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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 Number

Subject

Introduction Physiology Manual Blood Count Coulter Method Block Diagram Important Subsystem in Coulter Device Measurement of the parameter Histogram Calibration Maintenance Reference

3 3 4 7 8 10 13 17 19 19 22

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 . - Dont have a cell nucleus . - It is concaved disc-shaped cell. - The diameter of a typical human erythrocyte is 6-8 m. - Its 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 . - Its number (6 10)*1000 cells/mm.cubic . - The circulating life is 13 20 days . - 10m in diameter . Platelets are : - A nuclear and discoid . - Size 1.5 3.0 m. - The circulating life is 9 10 days . - produced in the bone marrow . - Its 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 cyanidecontaining 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 cells 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 its 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 ).