CBC .pdf

Complete Blood Count

1. NNNBJBJ

2. NNNBJBJ

3. NNNBJBJ
Interpretation of Parameters ,
Diagrams & Scattergrams in
Complete Blood Count
Alireza. A. Moghaddam
Hematology Supervisor at Pars Hospital
Laboratory

4. NNNBJBJ
CBC is one the most commonly ordered blood test in Clinical
medicine.
•Screening Test
•Provide useful information for diagnosis of some diseases
(Anemia , Thalassemia , Leukemia,…)
•Monitoring Disease & Follow up Treatment
•Differential between Bacterial & Viral Infections
CBC –Complete Blood Count
A major portion of the complete blood count is the measure of
the concentration of White blood cells (WBC ), Red
blood cells (RBC), and platelets (Plt) in the blood.

5. NNNBJBJ
Scatter Procedure

6. NNNBJBJ
PANDA
Peroxidase Activity
&
Nuclear Density
Assay

7. NNNBJBJ
Leukocytes : Parameters & Scattergrams
TWO separate channels to determine the white cell count:
1) Peroxidase channel
2) Basophil / lobularity channel
6-Part Differential Instruments
• LUC ( Large Unstained Cells )
• Lymphocyte
• Monocyte
• Basophil
• Neutrophil
• Eosinophil

8. NNNBJBJ
Peroxidase Channel ( Perox )
Specimen is diluted with Peroxidase reagents ,Red cells &
Platelets are lysed.
Dark precipitate forms in cells containing peroxidase:
• Neutrophils, Eosinophils, monocytes
• Lymphocytes, basophils, large unstained cells contain no
peroxidase and therefore do not stain
• A tungsten-based optical system is used to measure:
Individual impulses are counted to determine WBC
Stain intensity (absorbance) for each cell
Cell size (by forward light scatter)

9. NNNBJBJ
Cells are plotted on a scattergram: scatter (Size) on Y
axis and Peroxidase activity (Staining intensity) on
the X-axis.
• Each cell is classified based on this information
• In this channel, Basophils are classified with
Monocyte.
Peroxidase Channel ( Perox )

10. NNNBJBJ
PeroxidaseChannel (Perox)
Whole Blood + Lysant Change Cell Size
Nuclear Size ≈ Cell Size
50 -400 fl : White Blood Cell (WBC)
Yaxis : Cell Size ( fl )
Xaxis : Peroxidase Activity

11. NNNBJBJ
PeroxidaseChannel ( Perox )Cell Numbers X axis
(Peroxidase)
Y axis (Size ,fl)
(50-400 fl)
Cells
0 4+ 350 Promyelocyte
3000 3+ 320 Neutrophil
800 1+ 300 Monocyte
400 5+ 300 Eosinophil
0 0 280 Blasts
50 0 250 Atypical
Lymphocytes
2000 0 180 Lymphocyte
0 0 120 NRBC
1+

Basophil

12. NNNBJBJ
Peroxidase Activity
s
i
z
e

13. NNNBJBJ

14. NNNBJBJ

15. NNNBJBJ
Perox Disadvantages
1-Mono + Baso : diff as a one part
2-Decrease of Myeloperoxidase Enzyme
(Myeloid Leukemia , Myelodysplasia,…..)
Or Old Blood / Reagents

16. NNNBJBJ
WBC/ Baso Channel
Basophil:
•Large purple-black granules
•Unevenly distributed throughout the cytoplasm
•Metachromatic and contain histamine, heparin, cathepsin G,
major basic protein, lysophospholipase

17. NNNBJBJ
WBC / Baso Channel
An acidic reagent lyses the red blood cells and shrinks white blood
cells to bare nuclei, with the exception of basophils, which are not
affected. The resulting differences between basophils and other cells
are analyzed using forward and side scatter information.
(PH=3.3 )
> 3’

18. NNNBJBJ
• Laser light signal from a low-angle forward scatter
plot identifies basophils, which, due to their retained
cytoplasm, are larger than other cells
• A high-angle detector is sensitive to the shape and
structure (lobularity and density) of remaining nuclei
WBC / Baso Channel
WBC / Baso Channel
Mn
Pmn
Nuclear Density
LI= Pmn
Mn

19. NNNBJBJ
Erythrocytes: Parameters & Histogram

20. NNNBJBJ
Erythrocytes: Parameters & Histograms
• 36-200 fl in RBC / Plt aperture as Erythrocyte (RBC).
• A diluent is added that makes the red cells absorb fluid until
they are spherical in shape (total volume is unchanged)
• Cells pass one cell at a time through the laser beam
• A light scatter detector detects each cell as it passes in front
of the laser beam, using two different gain settings, one for
red cells and one for platelets
• Volume of RBCs and platelets is measured by comparing the
low angle and high angle light scatter created by each particle.
• Concentration of the Hb in each red cell is also determined:
Refractive index is determined by Hb concentration

21. NNNBJBJ

22. NNNBJBJ

23. NNNBJBJ
Bimodal Peak

24. NNNBJBJ
Erythrocyte’sIndices
Wintrobe’s
Indices
MCH
MCV
*
**

25. NNNBJBJ
CHCM
MCHC: Calculated Parameter
( Hb / HCT orMCH / MCV )
CHCM: Independent Parameter
( Measure Directly)

26. NNNBJBJ
RDW: Red Cell Distribution Width
RDW-CV% ≈ Anisocytosis , RDW-SD ≈ MCV ( fl )
(MCV= 90 , RDW-SD= 40 (90 +/-40 )
**
**
(n= RBC)
2 SD
3 SD
1SD
Giant
Plt
Lymph
&
NRBC

27. NNNBJBJ
RDW : Red Cell Distribution Width
RDWCV% = RDW SD
MCV

28. NNNBJBJ
Platelet

29. NNNBJBJ
Platelet :Parameters & Histograms

30. NNNBJBJ
RDW-CV% ≈
Giant Platelet≈
Cut off

31. NNNBJBJ
Platelet: Parameters & Histograms

32. NNNBJBJ
Platelet : Parameter & Histogram

33. NNNBJBJ
Immature platelet fraction ( IPF)
• Reticulated Platelet : Platelet + RNA
• Under condition of thrombocytopenia, Platelet RNA
content correlated directly with Megakaryocyte activity.
• Fluorescent dye stains the platelet membrane and Granules
Applications:
• Higher values in patients with ITP, DIC; normal values in patients
with decreased platelet production .
• An increase in immature platelet fraction indicates impending
platelet recovery; may precede recovery by several days; may allow
optimal perfusion of platelet concentrates.

34. NNNBJBJ
Immature Platelet fraction ( IPF )
■Laboratory diagnosis of Thrombocytopenia due to increase
peripheral platelet destruction, particularly autoimmune
thrombocytopenic purpura (AITP ) & thrombotic thrombocytopenic
purpura (TTP ) , Used to evaluate low platelet counts.
■IPF differentiates increased platelet consumption from bone marrow
failure/suppression in most cases
■May eliminate the need for bone marrow examination in some
patients, such as younger patients with high immature platelet fraction
(IPF) and clinical presentations suggesting idiopathic thrombocytopenic
purpura (ITP)

35. NNNBJBJ

36. NNNBJBJ
Reticulocyte: Parameters & Histograms
.

37. NNNBJBJ
Reticulocyte : Parameters & Histograms
• RBCs are stained with a fluorescent dye.
• Fluorescent intensity (RNA content ) and forward light scatter
(cell size) are plotted to distinguish reticulocytes from mature RBCs,
platelets, and WBC.
• Also measured in this channel:
–“Optical” platelet count
–Forward light scatter of mature RBCs (RBC-Y) and reticulocytes
(RET-Y)

38. NNNBJBJ
Reticulocyte Stages
The Reticulocyte count is used to estimate the degree
of effective erythropoiesis.
NRBC Retic (4 Stages) RBC
Retic I: filaments >>> granules ( BM , 1-1.2 d)
Retic II : filaments = granules ( BM , 1-1.2 d)
Retic III : granules > filaments ( BM , 1-1.2 d)
Retic IV : granules >>> filaments ( Blood , 1 d)

39. NNNBJBJ
Reticulocyte : Parameters & Histograms

40. NNNBJBJ
Reticulocyte: Parameters & Histograms
High Fluorescent Reticulocyte ( HFR) : Retic I
Mid Fluorescent Reticulocyte ( MFR) : Retic II , III
Low Fluorescent Reticulocyte ( LFR) : Retic IV ( Normal Retic )
B.M Daily Production
Immature Reticulocyte Fraction (IRF) : Retic I, II ,III (HFR + MFR )
(Polychromasia)

41. NNNBJBJ
RETICULOCYTE COUNT :
1stCORRECTION
adjusts the Reticulocyte count
based on the reduced number
of circulating red cells
provides an estimate of the
Retic corrected for Anemia
Corrected Retic =
Retic % Patient Hgb or Hct
* Normal Hgb or Hct
Example :
Retic 9%
hemoglobin 7.5 g/dl
hematocrit 23%
corrected Retic count
= 9 * (7.5 / 15)
or
(23 / 45)
= 4.5%

42. NNNBJBJ
RETICULOCYTE COUNT
2ndCORRECTION
for longer life of prematurely released
reticulocytes in the blood produces the
Reticulocyte Production Index
( Reticulocyte Maturation Rate in Blood )
Corrected Retic (4.5)
RPI= Retic % * ---------------------------= 2.25
Maturation Time ( 2 )
Maturation Time

43. NNNBJBJ
Reticulocyte : Parameters & Histograms
When Calculated RPI :
•Polychromasia in PBS : Retic I , II ,III ( IRF )
•HCT <35% ( Hb <11 g/dl)
RPI: < 2 Hypo Proliferation
> 2 Hyper Proliferation

44. NNNBJBJ
Reticulocyte: Parameter & Histogram

45. NNNBJBJ

46. NNNBJBJ
Thank you

47. NNNBJBJ

48. NNNBJBJ