Prepared By: Ashraf Shaqalaih

Bsc(MT), MSc(MT), CLS(H), CLSp(H)

Automated Hematology Cell Counters

1- Impedance Technology Hematology Counters
These counters are based on the Coulter principle, i.e.
electrical resistance principle, which depends on the fact that blood
cells are nonconductive to electricity, so when they pass through
an electrical field they will increase the electrical impedance
(resistance).
Well mixed blood is greatly diluted/suspended in an isotonic
electrolyte solution, so that cell sizes are not altered/changed in
terms of cell shrinkage nor cell swelling, this isotonic electrolyte
solution conduct electricity very well, while blood cells are non-
conductive. A counting chamber consists of (1) a beaker, (2) two
electrodes with a direct current pass through them, and an
orifice/small opening the aperture with specified dimensions; when
the suspended cells/particles passes through the aperture it will
displace its own volume of isotonic electrolyte solution and
increase the electrical resistance (impedance) because of their
non-conductivity between the two electrodes which are located
on each side of the aperture. This electrical resistance is
manifested as a pulse, each pulse means a cell/a particle, sum of
these pulses equals the total cell count. This pulse has a height
which is directly proportional to the cell size (i.e. pulse height
indicates cell volume/size). By this, the counter has counted and
sized the cells/ particles. In fact the counter has two chambers/
baths, one for red blood cell and platelet counting and sizing, while
the other one is for WBC counting and sizing. Each counting
chamber has 2 (+/-) electrodes, and an aperture with specified
dimensions for each, the RBC aperture is narrower than the WBC
aperture, why? Because the WBCs have larger sizes than red
blood cells and platelets.
From the above we can conclude that impedance hematology
counters actually does not identify the cells which are considered
here as particles, rather it counts and size them; but according to
their size cells/particles they can be discriminated into cell
types or populations.





Coulter Principle Counting Chamber







Let us now go step by step , as if we are inside the
counter. As soon as patient well mixed blood sample is aspirated
into the counter, blood is highly diluted with the isotonic solution
(the isotonic solution is called the diluent reagent), then this
dilution is divided into 2 portions/parts; first portion will be enforced
through the tubing toward the RBC chamber, where it is further
diluted with the isotonic solution, and then cells/particles are
passed through the aperture, in which red blood cells and platelets
are counted and sized according to the total number and heights
Coulter Principle
The Coulter principle of counting and sizing particles/cells is based on
measurable changes in electrical resistance produced/created by non-
conductive particles/cells suspended in conductive isotonic electrolyte
solution.
Beaker
Aperture
Electrodes
Cells
Cell passing through aperture
of created pulses. But your question here is, how the counter has
discriminated between platelets and red blood cells? Good
question, the answer is , cells/particles which have a volume
between 2-20 fl are considered and counted as platelets, while
cells between 36 to 360 fl are considered and counted as red
blood cells. You can assimilate from this point that the counter
actually didnt identify the cells as platelets or red blood
cells, but according to their size manifested by the generated
pulse height they are discriminated, right or not? I want you to
understand these points, so that you will feel that it is not strange
when we have microcytic red cells (decreased volume), or
fragmented red cells (schistocytes, which are characterized by
greatly reduced volume/size) they may be counted as platelets, on
the other hand, also you feel it is not strange that when we have
large platelets they may be counted as red blood cells, causing
erroneous counting results according to the defect. The second
portion of diluted blood sample will be moved through tubing
towards the WBC chamber, where it is further diluted with a lyse
reagent; not the diluent; this lyse reagent has several functions:
(1) will lyse the red blood cells, so that red blood cells will not be
counted or interfere with white blood cells; whereas in RBC
chamber WBCs are not lysed, but because of their low count
(WBCs, are in thousands/cumm) in comparison to the very high
RBC count which are in millions per cumm, they will not affect
significantly the total red blood cell count, only in cases of very
high WBC count as seen in leukemias, they may affect the RBC
total count, (2) lyse reagent contains Drabkins solution which is
provided for hemoglobin determination, when the red cells are
lysed they release hemoglobin. (3) lyse reagent will puncture the
WBC membranes so that they will collapse around the nucleus
with their granules. So, when the WBCs pass through the aperture
they are counted and sized in the same manner as red blood
cells and platelets are counted and sized, but the difference here
that the counter in counting and sizing the WBC nucleuses
because their membranes are punctured by the lyse reagent. Also,
the counter can perform three part WBC differential, i.e.
neutrophils, monocytes and lymphocytes, but how? Again through
their sizes, the particles/collapsed cells can be discriminated into
three zones, the first zone is considering cells with a volume of
35-90 fl - which are differentiated as lymphocytes, the second
zone with cell volumes between 90-160 fl, which are differentiated
as monocytes, and the third last zone with cell volumes between
160-450 fl, are differentiated as neutrophils. By this the counter
has counted the total WBC count, and performed three part
differential. You may ask, that we know that the monocytes are
bigger than neutrophils, but why the counter considered
neutrophils as bigger? Very good question; the answer is because
the lyse reagent puncture the cell membranes, with subsequent
collapse around the nucleus with the granules, the nucleus plus
granules size of neutrophils is bigger than monocyte nucleus
with its granules, the counter is not sizing the whole WBC cell
rather it is sizing the collapsed punctured WBC cell in which the
neutrophil collapsed cell is bigger than monocyte collapsed cell.
Actually the counter provide us with three part differential as said
before, but with the following sequence: (1) Lymphocytes, (2) MID
(which means middle cells, which are monocytes in normal states,
but may represent immature cells or variant lymphocytes in
abnormal states, but because of simplicity I explain it above as
monocytes), and (3) Neutrophils. One point also should be
considered here, that platelets are not lysed here, but because of
there small size they are not counted as WBCs, but when platelet
clumps or aggregated platelets are present they can be counted
as one big particle, they may be counted as WBCs and interfere
with WBC count. After the WBCs are counted, the WBC diluted
blood portion is moved toward the spectrophotometer which is
provided inside the counter for hemoglobin determination at 530-
540 nm, according to Drabkins method.
You may ask, can two or more cells enter at the same time to
the aperture and be sized and counted as one cell (as one high
pulse), giving a high pulse indicating a very large cell ?- the
answer is yes, and this is called the coincidence error. But this
error is minimized by the huge dilution, by decreasing the aperture
dimensions, and by the computer which statistically corrects for
this error. Also, one may ask since we can reduce it by the great
dilution, why we are not doing more and more dilutions to further
reduce this error?, this is because as you do more dilution, the
great error you have, because of the dilution error, right?
All of the above is supplied to the counter computer, which do
its calculations to give meaningful data. The counter is not only
supplying us with (1)RBC count,(2)Platelets count, (3) Hemoglobin
concentration, (4) WBC count, with its 3 part differential (5)
Neutrophils %, (6) Neutrophils absolute count, (7) MID %, (8)MID
absolute count, (9) Lymphocytes %, (10) Lymphocyte absolute
count, but the counter has extended hematology menu. It also
supply us with the (11) MCV, which is here is considered as a
measured parameter, not calculated parameter, and it is derived
from the RBC histogram (will be discussed later), (12) Hct, which
is a calculated parameter here; the counter does have a
hematocrit centrifuge inside it; it is derived from RBC total count
and the MCV, by applying the following formula (Hct = MCV x
RBC/10), (13) MCH, which is calculated from Hb and RBC count,
(14) MCHC, which is calculated from Hct , and Hb, (15) RDW, Red
cell Distribution Width, which measures the degree of anisocytosis;
RDW is a calculated parameter, RDW is helpful in many
instances, it is the first parameter to increase in cases of nutritional
deficiencies, especially iron deficiency anemia, it is also used to
differentiate between iron deficiency anemia (low MCV , high
RDW), and uncomplicated thalassemia minor (low MCV, normal
RDW). RDW has a normal range between 11.6 and 14.6, so
when RDW is normal it is expected to have red cells which are
homogeneous and exhibit very minimal anisocytosis on
peripheral blood films, but when RDW is high it is expected to find
red cells with altered sizes. (16) MPV, Mean Platelets Volume,
which analogues the MCV for red cells, MPV have a nonlinear
inverse relationship with platelets count, i.e. when platelet count is
low, MPV is high (platelets are large in size), a technical point
should be mentioned here, that when blood is extracted from the
patient, platelets change shape from discoid to spheroid, this
alteration in shape, causes an increase in MPV, this is why it is
advisable to analyze the blood specimens after 30 to 60 minutes
of blood extraction. When platelets count is low, and MPV is low, it
is expected that the bone marrow platelet production is defected,
because when platelets are low in number (thrombocytopenia) the
bone marrow will react by producing platelets with increased
volume/size(large platelets functions more than small platelets),
while if we have low platelets count with high MPV, the cause of
thrombocytopenia is expected to be peripheral not bone marrow in
origin, because here the bone marrow responded to
thrombocytopenia by producing and releasing large platelets, (17)
PDW, Platelet Distribution Width, is a measure of the uniformity of
platelet sizes, normal PDW is less than 20%, increased PDW is
associated with abnormal megakaryocytic development and
maturation, (18) Pct, Plateletcrit, this parameter represents an
estimate of the percent platelet mass, this parameter is especially
in Europe, to determine if the thrombocytopenic patient is of great
need of platelet concentrate transfusion or not, because patients
with reduced Pct may bleed spontaneously and are considered as
candidates for platelet concentrate transfusions. The counters
also display 3 histograms (for WBC, RBC, and PLT histograms),
these will be discussed in the following pages.
Hematology Parameter Menu Supplied By Impedance Technology Counters

No Abbreviation Description
1 WBC count Total white Blood Cell Count
2 Granulocyte % % Differential of Neutrophils
3 MID % % Differential of Middle Cells
4 Lymphocyte % % differential of Lymphocytes
5 Granulocyte Abs. Absolute Total Neutrophil Count
6 MID Abs. Absolute Total Middle Cells Count
7 Lymphocyte Abs Absolute Total Lymphocytes Count
8 RBC count Total Red Blood Cell Count
9 Hb Hemoglobin Concentration
10 Hct Hematocrit
11 MCV Mean Cell Volume
12 MCH Mean Cell Hemoglobin
13 MCHC Mean Cell Hemoglobin Concentration
14 RDW Red Cell Distribution Width
15 PLT Total Platelets Count
16 MPV Mean Platelet Volume
17 PDW Platelet Distribution Width
18 Pct Plateletcrit
19 RBC Histogram Red Blood Cell Histogram
20 WBC Histogram White Blood Cells Histogram
21 PLT Histogram Platelets Histogram



As said during our discussion above, that erroneous results
may be obtained by the Automated Hematology Counters, table
below summarize the frequent causes of erroneous CBC results
obtained with Automated Impedance Hematology Counters.
Erroneous Results That May Be Obtained With Automated Impedance Counters

Parameter Cause-Increase Cause Decrease
WBC
Nucleated RBC
Unlysed Red Cells
Platelet Clumping/
Aggregation
Cryoglobulin
Heinz Bodies
Malaria Parasite
Clotting
Smudge Cells
RBC
High WBC
Giant Platelets
Clotting
Microcytic cells
Hemolysis
Cold agglutinin
Hemoglobin
High WBC
Hyperlipidemia
High Protein
Clotting
Sulfhemoglobin
MCV
Autoagglutination
High WBC
Hyperglycemia
Giant Platelets

MCH
Autoagglutination
High WBC
High Hemoglobin
Low RBC count
Low Hemoglobin
High RBC
MCHC
Autoagglutination
High Hemoglobin
Low Hematocrit
High WBC
Low Hemoglobin
High Hematocrit
Platelets
Microcytic Red Cells
Red Cell Inclusions
White Cell Fragments
Dust Particles
Electronic/Electrical Noises
Hemolysis-Schistocytes
Clotting
Giant Platelets
Heparin
Platelet Clumping
Platelet Satellitosis



There are many brand name counters available in the market, they may use different
terminology, for example some counters will use large cell instead of granulocytes,
and use small cells instead of lymphocytes , you must always look and read the
counter manual to learn the used terminology in your Automated Hematology
Impedance Counter, but all of these counters have the same principles, described
above.
Examples of corrective actions can be taken by medical
technologists
Most of these erroneous results can be corrected by
competent medical technologists, as you.
Erroneous increase in hemoglobin can be corrected with the
preparation of sample blank
Erroneous increase in MCV , MCH, and MCHC, combined
with the erroneous decrease in RBC counts caused by cold
agglutinins, can be corrected by warming the patient specimen in
37 C water bath, for a short time, and then repeat CBC while the
specimen is worm, if problem still persists, you can extract blood
and place it in a prewarmed EDTA blood collection tube.
The above are examples of corrective actions that can be
taken/performed by medical laboratory technologists to correct the
error. Always, a blood film should be performed and visualized
whenever an abnormal results are obtained either they are true
or erroneous, and almost all erroneous results can be resolved
and clarified by visualizing a well made/stained blood film,
because the machine does not always detect what the eye does.
We have to mention that the counter consumes/utilizes three
types of reagents:(1)the diluent, (2) the lyse reagent, and (3) the
detergent, the first two reagents we have already talked about
above. The detergent has two functions:(1)to remove protein
precipitates from the tubing and the counting chambers, (2) is the
blank solution for the spectrophotometer (for hemoglobin
determination).
Automated Hematology Counter Histograms
Histograms are graphical representations of relative
cell/particle frequency versus cell/particle volume/size. Three
histograms are displayed; RBC, WBC, and platelets histogram.
Not only the histograms supply us with information about RBCs,
WBCs, and platelets frequency, their distribution, and average
sizes, but also depict the presence of cell subpopulations. Shifting
of the histogram in one direction or the other direction can be of
diagnostic importance. X-axis represents cell size , and theY axis
represents relative frequency of cells/particles.
Red Blood Cell Histogram
As discussed before, particles that are considered as RBC
have a cell size range from 36 to 360 fl (i.e. the counting zone for
red blood cells is between 36 and 360 fl). The RBC histogram
displays cells that are as small as 24 fl. The scale from 24 to 36 fl
allows us for the detection of RBC fragments, WBC fragments,
giant platelets or microcytic red blood cells, all of these may shift
the histogram to the left. The non lysed WBCs that are counted as
RBCs does not affect the RBC histogram curve because of their
low relative frequency, but the curve may be affected, when the
WBC count is very high as occurs in leukemias and infectious
leukemoid reactions. The curve may be shifted to the right
whenever high frequency of macrocytes are present, as seen in
cases of megaloblastic anemias, in cases of reticulocytosis and
polychromasia especially if accompanied with shifted
reticulocytosis, in cases of very high WBC, especially if anemia
complicates the case. The RBC histogram may be bimodal in
various conditions, and this may indicate the presence of two cell
populations. Bimodal curve may be seen in cold agglutinin
disease, in iron deficiency anemia with recent blood transfusion, in
sideroblastic anemia especially in the acquired forms, and in
megaloblastic anemia with recent blood transfusion. Automated
Hematology Counters extract MCV from the area under the curve
or may apply the following formula: Sum of pulse heights/ sum of
pulses.
White Blood Cell Histogram
WBC histogram displays the classification of WBCs according
to their sizes after cytoplasmic puncture by the lyse reagent, i.e.
does not display the native WBC sizes. From the histogram the
percent, absolute counts, and frequency distribution of the 3 part
WBC differential, i.e. lymphocytes, middle cells (normally
monocytes, abnormally may be immature cells such as
myeloblasts, and myelocytes), and granulocytes can be
determined. WBC histogram displays particles/cells as small as 30
fl, but only those cells which are greater than 35 fl are counted as
WBCs. The counter differentiate between lymphocytes, middle
cells (or you can use the term mononuclear cells), and
granulocytes. Mononuclear cells includes blasts, and other
immature cells, however, in normal specimens, monocytes
represents the mononuclear counting zone. On the histogram 4
regions are displayed at 35, 90, 160, and 450 fl. A valley should
be seen between the lymphocytes and mononuclear cells, and
between the mononuclear cells and granulocytes, the automated
counter determines the percentage of each cell type/
subpopulation according to these depression regions, if one or the
two valleys is/are absent this triggers an alert. The region below 35
fl zone should be clear, with no interfering cells, if this region is
not clear, it is expected to have either NRBC, or clumped/
aggregated platelets, or Heinz bodies, or cryoglobulin
1
, or unlysed
mature red blood cells, or malaria parasite, or any other causes, a
blood film should be made to clarify the reason for interference.
Cells that can trigger an alert (interfere) in the region between the
lymphocytes, and mononuclear cells are certain blast cell forms,
plasma cells, or in some cases eosinophilia and basophilia. Cells
that can trigger an alert in the region between mononuclear cells
and the granulocytes includes immature granulocytes, blasts, and
eosinophils. Cells that interfere in the far right side of the curve
usually indicates a high absolute granulocytic count and/or
presence of toxic granulation. Multiple region alerts may be
encountered , in one patient sample. Also, an alert may be
triggered at exactly 35 fl region, which is usually seen in cases of
chronic lymphocytic leukemia (CLL).
Platelets Histogram
Platelets histogram is useful in interpreting platelets sizes and
abnormal platelet morphology. Particles/Platelets that are between
2 to 20 fl are counted as platelets by the automated counter. The
platelets histogram x axis has a range from (0 to 35) fl. The lower
region between 0 to 2 fl can be interfered by air bubbles, dust,
electrical and electronic noises, whereas the upper region (over
20 fl) can be interfered by microcytic red cells, red blood cell
fragments (schistocytes), WBC fragments, giant platelets, clumped
platelets, and platelets satillitosis. So, platelet histogram flags
whenever the inverse relationship between the MPV and platelets
count is abnormal, which may be caused by the previously
mentioned causes.

Cryoglobulins are altered immunoglobulins, in which they become insoluble and precipitate to
varying degrees at temperatures below 37C. They are associated mostly with pathological conditions.
From the above discussion we can depict that the blood cell
histograms supplied by the automated hematology analyzers are
of great diagnostic and morphologic importance, also they alert us
if a patient sample need blood film preparation and examination or
not. Although practice and blood films examination will increase
the knowledge of interpreting and analyzing these blood cell
histograms.

Normal RBC Histogram


Curve to the left represents microcytic
red cells, whereas curve to the right
represents normocytic RBC curve.
The effect of microcytes on red cell and platelet histograms






The effect of giant platelets on RBC and PLT histograms