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 The methods described in this manual have been
developed in the author’s laboratory taking into account the facilities
available in the intermediate laboratories in developing countries, the
technical level of laboratory personnel and the availability of
chemicals/reagent kits at affordable cost. The validity of these methods with
reference to accuracy, precision, linearity, etc. has been well established.
These methods have been tried and tested and are being employed in a large
number of laboratories in India.
Numerous kits are available today manufactured
by several commercial firms, which contain all the reagents needed for
various biochemical tests. Details are given in the kit leaflet as to the
preparation and storage of reagents. It is important to follow the
manufacturer’s instructions carefully. With the ready availability of the
kits, very few laboratories are preparing their own reagents from the basic
chemicals. Even those laboratories using in-house reagents have the choice of
preparing the standard in their laboratories or buy from commercial
companies. It is very important to remember that whatever is the source, the
standard should be properly prepared. Use of improperly prepared standard
will drastically affect the quality of patients’ results.
(a) Generally the intermediate laboratories
are equipped with either a filter photometer or spectrophotometer to carry
out the analysis as a manual method. However, some laboratories employ
semiautomatic methods for which automated photometric systems are used.
(b) Two types of such systems are available for use in
the laboratory. A comparison of the specifications of these two types is
given in the following Table.
Comparision of Semiautomated
Systems
|
Specifications
|
Coventional Suction Cuvette
|
Flow-Through Sipper Systems
|
|
|
Example: BM 4010
(Boehringer Mannheim, Germany)
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Example: BM 4020
(Boehringer Mannheim Germany)
|
|
Programmable channels
|
None, Chemistries to be set as and when tests are done
|
Chemistries can be programmed into a defined number of
channels and can be recalled as and when tests are done.
|
|
Measuring temperature
|
25, 30, 370C
|
25, 30, 370C
|
|
Type of chemistries done
|
Absorbance, end point, two point,
kinetic.
|
Absorbance, end point, two point,
kinetic.
|
|
Modularity
|
Yes, the instrument has separate photometer, suction
device and incubating waterbath.
|
No, integrated. All the hardware is integrated into
one compact unit to save space.
|
|
Temperature maintenance
|
By circulating waterbath
|
Peltier element
|
|
Cuvette volume required for
measurement
|
500m l
|
Ranging from 20-80m l facilitating the use of small
volumes
|
|
Total reaction volume
|
1000m l
|
Ranging from 175m l to 500m l.
|
|
Cuvette material
|
Quartz
|
Quartz.
|
|
No of wavelength (nm)
|
340, 405, 546, 578, 623
|
340, 405, 505, 546, 570, 623, 700
|
|
Wavelength positioning
|
Manual rotation. Choice depends on the chemistry.
|
Automatic. Instrument automatically sets the
wavelength when the operator selects the chemistry to be done.
|
(c) General (common) laboratory glassware like
pipette, beaker, volumetric flask, measuring cylinder and test tube are
essential for carrying out the assays.
(d) Semiautomatic (push button) pipettes are now
employed in several laboratories. If a laboratory employs such pipettes,
regular maintenance must be carried out as per instructions provided by the
manufacturer. Calibration of pipettes should also be checked at definite
intervals.
The following Table shows a set of sample data
related to calibration of three different pipettes with volumes 50, 100 &
200 m l. The calibration procedure consists of weighing water delivered
through these pipettes into a preweighed beaker for
at least 10 times. From the data obtained, mean, SD, %CV and % error have
been calculated. %CV expresses the precision and % error expresses accuracy.
These data are well within the acceptable limit, i.e. 5%.
|
S. No
|
Weight
of 50 m l
of water (g)
|
Weight
of 100 m l
of water (g)
|
Weight
of 200 m l of water (g)
|
|
1
|
0.0505
|
0.0899
|
0.1900
|
|
2
|
0.0455
|
0.1000
|
0.1965
|
|
3
|
0.0455
|
0.0970
|
0.1957
|
|
4
|
0.0500
|
0.0958
|
0.1948
|
|
5
|
0.0466
|
0.0945
|
0.1960
|
|
6
|
0.0467
|
0.0954
|
0.1968
|
|
7
|
0.0500
|
0.0974
|
0.1956
|
|
8
|
0.0506
|
0.0993
|
0.1863
|
|
9
|
0.0477
|
0.0973
|
0.1897
|
|
10
|
0.0480
|
0.0976
|
0.1869
|
|
Mean
|
0.0481
|
0.0964
|
0.1928
|
|
SD
|
0.002
|
0.0028
|
0.0041
|
|
%CV
|
4.2
|
2.9
|
2.1
|
|
% error
|
1.8
|
3.6
|
3.6
|
|
|
|
|
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(e) Protection of laboratory personnel from
infectious diseases transmitted by blood, body fluids and tissue
A biohazardous condition is a
situation involving infectious agents, biological in nature such as hepatitis
B virus, human immunodeficiency virus (HIV), or the tuberculosis bacterium.
These infectious agents are present in human blood and blood products and in
other body fluids.
The major sources of contamination when handling
potentially infectious agents are:-
 Hand-to-mouth contact
Hand-to-eye contact
Direct contact with superficial cuts, open
wounds, and other skin conditions that permit absorption into subcutaneous
skin layer
Splashes or aerosol contact with skin and
eyes.
Specimens from patients known to have these infectious
diseases (Biohazard specimens) should be handled using special precautions.
However, since it is not possible to identify all carriers it is also
sensible to introduce a system of universal precautions whereby high-risk
procedures are identified as well as high-risk specimens. If laboratory staff
use the recommended precautions this should increase their protection.
Strictly adhere to the following procedures: -
Wear gloves when in contact with body fluids,
such as serum, plasma, urine or whole blood
Wash your hands when gloves are removed or
changed
Perform procedures carefully to minimize
aerosol formation
Wear protective clothing such as laboratory
coats or aprons when working with specimens
Keep your hands away from your face
Cover all superficial cuts before starting any
work
Dispose of specimens and other contaminated
materials according to your laboratory’s biohazard control procedure
Keep your work area disinfected, disinfect
tools and other items that have been in any contaminated area.
Do not eat or drink or apply cosmetics while
in the laboratory
Do not mouth pipette
any liquid, including water.
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