilling with the ADVIA 120 Sheath/Rinse

5. ADVIA 120 Sheath/Rinse readings (Baseline Mean)

The HGB Method

Calculating reported parameters

• HGB Hemoglobin (directly measured)

• MCH (HGB ÷ RBC) x 10

(Mean Corpuscular

Hemoglobin)

• MCHC (HGB ÷ [RBC x MCV]) x 1000

(Mean Corpuscular

Hemoglobin Concentration)

The HGB Method

Sample stream

Sheath streamShuttle chamber

FLOWCELL TECHNOLOGY

ADVIA 120 SHEATH encases the sample stream as the two fluids pass through the flowcell. Light detects the cells as they pass through the light path.

ADVIA 120 RBC/PLT contains:

- Sodium dodecyl sulfate, 0.035 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%- Buffer

Reaction:

- ADVIA 120 RBC/PLT reagent contains sodium dodecyl sulfate (SDS) and glutaraldehyde that causes sphering of the red blood cells and platelets. When red cells and platelets are isovolumetrically sphered, shape is eliminated as a variability factor.- RBCs and platelets are fixed

The RBC Method

No matterwhat your shape or size ....We can make you a SPHERE

The RBC Method

Low angle scatter 2o - 3o (Volume)

High angle scatter 5o - 15o (HGB concentration)

The RBC Method

The RBC Method

Referentie signaal

Laserdiode Sample stream Beamsplitter Dark stop Mirror

Absorption Low-angle High-angle detector scatter scatter

detector detectorFront view of the dark stop

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 along the x axis, and the low-angle light scatter (2° to 3°) is plotted along the y axis.

1. Low-angle light scatter (2° to 3°)

2. High-angle light scatter (5° to 15°)

3. Mie map containing RBCs

4. Platelets detected in RBC method

The Volume/Hemoglobin Concentration(V/HC) cytogram is a linear version of the RBC map that appears on the RBC cytogram. On the V/HC cytogram, hemoglobin concentration is plotted along the x axis and cell volume is plotted along the y axis. Only red blood cells appear on this cytogram.

1. 60 fL volume marker

2. 120 fL volume marker

3. 28 g/dL HC marker

4. 41 g/dL HC marker

The RBC Method

The RBC Method

28 41

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0

Microcytic

Macrocytic Hyperchrom

icHy p

och r

mic

Vo

lum

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MC

V

HGB Concentration - CHCM

NormocyticNormochromic

The RBC Method

Vo

lum

e -

MC

V

HGB Concentration - CHCM

28 41

6012

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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 shaped distribution with a mode channel between 60 fL and 120 fL. The mean corpuscular volume (MCV) and the red cell distribution width (RDW) are determined from this histogram.

• MCV is the mean of the of RBC Volume histogram.

• RDW is the coefficient of variation of the population.

The RBC Method

The RBC hemoglobin concentration (RBC HC) histogram represents the distribution of red blood cells by cellular hemoglobin concentration. The histogram has a range from 0 g/dL to 50 g/dL.

Normal samples have a bell-curve shaped Hgb concentration distribution with a mean channel between 28 g/dL and 41 g/dL.

The cell hemoglobin concentration mean (CHCM) and the hemoglobin distribution width (HDW) are obtained from this histogram. • CHCM is the mean of the RBC HC histogram.

• HDW is the standard deviation of the RBC HC histogram.

The RBC Method

The RBC CH (cellular hemoglobin) histogram represents the distribution of red blood cells by the amount of hemoglobin present in each cell independent of cell volume. The histogram has a range from 0 picograms to 100 picograms.

• Cellular Hemoglobin Content (CH) is the mean of the RBC CH histogram.

• Cell hemoglobin distribution width (CHDW) is the standard deviation of the RBC CH histogram.

The RBC Method


The RBC Method

• RBC Number of Red Cells (directly measured)

(Red Blood cel Count)

• MCV Mean of RBC Volume histogram

(Mean Corpuscular Volume)

• HCT (RBC x MCV) ÷ 10

(Hematocrit)

• MCH (HGB ÷ RBC) x 10

(Mean Corpuscular

Hemoglobin)

• MCHC (HGB ÷ [RBC x MCV]) x 1000 (Mean

Corpuscular Hemoglobin

Concentration)


The RBC Method

• CHCM Mean of RBC HC histogram

(Corpuscular Hemoglobin

Concentration Mean)

• CH Mean of RBC CH histogram

(Corpuscular Hemoglobin

content)

• RDW 100 x (SD of RBC Volume histogram ÷ MCV)

(Red cell volume Distribution

Width)

• HDW SD of RBC HC histogram

(Hemoglobin concentration

Distribution Width)

• %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 HGB


The RBC Method

The three severity levels are: +, ++ or +++ and are customized by Bayer for each customer site based on the technologists severity levels on the manual differentials

The 2-Dimensional platelet analysis (2D-PLT method) is based on the integrated analysis of red blood cell and platelet measurements.

Area of Platelet Analysis

The Platelet Method

Using the Mie theory of light scattering for homogeneous spheres , the low-angle and high-angle light scatter signals for each cell are transformed into volume and refractive index values.

The PLT Scatter cytogram is the graphical representation of two light-scatter measurements • (5° to 15°), scatter is plotted on the x axis • (2° to 3°), scatter is plotted on the y axis V

olu

me

= S

ize

Refractive Index = Platelet Content

The Platelet Method

PLATELET CYTOGRAM

1 Platelets 2 Large platelets3 Red blood cells4 RBC fragments 5 RBC ghosts

1

2

3

4

5

The Platelet Method

The Platelet Method

The 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.

• PLT 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)


The Platelet Method

• LPLT The percentage of large platelets (%LPLT) is greater than

(Large Platelets) 10% of the platelet count

• RBCF 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/ul

• RBCG 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

Morphology Flags

The three severity levels are: +, ++ or +++

The Platelet Method

The Retic Method

ADVIA 120 autoRETIC contains:

- Oxazine 750, 11.4 mg/L- Buffer- N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, 0.023 mmol/L

Reaction:

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 their RNA content.

The Retic Method

Referentie signaal



detector detector

Front view of the dark stop

The Retic Method

The Retic Method

The RETIC Scatter ABS cytogram is the graphical representation of the absorption and light-scatter measurements: • absorption (cell maturation) is plotted along the x axis • light 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 retics D High absorption retics E PlateletsF Coincidence events

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 mature RBCs 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)

The 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)

The Retic Method

Reticulocyte population (blue)

• %RETIC 100 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)

The Retic Method


• IRF-H 100 x (#HRetic ÷ RETIC Count)

(Immature Reticulocytes

Fraction High)

• IRF-M+H 100 x ([#HRetic + #MRetic] ÷ RETIC Count) (Immature

Reticulocytes Fraction Medium +

High)

These Parameters Not FDA Cleared For Reporting - Investigational Use Only

The Retic Method


Erythropoietin Treatment

Beginning After 4 days

After 2 weeks After 1 month

The Retic Method

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, 0.100 mmol/L- Buffer

Reaction:

- Surfactants (sodium dodecyl sulfate and Brij-35) in combination with thermal stress lyse the red blood cells.- Formaldehyde fixes the white blood cells.


- 4-Chloro-1-naphthol, 44.8 mmol/L- Diethylene glycol, 99.2%


- Stabilizer- Hydrogen peroxide, 0.3%

Reaction:

- The 4-Chloro-1-naphthol in ADVIA 120 PEROX 2 serves as a substrate that enables the hydrogen peroxide in ADVIA 120 PEROX 3 to form a dark precipitate at sites of peroxidase activity in the granules of white blood cells as described by the following equation: cellular peroxidase H2O2 + 4-chloro-1-naphthol dark precipitate within the cells

The Perox Method

The Perox Method

I’mmelting

PEROXSTAIN

Boy,your granuleslook great !

But youstill lookpale

If you have the granules - we have the stain

# gr

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les

Cell maturation

Blasts

PromyelocytesMyelocytes

Metamyelocytes

Band cellsMature PMN

Bone marrow Blood

Number of neutrophil granules

The Perox Method

Cytochemical classification according to peroxidase activity

Cel type Peroxidase

Myeloblasts -, sometimes ½+ (especially micromyeloblasts)Promyelocytes 3+Myelocytes 3+Metamyelocytes 3+Band cells 2-3+Neutrophils 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 -

The Perox Method

Tungsten lamp

Filter

Absorption detector

Sample streamScatter detector

Beam splitterDarkstop

The Perox Method

Scatter signal to measure the volume ofthe cells

Absorption signal for peroxidase activity measurement

Cells with medium peroxidase activity absorbs less light

than cells with high peroxidase activity

The Perox Method

The Perox Method

The 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.

Absorbed light = Peroxidase Activity

Ligh

t sc

atte

r =

Cel

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e

1 Noise2 Nucleated Red Blood Cells3 Platelet Clumps4 Lymphocytes and Basophils5 Large Unstained Cells6 Monocytes7 Neutrophils8 Eosinophils

The Perox Method

• WBCP 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)

The Perox Method


• %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)

The Perox Method


• 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)

The Perox Method

Morphology Flags


ADVIA 120 BASO contains:

- Hydrochloric acid, 9.00 mmol/L- Phthalic acid, 21.49 mmol/L- Preservative- Surfactant

Reaction:

- The ADVIA 120 BASO reagent contains phthalic acid and a surfactant which lyses the red cells, platelets, and the cytoplasm of all white cell types except basophils.

The Baso Method

The Baso Method

Referentie signaal



detector detector

Front view of the dark stop

The Baso Method

The Baso Method

When 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.

Cel

l Siz

e

Nuclear Configuration

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 Eosinophil nuclei)

The Baso Method

• WBCB RawWBC x (BasoCalFactor) (White Blood

cell Count Baso)

• %BASO 100 x (BASO Count ÷ BASO PHA Cells ) (%Basophils)

• #BASO (%BASO ÷ 100) x WBCB (#Basophils)

• %BLAST 100 x (Blasts ÷ BASO PHA Cells ) (%Blasts)

• %MN 100 x (MN ÷ BASO PHA Cells )

(%Mononuclear cells)

• %PMN 100 x (PMN ÷ BASO PHA Cells )

(%Polymorphonuclear cells)

• %BASO Suspect 100 x (BASO Suspect ÷ BASO PHA Cells )

(%BASO Suspect)

The Baso Method


The Baso Method

Morphology Flags


• BLASTS The presence of blasts is suspected.

(Blasts)

• LS The presence of nonsegmented neutrophils (bands) is suspected.

(Left Shift)

THE ENDTHE END

metodo, entrenamiento y principios del advia 120

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