1. From the 15TH
of June to the 20TH
of July 2014
Specialized Medical Center Hospital (SMCH)
Riyadh, Saudi Arabia.
Medical
Analysis
Internship Report 2014
Teaching Mentor: Bouyssi Didier
Professional tutor: Dr. Ali Hejazi
Supervisor : Bashar Kharfan
Sandra AOUF;2nd year chemical engineer student
2. 1
ACKNOWLEDGEMENTS
I would like to thank Dr. Ali Hejazi (Lab Director), for accepting me as intern
student in his lab. Also many thanks for his staff for their warm welcome and
support.
Finally, I thank the entire team for their advice, help and for the time they spent
with me all throughout this period, answering my questions
3. 2
Pages
I. Introduction 3
II. Machines and it’s principles 8
A) Machines 8
a. Cobas 6000-module c501 9
b. Cobas 6000-module e601 11
B) Technics 12
a. Photometry 12
b. Ions selective electrode 14
c. Immunoassay 14
III. Experimental aspect 16
A) Determine calcium level in a patient sample
(serum) by using the Photometry technic
16
B) Determine Vitamin D level in a patient sample
(serum) by using immune assay
18
C) Determine the presence of HBsAg by using Ab/Ag
reaction
21
IV. Results 24
V. Conclusion 25
Bibliography 26
Annex
Table of contents
4. 3
INTRODUCTION
My internship took place
in Specialized Medical
Center, in the clinical
laboratory department.
The laboratory
department is considered
as the first fully
automated lab in the
private field, in Saudi
Arabia, Fig (1).
This lab is open for 24 hours a day, seven days a week for inpatient and
outpatient services. The clinical Laboratory services cover all clinical sections:
Chemistry, special chemistry, Immunology, Hematology, Microbiology, Molecular
Biology, Pathology, and Blood Bank. I was trained in the Chemistry and special
chemistry section. The section provided STAT, Pre-Operation and routine testing
of whole blood, serum, plasma, and CSF (cerebral spinal fluid). In this hospital, the
laboratory technicians draw and collect the sample from the patients in certain
tubes.
Figure (1) : Showing the core lab which contain many equipment
and machines.
5. 4
Figure (2): pneumatic tube for normal cases.
Each patient’s sample is identified by the barcode which contains the MRN
(medical record number) and lab number of the patient. The samples are directly
sent from the extraction area to the main lab through the pneumatic system.
There are two pneumatic tubes in this lab, one that is used for normal cases, Fig
(2) and the other one for emergencies Fig (3).
There are many kinds of tubes that could be used in the clinical laboratory
department. Each of these tubes is used for a certain test or for a group of tests.
For efficiency all tubes are color coded to the corresponding usages. For example
if we need to determine the glucose level in a blood stream of a patient, a special
tube is used, which is made out of NaF/NaEDTA following the BD1
Diagnostics-
Preanalytical systems board.
1
Becton Dickinson, a medical company specialized in medical devices.
Figure (3): pneumatic tube for emergencies.
6. 5
Fig (4) : All kinds of tubes that can be used in a Clinical Laboratory.
The board
clearly
shows the
color code
of the tube,
its type or
what is it
made of,
their usage
or what they
determine
and the
times of
inversions.
The BD board, shown in Fig (4), is strategically placed in the extraction area where blood
is taken from patients. The most common type of tubes used in the SMCH lab is EDTA,
sodium citrated and plan tube. All the samples that are extracted in the “Extraction area”
will be sent to the processing
section in the core lab. In this
specific section, all samples are
double checked and distributed
to the different sections of the
lab. Fig (5).
Figure (5) : showing the processing Area; the area where the
samples and requests of the patients are revised and confirmed.
7. 6
Centrifugation
De
capping
Aliquiting Archiving
Once this is done, the
patient samples are ready to
be tested. The tests are
made by automated
machines; these machines
are MPA (Modular Pre-
Analytics) and Cobas 6000
analyzer series. Fig (6).
After the MPA process, the samples are automatically transferred from the MPA to
Cobas 6000 through the core module. Once the samples arrive to Cobas 6000 the
machine automatically run the tests of each sample in c 501 and/or e 601. The results
are displayed on a computer screen that is connected to the whole machine, Fig (7).
After the results are displayed on the computer, the system sends them automatically
to Physicians for verification, and then to the patient’s file.
Figure (6): showing the MPA machine. The main function of this
machine is to prepare patient‘s sample to be tested on Cobas 6000.
9. 8
Figure (8): showing the 6000 analyzer that is
divided in 2 units, the c 501module on the left
and the e 601 module on the right.
THE MACHINES AND THEIR PRINCIPLES.
A) Machines:
The Cobas 6000 analyzer series Fig (8) is a fully automated software-controlled
system for clinical chemistry and immunoassay analysis. It is designed for both,
quantitative and qualitative in vitro determinations using a large variety of tests
for analysis. It is ready to use 24 hours per day. The Cobas 6000 analyzer, use
serum/plasma, urine, CSF, supernatant and whole blood samples types. It
performs photometric assays and ion-selective electrode measurements on c 501
modules as well as electrochemiluminescence assays on e 601 modules.
The Cobas 6000 analyzer series comprises of a control unit, core unit cu 150 and
the following hardware units, which can be combined in various combinations:
- c 501 module
- e 601 module
10. 9
Figure (9): Showing the computers
that display the results of the tests.
The control unit uses a graphical user interface to control all instrument functions.
The Cobas link data station is used as the gateway for retrieving and distributing
information-such as important notes and test.
The following figure, Fig (9), shows the control unit together with the Cobas link
data station.
After the MPA step, the patient samples are automatically transferred from the
MPA to the core unit. The core unit contains a barcode reader which is used to
identify the tests and lab number for each sample tube. Using the information of
the barcode, core unit sends the sample to the c 501 module or to the e 601
module and selects the reagent that corresponds to the needed test.
The c 501 module comprises a photometric unit and an ISE unit (for ion-selective
electrode).
The photometric unit provides the analyzer with a flexible photometric method of
assaying up to 600 in vitro tests per hour on a wide range of analytes.
11. 10
Figure (10):
Showing the main
parts of the c 501
module:
sampling system,
reagent system
and reaction disk
system.
:
The sampling system takes a certain amount from the patient sample and loads it
into the reaction disk. The reagent system starts calculating the amount of
reagent that must be added to the patient sample which is present in the reaction
disk.
12. 11
Figure(12): Showing many types of the
washing buffer that are used to clean
the tubes of the e 601 after each
reaction.
Figure (11): Showing the main parts of the e 601
module: the reagent area, the measuring area , the
consumables area and the pre-wash area.
A reaction should occur giving a color; this is the principle of colorimetry. By using
the technic of photometry, the concentration of the sample is calculated.
The e 601 module is a fully automated analyzer for immunoassay analysis. It is
designed for both quantitative and qualitative in vitro determination using a large
variety of tests for analysis. Samples are transported to the e 601 module by the
core unit of the Cobas 6000 system. The e 601 module is a multi-test
immunoassay analyzer with random access with a capacity of up to 170 tests per
hour.
the reagent
area
the
measuring
area
the
consumables
area
the pre-wash
area
13. 12
B) TECHNICS
a)Photometry:
Photometry is the quantitative measurement of the reflection or transmission
properties of a material as a function of wavelength. Also defined as the study of
the transitions between molecular (or atomic) energy levels. Fig (13).
Photometric analysis for determining the amount of an inorganic compound in
solution involves a reaction between an organic reagent and an analyte to form a
colored complex. The reaction can be used to determine analyte concentrations
supposing the color intensity and absorbance is proportional to the analyte
concentration, the complex is stable, and the reagent does not significantly react
with other constituents thereby causing interferences. A photometer is the
specific device which measures the absorption of a monochromatic light beam by
a sample and added reagent. The experimental approach exploits Beer’s Law,
which predicts a linear relationship between the absorbance of the solution and
the concentration of the analyte. To determine the amount of an inorganic
compound in solution, photometric analysis is used. It includes a reaction
between an organic reagent and an analyte to form a colored complex.
Figure (13): Showing the principle of photometry with the light source, the cuvette, the sample,
the monochromator.
14. 13
BEER - LAMBERT LAW:
( )
" Is proportional to C (concentration of solution) and is
also proportional to L (length of light path through the solution)
with R = Transmittance
I0 = Original light intensity
I = Transmitted light intensity
with A = Absorbance
C = Concentration of the solution ( moles/Liter)
L = Thickness of the cell (cm)
As the cell thickness
increases, the
transmitted intensity of
light of "I" decreases
15. 14
b)ISE ( ion-selective electrode):
The c 501 module has an integrated ISE unit, which provides the analyzer with a
potentiometric method for assaying sodium, potassium and chloride samples. The
ISE unit can process up to 200 samples per hour. The following are the main
components of the ISE unit:
- ISE measuring compartment with measuring cartridges for Cl¯, K+ , Na+
and reference cartridge.
- ISE pipette
- ISE sipper
- IS bath
- ISE reagent compartment
c) Immunoassay:
Immunology's primary function is cheking presence of particular antibodies.
History:
Rosalyn Sussman Yalow and Solomon Berson established the first immunoassays
in the 1950s.
In the late 1960s, techniques for chemically linked enzymes to antibodies were
validated and lead to a simpler performance of immunoassays.
In 1993, radioactive iodine used in immunoassay was replaced with an acridinium
ester that makes its own light: chemiluminescence. This was completed by
Professor Anthony Campbell at Cardiff University. This kind of immunoassay is
now used in around 100 million clinical tests all around the world, allowing
clinicians to measure a wide range of proteins, pathogens and other molecules in
blood samples.
16. 15
Immunoassay definition:
A biochemical test that measures the presence or concentration of a
macromolecule in a solution through the use of an antibody or immunoglobulin.
The macromolecule detected by the immunoassay is often referred to as an
"analyte" and is in many cases a protein. Analytes in biological liquids such as
serum or urine are frequently measured using immunoassays for medical and
research purposes.
Immunoassays rely on the ability of an antibody to recognize and bind a specific
macromolecule in what might be a complex mixture of macromolecules. In
immunology the particular macromolecule bound by an antibody is referred to as
an antigen and the area on an antigen to which the antibody binds is called an
epitope.
In some cases an immunoassay may use an antigen to detect for the presence of
antibodies, which recognize that antigen, in a solution. In other words, in some
immunoassays, the analyte may be an antibody rather than an antigen.
In addition to the binding of an antibody to its antigen, the other key feature of all
immunoassays is a means to produce a measurable signal in response to the
binding. Most, though not all, immunoassays involve chemically linking antibodies
or antigens with some kind of detectable label. A large number of labels exist in
modern immunoassays, and they allow for detection through different means.
Many labels are detectable because they either emit radiation, produce a color
change in a solution, fluoresce under light, or because they can be induced to
emit light.
17. 16
EXPERIMENTAL ASPECT
A) DETERMINE CALCIUM LEVEL IN
PATIENT'S SAMPLE
Calcium
Intended use:
In vitro test for the quantitative determination of calcium in human serum,
plasma and urine on Roche cobas c systems.
Summary:
Calcium is the most abundant mineral element in the body with about 99% in the
bones primarily as hydroxyapatite. The remaining calcium is distributed between
the various tissues and the extracellular fluids where it performs a vital role for
many life sustaining processes. Among the extra skeletal functions of calcium are
involvement in blood coagulation, neuromuscular conduction, excitability of
skeletal and cardiac muscle, enzyme activation, and the preservation of cell
membrane integrity and permeability.
Test principle:
Method according to Schwarzenbach with o-cresolphthalein complexone.
Calcium ions react with o-cresolphthalein complexone (o-CPC) under alkaline
conditions to form a violet colored complex. The addition of 8-hydroxyquinoline
prevents interference by magnesium and iron.
18. 17
Reaction:
Ca2+ + o-CPC → calcium-o-CPC complexe
The color intensity of the complex formed is directly proportional to the calcium
concentration and is measured photometrically.
The Reagents:
R1: CAPS (3-[cyclohehaxylamino]-1-propanesulfonic acid) 525 mmol/L ;
NaOH: 400 mmol/L , pH 11.5 ; nonreactive surfactant
R2: o-cresolphthalein complexone : 0.5 mmol/L ; 8-hydroxyquinoline : 30
mmnol/L ; pH 1.1 ; Stabilizer
Limitations-Interferance :
Icterus , Hemloysis,Lipemia,Drogues : no significant interference.
Reference range:
Serum/plasma
Children (0-10days): 7.6 – 10.4 mg/dL
Children (10 days- 2 years): 9.0 – 11.0 mg/dL
Children (2-12 years): 8.8 – 10.8 mg/dL
Children (12-18 years): 8.4 – 10.2 mg/dL
Adults (18-60 years): 8.6 – 10.0 mg/dL
Adults (60-90 years): 8.8 – 10.2 mg/dL
Adults (> 90 years): 8.2 – 9.6 mg/dL
19. 18
B) Determine Vitamin D level on patient sample
(serum)
VITAMIN D total (25-Hydroxyvitamin D)
Summary:
Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in
the skin by exposure to sunlight. Vitamin D is biologically inert and must undergo
two successive hydroxylation in the liver and kidney to become the biologically
active 1,25-dihydroxyvitamin D.
The two most important forms of vitamin D are vitamin D3 (cholecalciferol) and
vitamin D2 (ergocalciferol). In contrast to vitamin D3, the human body cannot
produce vitamin D2 which is taken up with fortified food or given by supplements.
In human plasma vitamin D3 and D2 are bound to the vitamin D binding protein
and transported to the liver where both are hydroxylated to form vitamin D (25-
OH) , i.e. 25-hydroxyvitamin D. It is commonly agreed that vitamin D (25-OH) is
the metabolite to determine the overall vitamin D status as it is the major storage
form of vitamin D in the human body. This primary circulating form of vitamin D is
biologically inactive with levels approximately 1000-fold greater than the
circulating 1, 25-dihydroxyvitamin D.
The half life of circulating vitamin D (25-OH) is 2-3 weeks. More then 95% of
vitamin D (25-OH), measurable in serum, is vitamin D3 (25-OH) whereas vitamin
D2 (25-OH) reaches measurable levels only in patients taking vitamin D2
supplements. Vitamin D2 is considered to be less effective.
20. 19
Test principle:
Competition principle. Total duration of assay: 27 minutes.
-1st
incubation: By incubating the sample (15 µL) with pretreatment reagent 1 and
2, bound vitamin D (25-OH) is released from the vitamin D binding protein.
-2nd
incubation: By incubating the pretreated sample with ruthenium labeled
vitamin D binding protein, a complexe between the vitamin D (25-OH) and the
ruthenylated vitamin D binding protein is formed.
-3rd
incubation: After addition of streptavidin-coated microparticles and vitamin D
(25-OH) labeled with biotin, unbound ruthenium labeled vitamin D binding
proteins become occupied. A complex consisting of the ruthenylated vitamin D
binding protein and the biotinylated vitamin D ( 25-OH) is formed and becomes
bound to the solid phase via interaction of biotin and streptavidin.
- The reaction mixture is aspirated into the measuring cell where the
microparticles and magnetically captured onto the surface of the electrode.
Unbound substances are then removed with ProCell/ProCell M. Application of the
voltage to the electrode then induces chemiluminescent emission which is
measured by a photomultiplier.
- Results are determined via a calibration curve which is instrument-specifically
generated by 2-point calibration and a master curve provided via the reagent
barcode.
21. 20
Measuring Range:
Normal range
3.00 – 70.7 ng/mL or 7.50 – 175 nmol/L
If the levels < 30 ng/mL its considered insufficient or deficient.
The preferred level for vitamin D (25-OH) by many experts is now recommended
to be ≥ 30 ng/mL ( ≥ 75 nmol/L) . This is in line with the US National Osteoporosis
Foundation recommending level > 30 ng/mL to protect bone health.
22. 21
C) Determine the presence of HBsAg
Name of the test: ARCHITECT HBsAg Qualitative II.
Intended use:
The ARCHITECT HBsAg Qualitative II assay is a chemiluminescent microparticle
immunoassay (CMIA) for the qualitative detection of hepatitis B surface antigen
(HBsAg) in human serum and plasma.
The ARCHITECT HBsAg Qualitative II assay is intended to be used as an aid in the
diagnosis of HBV infection and as a screening test for donated blood and plasma.
SUMMARY AND EXPLANATION OF TEST:
The causative agent of serum hepatitis is hepatitis B virus (HBV) which is an
enveloped DNA virus. During infection, HBV produces an excess of hepatitis B
surface antigen (HBsAg), also known as Australia antigen, which can be detected
in the blood of infected individuals. It is responsible for binding the virus to the
liver cell and is the target structure of neutralizing antibodies. HBsAg is the first
serological marker after infection with HBV appearing one to ten weeks after
exposure and two to eight weeks before the onset of clinical symptoms. HBsAg
persists during this acute phase and clears late in the convalescence period.
Failure to clear HBsAg within six months indicates a chronic HBsAg carrier state.
HBsAg assays are used to identify persons infected with HBV and to prevent
transmission of the virus by blood and blood products as well as to monitor the
status of infected individuals in combination with other hepatitis B serological
markers. In most countries, testing for HBsAg is part of the antenatal screening
program to identify HBV infected mothers and to prevent perinatal HBV infection
by subsequent immunization.
23. 22
BIOLOGICAL PRINCIPLES OF THE PROCEDURE:
The ARCHITECT I 1000, Fig (14) HBsAg Qualitative II assay is a one-step
immunoassay for the qualitative detection of HBsAg in human serum and plasma
using CMIA technology, with flexible assay protocols, referred to as Chemiflex.
In the ARCHITECT HBsAg Qualitative II assay, sample, anti-HBs coated
paramagnetic microparticles , and anti-HBs acridinium labeled conjugate are
combined to create a reaction mixture. HBsAg present in the sample binds to the
anti-HBs coated microparticles and to the anti-HBs acridinium –labeled conjugate.
After washing, ancillary wash buffer is added to the reaction mixture. Following
another wash cycle, pre-trigger and trigger solutions are added to the reaction
mixture. The resulting chemiluminescent reaction is measured as relative light
units (RLUs).
A direct relationship exists between the amount of HBsAg in the sample and the
RLUs detected by the Architect; System optics.
The presence or the absence of HBsAg in the sample is determined by comparing
the chemiluminescent signal in the reaction to the cutoff signal determined from
an active calibration. If the chemiluminescent signal in the specimen is greater
than or equal to the cutoff signal, the sample is considered reactive for HBsAg.
24. 23
SPECIMEN COLLECTION AND PREPARATIOM FOR ANALYSIS:
Specimen Types:
-The specimen collection tubes listed below were verified for use with the
ARCHITECT HBsAg Qualitative II assay. Other specimen collection tubes have not
been tested with this assay.
- Human serum or plasma.
INTERPRETATION OF RESULTS:
ARCHITECT HBsAg Qualitative II Initial Result
Initial Result (S/CO) Instrument Interpretation Retest Procedure
< 1.00 NONREACTIVE No retest required
≥1.00 REACTIVE Retest in duplicate
Figure (14): showing the
ARCHITECT I 1000 machine
that is used for
Antibody/Antigen technic.
25. 24
RESULTS
Results of Calcium and Vitamin D tests runned on 10 samples.
Sample number Ca (mg/dL) Vitamin D (ng/mL)
1 8.39 16.64
2 8.82 9.72
3 8.03 4.04
4 9.20 11.96
5 11.04 3.00
6 9.00 21.59
7 8.26 5.01
8 8.83 5.37
9 8.15 3.00
10 9.13 17.17
I choose the Vitamin D and Calcium tests because these two are related.
Calcium is a mineral that is necessary for life. In addition to building bones and
keeping them healthy, calcium helps our blood clot, nerves send messages
and muscles contract. About 99 percent of the calcium in our bodies is in our
bones and teeth. Each day, we lose calcium through our skin, nails, hair, sweat,
urine and feces, but our bodies cannot produce new calcium. Food is the best
source of calcium. Dairy products, such as low-fat and non-fat milk, yogurt and
cheese are high in calcium. Certain green vegetables and other foods contain
calcium in smaller amounts.
26. 25
Vitamin D is as important as calcium for the maintenance of a healthy body and a
solid skeleton to hang it on. There are three ways to get vitamin D: sunlight, food
and supplements. Vitamin D aids the absorption of calcium, by facilitating the
intestinal absorption of calcium by mediating active calcium transport across the
intestinal mucosa.
CONCLUSION
To conclude this report I would like to say this internship, in this laboratory, gave
me an idea about the different fields that I can work in. I had the opportunity to
discover a new field in Chemistry which is the medical analysis field. To actually
work in one of these labs with these technics is a one of a kind experience. I spent
the two years of faculty learning theories and analysis methods in class, but when
I had the chance to see how to apply these lessons in real life, it expanded my
horizons.
I enjoyed working with these machines, not only because they were easy to use
and gave fast results, but also because all of the mathematical, physical and
chemical aspects it involved. The machines were used on a daily basis, for me at
first it was new but with time I began to understand better each machine and
relate all the chemical lessons I had at the university with the real life application I
saw in front of me. I found it really interesting how the machines have the
capacity to run big numbers of tests in a short time with a high accuracy.
27. 26
Knowing how to work in groups and co-operate with fellow colleagues was a new
experience for me. Finishing tasks, achieving goals and reaching the daily mission
were some of the few great lessons that the lab had to offer.
Working with advanced machines with high quality results was very interesting,
Also having a first-hand view of the differences between quantitative and
qualitative tests, understanding how each machine worked and how precise each
one was. All of that led to understanding how to be efficient and know which
machine to use when it comes to which analysis.
And I was fascinated to see the c 501 module applying the Photometry technic
because I learned to do it at University and it took me four hours to do it just with
one sample, but the c 501 module tests 1500 sample per hour.
I enjoyed my five week internship and hope that my next one will be in cosmetics
or pharmaceutical companies, I would like to expand my knowledge and widen
my horizons to see and learn new things. I will try to apply for other internships
that relate to chemistry so I can have a full overview of all subcategories of
chemistry and acquire as many knowledge and expertise as I can so that when the
day comes to be one of the working force citizens I will be ready.
28. 27
BIBLIOGRAPHY
-User Manual of the Cobas 6000 analyzer (by Roche)
- Google Images (for logos and for Images of technics)
- http://en.wikipedia.org/wiki/Immunoassay
- http://en.wikipedia.org/wiki/Spectrophotometry
- University Lectures.
29. 28
1. ANNEXES
Spectrum of radiation:
Electronic excitation:
Antibody/Antigen:
Energy
*
*
n
*
*
n*
n*
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
30. 29
Rack: Tubes that needs to be tested are put in Racks that go into the machine. All
Racks are numbered and have 5 positions numbered as well.
