Flow Cytometric Analysis o Blood Cells

FLOW CYTOMETRIC ANALYSIS OF BLOOD CELLS
CBC AND MORE
Mohamed Hassan Shaheen M.D.
The Red Cell
RBC
Retic
Reticulocyte Maturation
High Angle (5-15 degrees)
Maturity
Oxazine 750 Absorbance
Sheath Stream
huttle Chamber
Sample Stream
RBC Method
RBC Method
Mies theory of light scattering of spheres RBC reagent spheres and fixes the RBC and platelets using SDS and gluteraldehyde Reaction mix is passed through the flowcell in the laser optics where light scatter is used to determine the size and Hgb content of the cells
High angle detector (5o - 15o)

670nm Laser Diode
Low angle detector (2o-3o)
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
Reticulocyte Method
High angle detector (5o - 15 o )

670nm Laser Diode
Absorbance RNA Content

Oxazine 750 RNA Stain
Low angle detector (2o -3o )
RBC
Retic
PLT
Thank you
Macro (RBC Volume (fL

120fL
60fL 28g/dL 41g/dL
Micro
(Hgb Concentration (g/ dL Hypo Hyper
RBC Method
%Macro
120fl
60fl
%Micro
Hgb.conc. 28g/dl 41g/dl
%Hypo
%Hyper
The RBC Method
28
41
RDW
HDW
CHDW
CH
MCV
CHCM
Red Cell Indices

RBCs MCV MCH= HB/RBCs MCHC=HB/HCT = HB/MCVxRBC RDW
60 120 RBC Volume (V)
200 fL
28 41 50 g/ Hgb Concentration (HC)
dL
100 pg (Hgb Content (CH
Reticulocyte Indices
Retics % Retics # IFR MCVr CHr CHCMr
MCV
CHCM
CH
m
r
m r
CHr
RDWg CHCMg HDWg CHg MCVm RDWm CHCMm HDWm MCVr RDWr CHCMr HDWr
CHDWg MCVg CHm CHDWm CHr CHDWr
Cell Number
Hct 34.8 : Retic: 1.2% CH: 25.3 pg CHr: 5. 2 2 pg
Hct: 34.7 Retic: 2.9% CH: 25.8 pg CHr 31.3 : pg
CHr (pg)
25
50
Reticulocyte Method
The ADVIA 2120 provides simultaneous direct measurement of the haemoglobin content of the mature red cells (CH pg) and reticulocytes (CHr pg) These measurements can help optimise EPO and iron therapy.
Iron Deficiency Anemia
Ferritin 23 g/L
CHm 25.9 CHr 27.4
Ferritin 215 g/L

CHm 27.4 CHr 28.5
CHm 26.2 CHr 21.6
CHm 27.8 CHr 27.2
CHm 24.9 CHr 27.6
CHm 27.6 CHr 28.3
ASSESSMENT OF IRON STORES

I- Serum feritin
- Values < 15-30 ng/dl ; iron deficiency.
- Acute phase protein.

- Elevated in dialysis patients independent of Iron status (Poor sensitivity and specificity) cont.
II- Serum Transferrin Saturation

- Measures circulating iron
- Values less than 15% indicate iron deficiency
- Calculated: S.TSAT = Serum iron/ TIBC X 100

- Signifcant intermeasurement variability
III- Reticulocyte Hemoglobin Content (CHr)

- Direct measurement of iron in erythropoeitic pool
- Simultaneous measurement of volume and hemoglobin concentration in both red cell and reticulocyte on cellby-cell basis
AIHA
DIAGNOSTIC PERFORMANCE OF CHr
45 40 35 30 25 20 15 10 5 0 S.ferritin S.TSAT CHr
CHr. , cont..
- Direct measurement of iron in erythrocyte like ferritin and S.TSAT by iron related factors - Reticulocyte Iron Content (CHr), is a snapshot assay and reflects immediate iron status - Excellent senstivity and specificity - Optimizing rHuEpo and iron therapy
Date
8/30/99
9/06/99
9/13/99
9/20/99
10/04/99
11/01/99
RBC Scatter
RBC Vol (fL)
61.0
RBC-HC (g/dL)
68.3
73.4
75.4
76.9
78.8
24.4
RBC-CH (pg)
25.0
25.5
26.8
29.4
31.6
14.7
HGB (g/dL)
17.0 5.8
18.8 6.7
20.4 7.9
22.9 9.6
24.9 10.8
4.3
( Deficiency Anemia (IDA
-Thalassemia Trait
Deficiency Anemia IDA
-Thalassemia Trait
MCV = 66 Fl, M/H ratio = 2.05 Absence of Iron Deficiency -Thalassemia trait
Pre Warmed
RBC Method
(MCV < 80 (78.5) and M/H Ratio > 0.9 (1.5 Broad distribution width for RBC Volume and HC histograms Presence of Hyperchromia suggestive of sickle cell anemia
Normal
-Thalassemia Trait
( Deficiency Anemia (IDA
Sickle Cell Anemia
Immature Reticulocyte Fraction (IRF)

High Angle (5-15 degrees)
Maturity
Immature Reticulocyte Fraction (IRF)

Sensitive indicator of erythropoietic activity Used in a bivariate analysis with absolute retics. count Useful diagnostic and theraputic monitoring utility:
1 - Bone marrow transplantation 2 - Stem cell transplantation 3 - Hemodialysis patients 4 - Anemia
1- Bone Marrow Transplantation

Serial determinations of IRF in day 5 - 21 may indicate successful bone marrow engraftment The observation of rising peripheral blood granulocytes can be misleading (infection and GVH) Prophylactic platelet transfusion obscure Plt. Recovery Neither infection nor platelet transfusion have significant impact on IRF Reticulocyte count > 15 x 10^9 /L and IRF > 0.5 are associated with successful BM engraftment
2- Stem Cell Transpalntation

IRF values are cost-effective and relaible surrogate to CD34 stem cell quantitation
Monitoring patients on Hemopoietic Growth Factors and Cytotoxic Chemotherapy before harvesting the stem cells by aphersis
3- Hemodialysis Patients
IRF indicate response to the expensive anemia therapy , rHuEpo
When used with CHr , IRF optimize iron and rHuEpo requirements IRF should reduce the global cost of anemia mangment
4- Anemia Diagnosis And Monitoring

Aplasitc and Hypoplastic patients have both low Retics. and IRF values and recovery is associated with increased IRF Early erythropoeitic response in occult blood loss is an elevated IRF and normal Reticulocyte count Chronic erythropoietic response ( Hemolysis) ; both high Retics. and IRF
WBC
2 methods- Perox and Baso
Perox method uses tungsten optics Baso method uses laser optics Perox cytochemistry based method Baso method based on stripping cytoplasm from all cells except basophils
Perox Reaction
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
Baso Reaction
The Baso 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
Blasts (BLASTS)
Standard Software V5.2.7
Blast region
Blast region events to left of channel 9
%Blasts = 1.5-5% and %LUC 4.5% OR %Blasts 5%
%Blasts = 1.5-5% and %LUC 4.5% OR %Blasts 5 OR %BASO + %BASO Susp + %BASO Sat 10%
Baso Reaction
Basophils
Basophils
MN Cells PM Cells Blasts
MN Cells PM Cells Blasts
Cluster Analysis
Threshold Analysis
Baso Reaction
Low angle scatter is used to determine size
High angle scatter determines nuclear shape and density
Low Angle scatter - size
High Angle scatter density/ shape
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
Large Platelets
RBC Ghosts
Platelets
Debris
RBC Fragments
NRBC Method
NRBC methodology is based on physical characteristics of NRBC: the size and density of NRBC nuclei
NRBC Method
Manual Differential Neut 44 Band 4 Lymph 27 Mono 9 Eos Baso 1 Atyps Blasts 4 IG 11 NRBC 5
CONCLUSIONS
Flow Cytometric analysis of blood cells gives useful clinical information about the classification, differential diagnosis and monitoring of blood diseases
It also improves the diagnostic sensitivity and even allows minor changes to be detected with a degree of reliability , not previously possible
Thank You

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FLOW CYTOMETRIC ANALYSIS OF BLOOD CELLS

CBC AND MORE

Mohamed Hassan Shaheen M.D.

The Red Cell

Oxazine 750 Absorbance

High angle detector (5o - 15o)

Low angle detector (2o-3o)

The RBC Method

High angle detector (5o - 15 o )

Absorbance RNA Content

Low angle detector (2o -3o )

Macro (RBC Volume (fL

60fL 28g/dL 41g/dL

(Hgb Concentration (g/ dL Hypo Hyper

The RBC Method

Red Cell Indices

60 120 RBC Volume (V)

28 41 50 g/ Hgb Concentration (HC)

100 pg (Hgb Content (CH

CHDWg MCVg CHm CHDWm CHr CHDWr

Hct 34.8 : Retic: 1.2% CH: 25.3 pg CHr: 5. 2 2 pg

Hct: 34.7 Retic: 2.9% CH: 25.8 pg CHr 31.3 : pg

Iron Deficiency Anemia

Ferritin 215 g/L

CHm 26.2 CHr 21.6

CHm 27.8 CHr 27.2

CHm 24.9 CHr 27.6

CHm 27.6 CHr 28.3

ASSESSMENT OF IRON STORES

- Acute phase protein.

II- Serum Transferrin Saturation

- Calculated: S.TSAT = Serum iron/ TIBC X 100

III- Reticulocyte Hemoglobin Content (CHr)

DIAGNOSTIC PERFORMANCE OF CHr

45 40 35 30 25 20 15 10 5 0 S.ferritin S.TSAT CHr

RBC Vol (fL)

( Deficiency Anemia (IDA

Deficiency Anemia IDA

( Deficiency Anemia (IDA

Sickle Cell Anemia

Immature Reticulocyte Fraction (IRF)

Immature Reticulocyte Fraction (IRF)

1- Bone Marrow Transplantation

2- Stem Cell Transpalntation

4- Anemia Diagnosis And Monitoring

The Perox Method

Absorption detector Filter Scatter detector Tungsten lamp Sample stream

The Perox Method

Scatter signal to measure the volume of the cells

Absorption signal for peroxidase activity measurement

than cells with high peroxidase activity

The Baso Method

Absorption detector Front view of the dark stop

Low-angle scatter detector

High-angle scatter detector

Blast region events to left of channel 9

%Blasts = 1.5-5% and %LUC 4.5% OR %Blasts 5%

MN Cells PM Cells Blasts

MN Cells PM Cells Blasts

High angle scatter determines nuclear shape and density