BMS207 CLINICAL BIOCHEMISTRY

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BMS207
CLINICAL
BIOCHEMISTRY 1
Practical Manual
To be read in conjunction with the subject outline and with
instructions given for each practical
Please, have a laboratory coat, impervious shoes, safety
glasses, prescribed textbook and scientific calculator for
practicals.
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Clinical Biochemistry 1
BMS207 Practical Manual
Faculty of Science
Written and compiled by
Associate Professor Geoff McKenzie
Dr Phillip Bwititi
Acknowledgements
Ms Kim Day, Ms Kathy Shaw, Ms Rujuan Huang and Ms Marie Nakai for helpful
suggestions in compiling this manual
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Produced by Division of Learning and Teaching Services, Charles Sturt University, Albury – Bathurst – Wagga
Wagga, New South Wales, Australia.
First published 1984
Revised December 1998
Reprinted December 2000
Revised December 2001
Reprinted November 2002
Revised November 2003
Reprinted November 2004
Revised November 2005, November 2006
Reprinted November 2007, November 2008
Revised January 2010, December 2010, December 2011, December 2012, December 2014,
January 2016, January 2017, April 2018, March 2019, May 2020
Printed at Charles Sturt University
© Charles Sturt University
Previously published material in this book is copied on behalf of Charles Sturt University pursuant to Part VB of
the Commonwealth Copyright Act 1968.
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CONTENTS
Page
Introduction 5
Quality control, units, results, reference ranges, standards, samples 9
Write-up of experiments/ exercises and assessments in this subject 11
Aim of practicals 15
Experiment/exercise 1: handling and analysis of QC data 17
Experiment/exercise 2: electrolytes and acid base 18
Experiment/exercise 3: proteins 20
Plasma/serum total protein using biuret method 20
Plasma/serum total protein using bromocresol green method 23
Protein electrophoresis 25
Experiment/exercise 4: enzymes 30
Experiment/exercise 5: bilirubin using Jendrassik and Grof method 32
Experiment/exercise 6: glucose using hexokinase method & POCT 38
Experiment/exercise 7: lipids & POCT 41
Plasma/serum total cholesterol and HDL-cholesterol
using cholesterol oxidase 41
Plasma/serum triglycerides using lipase 47
Experiment/exercise 8: non-protein nitrogenous waste compounds 50
Plasma/serum urea using glutamate dehydrogenase 50
Plasma/serum creatinine using Jaffe’s method 53
Experiment/exercise 9: information on practical examination 55
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INTRODUCTION
Most of the experiments in this practical part of the subject are manual for strong educational
reasons, which include:
a. With automation, there is a tendency to treat them as ‘black boxes’. By using manual
methods, students understand the principles behind the methods and are able to
calculate results.
b. Many small and country hospital laboratories do not use highly automated
instruments and therefore skill in manual methods is essential.
c. Even in large laboratories with sophisticated instrumentation, machines break down
and skill in manual methods is an important stop-gap.
d. The principles of analyses in analysers and other equipment, in most cases are the
same as those in manual techniques.
e. Manual methods are cost effective especially for tests that are not frequently
requested.
f. Not all methods are automated.
For these reasons, it is important that we are familiar with manual techniques.
Laboratory rules
These rules are strictly enforced and non-compliance results in disciplinary action.
1. Closed-in, non-slip footwear must be worn in the laboratories at all times. Students
with bare feet, thongs or open-toed shoes are not be permitted to enter the laboratory.
2. Properly fastened laboratory coats must be worn in all laboratories where chemicals,
radioactive substances and biohazardous substances are being used, or if directed to
do so by an academic, demonstrator or laboratory staff. These are available for a
nominal fee from the Laboratories Store.
3. No horseplay or running is permitted in the laboratories.
4. Under no circumstances are unauthorised experiments to be conducted in the
laboratories.
5. No; smoking, answering of mobile phones, drinking, eating, and handling of food or
application of cosmetics is permitted in the laboratories.
6. Safety glasses must be worn when handling chemicals and/or biological samples or
when directed to do so by an academic, demonstrator or laboratory staff. These are
available for a nominal fee from the Laboratories Store.
7. No mouth pipetting is permitted at ANY time.
8. Disposable gloves must be worn when handling toxic and/or radioactive substances or
biological samples or if your hands contain wounds.
9. No unauthorised venepuncture or blood collection or collection of other bio-samples
is to be performed by an undergraduate student.
10. Sitting on laboratory benches is not permitted.
11. ALWAYS WASH YOUR HANDS BEFORE LEAVING THE LABORATORY.
12. All safety instructions given by an academic, demonstrator or laboratory staff must be
followed.
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General safety information

When in the laboratory, never adopt a casual attitude – always be conscious of
potential hazards.
Always take note of instructions given by academics, demonstrators and laboratory

staff.



Never drink from laboratory taps.
Regard all substances as hazardous unless there is definite information to the contrary.
Report ALL accidents – no matter how slight – to an academic, demonstrator or
laboratory staff.
Familiarise yourself with the locations of all fire exits and with the safety facilities
available in the laboratory:

o
there are first-aid kits available in each laboratory and emergency eye-wash
bottles located above or on each sink.

If you notice any fault with any piece of equipment, or a hazardous situation in the
laboratory, please report it immediately to an academic, demonstrator or laboratory
staff.

On completion of your practical session, ensure that your work areas are left clean
and tidy. If biohazardous materials have been used, the benches must be wiped with
disinfectant.
Do not attempt to adjust instrumentation or microscope light sources if you are not
familiar with them. Always ask a member of the laboratory staff for assistance.
All dirty glassware including test tubes is to be RINSED and placed in the tubs
provided. See special instructions for biohazardous material.
All dirty graduated and volumetric pipettes are to be placed TIPS DOWN in the
buckets provided. See special instructions for biohazardous material.
All pasteur pipettes, serology tubes and kahn tubes are disposable and should be




placed in the specially labelled ‘GLASS ONLY’ bins located in each laboratory. See
special instructions for biohazardous material.
Waste disposal
General:
Paper, plastic, non-contaminated material, etc., can be placed in the plastic
lined garbage bins.
Biohazards:
Contaminated slides, pipettes, pasteur pipettes etc., are to be placed in
disinfectant containers located in each work area. Serology tubes, agar
plates, dirty gloves and contaminated paper etc., are to be placed in
autoclave bags or in ‘BIOHAZARD WASTE’ bins located in each
laboratory.
Sharps:
Needles, scalpel blades etc., are to be placed in the special ‘SHARPS’
containers located in each laboratory.
Glass:
Broken glassware, uncontaminated pasteur pipettes and serology tubes,
etc., are to be disposed of in the ‘GLASS ONLY’ bins located in each
laboratory. A dustpan and broom is available from the prep room for
cleaning up broken glass. Under NO circumstances should broken glass be
picked up with your hands.
Hazardous
chemicals:
Waste bottles are placed in the fume hoods for organic, heavy metal and
other wastes that are not to be disposed of down the sink. If you are not
sure, ASK a demonstrator or the laboratory staff.
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Radioactive
waste:
Use in designated areas only and follow instructions given. Immediately
report all spills and contamination to a demonstrator or laboratory
staff. Dispose of wastes in appropriate vessels in your work area. If you
have ANY doubts, always ask a demonstrator or the laboratory staff.
Human experimentation
In this subject you perform experiments involving human subjects and/or human
samples. There are two important issues regarding the use of human subjects that you should
be aware of:

The ethical issues relating to the use of humans as experimental subjects. The issues
are outlined in the following section entitled Ethics in human experimentation;
The safety issues associated with working with materials of human origin, such as
blood, urine or saliva. The special precautions involved with such biohazardous

substances are included for those subjects using these materials in the section entitled
Biohazard laboratory safety rules.
Ethics in human experimentation
At Charles Sturt University, experiments involving human subjects are conducted according
to the guidelines set out by the Australian Government: National Health and Medical
Research Council entitled National Statement on Ethical Conduct in Human Research –
copies of these guidelines are available on online.
https://nhmrc.gov.au/about-us/publications/national-statement-ethical-conduct-humanresearch-2007-updated-2018
The guidelines are designed to apply to the conduct of research; however, most of the clauses
are relevant to the conduct of undergraduate experiments involving human subjects. One of
the clauses states:
Before research is undertaken, the free consent of the subject should be obtained. To this end,
the investigator is responsible for providing the subject (at his or her level of comprehension)
with sufficient information about the purpose, methods, demands, risks, inconveniences and
discomforts of the study. Consent should be obtained in writing, unless there are good
reasons to the contrary.
In the laboratory experiments that you perform or participate, please note that:


your participation as a subject in such experiments is entirely voluntary;
students who know that they have infectious/contagious diseases should not volunteer
to have e.g. specimens collected from them;
information about the purpose, risk, etc., is provided in the laboratory notes;
by agreeing to participate as subject for such experiments, you are giving your
informed consent – no written consent will be obtained.


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In the School of Biomedical Sciences, conduct of such experiments is under the control of the
CSU Ethics Committee. Should you have any concerns about the conduct of such
experiments, feel free to discuss the matter with the Governance Officer, Human Research
Ethics Committee: phone (02) 6933 4213; email RIECU@csu.edu.au or ethics@csu.edu.au.
Biohazard laboratory safety rules
Charles Sturt University has strict guidelines for the handling of materials of human
origin. These rules also apply to potentially infectious agents of animal origin. These
guidelines are based on the principle of Universal Precautions – that all materials are treated
as being potentially infectious.
The biohazard rules can be summarised as follows:

proper footwear and properly fastened laboratory coats must be worn at all
times. Laboratory coats are not be worn outside the laboratory; these may only be
taken from the laboratory in a sealed plastic bag;
disposable gloves should be worn whenever directed to do so;
no human venepuncture or blood collection or collection of other bio-samples are
performed by an undergraduate student, except for Medical Science, Pathology
students under appropriate supervision;
unless specified, for blood samples collected by finger prick or urine samples,
students must only work with their own sample;
pipetting by mouth is prohibited;
wherever practicable, biohazardous material are centrifuged in a sealed instrument or
container;
smoking, drinking, eating and placing articles (such as pens) in your mouth are
prohibited in the laboratory;
biohazardous material are placed in appropriate containers for either autoclaving or
disinfection before disposal;
when biohazardous materials are being used, the doors of the laboratory must be kept
closed, with signs on the outside indicating that biohazardous material is being used;
under these circumstances, unauthorised persons are not permitted in the laboratory;
at the end of each session, benches must be wiped with disinfectant;
on leaving the laboratory, laboratory coats must be removed and your hands washed
with disinfectant.










Marks are deducted from students who ignore these rules and/or students may be
excluded from carrying out practicals
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QUALITY CONTROL, UNITS, RESULTS, REFERENCE RANGES, STANDARDS,
SAMPLES
Quality control
Apart from the quality control project (separate experiment), the following procedures are
instituted for every assay or experiment carried out unless instructed not to:
1. In addition to the patients’ samples (specimens), two control sera are also normally
assayed in each experiment. Normally, one of these falls within the reference limits (a
‘normal’ control) and the other has elevated/reduced levels (‘abnormal’ control).
These control material can also come labelled as e.g. Level 1, 11 and 111.
2. After the experimentally determined levels for these control sera have been
ascertained, the assigned values for each control sera are checked. You will be given
the expected values of the controls.
Units
The systeme international (SI) of units is employed in clinical biochemistry. Only these units
are used in reporting results, with the following exceptions:
1. Enzyme activities are quoted in units/litre where 1 unit of enzyme activity converts 1
mmole of substrate to product in 1 minute.
2. Partial pressures of gases are often quoted in mm Hg rather than kPa.
3. Concentrations of proteins.
Results
Many test kits used in routine analysis provide conversion factors to convert raw instrument
readings to the final level of the constituent in sample. Such a procedure is NOT acceptable
in this subject, because this method does not indicate how the ‘factor’ is derived. In
calculating results, you use the logical steps provided with the notes for each estimation.
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Reference ranges
Below are the reference ranges that you use in this subject for practical write-ups. You can
also refer to reference ranges in your prescribed text. Reference ranges are affected among
others by sex, age and time of specimen collection: such factors are not given to allow
the student to consider all options (critical thinking) in the write-up or in interpretation
of data.
Serum
Total protein
Albumin
Triglycerides (fasting)
Total cholesterol (fasting)
HDL-cholesterol (fasting)
Total bilirubin
Conjugated bilirubin
Urea
60-85 g/L
35-55 g/L
<1.70 mmol/L
<5.5 mmol/L
>1.0 mmol/L
<26 µmol/L
<7 µmol/L
2.3-7.0 mmol/L
Creatinine
60-120 µmol/L
Plasma
Glucose (fasting) 3.3-5.8 mmol/L
Urine
Glucose
negative
Protein
negative
Bilirubin
negative
Standards
Standard and calibrator are the same and the value or concentration is given on the vial i.e.
container that has the standard.
Samples
Sample, test and specimen mean the same and will be labelled either 1 or 2 or A or B.
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WRITE-UP OF EXPERIMENTS/EXERCISES AND ASSESSMENTS IN THIS
SUBJECT
(Some of this information is also in your subject outline)
1. Submission time: (see subject outline: ‘Assessments’).
2. Content of report (see below and also subject outline: ‘Assessments information’)
except for the practical examination.
3. Dates for submission of practical write-ups are given in the subject outline. The
practical examination report is submitted at the end of the examination.
Medical laboratory science or pathology or laboratory medicine comprises investigative
medicine. As such, in investigations, information may be missing, incomplete, vague or even
false. All these need to be considered, you will not be provided will all the information on the
patients’ samples. This is to allow creativity or critical thinking on your part. Information on
e.g. age and sex may not be given and these affect pathology results. Please take such factors
into account when interpreting data.
In most of the experiments you will measure several analytes on each of the patients. For
pathology, several specimens may be taken and battery of tests ordered therefore we learn to
interpret results as a package. For example in lipids analysis: total cholesterol, HDLcholesterol and triglycerides levels are measured, the write-up is one i.e. do not write 3
separate experiments, one after the other. This information will be reinforced in the
practicals. Equally, because of time constraints, additional results on patients may be
provided in some of the practicals. This is because there will not be enough time to carry out
the tests.
Each experiment or exercise may have unique requirements and for the write-up, such
requirements are specified for each experiment. In addition to the specific requirements for
each experiment or exercise: your write-up should have clearly sub-headed and identifiable
sections:
(a) Name, ID & Title of assignment
(b) Introduction
The principles of the methods come in this section but if these are given in the practical
manual please do not repeat. Your ‘Introduction’ constitutes the biochemistry and physiology
of the metabolite or compound that you are measuring including the description of synthesis
and structure (diagrams are useful here). Explain what this metabolite or compound normally
does in the body i.e. its biochemical or physiological significance and the factors that control
it and also what it controls. In so-doing you are building a narrative or story towards the
‘Discussion’ as to what therefore happens when the level of this metabolite or compound is
changed in disease or what disease(s) arise when the level is changed.
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(c) Materials and methods
Materials and methods are normally given therefore do not repeat what is given. You will still
write this sub-heading but underneath just write ‘as per instruction or method provided’.
Should there be change(s) to the method then you simply highlight the change(s). The steps
in your methods are numbered, thus if for instance, in ‘step ‘5 you incubated for 20 minutes
instead of 30 minutes then in your write-up, simply highlight this by writing ‘as per
instruction but in step 5 incubation was for 20 minutes instead of 30 minutes’.
(d) Results
Results should be presented in a logical fashion. If a graph is used to derive the results then
the graph should have an informative title and the axes properly labelled. Normally, formulas
are given for calculation of results and if this is the case, please do not repeat the formula.
Again, just write that the formula is given and proceed to write the results. In most cases, you
have control(s) and 2 samples/specimens/patients therefore a summary of your results should
be in tubular form with an informative title and the rows and columns labelled.
Check that you have used the proper and correct units since units differ depending on the
measurement. If several analytes are measured on the same patient, make a Table with an
informative title to allow e.g. comparison between patients as shown below:
Measurement
Normal
control
Abnormal
control
Patient 1
Patient 2
Expected/reference
ranges
Triglycerides
(units)
Total
cholesterol
(units)
HDL
cholesterol
(units)
(e) Discussion
This is a continuation from ‘Introduction’ and ‘Results’. In each experiment, you analyse
controls (e.g. normal and abnormal) to see if your experiment worked. Therefore, this section
should start by discussing the control results i.e. performance of the experiment. Did your
experiment work and if not, what may have happened (recall your knowledge of errors) and
how can this can be corrected. This part calls for reflection and critical thinking, which is the
starting point of problem solving. Regardless of the control results i.e. the performance of
your experiment, you discuss the patients’ results. Even if your controls were out of range,
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you discuss the patients’ results as if your experiment worked and in this part, clearly
articulate:

if the results are pathological comparing with reference ranges (given in this manual
or in your text book;
the pathological states associated with the results;
other tests that can be done to confirm the results or in monitoring patients with the
disease;
answers to specific questions, where applicable.



(f) Conclusion
A conclusion is not a summary. From your discussion what do you conclude; you may say
for instance that the results point towards such a disease or the results are not conclusive but
from what you found it is most likely such a disease. Give reason(s) for the conclusion.
(g) References
Please use the APA referencing system.
(h) Other points
Having satisfied the above, some experiments/exercises may require special items or points
to be emphasized and if that is the case, these are indicated in the experimental procedure or
you will be told in class.
What does one-write up mean?
In investigative medicine of which pathology is, and as with other types of investigations you
look at several lines of evidence/presentations together i.e. as one. Besides pathology,
healthcare has other departments such as radiography that are also involved in diagnostic
medicine. Pathology has various departments (clinical biochemistry, haematology,
immunology, microbiology, immunology, etc.) and patients’ samples are sent to these
departments for investigation. The results from the various departments are analysed together
to make judgement. In clinical biochemistry, several tests are carried out on the same patient
and this requires that the results are also analysed together and not in isolation or one after the
other.
You will do practicals in which you measure different metabolites on the same patient. For
instance, you will measure total bilirubin and conjugated bilirubin levels. When discussing
these results in the Discussion section, please do not write a separate discussion of total
bilirubin then for conjugated bilirubin. The results are discussed as one since they are from
the same patient and in this case they are affected by the same mechanisms.
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Your discussion should therefore be e.g.:
In patient 1, total bilirubin and conjugated bilirubin levels in serum are high possibly
indicating obstructive jaundice …….
Patient 2, is presenting with high serum total bilirubin and slightly elevated conjugated
bilirubin concentration and this is associated with haemolytic jaundice ……
Assessment of the practical course
Each experiment is assessed by:
a. Accuracy of experimental results.
b. Content of the final report (presentation and discussion of results).
The relative weighting of these two components may vary according to the nature of the
experiment (see subject outline: ‘Assessments’).
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AIM OF PRACTICALS
The aim is to learn to accurately measure and analyse data of analytes/metabolites in clinical
biochemistry.
Specifically, students should be able to:
1. measure accurately and precisely levels of various constituents in biological fluids;
2. critically evaluate different methods for measuring a constituent;
3. carry out point of care testing (POCT);
4. employ appropriate quality control procedures in measurements;
5. report clearly and concisely on results;
6. interpret clinical biochemistry test results.
Depending on student numbers, students perform experiments individually or in groups (to
improve communication and collaborative skills) and according to a timetable provided at the
start of semester or residential school. The number of students in each group is determined
by student numbers and resources.
Practical are compulsory and this means that the student is expected to be present from start
to the end of the session and that students come on time to the practicals. Unless stated, ALL
practicals are written and submitted for marking.
Some of the practical sessions may be run as lectures, laboratory tutorials and/or
demonstrations.
Note: Technology is constantly changing and where possible CSU tries to expose students to
the latest and current techniques. This therefore means that the experiments in the practical
manual may change or may be modified by the time you carry out the experiment.
Preparation for practical sessions
Students are expected to read relevant material before each practical session. This includes
analytical principles behind the methods, which are in theory modules and in this practical
manual.
The experiments/assays
For most of the practicals you will measure ions or metabolites in biological fluids such as
serum or urine. Normally, 2 specimens/samples from patients and 2 control samples (normal
and abnormal) are provided. These are measured using a standard or a set of standards; you
are familiar with standard curves from previous subjects and if you have forgotten, please
revise.
Again, please note:
Sample and specimen mean the same.
Standard and calibrant mean the same.
* refers to concentration, which is the same as level.
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STD refers to the standard.
The value(s) of your standard(s) is/are given on the vial/container that has the standard.
After carrying out the experiment(s) you compare the results of patients to what is expected;
these are called reference values or ranges. If the references ranges are not given in the
method, please use the reference ranges given in your prescribed text.
Pipetting
Use the same pipette for pipetting the same solution or reagent.
Change pipette or pipette tips each time when pipetting standard, normal control, abnormal
control, sample 1 and sample 2.
Measurements are carried out in ‘single’ and not in ‘duplicate’.
Point of care (POCT)
Portable diagnostic devices and POCT are widely used in the laboratory, GP facilities, wards,
theatre, ambulances, at home, in the field e.g. disaster areas as well as in other various places.
Not all tests can be carried out by portable or point of care devices but the list of tests done
using such devices is growing due to advances in technology and improvements in health
care.
You will therefore carry out measurements using portable/point of care devices for selected
tests.
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EXPERIMENT/EXERCISE 1: HANDLING AND ANALYSIS OF QC DATA
This is a data handling exercise i.e. no experiment is carried out and the data is provided for
this exercise on the day.
The aim is to learn to handle QC data. Each analyte in clinical biochemistry has defined and
agreed imprecision limits, above and below which results are rejected. One way of
calculating these limits is to run a pooled sample multiple of times and calculating SD and
CV, in order to establish the limits.
a. On the day of the exercise, you are provided with data of serum urea levels that were
assayed several times in the same run, by the urease method. The mean and the range have
been calculated for you and are given.
Use this information to prepare Shewhart/Levey Jennings and cusum plots, which have
clearly marked on them, mean for the method and/or the accepted limits of imprecision (± 2
SD).
b. You are provided with urea levels assayed daily for 21 days, using the same (as
above) QC material from a QC program. Using this data and the limits/range from (a) plot:
i. Shewhart/Levey Jennings plots (2 plots).
ii. cusum plot.
c. You are also provided with data from 3 different laboratories on QC performance on
urea analysis using the same the method and same QC material over the same period. Plot the
performance of the 3 laboratories on the same graph paper using any of the two
Shewhart/Levey Jennings plots. Compare the weekly and overall performance of the 3
laboratories.
Write-up
Please see ‘Assessment information’ in the subject outline for more information on the writeup.
The write-up should among others include:
a. the concept of quality control, quality assurance and its significance in clinical
biochemistry (Introduction)
b. types of QC programs in clinical biochemistry (Introduction)
c. Shewhart and cusum plots for urea QC on separate graph papers (Section A of your
practical sheet with QC data) (Results)
d. graphs/plots (on one graph paper) using data from the 3 laboratories i.e. Laboratories
1 to 3 (Section B of your practical sheet with QC data). The student can use any of the
two Levey-Jennings/Shewhart plots covered (Results)
e. analyses of the data/graphs from the 3 laboratories and discussion including the
observed deviations and possible causes and remedies (Discussion)
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EXPERIMENT/EXERCISE 2: ELECTROLYTES AND ACID BASE
(To be run as theory in lecture, tutorial and/or demonstration)
Electrolytes
Na+, K+ and Cl- play a major part in maintaining the normal distribution of body water due to
the osmotic pressures which they exert. Together with bicarbonate they are the major
fractions of the cation-anion balance of the body. Hence the determination of these
electrolytes and blood gases is important in Clinical biochemistry.
Collection of blood samples for electrolyte analysis
Blood samples for K+, Na+ and Cl- estimations should be collected with the minimum of
stasis and the plasma separated within one hour of collection. Haemolysis must be avoided
and if the specimen is haemolysed this should be reported and noted. The sample must not be
taken from a limb into which an intravenous drip is running. Plasma is best since with serum,
marginally high K+ results are reported due to the break-up of platelets during coagulation
(remember that cell’s K+ levels are 10-20 times higher than extracellular fluid). If plasma is
used, the anticoagulant must not be a sodium or potassium salt.
Acid-base and blood gases (To be run as theory in lecture, tutorial and/or
demonstration)
Introduction
The laboratory assessment of a patient’s acid-base status is made by measurement of the
components of the bicarbonate – CO2 buffer system. This is the most important extracellular
buffer system in the body and maintains pH of blood within the limits of 7.36 and 7.44, in a
healthy person.
For many clinical purposes estimation of the plasma bicarbonate concentration is sufficient,
but when both respiratory and metabolic factors are involved in a disorder at least two of the
parameters must be measured to make a complete assessment of acid-base status.
The pH is related to the plasma bicarbonate concentration and to the partial pressure of
CO2 by the Henderson-Hasselbalch equation, which can be used to calculate one of the three
quantities if the other two are known.
Measurement of blood pH
Blood or plasma pH is determined on anaerobically collected heparin blood using a pH
meter.
Measurement of PCO2
This is the partial pressure of the CO2 of blood collected anaerobically and it is expressed in
mmHg or kilopascals and is related to the percentage CO2. (refer to gas laws).
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At 37°C the water vapour pressure is 47 mmHg (6.3 kPa) so at 750 mmHg (100 kPa) of
barometric pressure 5.7% CO2 (the normal percentage CO2 concentration in alveolar air)
corresponds to PCO2 of 40 mmHg or 5.3 kPa (refer to physiology subjects).
Collection of samples for pH and blood gas analysis
Four main factors govern the method of collection.
1. The PCO2 of air (0.2 mmHg) is much less than that of blood.
2. There is no measurable change in pH of a blood sample kept under anaerobic
conditions for up to 3 hours at 4°C.
3. Heparin has no significant effect on blood pH at the concentrations necessary to
prevent coagulation.
4. Respiration has no significant effect on blood pH in the short term.
Therefore blood is collected, transferred and manipulated under anaerobic conditions, using
heparin as an anticoagulant and keeping the sample at 4°C if there is any delay before
testing.
Arterial blood is preferable to venous blood because in the latter the O2 saturation may range
from 35% to 75% thus introducing an additional factor in the calculations of PCO2 and whole
blood buffer base. The usual sites of collection are the brachial, radial, or femoral arteries.
The pH difference between arterial and venous blood is not great in the majority of patients,
venous blood being more alkaline by usually less than 0.01 pH unit.
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EXPERIMENT/EXERCISE 3: PROTEINS
Total protein and albumin experiments are one, for the write up.
You write the electrophoresis practical separately but do not write an Introduction since it the
same as for total protein and albumin.
Plasma/serum total protein using the biuret method
Principle
Biuret method: Biuret is an organic compound and part of its structure is similar to the structure
around peptide bonds in proteins. In alkaline solution, copper ions complex with biuret reagent
forming a complex most likely as indicated. A similar reaction takes with proteins. The associated
colour changes have absorption maxima at around 330nm and 545nm and this is proportional to
peptide bonds (protein concentration). Amino acids do not react and dipeptides have a weak
reaction.
The complex formed by the biuret reaction means a compound that contains two of the following
groups linked by carbon to nitrogen atom will interfere; CONH2, CH2NH2, C(NH)NH2, CSNH2.
Biuret reaction (from Holme and Peck, Analytical Biochemistry: 1983)
Reagents
1. Biuret reagent:
16 mM NaK tartrate
6 mM CuSO4.5H2O
15 mM KI
in 0.2M NaOH
2. Standard: (the value of standard is written on the vial)
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Procedure
Total protein stock standard (100 g/L). If this value is different from what is written in the
vial, please use the value on vial.
Preparation of standards
Take 11 reaction/test tubes and using a marker, label the tubes as per the final concentration
of the total protein standard (g/L) i.e. 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100.
Pipette deionised water and stock total protein standard into tubes as follows:
Deionised water
(µL)
50
45
40
35
30
25
20
15
10
5

Protein standard
(µL)

5
10
15
20
25
30
35
40
45
50
Final
concentration
(g/L)

10
20
30
40
50
60
70
80
90
100
Mix well.
Instructions 1 and 5 are also written as a table.
1. Take another 5 reaction/test tubes and label the reaction tubes as reagent blank, normal
control, abnormal control, sample 1 and sample 2.
2. Then:
Into the reagent blank tube add 50 µL of deionised water
Into the normal control tube add 50 µL of normal control
Into the abnormal control tube add 50 µL of abnormal control
Into the sample 1 tube add 50 µL of sample 1
Into the sample 2 tube add 50 µL of sample 2
2. To each of the 16 tubes (11 for the standards and 5 for: reagent blank, normal control,
abnormal control, sample 1 and sample 2) add 2.5 mL of Biuret reagent.
Note: Your reagent blank and zero (0 g/L) standard have the same additions (do not worry as
this is what it should be).
4. Mix and incubate all tubes at room temperature for 30 minutes.
5. Read the absorbance at 545 nm of the standards, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer.
22
Reaction/test tubes
Reagent
blank
In each of
the
standards
Normal
control
Abnormal
control
Sample 1
Sample 2
Deionised
water
50 µL





Standard

50 µL




Normal
control


50 µL



Abnormal
control



50 µL


Sample 1




50 µL
Sample 2





50 µL
Biuret
reagent
2.5 mL
2.5 mL
2.5 mL
2.5 mL
2.5 mL
2.5 mL
Mix and incubate all tubes at room temperature for 30 minutes
Read the absorbance at 545 nm of the standards, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer
6. Draw a calibration/standard graph/curve of absorbance of standard on the y-axis and
concentration of standard on x-axis.
7. Read the concentration of normal control, abnormal control, sample 1 and sample 2
from the graph.
23
Plasma/serum albumin using bromocresol green method
Principle
Dye-binding methods: Proteins bind to various dyes and two of most widely used dye-binding
methods are for albumin using bromocresol green and bromocresol purple. The latter is a better
method except that animal based serum proteins do not react with equal sensitivity and specificity
as human serum proteins and this creates problems with standardisation and quality control.
Reagents
1. Working dye solution:
Bromocresol green: 0.08 mM in 0.05 M succinate buffer pH 4.15.
2. Standard: (the value of standard is written on vial)
Procedure
Instructions 1 and 5 are also written as a table.
1. Take 6 reaction/test tubes and using a marker, label the tubes as reagent blank,
standard, normal control, abnormal control, sample 1 and sample 2.
2. To each of the 6 tubes add 2 mL of working dye solution/reagent (bromocresol green).
3. Then:
Into the reagent blank tube add 20 µL of deionised water
Into the standard tube add 20 µL of standard
Into the normal control tube add 20 µL of normal control
Into the abnormal control tube add 20 µL of abnormal control
Into the sample 1 tube add 20 µL of sample 1
Into the sample 2 tube add 20 µL of sample 2
4. Mix and incubate all tubes at room temperature for 2 minutes.
5. Read the absorbance at 632 nm of the standard, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer.
24
Reaction/test tubes
Reagent
blank
Standard
Normal
control
Abnormal
control
Sample 1
Sample 2
Bromocresol
green reagent
2 mL
2 mL
2 mL
2 mL
2 mL
2 mL
Deionised
water
20 µL





Standard

20 µL




Normal
control


20 µL



Abnormal
control



20 µL


Sample 1




20 µL

Sample 2





20 µL
Mix and incubate all tubes at room temperature for 2 minutes
Read the absorbance at 632 nm of the standard, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer
6. Calculations
Calculation of albumin concentration in normal control
[Albumin in normal control]
=
absorbance of normal control x [STD]
absorbance of STD
Calculation of albumin concentration in abnormal control
[Albumin in abnormal control]
=
absorbance of abnormal control x [STD]
absorbance of STD
Calculation of albumin concentration in sample 1
[Albumin in sample 1]
=
absorbance of sample 1 x [STD]
absorbance of STD
Calculation of albumin concentration in sample 2
[Albumin in sample 2]
=
absorbance of sample 2 x [STD]
absorbance of STD
[] refers to concentration, which is the same as level
STD refers to standard
25
Electrophoresis of proteins
Principle
Molecules such as proteins can be separated based on their shape, size and charge in an electric
field. The various proteins carry different charges and have different sizes. In electrophoresis
proteins migrate in an electric filed and the rate and/or distance of migration of the different
proteins is influenced by the shape, size and charge that they carry. After separation the proteins
are stained to allow visualisation and quantitation. The following are requirements for
electrophoresis:
Gel or membrane: This is where the sample is applied for the proteins to be separated. The gel
or membrane for this purpose can be purchased but some institutions prepare the gel themselves.
Applicator: Samples for electrophoresis are very small in volume and this requires an applicator
to apply the samples onto the gel or membrane. Urines and other biological fluids contain very
small amounts of proteins hence such samples may need to be concentrated first. There are
facilities or equipment for concentrating proteins. Serum/plasma has high level of proteins and
the samples may need to be diluted first.
Electrophoresis chamber: This is special electrical equipment that provides the electrical
power to separate the proteins.
Staining apparatus: After separation the strips (gel/membrane) are stained (various stains are
available) to allow visualisation of the proteins.
De-staining apparatus: Excess stain is removed by e.g. alcohol so that only the proteins are
stained and the gel is clear.
Drying chamber/facility: The gel or membrane is dried mostly by gentle warm air.
Visualisation and quantitation: Although visualisation can be by naked eye special equipment
is available (densitometer) that converts the various areas stained (protein fractions) and density
of staining into a quantity.
Electrophoresis of serum proteins
I. Chamber preparation
1. The QuickGel must be plugged into a power supply. Set a timer for 8:00 minutes
and power at 400 volts. An electrophoresis time of 7:30 to 8:30 minutes is acceptable.
2. Snap the Electrophoresis Lid into place on the chamber.
3. Ensure that the chamber floor is cool (room temperature) before starting the test.
26
II. Sample application
A. QuickGel Applicator
Note: Application of Urine and CSF samples cannot be done with the Applicator Blades or
Cups packaged in the kit. Another Blade (Cat. No 1270) and Cup (Cat No. 1269) must be
purchased.
Sample Volume Blades Cups
Serum or control 45µl 1271 1259
Urine/Concentrated CSF 20µl 1270 1269
Specimens with insufficient volumes may be run using the QuickGel Accessory Kit (Cat. No.
3426).
1. Remove one QuickGel Applicator Blade from packaging. If testing more than 10
samples, remove two Applicator Blades from the packaging.
2. Urine and CSF samples must be applied on the gel three times. When testing
serum with urine or CSF samples, serum application is made after the second urine or
CSF application. Therefore the blade for serum application is not added until after the second
urine/CSF application.
3. Place an Applicator Weight on top of each Applicator Blade. If using two
Applicator Blades, place them into the vertical slots A and C of the Applicator. One blade
should be placed into the slot corresponding to cup placement.
4. Place the appropriate number of QuickGel Disposable Sample Cups into either
or both Row A and Row C of the Dispo Cup Tray into the Applicator Base.
5. Carefully cut open one end of the gel pouch. Remove one gel from the
protective packaging. Fold and tape the open end of the pouch to prevent drying of
the remaining gel. Remove the gel to be used from the plastic mould and discard the mould.
6. Dispense approximately 1 mL of REP prep onto the left side of the
electrophoresis chamber.
7. Place the left notch of the gel so that it fits the left pin of the chamber floor and
gently roll the gel to the right side fitting the right notch to the right pin of the chamber
floor. Use a lint free tissue to wipe around the edges of the gel backing to remove any excess
REP Prep. Make sure that no bubbles remain under the gel.
8. Using a QuickGel Blotter C, gently blot the entire gel using slight fingertip
pressure on the blotter. Remove the blotter.
27
9. If testing is only serum samples, follow steps 10 through 15. If testing serum and
urine or CSF on the same gel, perform steps 10 through 14 twice for the urine and CSF
samples. Place the Modified Blade for serum samples into the applicator, and repeat steps 10
through 14 for a total of 3 applications for urine and CSF samples and 1 application for serum
samples.
10. While holding the white Applicator knob, place the Applicator into the designated
slot on the Applicator Base aligning the small red dots on the Applicator with those on the
Base.
11. Slowly lower the Applicator Knob allowing the blades to enter the sample cups,
and immediately start a timer for 30 seconds.
12. After 30 seconds, lift the Applicator Knob. Immediately place the Applicator into
the slot on the chamber floor, aligning the red dots.
13. Slowly lower the Applicator Knob to apply sample to the gel. Set a timer for
30 seconds.
14. After the 30 seconds application, raise the Applicator Knob and remove
the Applicator from the chamber.
15. Close the lid, press the power switch to turn on the chamber and start the
power supply. Proceed to step III.
B. Sample Template Application
1. Serum specimens and controls are diluted 1: 4 (1 part serum with 3 parts
0.85% saline). Urine samples may be diluted, neat or concentrated. CSF samples must
be concentrated as instructed in SPECIMEN COLLECTION AND HANDLING.
2.
Carefully cut open one end of the gel pouch. Remove one gel from the
protective packaging. Fold and tape the open end of the pouch to prevent drying of
the
remaining gel. Remove the gel to be used from the plastic mould and discard the mould.
3. Dispense approximately 1 mL of REP prep onto the left side of the
electrophoresis chamber.
4. Place the left notch of the gel so that it fits the left pin of the chamber floor and
gently roll the gel to the right side fitting the right notch to the right pin of the chamber
floor. Use a lint free tissue to wipe around the edges of the gel backing to remove any excess
REP Prep. Make sure that no bubbles remain under the gel.
5. Using a QuickGel Blotter C, gently blot the entire gel using slight fingertip
pressure on the blotter. Remove the blotter.
28
6. Remove one QuickGel Sample Template from the package if only one row of samples
is tested. Remove two templates if two rows are tested. Hold the template so that the small
hole in the corner is toward the front right side of the chamber.
7. Carefully place the template on the gel aligning the template slits with the marks
on each side of the gel backing. The centre hole in the template should align with
the indention in the centre of the gel.
8. Apply slight fingertip pressure to the template making sure there are air bubbles under
it. NOTE: If wearing rubber gloves to perform this step, place a QuickGel Blotter A over the
template and then apply fingertip pressure to the template. Remove the blotter. Powder from
the gloves can produce gel artefacts.
9. Use the following chart to apply the appropriate serum dilution or
urine/CSF concentration to the template slits. After the last sample application, allow time
for the proper absorption. If two rows of samples are tested, start the timer for blotting the
first rows before applying samples to the second row. Then time the blot for the second row.
Sample Volume Absorption Time
Serum 1µL 3 minutes
Urine/CSF 3µL 7 to 10 minutes
Use the QuickGel Blotter A to gently blot the excess sample from the template. Carefully
remove the blotter and template. Dispose of templates as biohazardous waste.
Close the lid, press the power switch to turn on the chamber and start the power supply.
III Electrophoresis and staining
1. Electrophorese the gel for 8:00 minutes at 400 volts.
2. Turn off the Power Supply and QuickGel chamber.
3. Using the Gel Block Remover, remove the two gel blocks from the gel. Use a lint
free tissue to wipe around the edges of the gel backing to remove any excess moisture.
4. Replace the Electrophoresis Lid with the Drying Lid. Clean the two electrodes on the
Electrophoresis Lid with deionised water after each use. Wipe with lint free tissue. Close the
lid.
5. Turn on the QuickGel Chamber. Dry the gel for 15 minutes or until dry. When
drying is complete, turn the chamber off and remove the gel.
6. Fill a container with prepared stain. Fill another container with Destain solution.
7. Place the gel into the Staining Dish containing the prepared stain for 4
minutes. Remove the gel from the stain and allow it to drain on a blotter.
29
8. Destain the gel in three consecutive washes of Destain solution. Use a
gentle alternately rocking and swinging technique. Allow the gel to remain in each wash for 1
minute. The gel background should be completely clear. Tap the gel to remove the excess
destain solution.
9. Ensure that the chamber floor is clean. Re-place the gel onto the QuickGel
chamber. Dry the gel for 10 minutes or until dry. Turn off QuickGel Chamber and remove
gel.
IV Evaluation of protein bands
Qualitative evaluation: The urine and CSF samples run on the QuickGel Split Beta SPE Gel
can only be visually inspected for the presence of the bands.
Quantitative evaluation: Scan the QuickGel Split Beta Gel at 595 nm, agarose side down on
an EDC densitometer. Scan the agarose side up on other instruments. A slit size 5 is
recommended. If a quick scan 2 000 is used, scan on the acid blue setting.
Stability of end product
The completed, dried QuickGel Split Beta SPE gel is stable for an indefinite period.
As part of the write-up, first identify each of the bands observed on the strip and indicate if it
is normal or abnormal.
30
EXPERIMENT/EXERCISE 4: ENZYMES
(To be run as theory in lecture, tutorial and/or demonstration)
Please note:
(a) enzymes are used as analytical tools in the measurements of various metabolites e.g.
glucose, lipids and urea (analytical enzymology).
(b) enzymes are measured in diagnosis and in management of diseases (clinical enzymology).
Introduction
Clinical biochemistry laboratories measure the levels or activities of enzymes in biological
fluids, mainly serum. The reason for this is that the type and amount of enzymes in the blood
is an indicator of obstruction or damage to the tissues rich in these enzymes. Thus, measuring
serum enzyme level or activity is an important diagnostic tool in medicine.
Precautions required in measuring serum enzyme levels
The amount of enzyme in blood is small. Therefore, rather than measure the amount of
enzyme by chemical means, the biological activity of the enzyme is measured.
Activity is determined by comparing the rate of the reaction, which in most cases is the
amount of product formed or substrate destroyed in a given time. Conditions such as nature
of substrate, pH, temperature and concentration of metal ions must be strictly controlled to
permit comparison of reaction rates. Comparison under these conditions depends upon the
assumption that, if a given number of enzyme molecules are present in a certain reaction rate,
two or three times that number will increase the rate by a factor of two or three. This will
only apply if the substrate is present in sufficient excess to keep the enzyme active sites
saturated during the period of assay, i.e. the period of zero order kinetics with respect to
substrate. This appropriate [substrate] must be ascertained before routine use of a particular
enzyme assay is contemplated. The key to all accurate measurement of enzyme activities is
(i) optimization and (ii) standardization of assay conditions.
Methods available for measuring enzyme activities
Methods of determining the rates of enzymes reactions can be divided into two practical
categories. In the first category, the progress of the reaction is monitored continuously (e.g. in
a spectrophotometer) to give a reaction progress curve from which the initial rate of reaction
is derived; this is called ‘kinetic’ reaction. In the second category called ‘end point’, the
reaction is allowed to proceed for a fixed time and in this period it is assumed that the
reaction has gone to completion.
Examples of some of the analytical principles of these methods are discussed in Module 3 for
you to read before this session.
Alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γGGT), aspartate
aminotransferase (AST) and alanine aminotransferase (ALT), are mainly used in
31
investigations of hepatic diseases. Other enzymes that shall be covered include cardiac
enzymes such as lactate dehydrogenase (LD) and creatine kinase (CK).
Units of enzyme activity
An attempt has been made to standardize enzyme units by defining an international unit of
enzyme activity as that amount of enzyme which transforms 1mmole of substrate in 1
minute under conditions of measurement. It is obvious that the definition cannot go further
than this as optimum conditions for each enzyme vary widely. A standard temperature of
25°C was initially recommended but this has been increased to 30°C although there are few
laboratories which adhere to this temperature for all their enzyme assays. Indeed, some
laboratories perform enzyme assays at 37°C to maximize sensitivity. The use of temperature
conversion factors to compare enzyme activities from assays performed at different
temperatures is not recommended. Serum enzyme levels are expressed in international units
per litre (Iu/L) and the temperature at which they assay was performed should be stated.
* Note that the international unit is NOT a SI unit, which uses the second as the unit of
time. The SI unit of enzyme activity, the katal, is not commonly used.
Isoenzymes
In a number of investigations isoenzymes are measured using various techniques such as
immunoassays, selective inhibition (heat, chemical) and electrophoresis.
32
EXPERIMENT/EXERCISE 5: BILIRUBIN
Collection of plasma/serum samples for bilirubin estimation
Serum for the estimation of bilirubin should be obtained from blood collected in
absorptive state as many methods are performed on diluted serum without protein
precipitation, therefore turbidity due to lipaemia can interfere. Haemolysis must be avoided
and serum should be kept in the dark up to the time of testing as the pigment is extremely
photo labile. Exposure to direct sunlight at room temperature is said to decrease bilirubin
concentration by up to 50% after one hour. Photo lability of the pigment can be used as a
means of treating neonatal jaundice.
Principle
In 1883, Ehrlich introduced the “diazo” reaction for the detection of bilirubin in urine. Enrlich
showed that the diazo product (azobilirubin) behaved as an indicator appearing blue in alkali
and red in acid. Diazo compound has 2 linked nitrogen atoms (azo) as the terminal group
In 1913, Van Den Bergh and Snapper applied the diazo reaction to serum after removal of
proteins with ethanol.
A few years latter, Van Den Bergh and Muller discovered that two types of diazo reactions
could be distinguished in sera.
a. The direct reaction in which colour develops within 30 seconds in the absence of
alcohol.
b. The indirect reaction that requires the addition of alcohol for colour development.
By 1921, the diazo reaction referred to as the Van Den Bergh was further broken down as
follows:
a. Prompt direct colour development within several seconds without alcohol.
b. Delayed direct colour development only after one minute without alcohol.
c. Biphasic colour developing and increasing with time i.e. (a) and (b).
d. Indirect colour developing only after the addition of alcohol.
In the 1950’s the explanation for the Van Den Bergh test was that: in (c) variation in colour
development was due to the presence of accelerators of the Diazo reagent.
33
In (d) there are two different but closely related bilirubins.
Diazo reagent is prepared adding 0.3 mL solution B to 10 mL solution A.
Solution A:
Dissolve 1 g sulphanilic acid in 15 mL concentrated HCl and make up to 1 litre with distilled
H2O.
Solution B:
Dissolve 0.5 g sodium nitrite in distilled H2O and make up to 100 mL.
Method of Malloy and Evelyn
Serum is diluted with water and methanol added instead of ethanol in an amount insufficient
to precipitate proteins, yet sufficient to ensure all that bilirubin reacts with diazo reagent.
Since then, several other accelerating substances, which cause all the bilirubin to react, have
been found.
Jendrassik and Grof: Caffeine and mixture of sodium benzoate and sodium acetate.
Powell: Urea benzoate.
Michaelsohn: diphylline-sodium acetate.
Columbo et al: brijj 35.
Jendrassik and Grof method for determination of plasma/serum bilirubin levels
Reagents
1.
Diazo reagent:
sulphanilic acid
sodium nitrite
in 0.165 M HCl.
29 mM
0.22 mM
2. 0.165 M HCl
3.
Accelerator:
caffeine
sodium benzoate
0.26 M
0.52 M
4. Ascorbic acid: 0.2 M
5.
Alkaline tartrate:
NaK tartrate
in 2 M NaOH
0.93 M
34
6. Bilirubin standard: (the value of standard is written on the vial)
7. Standard: (the value of standard is written on vial)
Procedure
In the previous experiments you were using a ‘reagent’ blank. In other words in your blank
you had the reagent and not the sample (s). In this experiment, you are using a ‘sample’
blank. Sample blanks are useful when dealing with biological specimens that have an
inherent or endogenous colour, which obviously would interfere with spectrophotometric
measurements. We normally use sample blanks when measuring metabolites in urine or in
jaundiced or lipaemic samples.
Instead of having one blank tube for the whole experiment as we do with reagent blank, in
this experiment; standard, control (s) and each sample have their own blanks. Therefore, in
your test tube rack you should have two rows of test tubes; STD (test) and STD (blank),
Control (Test) and Control (blank) Sample (test) and Sample (blank) and so on. In addition,
since we are measuring total and conjugated bilirubin in the same experiment, we shall need
another two rows for conjugated bilirubin measurement. We can get away at least in this
experiment by having only one STD.
Instructions 1 and 10 are also written as a table.
1. Preparation of reaction/test tubes
Label with a marker as follows:
a. Total bilirubin standard test (TBST)
Total bilirubin standard blank (TBSB)
Conjugated bilirubin standard test (CBST)
Conjugated bilirubin standard blank (CBSB)
b. Total bilirubin normal control test (TBNCT)
Total bilirubin normal control blank (TBNCB)
Conjugated bilirubin normal control test (CBNCT)
Conjugated bilirubin normal control blank (CBNCB)
c. Total bilirubin abnormal control test (TBACT)
Total bilirubin abnormal control blank (TBACB)
Conjugated bilirubin abnormal control test (CBACT)
Conjugated bilirubin abnormal control blank (CBACB)
d. Total bilirubin sample 1 test (TBS1T)
Total bilirubin sample 1 blank (TBS1B)
Conjugated bilirubin sample 1 test (CBS1T)
Conjugated bilirubin sample 1 blank (CBS1B)
35
e. Total bilirubin sample 2 test (TBS2T)
Total bilirubin sample 2 blank (TBS2B)
Conjugated bilirubin sample 2 test (CBS2T)
Conjugated bilirubin sample 2 blank (CBS2B).
2. Addition of standard, normal control, abnormal control, sample 1 and sample 2:
Into all the standard tubes add 0.2 mL of standard
Into all the normal control tubes add 0.2 mL of normal control
Into all the abnormal control tube add 0.2 mL of abnormal control
Into all the sample 1 tubes add 0.2 mL of sample 1
Into all the sample 2 tubes add 0.2 mL of sample 2.
3. Into all the tubes marked total bilirubin test and conjugated bilirubin test add 0.2 mL of
diazo reagent.
4. Into all the tubes marked total bilirubin blank and conjugated bilirubin blank add 0.2
mL of 0.165 M HCl.
5. Into all the tubes marked total bilirubin test and total bilirubin blank add 1.0 mL of
accelerator.
6. Mix and incubate all tubes at room temperature for 10 minutes.
7. Into all the tubes add 0.1 mL of ascorbic acid.
8. Into all the tubes marked conjugated bilirubin add 1.0 mL of accelerator.
IMMEDIATELY
9. Into all the tubes add 1.0 mL of alkaline tartrate.
10. Read the absorbance at 600 nm of the standard test, normal control test, abnormal
control test, sample 1 test and sample 2 test using the respective reagent blank to zero the
spectrophotometer.
36
Reaction/test tubes
Total-bilirubin
Conjugated-bilirubin
Test
Blank
Test
Blank
Standard
0.2 mL
0.2 mL
0.2 mL
0.2 mL
Normal
control
0.2 mL
0.2 mL
0.2 mL
0.2 mL
Abnormal
control
0.2 mL
0.2 mL
0.2 mL
0.2 mL
Sample 1
0.2 mL
0.2 mL
0.2 mL
0.2 mL
Sample 2
0.2 mL
0.2 mL
0.2 mL
0.2 mL
Diazo reagent
0.2 mL

0.2 mL

0.165 M HCl

0.2 mL

0.2 mL
Accelerator
1.0 mL
1.0 mL


Mix and incubate all tubes at room temperature for 10 minutes
Ascorbic acid
0.1 mL
0.1 mL
0.1 mL
0.1 mL
Accelerator


1.0 mL
1.0 mL
IMMEDIATELY ADD TO ALL TUBES
Alkaline
tartrate
1.0 mL
1.0 mL
1.0 ml
1.0 mL
Read the absorbance at 600 nm of the standard test, normal control test, abnormal
control test, sample 1 test and sample 2 test using the respective reagent blank to zero
the spectrophotometer
11. Calculations
Calculation of total-bilirubin concentration in normal control
[Total-bilirubin in normal control]
=
absorbance of normal control test x [STD test]
absorbance of STD test
Calculation of total-bilirubin concentration in abnormal control
[Total-bilirubin in abnormal control]
=
absorbance of abnormal control test x [STD test]
absorbance of STD test
Calculation of total-bilirubin concentration in sample 1
[Total-bilirubin in sample 1]
=
absorbance of sample 1 test x [STD test]
absorbance of STD test
Calculation of total-bilirubin concentration in sample 2
[Total-bilirubin in sample 2]
=
absorbance of sample 2 test x [STD test]
absorbance of STD test
37
Calculation of conjugated-bilirubin concentration in normal control
[Conj-bilirubin in normal control]
=
absorbance of normal control test x [STD test]
absorbance of STD test
Calculation of conjugated-bilirubin concentration in abnormal control
[Conj-bilirubin in abnormal control] =
absorbance of abnormal control test x [STD test]
absorbance of STD test
Calculation of conjugated-bilirubin concentration in sample 1
[Conjugated-bilirubin in sample 1]
=
absorbance of sample 1 test x [STD test]
absorbance of STD test
Calculation of conjugated-bilirubin concentration in sample 2
[Conjugated-bilirubin in sample 2]
=
absorbance of sample 2 test x [STD test]
absorbance of STD test
[] refers to concentration, which is the same as level.
STD refers to the standard.
38
EXPERIMENT/EXERCISE 6: GLUCOSE & POCT
Whole blood, plasma or serum can be used but there are important differences in the results
obtained between whole blood and plasma or serum. In this practical, you familiarise with
glucose measurement in blood and urine and practice interpretation of data from blood and
urine glucose measurements.
Plasma/serum glucose measurement using hexokinase method
Principle
The hexokinase method for measurement of glucose was developed by Barthelmai and Czok
and modified by Richterich and Dauwalder. Glucose in the sample is phosphorylated by
hexokinase to glucose-6-phoshate in the presence of ATP as the phosphate donor. Hexokinase:
EC 2.7.1.1 requires Mg2+. Glucose-6-phosphate is then oxidised to 6-phosphogluconate with
simultaneous reduction of NAD+ to NADH2, which absorbs U/V light at 340nm. NAD+ does
not absorb therefore in this reaction the rate of increase in absorption as NADH2 is formed is
monitored.
D-glucose + ATP Hexokinase (Mg2+) Glucose-6-phosphate + ADP
Glucose-6-phosphate + NAD+ + H2O Glucose-6-phosphate dehydrogenase 6-
phosphogluconate + NADH2
The rate of increase in NADH2 formation is proportional to glucose concentration.
Reagents
This is derived from trace enzymatic glucose reagent (hexokinase). The final concentrations
of components are as follows:
1. Hexokinase reagent
ATP
NAD+
Hexokinase (yeast)
Glucose-6-P-dehydrogenase
1.3 μmol/L
0.65 μmol/L
1 500 u/L
2 500 u/L
in 50 μmol/L Triethanolamine buffer, pH 7.5
2. Standard: (the value of standard is written on the vial)
Procedure
In the previous experiments, although we incubated our reactions for a given period,
the time factor was not very important. In this experiment, we need to incubate each
tube for exactly 20 minutes at room temperature hence we need to concentrate.
39
1. Label small test tubes; blank, STD, normal control, abnormal control, sample 1 and
sample 2 accordingly.
2. Pipette 1.5 mL of glucose UV reagent into each of the labelled small test tubes.
3. Add 10 μL of deionised water into the blank tube and immediately start the stop watch
and at the same time mix thoroughly.
4. After exactly 1 minute, add 10 μL of STD to the appropriate tube and mix thoroughly.
5. Repeat this for normal control, abnormal control, sample 1 and sample 2 after every
minute.
6. When the stop watch strikes 20 minutes put the blank in the spectrophotometer and
zero the equipment at 340nm.
7. At 21 minutes read absorbance of STD and continue reading normal control, abnormal
control, sample 1 and sample 2 thereafter at every minute.
8. Calculations
Calculation of glucose concentration in normal control
[Glucose in normal control]
=
absorbance of normal control x [STD]
absorbance of STD
Calculation of glucose concentration in abnormal control
[Glucose in abnormal control]
=
absorbance of abnormal control x [STD]
absorbance of STD
Calculation of glucose concentration in sample 1
[Glucose in sample 1]
=
absorbance of sample 1 x [STD]
absorbance of STD
Calculation of glucose concentration in sample 2
[Glucose in sample 2]
=
absorbance of sample 2 x [STD]
absorbance of STD
* refers to concentration, which is the same as level.
STD refers to the standard.
Additional results
Both patients had positive glucose in their urine. Please take this into account when
interpreting the plasma/serum results.
40
Point of Care Test (POCT) for glucose level
Measure patients’ samples using the glucometer provided. The method for measurement is
given in the instructions/insert for the glucometer and a demonstration will also be done on
how to use the device.
With your working partner and with lecturer/demonstrator, compare and discuss the
glucometer results with the results that got using the hexokinase method.
Please note: this POCT exercise is not written/submitted for marking.
41
EXPERIMENT/EXERCISE 7: LIPIDS & POCT
Collection of samples for plasma lipid analysis
The preferred sample for these estimations is fresh plasma collected after the patient has
fasted for 12-16 hours. The patient should also have been on normal diet for the previous two
weeks. Samples for cholesterol and triglycerides may be stored frozen for several weeks prior
to analysis, but lipoprotein electrophoresis should be done the same day. If delay is
unavoidable the sample should be stored at 4°C. Plasma is preferred to serum as some
triglycerides are enmeshed in the clot and slightly lower results are obtained with serum.
As is the case with all large molecules, lipoprotein concentrations are affected by venous
stasis and posture. Plasma cholesterol concentration may be up to 10% higher in the upright
than in the recumbent position. Where serial estimations are performed it is important to use a
standardized collecting procedure.
Plasma/serum total cholesterol and cholesterol-HDL measurement using cholesterol
oxidase
Principle
Isolation of HDL
HDL cholesterol measurements are performed after first removing the other lipoproteins from
the sample and subsequently measuring the cholesterol content of the remaining HDLcontaining fraction. Several precipitation procedures are available for HDL isolation, using
e.g.:
Heparin-Mn++ (hep-Mn++)
Dextran sulphate-Mg++ (Dxtr)
Phosphotungstate-Mg++ (PTA)
Polyethylene glycol (PEG)
42
The cholesterol oxidase is specific for free cholesterol and cannot use cholesterol ester as a
substrate. It is therefore necessary to add cholesterol esterase, to convert cholesterol ester
present in the plasma into free cholesterol. Thus the reaction sequence for estimation of total
cholesterol is as follows:
This chemistry forms the basis of the colour reactions used in cholesterol measurements.
Please note that total cholesterol and HDL-cholesterol levels are being measured by the
same reaction and therefore we use the same standard and control(s) for total cholesterol
or HDL-cholesterol.
Reagents
1. Precipitating reagent – 200 g/L polyethylene glycol 6 000.
2. Cholesterol reagent – this is derived from Trace Cholesterol Reagent (Enzymatic –
Single Vial). The final concentration of components is as follows:
Sodium phenolate
4-aminoantipyrine
Peroxidase (horseradish)
Cholesterol esterase
Cholesterol oxidase
20 mmol/L
0.5 mmol/L
2 000 u/L
200 u/L
150 u/L
in 68 mmol/L phosphate buffer, pH 6.5.
3. Standard: (the value of standard is written on the vial)
43
Procedure
Instructions 1 and 13 are also written as a table.
1. Take 4 eppendorf tubes and using a marker, label the tubes as normal control, abnormal
control, sample 1 and sample 2.
Into the normal control tube add 200 µL of normal control
Into the abnormal control tube add 200 µL of abnormal control
Into the sample 1 tube add 200 µL of sample 1
Into the sample 2 tube add 200 µL of sample 2.
2. To all the tubes add 200 µL of polyethylene glycol.
3. Mix and incubate all the eppendorf tubes at room temperature for 5 minutes.
4. Centrifuge (see step 5) all the eppendorf tubes at 3 500 rpm for 10 minutes. After
centrifugation, separate the clear supernatant (see step 11).
5. While the eppendorf tubes are centrifuging, label 10 reaction/test tubes as:
reagent blank, standard
total cholesterol normal control, total cholesterol abnormal control, total cholesterol sample 1
and total cholesterol sample 2
HDL-cholesterol normal control, HDL-cholesterol abnormal control
HDL-cholesterol sample 1 and HDL-cholesterol sample 2
AFTER CENTRIFUGATION
6. Add 1.5 mL of colour reagent to all the 10 reaction tubes i.e.
reagent blank, standard
total cholesterol control, total cholesterol abnormal control, total cholesterol sample 1 and
total cholesterol sample 2
HDL-cholesterol normal control, HDL-cholesterol abnormal control
HDL-cholesterol sample 1 and HDL-cholesterol sample 2
7. To the reagent blank tube only; add 150 µL of deionised water.
8. To the standard tube add:
135 µL of deionised water
15 µL of standard.
9. To the tubes for total-cholesterol measurement i.e.:
total-cholesterol normal control, total-cholesterol abnormal control, total-cholesterol sample 1
and total-cholesterol sample 2
Add 135 µL of deionised water to each tube.
44
Then:
10. Into the total-cholesterol normal control tube add 15 µL of un-precipitated normal
control
Into the total-cholesterol abnormal control tube add 15 µL of un-precipitated abnormal
control
Into the total-cholesterol sample 1 tube add 15 µL of un-precipitated sample 1
Into the total-cholesterol sample 2 tube add 15 µL of un-precipitated sample 2
11. To the tubes for HDL-cholesterol measurement i.e.:
HDL-cholesterol normal control, HDL-cholesterol abnormal control,
HDL-cholesterol sample 1 and HDL-cholesterol sample 2
Into the HDL-cholesterol normal control tube add 150 µL of supernatant of normal control
from step 4
Into the HDL-cholesterol abnormal control tube add 150 µL of supernatant of abnormal
control from step 4
Into the HDL-cholesterol sample 1 tube add 150 µL of supernatant of sample 1 from step 4
Into the HDL-cholesterol sample 2 tube add 150 µL of supernatant of sample 2 from step 4
12. Mix and incubate all the tubes at 37°C for 10 minutes.
13. Using the reagent blank to zero the spectrophotometer read the absorbance at 500 nm
of the:
standard
total-cholesterol normal control
total-cholesterol abnormal control
total-cholesterol sample/test 1
total-cholesterol sample/test 2
HDL-cholesterol normal control
HDL-cholesterol abnormal control
HDL-cholesterol sample/test 1
HDL-cholesterol sample/test 2
45
Reagent blank
Total cholesterol
HDL-cholesterol
Colour reagent
1.5 mL
1.5 mL
1.5 mL
Deionised water
150 µL
135 µL

Un-precipitated standard

15 µL

Un-precipitated Normal
control

15 µL

Un-precipitated abnormal
control

15 µL

Un-precipitated sample 1

15 µL

Un-precipitated sample 2

15 µL

Supernatant from step
3 Normal control


150 µL
Supernatant from step
3 abnormal control


150 µL
Supernatant from step
3 sample 1


150 µL
Supernatant from step
3 sample 2


150 µL
Mix and incubate all the reaction tubes at 37OC for 10 minutes
Using the reagent blank to zero the spectrophotometer read the absorbance at 500 nm of the:
standard
total-cholesterol normal control, total-cholesterol abnormal control, total-cholesterol sample
1 test and total-cholesterol sample 2
HDL-cholesterol normal control, HDL-cholesterol abnormal control, HDL-cholesterol
sample 1 test and HDL-cholesterol sample 2
14. Calculations
Calculation of total-cholesterol concentration in normal control
[Total-chol in normal control]
=
absorbance of normal control test x [STD test]
absorbance of STD test
Calculation of total-cholesterol concentration in abnormal control
[Total-chol in abnormal control]
=
absorbance of abnormal control test x [STD test]
absorbance of STD test
Calculation of total-cholesterol concentration in sample 1
[Total-chol in sample 1]
=
absorbance of sample 1 test x [STD test]
absorbance of STD test
Calculation of total-cholesterol concentration in sample 2
[Total-chol in sample 2]
=
absorbance of sample 2 test x [STD test]
absorbance of STD test
46
[] refers to concentration, which is the same as level.
STD refers to the standard.
15. Using the same formula calculate HDL-cholesterol concentrations in: normal control,
abnormal control, sample 1 and sample 2.
Clearly show in your write-up how you worked the final concentration of HDLcholesterol.
47
Plasma/serum triglycerides measurement using lipase
Principle
In the body, degradation of triglycerides is by hydrolysis to FFAs and glycerol. The latter can
enter the glycolytic pathway.
Triglyceride + 3H2O  Glycerol + 3 Fatty acids
The reaction is catalyzed by intracellular lipases.
In this measurement the lipase is utilised in measurement:
Triglycerides -Lipase→ Glycerol + 3 Fatty acids
Glycerol + ATP –Glycerol kinase→ Glycerol phosphate + ADP
Glycerol phosphate + NAD –Glycerol phosphate dehydrogenase→ Dihydroxyacetone
phosphate +NADH2
NADH2 + INT –Diaphorase→ NAD+ + INT (reduced)
Reagents
1. Triglyceride Reagent – this is derived from Trace Triglycerides Reagent (INT,
Colorimetric). The final concentration of each component is as follows:
ATP
NAD+
EDTA
Mg2+
iodonitrotetrazolium violet
Lipase
2.5 mmol/L
2.5 mmol/L
1.4 mmol/L
2.0 mmol/L
0.3 mmol/L
150 000 u/L
Diaphorase
Glycerol kinase
Glycerol-1-P dehydrogenase
in 50 mmol/L phosphate buffer, pH 7.5.
3 000 u/L
900 u/L
6 000 u/L
2. Standard: (the value of standard is written on the vial)
Procedure
Instructions 1 and 5 are also written as a table.
1. Take 6 reaction/test tubes and using a marker, label the reaction tubes as reagent blank,
standard, normal control, abnormal control, sample 1 and sample 2.
2. To each of the 6 tubes add 1.5 mL of triglyceride solution/reagent.
48
3. Then:
Into the reagent blank tube add 30 µL of deionised water
Into the standard tube add 15 µL of standard
Into the normal control tube add 15 µL of normal control
Into the abnormal control tube add 15 µL of abnormal control
Into the sample 1 tube add 15 µL of sample 1
Into the sample 2 tube add 15 µL of sample 2.
To all tubes except reagent blank add 15 µL of deionised water
4. Mix and incubate all tubes at 37°C in a water bath for 10 minutes.
5. Read the absorbance at 500 nm of the standard, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer.
Reaction/test tubes
Reagent
blank
Standard
Normal
control
Abnormal
control
Sample 1
Sample 2
Triglycerides
reagent
1.5 mL
1.5 mL
1.5 mL
1.5 mL
1.5 mL
1.5 mL
Deionised
water
30 µL
15 µL
15 µL
15 µL
15 µL
15 µL
Standard

15 µL




Normal
control


15 µL



Abnormal
control



15 µL


Sample 1




15 µL

Sample 2





15 µL
Mix and incubate all tubes at 37°C in a water bath for 10 minutes
Read the absorbance at 500 nm of the standard, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer
6. Calculations
Calculation of triglycerides concentration in normal control
[Triglycerides in normal control]
=
absorbance of normal control x [STD]
absorbance of STD
Calculation of triglycerides concentration in abnormal control
[Triglycerides in abnormal control]
=
absorbance of abnormal control x [STD]
absorbance of STD
49
Calculation of triglycerides concentration in sample 1
[Triglycerides in sample 1]
=
absorbance of sample 1 x [STD]
absorbance of STD
Calculation of triglycerides concentration in sample 2
[Triglycerides in sample 2]
=
absorbance of sample 2 x [STD]
absorbance of STD
* refers to concentration, which is the same as level.
STD refers to the standard.
Point of Care Test (POCT) for total cholesterol level
Measure patients’ samples using the lipidometer provided. The method for measurement is
given in the instructions/insert for the lipidometer and a demonstration will also be done on
how to use the device.
With your working partner and with lecturer/demonstrator, compare and discuss the
cholesterol results with the results that got using the cholesterol oxidase method.
Please note: this POCT exercise is not written/submitted for marking.
50
EXPERIMENT/EXERCISE 8: NON-PROTEIN NITROGENOUS WASTE
COMPOUNDS
Plasma/serum urea measurement using glutamate dehydrogenase
Principle
H2NCONH2 + H2O Urease NH4+ + H2NCOOH (carbamate)
H2NCOOH  NH4+ + CO2
NH4+ + -ketoglutarate + NAD(P)H2 GLDH Glutamate + H2O + NAD(P)+
The reduction in absorbance at 340nm as NAD(P)+ formed is monitored and is proportional to
NH4+ level, which is proportional to urea concentration.
Reagent
1. Urea reagent
This is derived from Trace Enzymatic BUN Reagent (Blood Urea Nitrogen). The final
concentration of components is as follows:
2-oxoglutarate
ADP
5 mmol/L
3 mmol/L
NADH
Urease (jack bean)
Glutamate dehydrogenase
0.35 mmol/L
9 000 u/L
2 000 u/L
in 100 µmol/L phosphate buffer, pH 7.5.
2. Standard: (the value of standard is written on the vial)
51
Procedure
Instructions 1 and 5 are also written as a table.
1. Take 6 reaction/test tubes and using a marker, label the reaction tubes as reagent blank,
standard, normal control, abnormal control, sample 1 and sample 2.
2. To each of the 6 tubes add 1.5 mL of urea/BUN solution/reagent.
3. Then:
Into the reagent blank tube add 5 µL of deionised water
Into the standard tube add 5 µL of standard
Into the normal control tube add 5 µL of normal control
Into the abnormal control tube add 5 µL of abnormal control
Into the sample 1 tube add 5 µL of sample 1
Into the sample 2 tube add 5 µL of sample 2.
4. Mix and incubate all tubes at 37OC in a water bath for 10 minutes.
5. Read the absorbance at 340 nm of the reagent blank standard, normal control, abnormal
control, sample 1 and sample 2 using the water to zero the spectrophotometer.
Reaction/test tubes
Reagent
blank
Standard
Normal
control
Abnormal
control
Sample 1
Sample 2
Urea/BUN
reagent
1.5 mL
1.5 mL
1.5 mL
1.5 mL
1.5 mL
1.5 mL
Deionised
water
5 µL





Standard

5 µL




Normal
control


5 µL



Abnormal
control



5 µL


Sample 1




5 µL

Sample 2





5 µL
Mix and incubate all tubes at 37OC in a water bath for 10 minutes
Read the absorbance at 340 nm of the reagent blank, standard, normal control,
abnormal control, sample 1 and sample 2 using deionised water to zero the
spectrophotometer
52
6. Calculations
To get absorbance values subtract each of the absorbancies i.e. standard, normal control,
abnormal control, sample 1 and sample 2 from the absorbance of reagent blank.
Calculation of urea concentration in normal control
[Urea in normal control]
=
absorbance of normal control x [STD]
absorbance of STD
Calculation of urea concentration in abnormal control
[Urea in abnormal control]
=
absorbance of abnormal control x [STD]
absorbance of STD
Calculation of urea concentration in sample 1
[Urea in sample 1]
=
absorbance of sample 1 x [STD]
absorbance of STD
Calculation of urea concentration in sample 2
[Urea in sample 2]
=
absorbance of sample 2 x [STD]
absorbance of STD
* refers to concentration, which is the same as level.
STD refers to the standard.
53
Plasma/serum creatinine using Jaffe’s method
Principle
In 1886, Jaffe reported that creatinine reacted with picric acid in alkaline solution to produce
creatinine-picrate complex, which is orange-red in colour. The structure of the product of
Jaffe’s reaction is still a matter of conjecture.
Reagents
1. Alkaline picrate reagent: 10mM picric acid in 1.25M NaOH.
2. 1.2M trichloroacetic acid (TCA).
3. Standard: (the value of standard is written on the vial)
Procedure
Instructions 1 and 6 are also written as a table.
1. Take 6 centrifuge tubes and using a marker, label the tubes as reagent blank, standard,
normal control, abnormal control, sample 1 and sample 2.
Into the reagent blank tube add 0.8 mL of deionised water
Into the standard tube add 0.8 mL of standard
Into the normal control tube add 0.8 mL of normal control
Into the abnormal control tube add 0.8 mL of abnormal control
Into the sample 1 tube add 0.8 mL of sample 1
Into the sample 2 tube add 0.8 mL of sample 2.
2. To all the tubes add 0.8 mL of trichloroacetic acid (TCA).
3. Mix and centrifuge at 2 000 rpm for 10 minutes.
4. In the meantime:
Take 6 reaction/test tubes and using a marker, label the tubes as reagent blank, standard,
normal control, abnormal control, sample 1 and sample 2.
AFTER CENTRIFUGATION
5. Remove 1.0 mL of supernatant into appropriately labelled reaction tubes and add 1.0
mL alkaline picrate reagent.
6. Mix and incubate all the tubes at room temperature for 10 minutes.
7. Read the absorbance at 520 nm of the standard, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer
54
Centrifuge tubes
Reagent
blank
Standard
Normal
control
Abnormal
control
Sample 1
Sample 2
Deionised water
0.8 mL





Standard

0.8 mL




Normal control


0.8 mL



Abnormal
control



0.8 mL


Sample 1




0.8 mL

Sample 2





0.8 mL
Trichloroacetic
acid
0.8 mL
0.8 mL
0.8 mL
0.8 mL
0.8 mL
0.8 mL
Mix and centrifuge at 2 000 rpm for 10 minutes
Reaction/test tubes
Supernatant
1.0 mL
1.0 mL
1.0 mL
1.0 mL
1.0 mL
1.0 mL
Alkaline picrate
1.0 mL
1.0 mL
1.0 mL
1.0 mL
1.0 mL
1.0 mL
Mix and incubate all tubes at room temperature for 10 minutes
Read the absorbance at 520 nm of the standard, normal control, abnormal control,
sample 1 and sample 2 using the reagent blank to zero the spectrophotometer
7. Calculations
Calculation of creatinine concentration in normal control
[Creatinine in normal control]
=
absorbance of normal control x [STD]
absorbance of STD
Calculation of creatinine concentration in abnormal control
[Creatinine in abnormal control]
=
absorbance of abnormal control x [STD]
absorbance of STD
Calculation of creatinine concentration in sample 1
[Creatinine in sample 1]
=
absorbance of sample 1 x [STD]
absorbance of STD
Calculation of creatinine concentration in sample 2
[Creatinine in sample 2]
=
absorbance of sample 2 x [STD]
absorbance of STD
* refers to concentration, which is the same as level.
STD refers to the standard.
55
EXPERIMENT/EXERCISE 9: INFORMATION ON PRACTICAL EXAMINATION
Distance education students carry out the practical test on the last day of residential school.
Internal students carry out the practical test in the last practical session on your practical time
table.
In case there are changes to venue, dates and time of practical test, students will be notified
on the interact2 site.
Methods and instructions are provided in practical examination.
TIME ALLOCATED: 1 to 2 (one to two) HOURS.
Practical and write up to be competed in the time allocated.
No books or literature are allowed into the practical examination.
Materials allowed: pencil, ball pen, rubber, ruler, non-programmable calculator with no text
facilities.
Material supplied: Practical method(s), reagents and equipment and writing paper.
The practical exam tests your ability to carry out a procedure accurately and safely. You will
write the results inclusive of calculations of your experiment(s) and not the Introduction or
Discussion.

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