CBC Histogram DR NARMADA PRASAD TIWARI

HISTOGRAM

1. WBC Composite Histogram
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INTERPRETATION OF

HISTOGRAM

2. Histograms are graphic representation of
cell frequencies verses size.
Histogram provide information about
erythrocytes ,leukocytes and platelet
frequency and distribution as well as
presence of subpopulation.
Shift in one direction or another can be of
diagnostic importance.

3. Produced from thousands/millions of
signals generated by the cells passing
through detector where they are
differentiated by:
Their size
Frequency of occurrence in the population

4. 3-part differential usually cont
Granulocytes or large cells
Lymphocytes or small cells
Monocytes(mononuclear cells) or (middle cells)
5-part classify cells to
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes

5. A sixth category designated “large unstained
cells” include cells larger than normal and lack the
peroxidase activity this include
◦Atypical lymphocytes
◦Various other abnormal cells.
Other counters identifies 7 categories including
◦Large immature cells(composed of blasts and
immature granulocytes)
◦Atypical lymphocytes(including blast cells).

6. Hematology analyzer provide mathematical
results obtained by electrical and light
signals generated when blood cells pass
through sensing zone of the machine.
Two method-
1- electrical impedance counting
2- light scatter method.

7. Cell counting
Coulter Principle
Dilution
Vacuum and
pressure
Electrical
impedance
Reagent systems

8. Sensing Zone
Red Blood Cell
The Coulter PrincipleThe Coulter Principle
A red cell
passes through
RBC aperture
Oscilloscope

9. Sensing Zone
Neutrophil
Oscilloscope
The Coulter PrincipleThe Coulter Principle
A white cell
passes through
WBC aperture

10. Before adding lysing reagent After
Cell diameter in μm fl
Neutrophils 10 - 15 120 - 250
Basophils 9 - 14 70 - 130
Eosinophils 11 - 16 80 - 140
Monocytes 12 - 20 60 - 120
Lymphocytes 7 – 12 30 - 80

11. Discriminations thresholds
Platelet- with a volume of 8-12 fl are
counted from 2-30 fl.
RBC- with volume of 80-100 fl is detected
from 30 -250 fl.
WBC- RBC are lysed by lytic reagent .the
different WBC discriminator set at different
levels between the ranges of 30-450 fl.

12. Normal Histogram
Three parts differential white blood cells:
30 to 125µ3 : lymphocytes
125 to 160µ3 : monocyte
160 to 450 µ3 : granulocytes

13. WBC Composite Histogram
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14. Eos
Baso
Coulter WBC Histogram
Lymphs
30 – 90
fL
Monos
90 -160
fL
Neuts
160 - 450
fL

16. RBC HISTOGRAM
NORMAL
NORMAL

17. RBC HISTOGRAM
RBC FRAGMENTS, MICROCYTIC RBCs, Giant PLTRBC FRAGMENTS, MICROCYTIC RBCs, Giant PLTDI RBCs
DI RBCs
MACROCYTIC, TARGET CELLS, DI RBC
MACROCYTIC, TARGET CELLS, DI RBC
COLD AGGLUTININ
COLD AGGLUTININ
Post Transfusion
Post Transfusion

18. PLT HISTOGRAMS
NORMAL
NORMAL

19. PLT HISTOGRAMS
Giant Platelets
Small Platelets

20. WBC HISTOGRAMS
ImmNE2
Eosinophilia
ImmNE1 & ImmNE2
Blasts
Lymphocytosis
ImmNE1 = band formsImmNE2 = immature neutrophils :
metamyelocytes, myelocytes, promyelocytes

21. WBC
WBC Adults 4-10 x 103/μl
Childs till 12 x 103/μl
Newborns till 15 x 103/μl

22. Lymph. Adults -25-40 %
Childs, Newborns- till 70 %
MXD - Adults 3-13 %
Neutro. - Adults 50-70 %

23. Red Blood Cell Count
RBC Men 4.6-6.2 x 106/μl
Women 4.2-5.4 x 106/μl
HGB Men 14-18 g/dl
Women 12-16 g/dl
HCT Men 43-49 %
Women 36-46 %

24. MCV- 85-95 fl
MCH -27-33 pg
MCHC- 32-36 g/dl
RDW-SD 37-46 fl (Width in 20% of the Peak
hight)
RDW-CV 11-16 % (calc. width of the 68 %
Peak hight)

25. PLATELET
PLT 150-400 x 103/μl x 109/l
PDW 9-14 fl (Width in 20% of the Peak hight)
MPV 8-12 fl
P-LCR 15-35 %

26. Anemia is not yet apparent
MCV still is in the normal
range
Peripheral Smear shows mild
Anisocytosis
BUT
RDW is increased (Earliest
Indicator)
Histogram is Unimodal
but is wider
Increased RDW combined
with normal RBC values
(MCV , Hb , Hct )
distinguishes
Iron deficiency from Normal
Subjects 10

27. Anemia is present, MCV is
very low, and the smear is
very abnormal
RDW is abnormally high;
Histogram remains abnormal.
The diagnosis is easily made
at this point, but earlier
identification would improve
management

28. The red cell count is
increasing,
MCV is not yet normal,
and
Two populations of red
cells are seen-preexisting
microcytes, and newly
formed normocytes.
The two populations are
distinguished easily on the
red cell histogram but not
so easily on the peripheral
blood smear.

29. EARLY FOLATE
DEFICIENCY-
• The MCV is still normal RBC
count and Hb slightly reduced
but
• RDW is clearly increased ,
even before apparent anemia.
SEVERE FOLATE
DEFICIENCY –
• RBC Count is low.
• MCV is high.
•RDW is increased

30. Normocytic recovery
a small peak of cells in the
normal range
• RDW is higher than untreated
megaloblastic anemia due to
two cell population contributing
to the heterogeneity.
Microcytic recovery
Two Cell population is clearly
seen in this histogram – old
macrocytes and newly produced
microcytes .
Concomitant iron deficiency has
been unmasked.
RDW is markedly increased..

31. Case -
12 yr old boy with purpura,
marked pallor, fever
•Pancytopenia
•MCV 100.5, RDW 15.9%
•RBC histogram skewed to
right
•WBC histogram:
lymphocyte peak, faint
dome of neutrophils
•PLT histogram- abn
shape,descending slope not
touching baseline
•BM Bx confirmed AA

32. Case -
WBC
LYM%
MXD%
NEUT%
+ 23.8 x 109/L
8.1%
7.9%
84.0%

34. Case -
WBC 7.9 x 109/L
LYM% + 64.7%
MXD% 15.8%
NEUT% – 19.5%

36. Case
WBC 7.7 x 109/L
LYM% F1 * 13.2%
MXD% F2 * 37.7%
NEUT% 49.1%

38. Case
WBC 4.3 x 109/L
LYM% 18,3%
MXD% + 62,2%
NEUT% 19.5%

39. Case -

40. Case
WBC 2.3 x 109/L
LYM% 39.7%
MXD% 32.2%
NEUT% 28.1%

42. Case -6
RBC 4.48 x1012/L
HGB 8.8g/dl
HCT 29.3%
MCV 65.4fl
MCH 19.6pg
MCHC 30.0g/dl
RDW-CV 18.2%

44. Case-
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
1.64 x1012/L
6.2g/dl
18.2%
110.0fl
37.8pg
34.1g/dl
15.2%

46. Case
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
4.15 x1012/L
14.0g/dl
40.8%
98.3fl
33.7pg
34.3g/dl
22.7%

47. Anisocytosis

48. Case
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
3.62 x1012/L
11.1g/dl
31.9%
88.1fl
30.7pg
34.8g/dl
+ 25.5%

49. Poikilocytosis

50. Case
PLT
PDW
MPV
P-LCR
71 x109/L
PU
DW
DW

51. Giant platelet

52. Although the wide distribution on the PLT
histogram suggests the appearance of large
platelets, the distribution curve intersects
the discrimination line at a high point

53. Case
WBC
LYM%
MXD%
NEUT %
PLT
PDW
MPV
P-LCR

6.0 x109/L
27.5%
7.9%
64.4%
86 x109/L
18.6fl
12.8fl
43.7%

55. Platelet Aggregation
The smear clearly shows that platelets are
aggregating. The WBC histogram shows a
peak in the ghost area ( ) ,
PLT histogram shows a wide distribution.
Although these large particles usually affect
the leucocyte counts, the leukocytes
distribution of case 1 is well separated from
the ghost area on the WBC histogram,
probably without any effect of small
particles in the ghost area. There is no WL
Alarm given .

56. Case
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
2.23 x1012/L
14.4g/dl
24.9%
111.7fl
64.6pg
57.8g/dl
25.4fl

57. Cold Agglutinins

58. Incubation 30 min
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
4.35 x1012/L
14.5g/dl
43.5%
100.0fl
33.3pg
33.3g/dl
14.7fl

60. Because in this case erythrocytes have
passed through the detector as clusters of
several cells, the RBC, HCT,MCH, MCV,
MCHC and RDW values are abnormal. The
RBC histogram shows a second peak.
After the clusters have been dissolved by
incubation, all erythrocytes aredetected as
single cells. Therefore the second peak on
the RBC histogram doesnot appear and the
RBC, HCT, MCV, MCH, MCHC and RDW
values are

61. Case
WBC
LYM%
MXD%
NEUT %
49.4 x109/L
-.---
-.---
-.---

62. Insufficient Lysing of Erythrocytes

63. The histogram show On the WBC
histogram the distribution curve intersects
the WBC lower discrimination line at an
abnormally high point.

64. This is frequently seen with blood samples
taken from hepatic disease patients or
newborns. These problems are solved by
diluting the sample or replacing plasma
with cellpack.
The smear photo shows large platelets and
acantocytes, suggesting hepatic diseases

66. RL: Abnormal height at lower discriminator
of RBC Histogram (LD)
RU: Abnormal height at upper discriminator
of RBC Histogram (UD)
MP: Multiple peaks: Distinguish ?? of two
RBC Populations
DW:The distribution (RDW) can not be
detected because the Histogram does not
cross the 20 % limit twice

67. WL: Abnormal height at lower discriminator
of WBC Histogram (LD)
WU: Abnormal height at upper
discriminator of WBC Histogram (UD)
T1: Valley 1 not found
T2: Valley 2 not found
F1, F2, F3: Abnormal height at the points
T1 or T2; adjacent fractions are marked

68. PL: Abnormal height at lower discriminator
of PLT Histogram (LD)
PU: Abnormal height at upper discriminator
of PLT Histogram (UD)
MP: Multiple Peaks found
DW:The distribution (PDW) can not be
detected because the Histogram does not
cross the 20 % limit twice

69. Mark “ RL “, abnormal height
at lower discriminator
Possible causes:
• Giant Platelets
• Micro-Erythrocytes
• Platelet Clumps

70. Mark “ RU “, abnormal height at the upper
discriminator
Possible causes:
Cold Agglutinins (check MCHC > 40 g/dl)
Erythroblasts / Normoblasts

71. MP “, multiple peaks found
Possible causes:
Iron deficiency in therapy
Infection or Tumor Anemia (visceral iron
deficiency)
Transfusions

72. “DW “, abnormal histogram distribution
Distribution curve does not cross 20% level
twice.
The overall height of the curve is always
100 %. The width is calculated on the 20 %
height of the curve.
Hint for extreme Aniso- or. Poikilocytosis

73. Thrombocyte-Histogram
MPV (mean PLT volume) Ref range: 8 - 12 fl
P-LCR (ratio of large platelets)
Ref range: 15 - 35 %
Increase could be a sign for:
• PLT Clumps
• Giant PLT
• Microerythrocytes

74. PDW, (platelet distribution width at 20 % of
peak height Ref range: 9 - 14 fl
Increase could be a sign for:
PLT Clumps
Microerythrocytes
Fragments

75. Mark “ PL “, abnormal height at lower
discriminator
Possible cause:
High blank value
Cell fragments

76. Mark “ PU “, abnormal height at upper
discriminator
Possible Cause :
• PLT Clumps EDTA-Incombatibility Clotted
sample
• Giant Platelets
• Microerythrocytes

77. Mark “ MP “, Multi Peaks found
Possible Cause:
Platelet transfusion

78. Mark “ DW “, Distribution With
The distribution can not be detected
because the Histogram does not cross the
20 % limit twice.
• This curve in only an example but could
also show another course.
• The overall height of the curve is always
100 %. The width is calculated on the 20 %
height of the curve.

79. Leukocyte-Histogram
Flag “ WL “, Curve does not begin at the basis line
Possible causes :
• PLT Clumps EDTA-
Incombatibility coagulated
Sample
• high osmotic resistant
(Erythrocytes not lysed)
• Erythroblasts
• cold agglutinate

80. RBC Histogram
ABN / INDICATORPROBABLE CAUSE COMMENT
Left of curve does
not touch baseline
Schistocytes and
extremely small red
cells
Review smear CBC
and Platelet
histogram
Bimodal peak Transfused cells,
therapeutic response
Review Smear
Right portion of
curve extended
Red cell
autoagglutination
Review CBC &
Smear
Left shift of curveMicrocytes Review smear &
CBC
Right shift of curveMacrocytes Review smear &
CBC

81. WBC Histogram
ABN / INDICATORPROBABLE CAUSE COMMENT
Trail extending downward
at extreme left, or lymph
peak not starting at
baseline
NRBC, Plt clumping,
unlysed RBC,
cryoproteins, parasites
Review smear and correct
WBC for NRBC
Peak to the left of lymph
peak or widening of
lymph peak towards left
NRBC Review smear & correct
WBC for NRBC
Widening of lymph peak
to right
Atypical lymphs, blasts,
plasma cells, hairy cells,
eosinophilia, basophilia
Review smear
Wider mono peak Monocytosis, plasma
cells, eosinophilia,
basophilia, blasts
Review smear

82. WBC Histogram
ABN / INDICATORPROBABLE CAUSE COMMENT
WBC histogram
(lymph peak) does
not start at baseline
Giant platelets,
NRBC, Plt clumping
Review smear,
correct WBC for
NRBC
Elevation of left
portion of
granulocyte
Left Shift Review smear
Elevation of right
portion of
granulocyte peak
Neutrophilia Review smear

83. Platelet Histogram
ABN / INDICATORPROBABLE CAUSE COMMENT
Peak or spike at left
end of histogram (2-
8 fl)
Cytoplasmic
fragments
Review smear
Spike towards right
end of histogram
Schistocytes,
microcytes, giant
platelets
Review smear + CBC
( MCV &  RDW)
( MPV &  PDW)
Bimodal peak Cytoplasmic
fragments
Review smear

84. R1- RBC precursors, Giant or clumped
platelets, cryoglobulins.
R2- Blast, basophilia, eosinophilia,
monocytosis,plasma cells and abnormal size
lymphocytes.
R3-eosinophilia and immature granulocytes.
R4-absolute granulocytosis.

85. CONCLUSION
Histogram in conjunction with absolute
counts give valuable information about the
abnormality of the sample & the need for
follow up peripheral blood
examination.Histogram should be used as
quality check but not diagnostic for any
pathological condition.The manual blood
film remains the definitive tool for
complete haematological analysis.

86. Take home messages
Shapes of histograms identified pathology
before the blood smear could be examined.
Newer parameter like RDW and PDW have
added new dimension to understand blood
cells and classify there abnormality.
The manual blood film remains the
definitive tool for complete
haematological analysis.

87. Histograms
Interpretation
LYMPH% 31,2%
MXD% 6,8%
NEUT% 62,0%
LYMPH# 1,8x10
3
/µl
MXD# 0,4x10
3
/µl
NEUT# 3,6x10
3
/µl
250
RBC
RDW-SD 40,0fl
40
PLT
PDW 13,1fl
MPV 10,4fl
P-LCR 28,1%
WBC
300THANYOU
SPEAKER- DR NARMADA PRASAD
TIWARI

88. Known interfering substance
RBCs
• High WBCs esp if RBCs is low ®
­RBCs
• Agglutinated RBCs ® ¯ RBCs
Hb
Turbidity of the blood sample ® ­ Hb
•Elevated WBCs
•Elevated lipids
•Fetal bloods

89. MCV
Red cell agglutination
­ number of large platelets
HT
Red cell agglutination
RDW
Agglutination of RBCs
Nutritional deficiency
Blood transfusion

90. WBCs interferring subs.
Normoblasts ® ­ WBCs
Unlysed RBCs ® ­ WBCs
MM ® ­ WBCs (ppt protein)
Hemolysis ® ­ WBCs (red cell stroma)
Leukemia ® ¯ WBCs (­ cell fragility)
® In CLL small lymph not counted
Cryoglobulin ® ­ all parameters of blood

91. Platelets
RBCs fragments ® ­ plat (microcytes)
WBCs fragments ® ­ plat (microcytes)
Chemotherapy ® ¯ plat (­ plat. fragments)
Hemolysis ® ­ Plat (red cell strom)
ACD blood ® ¯ plat (plat. Aggregation)
RBCs inclusion ® ­ plat. (Malaria, H.j bodies)
Plat. agglutination ® ¯ plat

92. Lymphocytes
Nucleated RBCs ® ­ lymph
Parasites ® ­ lymph
Resistent RBCs ® ­ lymph
Monocytes
­ in large lymphocytes, atypical lymph,
blasts and basophils
Granulocytes
­ in eosinophilia, blasts, promyelo, myelo,
metameyl and plasma cell