Automated CBC

Prepared by:
• Professor / Manal Hashim Fayek
• Assistant Professor / Botheina Ahmed Thabet Farweez

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Component of automated CBC
Blood count basic parameters:
Hb, Hct, RBC, WBC, platelets.
Red cell indices:
MCV, MCH, MCHC, RDW
WBC differentials
histogram or Scatter-gram
Reticulocyte count

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CP6006a
Principles of Automated Cell
Count
• Electronic Impedance (Low VoltageDirect current Resistance(DC))
• Light Scatter at various angles
• Conductivity Radiofrequency(RF) ( high voltage electromagnetic
current resistance) gives idea about internal cell structure density
• Fluorescence combined with light scatter

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Electric Impedance
principle
- Cells (diluted in saline) traversing
an aperture through which an
electric current is flowingcreate
electric resistance which are
counted and measured as pulses.

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While number of pulses indicate particle count their amplitude reflect cell
size.
Each pulse is stored in a computer memory and a histogram is created
according to their sizes.
X axis represents cell size while Y axis represents relative number of
cells.
Thus average size and SD in size can be determined.

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Light
scattering
• A suspension of cells is passed through a flow chamber
and a laser light beam is focused into it
• The scatter of the laser light beam at different angles is
recorded, giving information about cell size, structure,
internal structure, and granularity.
• Low angle light scatter: cell size
• High angle light scatter: internal structure (lobularity
and graularity)

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In such instruments, whole blood is aspirated, diluted, and then divided into two samples. One sample is used to analyze
the red blood cells and platelets while the second sample is used to analyze the white blood cells and hemoglobin .

sample

RBCs & Hb &

platelets CP6006a
WBCs 8
In such instruments, whole blood is aspirated, diluted, and then divided into two samples. One sample is used to analyze
the red blood cells and platelets while the second sample is used to analyze the white blood cells and hemoglobin .

sample

RBCs & Hb &

platelets CP6006a
WBCs 9
Instrument
Flags
• Many normal samples according to normal ranges and distribution
are computed.
•Any abnormality whether quantitative or qualitative is flagged.
NB;
• 50% of patient samples are flagged in most centers.

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Types of
Numerical Flags
flags
1. Back light / underlined or check limit flags (H/L flags).
Incomplete data flags(beyond instrument linearity limits).
Suspect (morphological) flags: eg.,
1. Region (R) flags(means interference)
- Dimorphic reds
- Blast cells
- Variant lymphocytes

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RBC
Histogram
• The RBC histogram reflects the native red cell size.

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• RBC histogram and count. The shaded area represents those cells used in the RDW (i.e., RDW-CV)
calculation. The excluded cells can represent large platelets, platelet clumps, or electrical interference
on the left and RBC doublets, RBC triplets, RBC agglutinates, or aperture artifacts on the right

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RBC
HISTOGRAM

COLD AGGLUTININ MACROCYTIC, TARGET CELLS, DI RBC

RBC FRAGMENTS, MICROCYTIC RBCs, Giant PLT DI RBCs Post Transfusion

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Red cell parameters
Direct Measurement
Erythrocyte Concentration (RBC) x 106/ml
Mean Corpuscular Volume (MCV) Femtolitre (fl)?
HCT?
Hemoglobin (Hb) Gram/decilitre (g/dl)
Indirect Measurement
Hematocrit (Hct) = RBC x MCV/10 % ?
MCV?
Mean Corpuscular Hemoglobin (MCH)
= HB x 10 / RBC (pg)
Mean Corpuscular Hemoglobin
Concentration (MCHC)= Hb/Hct x 100
(g/dl) CP6006a 15
Classification of anemia according to MCV
&RDW
Normal MCV High MCV Low MCV

Normal RDW Other causes of Aplastic anemia Alpha & beta

n n anemia thal trait

High RDW Double Megaloblastic Iron deficiency

population anemia anemia

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 WBCs histograms
2 part differential: passage of cells with RBC lyse results in two peak
histogram.

3 part differential: the WBC histogram displays white cell size after partial
lysing with a specific lyse which results in shrinkage of cell membrane
around nucleus thus it does not display the native size but relative sizes
5/6 part differential

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CP6006a 18
Region
flags

Sites of different types of region flags

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Coulter WBC
Histogram Monos
Neuts
Lymphs 90 -160 fL Eos
50 – 90 160 - 450
fL fL
Baso

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 Presence of abnormal cells in a significant number leads to change in the shape of
the curve.

R1 flags (far left region flags):

Indicate either the presence of:
1. Clumped or giant platelets. 2. Nucleated RBCs.
3. Non-lysed RBCs 4. Paraprotein.
5. Cryoglobulin. 6. Malaria parasite.
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R2 flags region between lymphocytes and mononuclear
cells
Indicats the presence of either:
1-Varint lymphocytes 2- Abnormal lymphocytes.
3-Blast cells 4- Plasma cells.

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 R3 flags(region between mononuclear cells and granulocyte curve):

Indicate the presence of either:

1- Immature granulocytes.
2-Abnormal cell population
3-Eosinophilia.
4-Basophilia.

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R4 flags (in the far right
region)the presence of either
Indicate
1. High absolute granulocyte count.
2. Right shift.
RM flags = Multiple region flags

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WBCs (Light Scatter
Differential)

• WBC scatterplot. WBC scatterplot graphs volume versus light scatter and reveals the locations of four
leukocyte populations in two-dimensional space. The basophil population is located behind the
lymphocytes, but in three-dimensional space is clearly delineated

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Combining Impedance& Optical
Principles

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Automated 5 Part
Diff

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3-D Cellular Analysis - VCS
VOLUME (Y)
CONDUCTIVITY (Z)

Eos
Monos
LIGHT SCATTER (X)

The 3 probes (DC, RF and Scatter) interrogate

each of the 8192 cells simultaneously. Neuts
Every cell is treated in the same manner and Lymphs Basos
each cell is given an X, Y, and Z coordinate on
the dataplot; with 16 million points in the matrix.
ALL cell populations are DIRECTLY
measured CP6006a 40
Platelet histogram

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Platelet
Histogram
• Platelet counting and sizing in both the electrical impedance and
optical systems reflect the native cell size.
• Counting and sizing take place in the RBC aperture.
• The computer classifies particles that are greater than 2 fL or less
than 20 fL as platelets,

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 Platelet
•Histogram
The raw data is sorted and histograms are then smoothed (smooth curve) and tested
against mathematical criteria that eliminate nonplatelet particles and finally fitted to
a log-normal distribution curve.
• This distribution curve has a range of 0 to 70 fL (fitted curve). The final
platelet count is derived from the integrated area under this “best fit” log-
normal curve.
• On the Coulter, a minimum of 400 particles per aperture must be detected. If an
insufficient number of particles are present in the 2–20 fL range, a “no-fit” condition
is reported.

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Platelet
Histogram
• Small particles, such as bubbles or dust, can overlap at the low end of
the histogram. Microcytic erythrocytes can interfere at the upper
end.
• If the histogram does not return to the baseline at both the right and
left of the peak, either there is severe thrombocytopenia or
nonplatelets are being counted. Either erythrocyte or leukocyte
fragments may be responsible.
• In such cases, the platelet count and derived parameters are
not reliable

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PLT
HISTOGRAMS

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Sources of error in cell
counts
Cold agglutinins - low red cell counts and high MCVs can be caused by red cell
agglutinates. If agglutinated red cells are present, the automated hematocrits, MCH
and MCHCs are also incorrect. Cold agglutinins cause agglutination of the red cells as
the blood cools.
1. Cold agglutinins can be present in a number of disease states, including infectious
mononucleosis and mycoplasma pneumonia infections.
How To Solve
If red cell agglutinates are seen on the peripheral smear, warm the sample in a 37
degrees C heating block and mix and test

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Sources of error in cell
counts
2. Fragmented or very microcytic red cells these may cause red cell
counts to be decreased and may flag the platelet count as the red cells
become closer in size to the platelets and cause an abnormal platelet
histogram. The population is visible at the left side of the red cell
histogram and the right end of the platelet histogram

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Fragmented or very microcytic
RBCs

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Sources of error in cell
counts
3. Platelet clumps and platelet satellitosis - these cause falsely decreased
platelet counts. Platelet clumps can be seen on the right side of the
platelet histogram. Decreased platelet counts are confirmed by reviewing
the peripheral smear. Always scan the edge of the smear when checking
low platelet counts

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4. Giant platelets - these are platelets that approach or exceed the size
of the red cells. They cause the right hand tail of the histogram to
remain elevated and may be seen at the left of the red cell histogram.

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• Spurious elevation of automated platelet counts in secondary
leukemia associated with tumor lysis syndrome
• Enumeration of yeast forms as platelets
• Enumeration of bacteria as platelets

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• 5. Nucleated red blood cells - these interfere with the WBC on some
instruments by being counted as white cells/lymphocytes

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Factors known to cause spurious laboratory results in hematology analyzers

Parameter Spuriously increased Spuriously deccreased

Hb hyperlipidemia, hyperbilirubinemia Clotting

High total WBCs

RBCs WBC >50 000/mm3 Clotting

Giant platelets
MCV Cold agglutinins, hyperglycemia,
WBC >50 000/mm3

MCHC Hyperlipidemia, cold agglutinins WBC >50 000/mm3

RDW Post transfusion

WBCs Nucleated red cells, Platelet clumps, Clotting

Unlysed red cells,
Cryoglobulins

Platelets RBCs, WBC fragmentation, Severe Satellitism, clumping

microcytosis,
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Cryoglobulins