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Automated Hematology Cell Counters

Analyzer
BC-5800
Special feature

 5-part differentiation, 29 parameters,2 histograms + 2 scattergrams


 Up to 90 samples per hour
 Independent channel and optical method for Basophil measurement
 Powerful capability to flag abnormal cells
 Optional autoloader, barcode scanner
 Large touch screen
 Large storage capacity: up to 40,000 samples
 Recommended or customizable decision rules to re-exam abnormal samples

Chemical dye method by original reagents

The basophils and eosinophils can be accurately differentiated by the unique chemical dye
method. The Mindray original reagents , control and calibrator is a complete system to ensure
high accuracy.
M 58 LEO (I) lyse –it breaks down red cell membrane and co-operates with the M 58 LEO
(II) lyse to differentiate WBC s.
M 58 LEO (II) lyse – it co-operates with the M 58 LEO (I) lyse to differentiate WBC s.
M 58 LBA lyse - it breaks down red cell membrane and differentiate WBC s.
M 58 LH lyse - it breaks down red cell membrane and converts Hb to a haemoglobin
complex to determine the HGB.

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Two separate channels to separate WBC

BC-5800 counts lymphocytes , monocytes, neutrophils and eosinophils in different channel,


and basophils in a separate baso channel. Optical method to test basophil: more information
about cell physiology, more reliable.

Digital sheath flow

This can monitor every cell’s status while going through the aperture, this accurately
measures the volume of cells, giving a standardized histogram shape.
Re- exam

Decision rules recommended by Mindray can help remind to re- exam abnormal samples,
reduce post analytical errors and review rate. Decision rules can also be customerized with
new input support to fit any standard laboratory practices.

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Data management software (DMS)

With DMS , data can be transmitted to PC. DMS is a standard configuration of BC – 5800 to
meet different laboratories’ demand , and offer more customization on report , data editing
and net connection.

Principle

Impedance technique

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 nonconductive.

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

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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, Because the WBC’s 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.

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;

o 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.

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Cell passing through a perture of created pulses. 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.

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 when we have large platelets they may be counted as red
blood cells, causing erroneous counting results according to the defect.

o 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 WBC’s are not lysed, but because
of their low count (WBC’s, 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 Drabkin’s 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 WBC’s 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.

e.g.:- three part WBC differential

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.

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 We know that the monocytes are bigger than neutrophils, but why the counter
considered neutrophils as bigger?

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 it’s 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.

o One point also should be considered here, that platelets are not lysed here, but because
of there small size they are not counted as WBC’s, but when platelet clumps or
aggregated platelets are present they can be counted as one big particle, they may be
counted as WBC’s and interfere with WBC count.

Hb determination

After the WBC’s 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 Drabkin’s method. Hb is measured using modified
cyanomethhaemoglobin method. Non ionic detergent is added to ensure rapid cell lysis and
reduced turbidity caused by membrane. Measurement of absorbance is done at a set time
interval after mixing the reagent with blood but before the reaction is completed.

White cell differential count

Differential white cell count is based on various physical characteristics of white cells. Three
simultaneous measurements are done on each white cells ( two electrical and one optical
measurements)

I. Impedance measurement
II. Conductivity measurement
III. Light scattering

Impedance measurement depends on the cell volume. Conductivity measurement depends on


the internal structure of the cell , nuclear cytoplasmic ratio, nuclear density and nuclear
granularity.

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Light scattering depends on the cell structure , shape and reflectivity of cells.

Light scattering measurements

A solution containing blood cells are allowed to flow through a cuvette .A beam of light is
focused on to this cuvette. Then this light be is pass through it to a dark field. When a light
beam is interrupted by a cell , it causes the light to be scattered.

These scattered light rays radiate out beyond the periphery of dark field disk. These are
collected by photomultiplier tube.

It generates electrical pulse and then it is counted. When no cell flow through the cuvette, the
dark field disc catches all light rays and therefore no pulses are generated by photomultiplier
tube.

BC-5800 haematology analyser utilizes the semi conductor laser for the flowcytometry
system. Through calculations of different angles via laser scatters, the instrument provides
complete analysis including cell size, granularity and complexity.
Differential white cell count is based of the different light absorption and scattering
characteristics of each white cell type.

Eg;-

00 –indicator of cell size

100 –indicator of the structure and complexity.

900 – indicates nuclear lobularity and cell granularity.

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Conductivity measurements

This is done by alternating current in the radio frequency range.It circuits the bipolar lipid
layer of the cell membrane allowing the energy to penetrate the cell. Powerful probe is used
to collect information about cell size and internal structures including chemical composition
and nuclear volume.

Errors in auto analyzers.

 Red cell agglutinations can be counted as one cell.


 Cell deformities- appear smaller
 Cells which recirculation causes for the aberrant impulse
 Cells which pass simultaneously counted as a single cell.

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. This is called the
coincidence error. But this error is minimized by the huge dilution ( but more and more
dilutions, causes dilution error.), by decreasing the aperture dimensions, and by the
computer which statistically corrects for this error.

 Counting of bubbles, lipid droplets and micro organisms as cells.

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Causes for inaccurate automated blood counts.

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 FBC while the specimen is worm, if
problem still persists, we can extract blood and place it in a prewarmed EDTA blood
collection tube.

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.

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Automated Hematology Counter Histograms

Histograms are graphical representations of relative cell/particle frequency versus


cell/particle volume/size. Normally three histograms are displayed; RBC, WBC, and
platelets histogram. Not only the histograms supply us with information about RBC’s,
WBC’s, 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

Fig: Normal RBC histogram

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.

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Fig: The effect of microcytes on red cell and platelet histograms

The non lysed WBC’s that are counted as RBC’s 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 bi-modal in various conditions, and this may indicate the
presence of two cell populations. Bi-modal 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.

White Blood Cell Histogram

WBC histogram displays the classification of WBC’s 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 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 WBC’s. 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,

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or cryoglobulin, 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.

Fig:The effect of giant platelets on RBC and platelet histograms

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Cryoglobulins are altered immunoglobulins, in which they become insoluble and
precipitate to varying degrees at temperatures below 37°C. They are associated mostly with
pathological conditions.

From the above discussion we can conclude 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.

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Quality control

 Mindray original quality control and calibrator. Provides 60 QC files, each file can
save 310 QC results.
 Import QC information including lot No, level,expire date, mean and range value
through USB disk.
Preparation of stabilized whole blood control
Formaldehyde 40% - 6.75 ml
Dluteraldehyde 5% - 0.75 ml
Tri sodium citrate - 26 g
Distilled water
I. Obtain whole blood in CPD or ACD.
II. Add broad spectrum antibiotic 1 mg penicillin per 100 ml and add 5 mg gentamicin
per 100ml.
III. Mix well and add 1 volume of reagent to 50 volums of cell suspension.
IV. Mix in a mechanical mixture for one hour at room temperature.
V. Leave for 24 h at 4 0C.
VI. With continuous mixing dispence in to a sterile container.
VII. Cap tightly and seal. Refrigerate at 4 0C.
VIII. Keep ½ h at room temperature before analysis mix on a roler mixer, assign values
doing testing two times. Draw control charts.

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Report-A

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Report-B

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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 5 part differential,

(5) MCV, which is here is considered as a measured parameter, not calculated parameter,
and it is derived from the RBC histogram.

(6) 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),

(7) MCH, which is calculated from Hb and RBC count,

(8) MCHC, which is calculated from Hct , and Hb,

(9) 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.

(10) 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

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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,

(11) 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.

(12) 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.

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