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