Post on 08-Feb-2016
description
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 ).