Palestine polytechnic university

College of engineering and technology

Electrical and computer engineering department

Medical Instrumentation.

Project In:

Hematology analyzer (Coulter Method)

Worked By:

Nadia AL-Manasra.

Nabeel AL-Zubidi.

Supervised By:

Eng.Abdullah Arman.

2006.

Contents .

Page

NumberSubject

3Introduction

3Physiology

4Manual Blood Count

7Coulter Method

8Block Diagram

10Important Subsystem in Coulter Device

13Measurement of the parameter

17Histogram

19Calibration

19Maintenance

22Reference

Chapter One.

Introduction.

The blood consists of formed elements ,substances in solution

and water .on the other words blood consist of hematocrit and

serum .many diseases cause characteristic variations in the

composition of blood , Certain disease states are defined by an

absolute increase or decrease in the number of a particular

type of cell in the bloodstream.Many disease states are

heralded by changes in the blood count. any tests are

considered to determine the state of the patient .Some tests

applicable on serum include Glucose ,Uric Acid,…etc .In

this project we interest in determining blood cells .The cell

blood counting can be determined by using hematology

analyzers.

Physiology :

Blood have the following types of cells:

Red Blood Cell : RBC

- The main purpose is the transport of oxygen to the tissue

and pickup CO2 .

- Don’t have a cell nucleus .

- It is concaved disc-shaped cell.

- The diameter of a typical human erythrocyte is 6-8 µm.

- It’s number 4.5 – 5.5 million cells/ mm. cubic .

- Each red blood cell contains four iron atoms in structure

know as the hemoglobin Hgb.

White Blood Cell: WBC

- Act as immune cells and fight infection .

- Normally between 4G & 11G WBC in a liter of healthy

adult blood .

- Have nucleus .

- It’s number (6 – 10)*1000 cells/mm.cubic .

- The circulating life is 13 – 20 days .

- 10µm in diameter .

Platelets are :

- A nuclear and discoid .

- Size 1.5 – 3.0 µm.

- The circulating life is 9 – 10 days .

- produced in the bone marrow .

- It’s number (200 – 800)*1000 cells /mm.cubic .

- A normal platelet count in healthy person is between (150

- 400)*G /L of blood .

- Responsible for coagulation and clotting .

Manual blood count:

Manual blood cell counts are performed by using a

microscope.

Counting chambers that hold a specified volume of diluted

blood (as there are far too many cells if it is not diluted) are

used to calculate the number of red and white cells per litre of

blood.

To identify the numbers of different white cells, a blood film is

made, and a large number of white cells (at least 100) are

counted. This gives the percentage of cells that are of each

type. By multiplying the percentage with the total number of

white blood cells, the absolute number of each type of white

cell can be obtained.

The advantage of manual counting by a medical technologist

is that blood cells that may be misidentified by an automated

counter can be identified visually. It is, however, subject to

human error because so few cells are counted compared with

automated analysis.

Nowadays, this process is generally automated by use of an

automated analyse , with only specific samples being examined

manuallyr

A complete blood count (CBC) or full blood count (FBC) is a

test requested by a doctor or other medical professional that

gives information about the cells in a patient's blood. A CBC is

also known as a "hemogram".

The basic principles of particle counting in the laboratory

generally fall into two categories:

a. Optical method

b. Coulter method (in which we interest ).

General Principle of Coulter Method :

Electrical impedance: resistance or change in current

when cell passes between two electrodes in NaCl solution.

The coulter is an automated hematology analyzer device

that is used for count and sizes cells for blood . These

systems determine the following hematologic parameters

of whole – blood specimens .

WBC: white blood cell (leukocyte count)

RBC : red blood cell (erythrocyte count )

Hgb : hemoglobin concentration

Hct :hematocrit (relative volume of erythrocytes )

MCV :mean corpuscular (erythrocyte ) volume

MCH : mean corpuscular (erythrocyte ) hemoglobin .

MCHC: mean corpuscular (erythrocyte ) hemoglobin

concentration .

Plt : platelet or thrombocyte count .

Chapter Two.

Coulter device “ CBC ” .

Coulter Method (Impedance)

In this method cells are made to pass through an aperture,

in which there is an electrical current flowing. This is done by

suspending electrodes in the diluent, one electrode inside the

aperture tube, and one electrode outside the tube in the sample

cup. The aperture in the aperture tube is approx. 100 microns

in diameter and the diluent containing the suspended particles

(cells) is drawn up into the aperture tube through the aperture.

Another property of cells and particles, other than their

opacity, is their increased resistance to the flow of an electric

current, compared to the suspending fluid. Therefore, as each

cell passes through the aperture, there is a momentary drop in

the current flowing between the two electrodes. This causes a

temporary drop in the circuit current and is interrupted in the

way previously described. Again, the size of the current drop

is proportional to the size of the particle.

Figure 2.1 : some models of coulter device .

A simple block diagram for coulter model of CBC

Methods

Samples

Blood is taken in a test tube containing an anticoagulant

(EDTA, sometimes citrate) to stop it from clotting, and

transported to a laboratory.

Automated blood count

The blood is well mixed (though not shaken) and placed on a

special rack on the analyzer. This instrument has many

different components to analyze different elements in the

blood. The cell counting component counts the numbers and

types of different cells within the blood. The results are printed

out or sent to a computer for review by a technologist.

Blood counting machines aspirate a very small amount of the

specimen through narrow tubing. Within this tubing, there are

sensors that count the number of cells going through it, and

can identify the type of cell. The two main sensors used are

light detectors, and electrical impedance. One way the

instrument can tell what type of blood cell is present is by size.

Other instruments measure different characteristics of the

cells to categorize them.

Because an automated cell counter samples and counts so

many cells, the results are very precise. However, certain

abnormal cells in the blood may be identified incorrectly, and

require the trained eye of a medical technologist. Medical

technologists are specially trained to review the instrument's

results and identify any abnormal cells the instrument could

not categorize.

In addition to counting, measuring, and analyzing red blood

cells, white blood cells and platelets, automated hematology

analyzers also measure the amount of hemoglobin in our

blood and within each red blood cell. This information can be

very helpful to a physician who, for example, is trying to

identify the cause of a patient's anemia. If the red cells are

smaller or larger than normal, or if there's a lot of variation in

the size of the red cells, this data can help guide the direction

of further testing and expedite the diagnostic process so

patients can get the treatment they need quickly.

Important subsystems in coulter device :

1. The diaphragm pump subsystem :

Coulter device uses diaphragm pump to dispense reagents

A& C ,aspirate whole blood samples ,and backwash the

sampling lines . A pump shown in figure 2.2 is composed of

tow chambers seoarated by an elastic diaphragm .the

application of presure (figure 2.2a ) or vacum (figure 2.2b ) to

the pneumatic chamber of the pump decrease or increase

,respictivly ,the volume of the fluidic chamber by movment of

the diaphragm .thus , liquid supplied to the chamber would

alternately be pumped out and drawn in by the alternating

application of preasure and vacum to the pneumatic chamber .

2. Reagent subsystem.

The operation of coulter device requires the use of reagents

that have well-controlled properties .

Reagent A:

In a counting system highly sensetive to the volume of

individual particles being counted ,the conductive liquid in

which the particles or cells are suspended must have a

minimum infuence on their biological integrity ,and ,hence ,on

their size .

The diluents used for leukocyte counting in the electronic

impedance counter must have the capability of destroying

erythrocytes without significantly affecting leukocyte nuclei

.this must be done rapidly enough to satisfy the short

processing time used in the fully automatic ,multiparameter

cell counter .

- reagent A intended to dilute whole blood sample .

Reagent B:

Coulter manufactures a cleaning agent .this azide-free

reagent is premixed with reagent A .the blended solvent and

cleaning action of reagent B effectively cleans and rinses the

cloulter components and tubing .It removes blood components

and residue ,and reduces the residual particulate count to an

insignificant level.

Reagent C :

This azide –free lytic reagent rapidly lyses erythrocytes,

freeing native Hgb and reducing the size of cellular debris to a

level that does not interfere with leukocyte counts .it also

causes a substantial conversion of Hgb to a stable cyanide-

containing pigment ,the absorbance of which is directly

proportional to the Hgb concentration over the clinical range.

Cell Control :

A stable cell cotrol is required when establishing standareds

for quality control for the performance of the coulter device .

Calibrator :

Coulter manufactures the S-CAL Kit as an acceptable

alternative to the reference calibration method using whole

blood .

3.Aperture subsystem:

the coulter method of cell counting and sizing is based on the

detection and measurment of changes in electrical resistance

produced in coductive liquid traversing a small aperture .

when cells are suspended in aconductive liqiud (diluent) , they

function as discrete insulators .when a dilute suspension of

cells is drawn through a small cylindrical aperture , the

passage of each individual cell momentarily increase the

resistance of the electrical path between two submerged

electrodes located on each side of the aperture .figure 2.3

illstrates the passage of cell through an aperture . an electrical

pulse , suitable for counting and sizing , results from the

passage of each cell through the aperture .

Figure 2.3 :coulter method of counting and sizing .

While the number of pulses indicates particle count , the

amplitude of the electrical pulse produced depend on the cell’s

volume. The effective resistance between the electrodes is due

to the resistance of the conductive liquid within the boundaries

of the aperture .the presence within the aperture boundareis of

a cell , or other particle ,raises the resistance of the conductive

pathway by an amount that depends on the cell volume

.theoritical analysis of the behavior of particles within an

aperture shows that the high of the electrical pulse produced

by the cell is characteristic which most nearly exhibits

proportional to the cell volume . this method permits the

selective counting of cells within very narrow size –

distrubution ranges by electronic selection of the pulses they

generate .

*Ocaionally ,more than one cell is within the boundaries of

an aperture at the same time (coincidence ). When this occurs

,only one one large pulse is generated . this results in low cell

count and high cell volume measurments. However , the

frequency of coincidence is a statistically predictable function

of cell concentration , and is corrected by the instrument .

measurment of the parameter :

WBCcount :

A leukocyte is measured as aparticle with a volue

greater than 35 fL remaining test suspension after the

erythocytes have been lysed .after the statistical test for

precision of the three WBC counts ( voting ) , and after the

correction of coincidence error ,the count is scaled by the

calibration factor to express its value in the conventional units

WBC = n *1000 cells / mm(cubic).

WBC Counter .

RBC count:

An erythrocyte is measured as a cell having a

volume of 36 fL or greater . after the statistical test for

precision of the three RBC counts (voting ) , and after

correction of coincidence error , the count is scaled by a

calibration factor to express its value in the conventional units

RBC count = n *1000(1000) cells / mm(cubic ).

RBC Count .

Hgb :

The lytic reagent (eragent C ) not only lyses the

erythrocytes , but converts a substantial portion of the

hemoglobin released by hemolysis to a stable cyanide –

containig pigment .the formation of this pigment has reached

equilibrium when the photometric measurment is made .

A beam of white light from an incandescent lamp passes

through the WBC aperture bath and then through an optical

filter that has a center transmission wavelengh of 525 nm .

light passing through the filter falls on a photodiode . the

photocurrent thus generated is proportional to the

transmittace of the contents of the WBC apearture path at the

chosen wavelenth . a significant refinement included in the

coulter counter system is the introduction of a reagent blank

into the WBC aperture path during each operating cycle . the

reagent –blank signal level provides a reference ad\gainst

which the sample signal is compared .

The reference and sample voltages(VR and Vs ,respectively)

generated by the photocurrent circuitry to measure

hemoglobin concentration (Hgb ) are used in the following

equation :

Hgb (g/dL) = constant * Absorbance

Where :

Absorbance = log VR / Vs

And the constant is the scaling calibration factor .

Hgb Cocentration measurement .

MVC :

This is derived by integrating the RBC pulse – height

values and averaging them over three periods of 4 s each .

then is corrected for coincidence error and scaled by a

calibration factor to produce a final result expresed in

femtoliters .

Hct:

After being scaled to conventional units , the MCV values

are entered into the following equiation :

Hct (%) =( RBC * MCV)/ 10

Plt :

Cells in the RBC path that are 2.0 to 20.0 fL are classified

as platelets (volume calibration referenced to spherical latex

particles ) . after the statistical test for precision of the three

Plt counts (three average counts when counting is extended ),

after correction of coincidence error , and after size

distribution analysis , the Plt count is scaled by a calibration

factor to express its value in conventional units :

Plt count = n * 1000 ells /mm (cubic).

* Hitogram

It represent the waveform of the electrical pulses that

produced from the passage of the blood cells through an

aperture .

- The given pulse depend on :

1. Number of passing cells # of pulses .

2. Volume of cell ( cell characteristics ) The amplitude of

the pulse .

Figure 2.4: Example of general histogram .

X- axis : cells size in femtoliter ( fL) .

Y- axis : # of cells .

Chapter Three .

Calibration of the device

Calibration procedures should be performed to maintain your

coulter device within optimum operation tolerances . coulter

recommend the S-cal Kit or an exact equivalent for calibration

.coulter recommends that you verify calibration monthly with

the S-CAL Kit .An alternate to the S-CAL Kit calibration is

whole –blood calibration with normal , fresh ,whole blood .

Initial adjustments and the total instrument check are essential

for a complete verification of all functions prior to calibration.

Recalibration is necessary when replacing any component that

involves the dilution characteristics (such as the blood

sampling valves) or the primary measurments ( such as an

aperture ).

Although the coulter device is relatively intensive to room

temperature changes, the calibration should be performed

when the room temperature is within its normal temperature

range . if the room temperature varies by more than 1 celicus

,then verifiction and possibly recalibration is necessary .

Calibration include the folloeing :

1. Preliminary procedures .

They are required when the calibration is not performed

immediately after the coulter service representative has

installed your instrument .

* Clean those compnenets :

- blood sampling valve

- aperture

* Check that you have a sufficient supply of reagents to

complete this procedure .

* perform daily startup procedures .

* perform the total instrument check .

2. Total instrument check .

Performed to verify the power supply subsystem voltages are

within their acceptable ranges .

3. S-CAL Kit Calibration .

The S-CAL Kit is intended for yhe quantitive determination of

adjustment factors to be used for the calibration of the coulter

device .Calibration instructions are provided in the S-CAL Kit

package insert .the calibration adjusment is made

automatically as a part of AUT-CAL program .

4. AutoCAL .

5. CAL Log Sheet .

6. Whole –Blood Calibration

It is used as an alternative to calibration with the S-CAL Kit .

Whole-Blood calibration requires that you obtain 20 fresh

,normal whole-Blood specimens .each specimen must be of

suffecient quantity (at least 2 mL ) to obtain reference method

values for each directly measured parameter from the three

samples on the coulter model .

Maintenance of coulter device

We will interest in our project in the meantenance procedure

in which the operator is responsible which include : cleaning

,replacment ,and adjustment procedures. while the big

troubleshootings which need the technitions to solve them by

it’s experince and returns to the survice manual.

Cleaning :

- Blood sample valve (BSV):

Clean the BSV weekly or as necessary .signs that indicate

that the BSV needs cleaning include :binding or irregular

motion of the BSV , erratic results , imprecision ,or failure to

cover control values .

-Bleach Apertures :

Bleach the apertures every two weeks or as necessary .

bleaching removes the protin buildup at the apertures that

restricts proper sample flow . signs that the apertures require

bleaching include : increase voteouts , icreased MCV values

and decreased cell counts, or failure to recover control values .

before bleaching check that the aperture is not clogged .

-Cleaning the external surface :

The external surface of the main unit should be cleaned with

warm , soapy water to prevent the buildup of corrosive

deposits .

Replacing :

- Printer paper .

- Aperture –viewing screen image adjustment (mirror ).

- check valve : which allow the flow of fluid or air within a

tube in one direction .

- Dispenser pumps:

Which include RBC dispenser pump,WBC dispenser pump,

reagent C pump ,backwash pump ,and aspiratin pump .

-Optic lamp:

If the optic lamps do not illstrate , no image will appear on

the RBC &WBC apertue –viewing screens .

Reference List .

1. Medical Instrumentation “Application and Design ”,Second

Edition .John G.Webster ,Editor .

2. Biomedical Instrumentation and Measurements .Second

Edition .Leslie Cromwell .

Fred J . Weibell .

Erich A . Pfeiffer .

3.Reference Manual for the Coulter Counter . MODEL T660 .

Issue A :July 186 . Produced By Technical Communications

Coulter Electronics , INC .

4. InterNet : Google

PubMed

( Some Papers .Some PDF Reviews ).