INTERPRETATIO

N OF
WBC Composite Histogram

90.0
80.0
70.0

Count

60.0
50.0
40.0
30.0

HISTOGRAM

20.0
10.0
0.0
0

50

100

150

200

250

Channel

300

350

400

450

500

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.

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

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

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).

Hematology analyzer provide

mathematical results obtained by
electrical and light signals
generated when blood cells pass
through sensing zone of the machine.
Two method1- electrical impedance counting
2- light scatter method.

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

The Coulter Principle

Red Blood Cell

A red cell
passes
through RBC
aperture

Sensing
Zone
Oscilloscope

The Coulter Principle

Neutroph
il

A white cell
passes
through WBC
aperture

Sensing Zone
Oscilloscope

Before adding lysing reagent

Cell diameter in m

Neutrophils
Basophils
Eosinophils
Monocytes
Lymphocytes

10 - 15
9 - 14
11 - 16
12 - 20
7 12

After
f

120 - 250
70 - 130
80 - 140
60 - 120
30 - 80

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

Normal Histogram
Three parts differential white
blood cells:
30 to 1253

lymphocytes

125 to 1603

monocyte

160 to 450 3

: granulocytes

WBC Composite Histogram

90.0
80.0
70.0

Count

60.0
50.0
40.0
30.0
20.0
10.0
0.0
0

50

100

150

200

250

Channel

300

350

400

450

500

Coulter WBC Histogram

Monos
Lymphs
30 90
fL

90 -160
fL

Baso

Neuts

Eos

160 - 450
fL

RBC HISTOGRAM

NORMAL
NORMAL

RBC HISTOGRAM

MACROCYTIC,
COLD
MACROCYTIC, TARGET
TARGET CELLS,
CELLS, DI
DI
COLD AGGLUTININ
AGGLUTININ

DI
RBCs
Post
Transfusion
DI
RBCs
Post
Transfusio
AGMENTS,
MICROCYTIC
RBCs,
Giant
PLT
AGMENTS, MICROCYTIC RBCs, Giant PLT

PLT HISTOGRAMS

NORMAL
NORMAL

PLT HISTOGRAMS

Giant Platelets

Small Platelets

WBC HISTOGRAMS

ImmNE1&ImmNE2

Lymphocytosis

ImmNE2
Eosinophilia
Blasts
ImmNE2 = immature neutrophils :
ImmNE1 = band forms

WBC
WBC Adults
Childs till
Newborns till

4-10 x 103/l
12 x 103/l
15 x 103/l

Lymph.

Adults
-25-40 %
Childs, Newborns- till 70

%
MXD
- Adults 3-13 %
Neutro. - Adults 50-70 %

Red Blood Cell Count

RBC Men
4.6-6.2 x 106/l
Women 4.2-5.4 x 106/l
HGB
HCT

Men 14-18 g/dl

Women 12-16 g/dl
Men 43-49 %
Women 36-46 %

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

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

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

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

The red cell count is

increasing,
MCV is not yet
normal, and
Two populations of
red cells are seenpreexisting
microcytes, and
newly formed
normocytes.
The two populations
are distinguished
easily on the red cell
histogram but not so
easily on the

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

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

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

Case WBC
LYM%
MXD%
NEUT%

+ 23.8 x 109/L
8.1%
7.9%
84.0%

Case WBC
LYM%
MXD%
NEUT%

7.9 x 109/L
+ 64.7%
15.8%
19.5%

Case
WBC
LYM%
MXD%
NEUT%

7.7 x 109/L
F1 * 13.2%
F2 * 37.7%
49.1%

Case
WBC
LYM%
MXD%
NEUT%

4.3 x 109/L
18,3%
+ 62,2%
19.5%

Case -

Case
WBC
LYM%
MXD%
NEUT%

2.3 x 109/L
39.7%
32.2%
28.1%

Case -6
RBC
HGB
HCT
MCV
MCH
MCHC
RDW-CV

4.48 x1012/L
8.8g/dl
29.3%
65.4fl
19.6pg
30.0g/dl
18.2%

CaseRBC
HGB
HCT
MCV
MCH
MCHC
RDW

1.64 x1012/L
6.2g/dl
18.2%
110.0fl
37.8pg
34.1g/dl
15.2%

Case
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

4.15 x1012/L
14.0g/dl
40.8%
98.3f
33.7pg
34.3g/dl
22.7%

Anisocytosis

Case
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

3.62 x1012/L
11.1g/dl
31.9%
88.1f
30.7pg
34.8g/dl
+ 25.5%

Poikilocytosis

Case
PLT
PDW
MPV
P-LCR

71 x109/L
PU
DW
DW

Giant platelet

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

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

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

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 .

Case
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

2.23 x1012/L
14.4g/dl
24.9%
111.7fl
64.6pg
57.8g/dl
25.4f

Cold Agglutinins

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
14.7f

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

Case
WBC
LYM%
MXD%
NEUT %

49.4 x109/L
-.---.---.---

Insufficient Lysing of
Erythrocytes

The histogram show On the WBC

histogram the distribution curve
intersects the WBC lower
discrimination line at an
abnormally high point.

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

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

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

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

Mark RL , abnormal
height at lower
discriminator

Possible causes:
Giant Platelets
Micro-Erythrocytes
Platelet Clumps

Mark RU , abnormal height

at the upper discriminator
Possible causes:
Cold Agglutinins (check MCHC >
40 g/dl)
Erythroblasts / Normoblasts

MP , multiple peaks found

Possible causes:
Iron deficiency in therapy
Infection or Tumor Anemia
(visceral iron deficiency)
Transfusions

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

ThrombocyteHistogram
MPV (mean PLT volume) Ref range: 8 - 12 f
P-LCR (ratio of large platelets)
Ref range: 15 - 35 %
Increase could be a sign for:
PLT Clumps
Giant PLT
Microerythrocytes

PDW, (platelet distribution width at 20

% of peak height Ref range: 9 - 14 f
Increase could be a sign for:
PLT Clumps
Microerythrocytes
Fragments

Mark PL , abnormal height at

lower discriminator
Possible cause:
High blank value
Cell fragments

Mark PU , abnormal height at

upper discriminator
Possible Cause :
PLT Clumps EDTA-Incombatibility
Clotted sample
Giant Platelets
Microerythrocytes

Mark MP , Multi Peaks found

Possible Cause:
Platelet transfusion

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.

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

RBC Histogram
ABN / INDICATOR
Left of curve does
not touch baseline

PROBABLE CAUSE
Schistocytes and
extremely small red
cells

COMMENT
Review smear CBC
and Platelet
histogram

Bimodal peak

Transfused cells,
Review Smear
therapeutic response

Right portion of
curve extended

Red cell
autoagglutination

Review CBC &

Smear

Left shift of curve

Microcytes

Review smear &

CBC

Right shift of curve

Macrocytes

Review smear &

CBC

WBC Histogram
ABN / INDICATOR

PROBABLE CAUSE

COMMENT

Trail extending downward NRBC, Plt clumping,

at extreme left, or lymph unlysed RBC,
peak not starting at
cryoproteins, parasites
baseline

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

WBC Histogram
ABN / INDICATOR

PROBABLE CAUSE

COMMENT

WBC histogram
(lymph peak) does
not start at baseline

Giant platelets,
Review smear,
NRBC, Plt clumping correct WBC for
NRBC

Elevation of left
portion of
granulocyte

Left Shift

Review smear

Elevation of right
portion of
granulocyte peak

Neutrophilia

Review smear

Platelet Histogram
ABN / INDICATOR
PROBABLE CAUSE
Peak or spike at left Cytoplasmic
end of histogram (2- fragments
8 fl)

COMMENT
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

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.

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.

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.

Histograms

Interpretation

WBC

THANYOU

300
LYMPH%
MXD%
NEUT%
LYMPH#
MXD#
NEUT#

31,2
6,8
62,0
1,8
0,4
3,6

SPEAKER- DR NARMADA
PRASAD TIWARI

%
%
%
x103/l
x103/l
x103/l
RBC

250

RDW-SD

40,0 f
PLT

40

PDW
MPV
P-LCR

13,1 f
10,4 f
28,1 %

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

MCV
Red cell agglutination
number of large platelets

HT
Red cell agglutination

RDW
Agglutination of RBCs
Nutritional deficiency
Blood transfusion

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

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

Lymphocytes
Nucleated RBCs lymph
Parasites lymph
Resistent RBCs lymph

Monocytes
in large lymphocytes, atypical lymph,
blasts and basophils

Granulocytes
in eosinophilia, blasts, promyelo, myelo,