This document provides information about interpreting histograms generated by hematology analyzers when performing complete blood count (CBC) tests. It includes histograms showing normal and abnormal patterns for red blood cells, white blood cells, and platelets. Abnormal indicators are described that may indicate issues like schistocytes, transfusion, autoagglutination, microcytosis, macrocytosis, nucleated red blood cells, atypical lymphocytes, and more. The document emphasizes the importance of correlating histogram findings with peripheral blood smear examination to make an accurate diagnosis.
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.
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.
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 Smearshows mild
Anisocytosis
BUT
RDWis increased (Earliest
Indicator)
Histogramis Unimodal
but is wider
Increased RDWcombined
with normal RBC values
(MCV , Hb , Hct )
distinguishes
27. Anemia is present, MCV is
very low, and the smearis
very abnormal
RDWis 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.
Thetwo populations are
distinguishedeasilyonthe
redcell histogrambut not
so easilyontheperipheral
bloodsmear.
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 peakof cells in the
normal range
• RDWis higherthan 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.
RDWis markedly increased..
31. Case -
12 yrold boy with purpura,
marked pallor, fever
•Pancytopenia
•MCV 100.5, RDW15.9%
•RBC histogram skewed to
right
•WBC histogram:
lymphocyte peak, faint
dome of neutrophils
•PLT histogram- abn
shape,descending slope not
touching baseline
•BMBx confirmed AA
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
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 .
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
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
65.
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 / INDICATOR PROBABLE 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 curve Microcytes Review smear &
CBC
Right shift of curve Macrocytes Review smear &
CBC
81. WBC Histogram
ABN / INDICATOR PROBABLE 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 / INDICATOR PROBABLE 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 / INDICATOR PROBABLE 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.
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,
Notes de l'éditeur
Moderator- DR. SHALIENDRA SINGH THAKUR
When automation was first introduced, the Coulter principle was the first mechanism to be put into widespread use. The Coulter principle is based on the following:
Particles suspended in an isotonic diluent, when drawn through an aperture which has an electric current flowing through it will cause a measurable drop in voltage which is proportional to the size of the particle passing through the aperture. The pulses produced can then be counted and if the flow through the aperture is constant the particles can be quantified per unit volume. This is also called electrical impedance. This is the mechanism employed in most hematology analyzers in one way or another. Most analyzers use a combination of high and low vacuum and pneumatic pressure to push and pull the sample through the analyzer.
What results is a histogram. This is an example of a NORMAL WBC histogram. The lymphocytes, being the smallest cell fall to the far left of the histogram. The lymphocytes are followed by the Basos, Monos Eos and Neutrophils. Have you ever wondered why basophils, which when you look through the microscope appear to be about the same size as a neutrophil or eo appear between the lymphs and monos?? Well…basophilic granules are water soluble and try as hard as we might to create the “perfect” isotonic solution, you still lose some of the granules when the basophils are put in solution. When this happens, the cell cytoplasm shrinks down around the nucleus and the few remaining granules and makes the cell appear smaller than it is in vivo.
This is an example of a NORMAL RBC histogram. Most RBCs fall between 80 and 100 fl. The histogram should start at the baseline on the left and a small “tail” may be evident on the right. This represents doublets and triplets (cells that go through the aperture in twos and threes). These are excluded from the RBC MCV by algorithms but may be seen on the histogram.
These are a variety of examples of abnormal RBC histograms but certainly does not represent all the possibilities. Like the WBC histogram, RBCs do not always follow the “textbook” so some of the things represented here may look different with different specimens.
This is a NORMAL PLT histogram. The PLT histogram has two curves. One is the curve created from the directly measured PLT count and the other is Coulter’s patented curve fitting process which allows an accurate PLT count without interference from microcytic RBC or RBC fragments.
Here are a couple of examples of abnormal PLT histograms. One represents the presence of large or giant PLTs. Note how the directly measured curve (blue line) does not come back down to the baseline at 20 fl. The instrument then extrapolates this curve to eliminate any interference but account for the large or giant PLTs still out on the far right hand edge. The other histogram indicates a patient with small PLTs (small MPV). Note there is no need for the curve fitting process in this case as the PLTs all fall withing the directly measured region.
These are examples of other WBC histograms and the corresponding suspect flag. Keep in mind, these are examples of the most common scenarios and unfortunately, cells do not always follow the “textbook” so occasionally you may see a certain suspect flag and it may not be exactly the same as the examples noted here depending on what is going on with the patient.