Automated Hematology Cell Counters

Analyzer

BC-5800Special 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

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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.

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

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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.

Data management software (DMS)

With DMS , data can be transmitted to PC. DMS is a standard

configuration of BC – 5800 to meet different laboratories’

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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

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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, 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.

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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.

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

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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.

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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.

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.

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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.

Causes for inaccurate automated blood counts.

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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

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blood film, because the machine does not always detect what

the eye does.

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

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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.

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.

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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

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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, 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.

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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

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.

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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.

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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.

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

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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 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

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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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