ADVIA 120

TECHNOLOGY

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

ADVIA 120

METHODS

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 form
the reaction product.
CYANISATION
HEMOGLOBIN + HGB reagent
Fe++

METHEMOGLOBIN
Fe+++

CYANIDE HGB
Fe+++.CN

The HGB Method

HGB reaction chamber

Lamp
assembly

UFC

Filter + Photodiode

Optical readings are obtained colorimetrically at 546 nm.

The HGB Method

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
seconds

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
(Mean Corpuscular
Hemoglobin Concentration)

(HGB [RBC x MCV]) x 1000

FLOWCELL TECHNOLOGY

Shuttle chamber

Sheath stream

Sample stream
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.

The RBC Method

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 matter
what 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

Laserdiode

Sample stream

Beamsplitter

Dark stop

Mirror

Referentie signaal
Absorption
detector
Front view of the dark stop

Low-angle
scatter
detector

High-angle
scatter
detector

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 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

Hypochrmic

120
60

Volume - MCV

Normocytic
Normochromic

Microcytic
28

41

HGB Concentration - CHCM

Hyperchromic

Macrocytic

Volume - MCV

The RBC Method

HGB Concentration - CHCM

60

120

The RBC Method

28

41

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

Calculating reported parameters

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
(Mean Corpuscular
Hemoglobin Concentration)

(HGB [RBC x MCV]) x 1000

The RBC Method

Calculating reported parameters

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)

The RBC Method

Calculating 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 HGB

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 Platelet Method

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

Volume = Size

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

Refractive Index = Platelet Content

The Platelet Method

PLATELET CYTOGRAM

2
5

3
1
4

1
2
3
4
5

Platelets
Large platelets
Red blood cells
RBC fragments
RBC ghosts

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.

The Platelet Method

Calculating reported parameters

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
(Large Platelets)

Platelets with volumes greater than 20 fL

The Platelet Method

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

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

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

The Retic Method

Laserdiode

Sample stream

Beamsplitter

Dark stop

Mirror

Absorption
detector

Low-angle
scatter
detector

High-angle
scatter
detector

Referentie signaal

Front view of the dark stop

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
2
3
4
5
A
B
C
D
E
F

RTC Platelet threshold

RTC Coincidence threshold
RTC threshold
Low/Medium RTC threshold
Medium/High RTC threshold
Mature RBCs
Low absorption retics
Medium absorption retics
High absorption retics
Platelets
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 Method

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

Calculating reported parameters

%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

Calculating reported parameters

IRF-H

100 x (#HRetic RETIC Count)

(Immature Reticulocytes

Fraction High)

IRF-M+H

100 x ([#HRetic + #MRetic] RETIC Count)

Reticulocytes

Fraction Medium + High)

(Immature

These Parameters Not FDA Cleared For Reporting - Investigational Use Only

The Retic Method

Erythropoietin Treatment

Beginning

After 2 weeks

After 4 days

After 1 month

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.

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 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

If you have the granules - we have the stain

Im
melting

PEROX
STAIN

But you
still look
pale
Boy,
your granules
look great !

The Perox Method

Number of neutrophil granules

Bone marrow

Blood

# granules

Promyelocytes
Myelocytes
Metamyelocytes
Band cells
Mature PMN

Blasts
Cell maturation

The Perox Method

Cytochemical classification according to peroxidase activity
Cel type

Peroxidase

Myeloblasts
Promyelocytes
Myelocytes
Metamyelocytes
Band cells
Neutrophils
Eosinophils
Basophils
Lymphoblasts
Prolymphocytes
Lymphocytes
Atypical lymphocytes
Monoblasts
Promonocytes
Monocytes
Plasma cells
Nucleated red blood cells

-, sometimes + (especially micromyeloblasts)

3+
3+
3+
2-3+
2+
4+
-1+ (stay unstained in the ADVIA 120)
-1+
1+
-

The Perox Method

Absorption detector
Filter
Scatter detector
Tungsten lamp

Sample stream

Beam splitter

Dark
stop

The Perox Method

Scatter signal to measure the volume of
the cells

Absorption signal for peroxidase activity

measurement
Cells with medium peroxidase activity
absorbs less light

than cells with high peroxidase activity

The Perox Method

Light scatter = Cell Size

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-bysample basis.
Absorbed light = Peroxidase Activity

1
2
3
4
5
6
7
8

Noise
Nucleated Red Blood Cells
Platelet Clumps
Lymphocytes and Basophils
Large Unstained Cells
Monocytes
Neutrophils
Eosinophils

The Perox Method

The Perox Method

Calculating reported parameters

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
(#Monocytes)

(%MONO 100) x WBC

The Perox Method

Calculating 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)

The Perox Method

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

ATYP The presence of atypical lymphocytes is suspected.

IG

(Atypical Lymphocytes)

The presence of immature granulocytes is suspected.

(Immature

Granulocytes)

MPO Sample is a weak peroxidase stainer.

NRBC

(Myeloperoxidase deficiency)

The presence of nucleated red blood cells is suspected. (Nucleated Red

Blood Cells)

PLT-CLM

Presence of clumped platelets is suspected.

(Platelet Clumps)

The Baso Method

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

Laserdiode

Sample stream

Beamsplitter

Dark stop

Mirror

Absorption
detector

Low-angle
scatter
detector

High-angle
scatter
detector

Referentie signaal

Front view of the dark stop

The Baso Method

Cell Size

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.
The BASO cytograms is representative of a patient specimen.

Nuclear Configuration

1
2
3
4
5
6
7

Noise
Blast cell nuclei
Mononuclear WBCs (Monocyte and Lymphocyte nuclei)
Basophils
Baso Suspect
Saturation
Polymorphonuclear WBCs (Neutrophil and Eosinophil
nuclei)

The Baso Method

The Baso Method

Calculating reported parameters

WBCB

RawWBC x (BasoCalFactor)

(White Blood cell Count

Baso)

%BASO

100 x (BASO Count BASO PHA Cells )

#BASO

(%BASO 100) x WBCB

%BLAST

100 x (Blasts BASO PHA Cells )

%MN 100 x (MN BASO PHA Cells )

%PMN100 x (PMN BASO PHA Cells ) (%Polymorphonuclear cells)

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

(%Basophils)

(#Basophils)
(%Blasts)

(%Mononuclear cells)
(%BASO Suspect)

The Baso Method

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

BLASTS

The presence of blasts is suspected.

(Blasts)

LS
(Left Shift)

The presence of nonsegmented neutrophils (bands) is suspected.

THE END