Advia 120 Methods

Denise Pegnall
The Retic Method Meeting Information
Meeting Date
Reticulocyte Count
Determines the Percentage of Young RBC’s in the Peripheral Blood
Why do we do Retics?

Assess the Red Blood Cell Production

Distinguish Between Anaemia’s Caused by Blood Loss
and those Caused by Bone Marrow Failure

Evaluate Patient Response to Chemotherapy and Radiation


Assess the Return of Bone Marrow Function after Autologous
Bone Marrow Transplant

Monitoring the Response of Erythropoeitin Therapy
 The Retic Method
 Advia 120 autoRETIC Reagent 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

 Utilizes Same Technology as RBC


Individual Cell Size

Individual Cell Hgb Content

Individual Cell Absorption
 Nucleic Acid Dye

Oxazine 750
The Retic Method
Laser Bench
Sample Dark
Laser diode Beam-splitter Mirror
Stream Stop

Absorption Low-angle
Absorption Low-angle High-angle
High-angle
Detector
detector Detector
scatter Detector
scatter
detector detector

Front view of dark stop

Reticulocyte Analysis
High Angle Detector
Measures Retic
Density/Hgb content

Absorption
Laser detector
Zero Angle

Low Angle Detector

Measures size
 The Retic Absorption Cytogram
The Retic Scatter Abs Cytogram is the Graphical Representation of the
Absorption and Light-Scatter Measurements:
Y 1 Retic Platelet Threshold
2 Retic Coincidence Threshold
3 Retic Threshold
4 Low / medium Retic Threshold
High Angle Low Gain

5 Medium / high Retic Threshold

A Mature RBC’s
B Low Absorption Retics
Cell Size

C Medium Absorption Retics

D High Absorption Retics
E Platelets
High Gain, Absorption X
Cell Maturation F Coincidence Events
 The Retic Scatter Cytogram

Y  The Retic Scatter Cytogram is the Graphic

Representation of Two Light-scatter
Measurements
Low Angle Low Gain

 Cells Identified as Mature RBC’s are

Colored Red

 Cells Identified As Reticulocytes Are

Colored Cyan.

High Angle, Low Gain X

Retic Volume/Haemoglobin Concentration Cytogram

Y
 Cells Identified as Mature RBC’s are Red
Volume

 Cells Identified as Reticulocytes are Cyan

Haemoglobin Concentration X
Retic Volume & Haemoglobin Conc. Histograms
The Retic Volume Histogram Represents the
Overlaid Distributions of Mature RBC’s and
Reticulocytes by Cell Size only
(The Displayed Data Includes the MCVg
Calibration Factor)

0fL
Mature RBC Population (Red)
200fL
Reticulocyte Population (Blue)
The Retic Haemoglobin Concentration Histogram
Represents the Overlaid Distributions of Mature
RBC’s and Reticulocytes by Cellular Haemoglobin
Concentration only

(The Displayed Data Includes the CHCMg

Calibration Factor)
0g/dL 50g/dL
The Retic Cellular Haemoglobin Histogram
The Retic Cellular Haemoglobin (CHr)
Histogram Represents the Overlaid
Distributions of Mature RBC’s and
Reticulocytes by the Actual Weight or Mass
of Haemoglobin Present in Each Cell.
CHr is the Haemoglobin Content of a Retic

0 pg 100 pg

Mature RBC Population (Red)

Reticulocyte Population (Blue)
Retic Absorption & Abs. Flatness Histograms
The Retic Absorption Histogram Represents the
Distribution of Mature RBC’s & Reticulocytes According
to their Corrected Absorption Values. Absorption
Correction Compensates for Drift in the Absorption
Channel Optics & for Scattered Light Not Collected.

The Retic Absorption Flatness Histogram Data

Consists of 50 Points, one taken every 200
Milliseconds. Each Point Represents the Mean
Absorption for the Last 200 Milliseconds. This
Histogram Provides a Visual Indication of the
Absorption Signal “Flatness” to Monitor the Laser
Performance. Laser Oscillation can cause an Erratic
Presentation.
The Retic Rate Histogram

 The Retic Rate Histogram Shows the

Uniformity of the Cell-Counting Rate.

 The Rate Histogram Data Consists of

50 Points, one taken Every 200
Milliseconds.

 Each Point Represents the Number of

Valid Cells Counted During the Last 200
Milliseconds.
 The Retic Scatter Absorption Cytogram

High Angle, Low Gain

RBC’s Retics

Volume
Low Med High

High Gain Absorption

 Fully Automated Reticulocyte Analysis Decreasing Maturity
 On-line Reagent
 Diagnosis / Monitoring Tools
 IRF (Immature Retic Fraction)
 Reticulocyte Cellular Hgb
Calculating Reported Parameters

 %Retic 100 x (Retic Count ÷ #Rtc Gated Cells)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 Haemoglobin Population Content Reticulocytes)

Calculating Reported Parameters

 CHCMr Mean of the Retic HC Histogram for the Retic Population

(Cell Haemoglobin Concentration Mean Reticulocytes)

 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)

Erythropoietin Treatment
Normal CHr

Normal CH
28pg

28pg Beginning After 4 Days

After 2 Weeks After 1 month