Students' Industrial Training Report on Clinical Chemistry Tests

A REPORT OF THE STUDENTS’ INDUSTRIAL WORK EXPERIENCE
SCHEME (SIWES)
UNDERTAKEN AT
BENUE STATE UNIVERSITY TEACHING HOSPITAL (BSUTH)
KILOMETER 3, MAKURDI-GBOKO ROAD MAKURDI-NIGERIA
FROM APRIL 2016 TO JUNE 2016
BY
OCHIJELE IDOKO VICTOR
UE/30067/14
SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES,
UNIVERSITY OF AGRICULTURE, MAKURDI
AUGUST 2016

2
APPROVAL
This SIWES report has been approved for the department of Biological sciences,
University of Agriculture, Makurdi.
By
Dr. Mrs. Abulu C.O Prof. Iheukwumere C.C
(Departmental SIWES Coordinator) (Head of Department)

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CERTIFICATION
This is to certify that this technical report was written by OCHIJELE IDOKO VICTOR
with matriculation number UE/30067/14 of the Department of Biological sciences,
Federal University of Agriculture, Makurdi. The report meets the requirement for the
degree of Bachelor of Science in Biochemistry. The work embodied in this report has not
been submitted in part or in full for any other diploma or degree of this or any other
University.
_____________________ _______________________
Dr. Mrs. Abulu C.O Ochijele Idoko Victor
(Departmental SIWES Coordinator) (Student)

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DEDICATION
I dedicate this report to God Almighty, who saw me through the period of my SIWES
training programme and ordered my steps all the way. I also dedicate this report to my
family.

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ACKNOWLEDGEMENTS
My sincere gratitude goes to the Almighty God for His love, mercy, favour and
protection upon me throughout the period of my Industrial Training.
My unreserved gratitude goes to Mr. and Mrs. Danjuma Theophilus for their parental,
moral and financial support. Thank you for raising a world leader.
I am greatly indebted to my siblings Adama Inalegwu, Adama Patience, Mary Ogweba,
Joy Ogweba and my little angel, Danjuma Dorcas. I thank you all for your moral,
spiritual and financial support.
Also my unalloyed thanks goes to my uncles Mr. Ogbole Elijah and Mr. Innocent
Adama for their love and care towards ensuring I am always on the right path in my
every dealings in life.
I express my sincere gratitude to Mr. Samuel Atabo and all the lecturers in the
Department of Biological Sciences, FUAM for the support and encouragement given to
me and not forgetting the knowledge they imparted in me without hesitation.
I appreciate my industry based supervisor, Mrs. Agbinya Alice and all BSUTH staff in
the chemical pathology department. To my co-interns Uroko Fatima, Kurga Doris,
Asemah Emmanuella, Asemave Solomon, Tertsea Judith and Aondowase Stephen I say
thanks for making my stay at BSUTH a fun filled one.
Finally, in no particular order, a very special gratitude goes to Owoicho Ngbede , Tar
Leonard, Ademu Blessing, Ugwuoke Paul, Owoicho Alex, Uche Lucy, Owoicho Samson,
Eze Emmanuel, Edeh Sunday, Ogar Michael, Margaret Ogweba and Ihudu Agbo. The
definition of friendship is incomplete without you all. Thank you for the words of
encouragement and inspiration. Love you big time. God bless you richly.

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ABSTRACT
This is a comprehensive report on my SIWES program at the Benue State University
Teaching Hospital (BSUTH). BSUTH is a health institute mainly focused on quality
health care delivery, research, rigorous education, management of patients and medical
personnel in the health sector, providing state of the art equipments such as vitros 350,
dialysis machine, CT scan for medical research and applications. The report covers the
work done during my stay with the hospital and the experience gained from the
laboratory unit. The major work presented in this report includes: standard WHO
practices in phlebotomy, chemical analysis of urine (urinalysis), separation of
plasma/serum from whole blood, in vitro quantitative determination of Creatinine, Uric
acid, Direct bilirubin, Alkaline phosphatase, cholesterol, Alanine aminotransferase,
Aspartate aminotransferase, Albumin, Triglycerides, HDL cholesterol in serum or plasma
using Agappe and Randox reagent kits as well as their clinical significances. Included in
this report also are challenges encountered and solutions.

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TABLE OF CONTENTS
Title Page -------------------------------------------------------------------------------------- i
Title and Requirement Declaration Page -------------------------------------------------- ii
Approval Page -------------------------------------------------------------------------------- iii
Certification ----------------------------------------------------------------------------------- iv
Dedication ------------------------------------------------------------------------------------- v
Acknowledgement ---------------------------------------------------------------------------- vi
Abstract ---------------------------------------------------------------------------------------- vii
Table of Contents --------------------------------------------------------------------------- vii-ix
List of Figures ---------------------------------------------------------------------------------- x
List of Tables ------------------------------------------------------------------------------------ xi
CHAPTER ONE
1.0 Students Industrial Work Experience Scheme (SIWES) ----------------------- 1
1.1 Aims and Objectives of SIWES ----------------------------------------------------2
1.2 SIWES Implementation -------------------------------------------------------------2
1.3 Importance of SIWES----------------------------------------------------------------2

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CHAPTER TWO
2.0 A Brief Background of BSUTH ----------------------------------------------------3
2.1 Location --------------------------------------------------------------------------------3
2.2 Leadership------------------------------------------------------------------------------3
2.3 BSUTH capacity-----------------------------------------------------------------------3
2.4 Departments----------------------------------------------------------------------------3
2.5 Vision of BSUTH ---------------------------------------------------------------------3
2.6 Mission of BSUTH -------------------------------------------------------------------3
2.7 Core values of the institute ----------------------------------------------------------4
2.8 Organizational Structure of BSUTH ---------------------------------------------- 4-5
2.9 Chemical pathology department-----------------------------------------------------7
2.9.1 Organizational Structure of Chemical pathology ---------------------------------8
CHAPTER THREE
3.0 Personal Involvement in each Division ---------------------------------------------8
3.1 Personal Involvement with the phlebotomy unit-----------------------------------8-32
3.2 Personal Involvement with the Wash up/Separation unit -----------------------33-49
3.3 Personal Involvement with the Chemical Pathology Main laboratory---------50-62
CHAPTER FOUR
4.0 Skills and Knowledge Acquired ----------------------------------------------------- 63
4.1 Experiences gained during my industrial attachment-------------------------------63

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CHAPTER FIVE
5.0 challenges, recommendations and conclusions ------------------------------------ 64
5.1 Challenges Encountered -------------------------------------------------------------- 64
5.2 Recommendations --------------------------------------------------------------------- 64
5.3 Conclusions----------------------------------------------------------------------------- 65
5.4 References-------------------------------------------------------------------------------65
List of Figures
Fig 2.8: Laboratories in BSUTH-------------------------------------------------------------5
Fig 2.9: BSUTH organogram-----------------------------------------------------------------6
Fig 3.0: BSUTH laboratory layout showing units in chemical pathology--------------8
Fig 3.1: Order of draw for multiple tube collection---------------------------------------16
Fig 3.2: Illustrations for best practices in phlebotomy---------------------------------19-22
Fig 3.4: Blood-sampling systems-----------------------------------------------------------25
Fig 3.5: Illustrations for paediatric and neonatal blood sampling--------------------28-32
Fig 3.6: urinalysis colour chart--------------------------------------------------------------34
Fig 3.7: pregnancy test strip result indicator-----------------------------------------------39
Fig 3.8: Blood components after centrifugation-------------------------------------------48
Fig 3.9: cryogen vials-------------------------------------------------------------------------48
Fig 4.0: Ion separation electrode (ISE) ----------------------------------------------------48
Fig 4.1: Automatic Micropipette------------------------------------------------------------62
Fig 4.2: The effect of pipetting position----------------------------------------------------62
Fig 4.3: Evolution 3000 Spectrophotometer-----------------------------------------------62
Fig 4.4 Ion Separation electrode (ISE) ----------------------------------------------------- 62

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List of Tables
Table 2.1 Elements of quality assurance in phlebotomy---------------------------------9-10
Table 2.2 Infection prevention and control practices--------------------------------------12
Table 2.3 Recommended order of draw for plastic vacuum tubes-----------------------17
Table 2.4 Recommended needle gauge, length and device for routine injection and
phlebotomy procedures for different age groups------------------------------------------24-25
Table 2.5 creatinine test procedure-----------------------------------------------------------51
Table 2.6 creatinine test procedure-----------------------------------------------------------52
Table 2.7 Alkaline phosphatase test procedure---------------------------------------------54
Table 2.8 Cholesterol test procedure---------------------------------------------------------56
Table 2.9 SGPT test procedure----------------------------------------------------------------57
Table 3.0: SGOT test procedure------ --------------------------------------------------------58
Table 3.1: Triglycerides test procedure-------------------------------------------------------60
Table 3.2: HDL Cholesterol precipitation----------------------------------------------------61
Table 3.3: HDL Cholesterol test procedure --------------------------------------------------61

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CHAPTER ONE
1.0 Student Industrial Work Experience Scheme (SIWES)
Student Industrial Work Experience Scheme (SIWES) was established in 1973/1974
session. Prior to the establishment of the scheme, there was a growing concern among our
industrialists that graduates of our institutions of higher learning lacked adequate
practical background studies preparatory for employment in the industries. It is against
this background that the rationale for initiating and designing the scheme was hinged.
Consequently, the scheme affords students the opportunity of familiarizing and exposing
themselves to the needed experience in handling equipment and machinery that are
usually not available in their institutions. The growing concern led to the formation of
Students Industrial Work Experience Scheme (SIWES) by ITF in 1993/1994
(Information and Guideline for SIWES 2002). SIWES in Nigeria is organized and
coordinated by the Industrial Training Fund (ITF) for a period of Three (3) months to
One year, depending on the Institution or Faculty involved. ITF’s mandate is to promote
and encourage the acquisition of skills in Commerce and Industry with the view of
generating numerously trained man power, which will gather basic practical knowledge
needed in the industrial world out there.
Here in the College of sciences, Federal University of Agriculture Makurdi, the SIWES
Program is expected to last a period of three (3) months each at the end of second
semester for qualified candidates in the third and fourth year classes. As a Semester
Course, it is awarded three (3) Credit units in the Department of biological sciences.
It is from the foregoing that I hence present this report – a summary of my Work
experience at the Benue State University Teaching Hospital (BSUTH), Makurdi-Benue
state, the health institution in which I gained Industrial Work Experience.

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1.1 Aims and Objectives of SIWES
Students Industrial Work Scheme aims at the following:
1. Provide an avenue for students in institutions of higher learning to acquire industrial
skills and experience in their approved course of study.
2. Prepare students for the industrial work situation which they are likely to meet after
graduation.
3. Expose students to work methods and techniques in handling equipment and
machinery in their institutions.
4. Provide students with an opportunity to apply their knowledge in real work situation
thereby bridging the gap between theory and practical.
5. Enlist and strengthen employers’ involvement in the entire education process and
prepare students for employment in industry and commerce.
6. Make transition from the various institutions to the world of work easier and thus,
enhance students contact for job placement.
1.2 SIWES Implementation
Various organs such as the Federal Government, the Industrial Training Fund (ITF) in
collaboration with agencies such as the National Universities Commission (NUC),
Employers of labour and institutions had roles assigned to them in the management of
SIWES for effective performance and continued sustenance of the scheme.
1.3 Importance of SIWES
SIWES has a lot of importance amongst which are:
1. It exposes students to real life situation, thus supplementing the theoretical lesson.
2. It helps to improve the quality of skilled manpower of the students.
3. It gives students practical knowledge of course of study.
4. It provides a forum for industries to evaluate prospective employers and gives
feedback to institutions.
5. It establishes a close collaboration between institutions and industries, a factor which is
essential for preparing student for the workforce.

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CHAPTER TWO
2.0 A Brief Background of BSUTH
The BSUTH project started sometimes in the year 2008 with the management board
inaugurated on 27th
August, 2008 by the executive governor of Benue state, Rt. Hon. Dr.
Gabriel Torwua Suswam with Prof. Peter O. Obekpa as chairman. The hospital was
officially commissioned by the president of the federal republic of Nigeria on 9th
March
and began services officially on 26th
March 2012. BSUTH is the first teaching hospital of
a state university in the northern part of Nigeria, situated in the southern part of North
central Nigeria.
2.1 Location
Benue state university teaching hospital is located in Makurdi the Benue state capital;
Kilometer 3, Makurdi-Gboko road. P.M.B 202131 Makurdi-Nigeria.
2.2 Leadership
The teaching hospital is headed by the chief medical director (CMD), Prof. Abraham O.
Malu, with two main directorates: directorates of clinical services headed by the
chairman Medical advisory committee (CMAC), Dr. Samuel Kuma Hemba-Hilekaan,
and directorates of administration headed by Mr. Tekaa Luga.
2.3 BSUTH capacity
The hospital has bed space capacity of over 500
Staff strength of over 800
Doctors – 151(88 consultants, 63 residents)
Nurses – 259
Pharmacists – 17
Laboratory scientists – 29, others are health assistants, record officers.
Administrative and finance support staff
5 functional theatres with a supportive CSSD
Laboratory complex housing Microbiology, Haematology, Chemical Pathology
and Histopathology with each unit headed by pathologists.

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An academic block accommodating consultant offices, ICT offices and a library.
2.4 Departments
There are basically two major groups of departments that constitute the main workforce
of the hospital for service delivery; these are administrative departments and clinical
departments.
2.5 Vision of BSUTH
To be a leading tertiary health care institution applying modern technology in research,
teaching and the management of patients, and the preferred environment for all categories
of health care workers seeking fulfillment of their professional dreams.
2.6 Mission of BSUTH
To provide excellent, relevant and affordable tertiary health care services to the people of
Benue state in particular and Nigeria in general, with conducive environment for research
and training of healthcare professionals for the improvement of the health of the people
and advancement of health related knowledge.
2.7 Core Values
• We place high premium on, and value our patients and relatives, providing succor
and treating them with dignity.
• We respond promptly and effectively to the needs of our patients and relatives.
• We strive to provide a conducive environment that is in itself healing to our
patients.
• We are ethical in our patient care and research.
• We value our staff and provide enabling environment and tools.
• We strive to produce healthcare professionals who will compare with the best in
learning, character and skill.
• We work as a team for the common good of our patients, students and staff.
• We are accountable to our patients and people of Benue state.

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2.8 Organizational structure of BSUTH
The hospital is basically divided into finance/supply, audit, security, legal unit etc with
two directorates namely directorate of clinical services and directorate of administration
with the chief medical director as the superior head.
The laboratory section of the directorate of clinical services is where I received my
industrial training. The laboratory section houses the
1. Haematology department
2. Histopathology department
3. Chemical pathology department
4. Microbiology department

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Fig 2.8. Laboratories in BSUTH
MICROBIOLOGY
HISTOPATHOLOGY
CHEMICAL
PATHOLOGY
HAEMATOLOGY
LABORATORY

17
Fig 2.9 BSUTH organogram
I got my experience at the chemical pathology department of the laboratory unit

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2.9 Chemical Pathology Department
Chemical Pathology is the branch of pathology dealing with the biochemical basis of
disease and the use of biochemical tests for screening, diagnosis, prognosis and
management. Chemical pathology (also known as clinical biochemistry) involves also the
biochemical investigation of bodily fluids such as blood, urine and cerebrospinal fluid.
By discovering how and where the body’s chemistry has changed, diseases can be
diagnosed and monitored.
The department which is an integral part of the four laboratory units is well equipped
with ultra modern equipments such as vitros 350/250/250AT, ISE 4000, Evolution 3000
spectrophotometer etc. The department which is headed by Professor Ogagbon H.U is
well staffed with five qualified medical laboratory scientists and four technicians. All
tests performed in this laboratory are done using blood plasma and they include: Liver
function test (LFT), Fasting Lipid Profile (FLP), Fasting Blood Glucose/sugar
(FBG/FBS), Random Blood Glucose/Sugar (RBG/RBS), Electrolytes (Na+
, K+
, Ca2+
,
PO4
2-
,), Urea, Creatinine, protein test, Oral Glucose Tolerance Test (OGTT), Two hours
post prandia glucose (2HrPP), cerebrospinal fluid biochemistry.

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Fig 3.0 BSUTH laboratory layout showing units in chemical pathology
CHAPTER THREE
3.0 PERSONAL INVOLVEMENT IN EACH DIVISION
During my training, I was attached to various division of the department where I was
involved personally in some activities and gained useful experience. Though, I was not
allowed access to operations in the main laboratory due to the sensitive and professional
nature of the tests performed there, and also due to the technical nature of the machines
used there.
Personal involvements with the phlebotomy unit
Phlebotomy is the practice of drawing blood from patients and taking the blood
specimens to the laboratory to prepare for testing.
Background information on best practices in phlebotomy
Best practices in phlebotomy involve the following factors:
• planning ahead;
LABORATORY
HAEMATOLOGY CHEMICAL
PATHOLOGY
MICROBIOLOGY HISTOPATHOLOGY
PHLEBOTOMY
UNIT
STAFF LOUNGE
CHEMICAL
PATHOLOGY
MAIN
LABORATORY.
ION SEPARATION
ELECTRODE (ISE)
ROOM
WASH UP &
SEPARATION UNIT
RECEPTION/
RESULTS
SCIENTIST
LOUNGE
PAY POINT

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• using an appropriate location;
• Quality control;
• Standards for quality care for patients and health workers, including
–– availability of appropriate supplies and protective equipment;
–– availability of post-exposure prophylaxis (PEP);
–– avoidance of contaminated phlebotomy equipment;
–– appropriate training in phlebotomy;
–– cooperation on the part of patients;
• Quality of laboratory sampling.
Planning ahead
This is the most important part of carrying out any procedure, and is usually done at the
start of a phlebotomy session.
Using an appropriate location
The phlebotomist should work in a quiet, clean, well-lit area, whether working with
outpatients or inpatients.
Quality control
Quality assurance is an essential part of best practice in infection prevention and control.
In phlebotomy, it helps to minimize the chance of a mishap. Table 2.1 lists the main
components of quality assurance, and explains why they are important.
Table 2.1 Elements of quality assurance in phlebotomy
Elements Notes
Education and training Education and training is necessary for all
staff carrying out phlebotomy. It should
include an understanding of anatomy,
awareness of the risks from blood
exposure, and the consequences of poor
infection prevention and control.
Standard operating
procedures (SOPs)
SOPs are required for each step or
procedure. They should be written and be
readily available to health workers.

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Correct identification of
the patient
Identification should be through matching
to the laboratory request form.
• For blood donation, the identity of the
donor should be accurately matched to the
results of screening tests.
• For blood sampling, after samples have
been taken from a patient or donor, a
system of identification and tracking is
essential to ensure that the sample is
correctly matched with the result and with
the patient or donor.
The condition of the sample The condition of the sample should be such
that the quality of the results is satisfactory.
Safe transportation Making safe transportation of blood or
blood products part of best practices will
improve the quality of results from
laboratory testing.
An incident reporting system A system is required for reporting all
adverse events. A log book or register
should be established with accurate details
of the incident, possible causes and
management of adverse events
Avoidance of contaminated phlebotomy equipment
Tourniquets are a potential source of methicillin-resistant Staphylococcus aureus
(MRSA), with up to 25% of tourniquets contaminated through lack of hand hygiene on
the part of the phlebotomist or reuse of contaminated tourniquets. In addition, reusable
finger-prick devices and related point-of-care testing devices (e.g. glucometers)
contaminated with blood have been implicated in outbreaks of hepatitis B.
To avoid contamination, any common-use items, such as glucometers, should be visibly
clean before use on a patient, and single-use items should not be reused.

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Patient cooperation
One of the essential markers of quality of care in phlebotomy is the involvement and
cooperation of the patient; this is mutually beneficial to both the health worker and the
patient. Clear information – either written or verbal – should be available to each patient
who undergoes phlebotomy. Annex F provides sample text for explaining the blood-
sampling procedure to a patient.
Quality of laboratory sampling
Factors that influence the outcome of laboratory results during collection and
transportation include:
• Knowledge of staff involved in blood collection;
• Use of the correct gauge of hypodermic needle (see Table 3.1 in Chapter 3) to prevent
haemolysis or abnormal results;
• The anatomical insertion site for venepuncture;
• The use of recommended laboratory collection tubes;
• Patient–sample matching (i.e. labeling);
• Transportation conditions;
• Interpretation of results for clinical management.
Practical guidance on best practices in phlebotomy
Provision of an appropriate location
• In an outpatient department or clinic, provide a dedicated phlebotomy cubicle
containing:
–– a clean surface with two chairs (one for the phlebotomist and the other for the patient);
–– a hand wash basin with soap, running water and paper towels;
–– alcohol hand rub.
• In the blood-sampling room for an outpatient department or clinic, provide a
comfortable reclining couch with an arm rest.
• In inpatient areas and wards:
–– at the patient’s bedside, close the bed curtain to offer privacy
–– ensure that blood sampling is done in a private and clean manner.
Provision of clear instructions

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Ensure that the indications for blood sampling are clearly defined, either in a written
protocol or in documented instructions (e.g. in a laboratory form).
Procedure for drawing blood
At all times, these given strategies are followed for infection prevention and control listed
in Table 2.2
Table 2.2 Infection prevention and control practices
Do Do not
DO carry out hand hygiene (use soap and
water or alcohol rub), and wash carefully,
including wrists and spaces between the
fingers for at least 30 seconds.
DO NOT forget to clean your hands
DO use one pair of non-sterile gloves per
procedure or patient
DO NOT use the same pair of gloves for
more than one patient.
DO NOT wash gloves for reuse
DO use a single-use device for blood
sampling and drawing
DO NOT use a syringe, needle or lancet for
more than one patient
DO disinfect the skin at the venepuncture
site
DO NOT touch the puncture site after
disinfecting it
DO discard the used device (a needle and
syringe is a single unit) immediately into a
robust sharps container
DO NOT leave an unprotected needle lying
outside the sharps container
Where recapping of a needle is
unavoidable, DO use the one-hand scoop
technique
DO NOT recap a needle using both hands
DO seal the sharps container with a tamper-
proof lid.
DO NOT overfill or decant a sharps
container

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DO place laboratory sample tubes in a
sturdy rack before injecting into the rubber
stopper.
DO NOT inject into a laboratory tube while
holding it with the other hand.
DO immediately report any incident or
accident linked to a needle or sharp injury,
and seek assistance; start PEP as soon as
possible, following protocols
DO NOT delay PEP after exposure to
potentially contaminated material; beyond
72 hours, PEP is NOT effective
Post-exposure prophylaxis (PEP); WHO, World Health Organization.
Step 1 – Assemble equipment
Collect all the equipment needed for the procedure and place it within safe and easy reach
on a tray or trolley, ensuring that all the items are clearly visible. The equipment required
includes:
• A supply of laboratory sample tubes, which should be stored dry and upright in a rack;
blood can be collected in
–– sterile glass or plastic tubes with rubber caps (the choice of tube will depend on what
is agreed with the laboratory);
–– vacuum-extraction blood tubes; or
–– glass tubes with screw caps;
• A sterile glass or bleeding pack (collapsible) if large quantities of blood are to be
collected;
• Well-fitting, non-sterile gloves;
• An assortment of blood-sampling devices (safety-engineered devices or needles and
syringes, see below), of different sizes;
• A tourniquet;
• Alcohol hand rubs;
• 70% alcohol swabs for skin disinfection;
• Gauze or cotton-wool ball to be applied over puncture site;
• Laboratory specimen labels;
• writing equipment;
• Laboratory forms;

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• Leak-proof transportation bags and containers;
• A puncture-resistant sharps container.
Ensure that the rack containing the sample tubes is close to you, the health worker, but
away from the patient, to avoid it being accidentally tipped over.
Step 2 – Identify and prepare the patient
Where the patient is adult and conscious follow the steps outlined below.
• Introduce yourself to the patient, and ask the patient to state their full name.
• Check that the laboratory form matches the patient’s identity (i.e. match the patient’s
details with the laboratory form, to ensure accurate identification).
• Ask whether the patent has allergies, phobias or has ever fainted during previous
injections or blood draws.
• If the patient is anxious or afraid, reassure the person and ask what would make them
more comfortable.
• Make the patient comfortable in a supine position (if possible).
• Place a clean paper or towel under the patient’s arm.
• Discuss the test to be performed (see Annex F) and obtain verbal consent. The patient
has a right to refuse a test at any time before the blood sampling, so it is important to
ensure that the patient has understood the procedure..
Step 3 – Select the site
General
• Extend the patient’s arm and inspect the antecubital fossa or forearm.
• Locate a vein of a good size that is visible, straight and clear. The diagram in Section
2.3 shows common positions of the vessels, but many variations are possible. The median
cubital vein lies between muscles and is usually the most easy to puncture. Under the
basilica vein runs an artery and a nerve, so puncturing here runs the risk of damaging the
nerve or artery and is usually more painful. DO NOT insert the needle where veins are
diverting, because this increases the chance of a haematoma.
• The vein should be visible without applying the tourniquet. Locating the vein will help
in determining the correct size of needle.
• Apply the tourniquet about 4–5 finger widths above the venepuncture site and re-
examine the vein.

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Hospitalized patients
In hospitalized patients, do not take blood from an existing peripheral venous access site
because this may give false results. Haemolysis, contamination and presence of
intravenous fluid and medication can all alter the results. Nursing staff and physicians
may access central venous lines for specimens following protocols. However, specimens
from central lines carry a risk of contamination or erroneous laboratory test results. It is
acceptable, but not ideal, to draw blood specimens when first introducing an in-dwelling
venous device, before connecting the cannula to the intravenous fluids.
Step 4 – Perform hand hygiene and put on gloves
• Perform hand hygiene; that is
–– wash hands with soap and water, and dry with single-use towels; or
–– if hands are not visibly contaminated, clean with alcohol rub – use 3 ml of alcohol rub
on the palm of the hand, and rub it into fingertips, back of hands and all over the hands
until dry.
• After performing hand hygiene, put on well-fitting, non-sterile gloves.
Step 5 – Disinfect the entry site
• Unless drawing blood cultures, or prepping for a blood collection, clean the site with a
70% alcohol swab for 30 seconds and allow to dry completely (30 seconds).
Note: alcohol is preferable to povidone iodine, because blood contaminated with
povidone iodine may falsely increase levels of potassium, phosphorus or uric acid in
laboratory test results
• Apply firm but gentle pressure. Start from the centre of the venepuncture site and work
downward and outwards to cover an area of 2 cm or more.
• Allow the area to dry. Failure to allow enough contact time increases the risk of
contamination.
• DO NOT touch the cleaned site; in particular, DO NOT place a finger over the vein to
guide the shaft of the exposed needle. It the site is touched, repeat the disinfection.
Step 6 – Take blood
Venepuncture
Perform venepuncture as follows.

27
• Anchor the vein by holding the patient’s arm and placing a thumb BELOW the
venepuncture site.
• Ask the patient to form a fist so the veins are more prominent.
• Enter the vein swiftly at a 30 degree angle or less, and continue to introduce the needle
along the vein at the easiest angle of entry.
• Once sufficient blood has been collected, release the tourniquet BEFORE withdrawing
the needle. Some guidelines suggest removing the tourniquet as soon as blood flow is
established, and always before it has been in place for two minutes or more.
• Withdraw the needle gently and apply gentle pressure to the site with a clean gauze or
dry cotton-wool ball. Ask the patient to hold the gauze or cotton wool in place, with the
arm extended and raised. Ask the patient NOT to bend the arm, because doing so causes a
haematoma.
Step 7 – Fill the laboratory sample tubes
• When obtaining multiple tubes of blood, use evacuated tubes with a needle and tube
holder. This system allows the tubes to be filled directly. If this system is not available,
use a syringe or winged needle set instead.
• If a syringe or winged needle set is used, best practice is to place the tube into a rack
before filling the tube. To prevent needle-sticks, use one hand to fill the tube or use a
needle shield between the needle and the hand holding the tube.
• Pierce the stopper on the tube with the needle directly above the tube using slow, steady
pressure. Do not press the syringe plunger because additional pressure increases the risk
of haemolysis.
• Where possible, keep the tubes in a rack and move the rack towards you. Inject
downwards into the appropriate coloured stopper. DO NOT remove the stopper because
it will release the vacuum.
• If the sample tube does not have a rubber stopper, inject extremely slowly into the tube
as minimizing the pressure and velocity used to transfer the specimen reduces the risk of
haemolysis. DO NOT recap and remove the needle.
• Before dispatch, invert the tubes containing additives for the required number of times
(as specified by the local laboratory).
Step 8 – Draw samples in the correct order

28
Draw blood collection tubes in the correct order, to avoid cross-contamination of
additives between tubes. As colour coding and tube additives may vary, verify
recommendations with local laboratories. For illustration purposes, Table 2.3 shows the
revised, simplified recommended order of draw for vacuum tubes or syringe and needle,
based on United States National Committee Clinical Laboratory Standards consensus in
2003
Fig 3.1: Order of draw for multiple tube collection
Table 2.3 Recommended order of draw for plastic vacuum tubes
Order of use Type of tube/
usual colour
Additive Mode of action uses
1 Blood culture
bottle (yellow-
black striped
tubes)
Broth mixture Preserves
viability of
microorganisms
Microbiology –
aerobes, anaerobes,
fungi
2 Coagulation tube
(light blue top)
Sodium citrate Forms calcium
salts to
remove calcium
Coagulation tests
(protime and
prothrombin time),
requires full draw
3 Clot activator (red Clot activator Blood clots,
and the
Chemistries,
immunology and

29
top) serum is
separated by
centrifugation
serology, blood bank
(cross-match)
4 Serum separator
tube
(red-grey tiger top
or
gold)
None Contains a gel
at the
bottom to
separate
blood from
serum on
centrifugation
Chemistries,
immunology and
serology
5 Sodium heparin
(dark
green top)
Sodium heparin
or
lithium heparin
Inactivates
thrombin and
thromboplastin
For lithium level use
sodium heparin, for
ammonia level use
either
6 PST (light green
top)
Lithium
heparin
anticoagulant
and a
gel separator
Anticoagulants
with
lithium,
separates
plasma with
PST gel at
bottom of tube
Chemistries
7 EDTA (purple
top)
EDTA Forms calcium
salts to
remove calcium
Haematology, Blood
Bank (cross-match)
requires full draw
8 Oxalate/fluoride
(light grey top)
Sodium
fluoride and
potassium
oxalate
Antiglycolytic
agent
preserves
glucose up to
five days
Glucoses, requires
full draw (may cause
haemolysis if short
draw)
ACD, acid-citrate-dextrose; DNA, deoxyribonucleic acid; EDTA,
ethylenediaminetetraacetic acid; HLA, human leucocyte antigen; PST, plasma separating
tube.
a. Colour codes may differ with laboratories.
b. Gently invert tubes with additives to mix thoroughly; erroneous test results may be
obtained when the blood is not thoroughly mixed with the additive.
c. If a routine coagulation assay is the only test ordered, then a single light blue top tube
may be drawn. If there is a concern about contamination by tissue fluids or
thromboplastins, then a non-additive tube can be drawn before the additive tube. The PST

30
tube contains lithium heparin anticoagulant and a gel separator; if used, it is drawn in the
order shown.
Step 9 – Clean contaminated surfaces and complete patient procedure
• Discard the used needle and syringe or blood sampling device into a puncture-resistant
sharps container.
• Check the label and forms for accuracy. The label should be clearly written with the
information required by the laboratory, which is typically the patient’s first and last
names, file number, date of birth, and the date and time when the blood was taken.
• Discard used items into the appropriate category of waste. Items used for phlebotomy
that would not release a drop of blood if squeezed (e.g. gloves) may be discarded in the
general waste, unless local regulations state otherwise.
• Perform hand hygiene again, as described above.
• Recheck the labels on the tubes and the forms before dispatch.
• Inform the patient when the procedure is over.
• Ask the patient or donor how they are feeling. Check the insertion site to verify that it is
not bleeding, then thank the patient and say something reassuring and encouraging before
the person leaves.
Step 10 – Prepare samples for transportation
• Pack laboratory samples safely in a plastic leak-proof bag with an outside compartment
for the laboratory request form. Placing the requisition on the outside helps avoid
contamination.
• If there are multiple tubes, place them in a rack or padded holder to avoid breakage
during transportation.
Step 11 – Clean up spills of blood or body fluids
If blood spillage has occurred (e.g. because of a laboratory sample breaking in the
phlebotomy area or during transportation, or excessive bleeding during the procedure),
clean it up. An example of a safe procedure is given below.
• Put on gloves and a gown or apron if contamination or bleaching of a uniform is likely
in a large spill.

31
• Mop up liquid from large spills using paper towels, and place them into the infectious
waste.
• Remove as much blood as possible with wet cloths before disinfecting.
• Assess the surface to see whether it will be damaged by a bleach and water solution.
• For cement, metal and other surfaces that can tolerate a stronger bleach solution, flood
the area with an approximately 5000 parts per million (ppm) solutions of sodium
hypochlorite (1:10 dilution of 5.25% chlorine bleach to water). This is the preferred
concentration for large spills. Leave the area wet for 10 minutes.
• For surfaces that may be corroded or discoloured by strong bleach, clean carefully to
remove all visible stains. Make a weaker solution and leave it in contact for a longer
period of time. For example, an approximately 525 ppm solution (1:100 dilution of
5.25% bleach) is effective.
• Prepare bleach solution fresh daily and keep it in a closed container because it degrades
over time and in contact with the sun.
If a person was exposed to blood through nonintact skin, mucous membranes or a
puncture wound, complete an incident report. For transportation of blood samples outside
a hospital, equip the transportation vehicle with a blood spillage kit.
Fig 3.2: Illustrations for best practices in phlebotomy

35
3. Blood-sampling systems
Several blood-sampling systems are available for phlebotomy. The system most
appropriate for the procedure should be chosen. I will provide detailed information on all
the systems available for drawing blood, and outline the advantages and disadvantages of
each device.
Background information on blood-sampling systems
Closed systems
Closed systems for blood sampling are preferable because they have proven to be safer
than open systems.

36
Needle and syringe
The use of a hypodermic needle and syringe is the most common means of blood
sampling.
Choice of gauge
If the needle is too large for the vein for which it is intended, it will tear the vein and
cause bleeding (haematoma); if the needle is too small, it will damage the blood cells
during sampling, and laboratory tests that require whole blood cells, or haemoglobin and
free plasma, will be invalid.
Blood collection for transfusion requires a larger gauge than is used for blood drawing.
Vacuum extraction systems
The use of vacuum extraction tube systems as closed systems for blood collecting
reduces the risk of direct exposure to blood and has made it easier to take multiple
samples from a single venepuncture.
Vacuum extraction systems are widely available in most well-resourced countries. These
are recommended, but users should check their own country’s recommendations.
Although vacuum extraction systems are safe, training and skill is required for their use.
Double-ended needles are available in several recommended gauge sizes. The end
covered by a rubber cuff is screwed into the barrel (also known as the tube holder,
evacuated tube needle holder or bulldog). A thread separates the two ends, and this is
where the barrel is screwed into place. The barrel holds the sample collection tube in
place and protects the phlebotomist from direct contact with blood. The sample tube is
under vacuum. Once the needle is in the vein, the tube is pressed on to the needle and the
blood is drawn automatically into the sample tube by vacuum until the required amount is
collected. This system comes complete with needle, barrel and the laboratory sample
tubes with appropriately coloured tops for different types of samples.
Tubes for adult and paediatric specimens are available.
Discard the barrel and syringe as a single entity where possible. If there is a need to reuse
the barrel, use a one-hand scoop technique to cover the sharp end of the needle and thus
to safely remove the needle from the barrel. Alternately, use a sharps container with a
needle removal hold, again employing a one-handed technique.

37
Some systems have a mechanism that can be activated once the needle has been used; the
mechanism retracts the used needle into the barrel and snaps it shut. Others have a quick
release mechanism to dislodge the used needle into the sharps container.
Vacuum systems may also be used with a winged butterfly needle and luer-lock
connectors.
Winged butterfly needles are also available with safety-engineered devices.
The sharps container must be within arm’s reach and clearly visible, to ensure safe
disposal of sharps.
Open systems
Open systems include hypodermic needle and syringes, as well as winged steel needles
attached to a syringe.
Practical guidance on blood-sampling systems
Needle and syringe
To use a needle and syringe system:
• open the packaging of the hypodermic needle from the hub end (back of the needle),
keeping it capped;
• open the sterile packaging of the syringe from the plunger end (back of the syringe),
keeping the nozzle protected in the packaging;
• Carefully remove the syringe from the packaging and insert the nozzle of the syringe
firmly into the exposed hub of the capped hypodermic needle;
• leave the needle and syringe in place until ready for use.
Choice of gauge
Choose the gauge of hypodermic needle that fits comfortably into the most prominent
vein with little discomfort (Table 2.4).
Table 2.4 Recommended needle gauge, length and device for routine injection and
phlebotomy procedures for different age groups
Needle gauge Patient population Procedure
Adult Paediatric,
elderly, small
veins
Neonatal

38
16-18 yes blood
donation
19-20 NA NA NA
20-21 (1-1.5 inch or
2.54cm)
NA NA
22 (1 inch or 2.54cm) (1 inch or 2.54cm) NA
23 (1-1.5 inch or
2.54cm)
(Winged set
(Butterfly); 0.5inch
or 0.75cm
(Winged set
(Butterfly); 0.5inch
or 0.75cm
NA-not applicable
3.3 Illustrations for blood-sampling systems
Fig 3.4: Blood-sampling systems

39
Paediatric and neonatal blood sampling
The information given here supplements that given in Chapters 2 and 3. Users of these
guidelines should read Chapters 2 and 3 before reading the information given below. This
part of the report covers background information (Section 6.1), practical guidance
(Section 6.2) and illustrations (Section 6.3) relevant to paediatric and neonatal blood
sampling.
Background information on paediatric and neonatal blood sampling
This chapter discusses aspects specific to paediatric and neonatal blood sampling.
Anyone taking blood from children and neonates must be well trained and practiced in
venepuncture techniques. A uniform sampling technique is important to reduce pain and
psychological trauma.
Choice of procedure and site
The choice of site and procedure (venous site, finger-prick or heel-prick – also referred to
as “capillary sampling” or “skin puncture”) will depend on the volume of blood needed
for the procedure and the type of laboratory test to be done. Venepuncture is the method
of choice for blood sampling in term neonates; however, it requires an experienced and
trained phlebotomist. If a trained phlebotomist is not available, the physician may need to
draw the specimen. Section 7.1 provides information on when a capillary blood specimen
from a fingerprick or a heel-prick is appropriate. The blood from a capillary specimen is
similar to an arterial specimen in oxygen content, and is suitable for only a limited
number of tests because of its higher likelihood of contamination with skin flora and
smaller total volume.
Finger and heel-prick
Whether to select a finger-prick or a heel-prick will depend on the age and weight of the
child. Section 4.2 explains which procedure to select, based on these two elements.
Patient immobilization is crucial to the safety of the paediatric and neonatal patient
undergoing phlebotomy, and to the success of the procedure. A helper is essential for
properly immobilizing the patient for venepuncture or finger-prick, as described in
Section 6.2.
Practical guidance on paediatric and neonatal blood sampling
Patient identification

40
For paediatric and neonatal patients, the methods described below are used to ensure that
patients are correctly identified before taking blood.
• Use a wrist or foot band only if it is attached to the patient; DO NOT use the bed
number or a wrist band that is attached to the bed or cot.
• If a parent or legal guardian is present, ask that person for the child’s first and last
names.
• Check that the names, date of birth and hospital or file number are written on the
laboratory form, and match them to the identity of the patient.
Venepuncture
Venepuncture is the preferred method of blood sampling for term neonates, and causes
less pain than heel-pricks.
Equipment and supplies for paediatric patients.
• Use a winged steel needle, preferably 23 or 23 gauge, with an extension tube (a
butterfly):
–– avoid gauges of 25 or more because these may be associated with an increased risk of
haemolysis;
–– use a butterfly with either a syringe or an evacuated tube with an adaptor; a butterfly
can provide easier access and movement, but movement of the attached syringe may
make it difficult to draw blood.
• Use a syringe with a barrel volume of 1–5 ml, depending on collection needs; the
vacuum produced by drawing using a larger syringe will often collapse the vein.
• When using an evacuated tube, choose one that collects a small volume (1 ml or 5 ml)
and has a low vacuum; this helps to avoid collapse of the vein and may decrease
haemolysis.
• Where possible, use safety equipment with needle covers or features that minimize
blood exposure. Auto-disable (AD) syringes are designed for injection, and are not
appropriate for phlebotomy.
Preparation
Ask whether the parent would like to help by holding the child. If the parent wishes to
help, provide full instructions on how and where to hold the child; if the parent prefers
not to help, ask for assistance from another phlebotomist.

41
Immobilize the child as described below.
• Designate one phlebotomist as the technician, and another phlebotomist or a parent to
immobilize the child.
• Ask the two adults to stand on opposite sides of an examination table.
• Ask the immobilizer to:
–– stretch an arm across the table and place the child on its back, with its head on top of
the outstretched arm;
–– pull the child close, as if the person were cradling the child;
–– grasp the child’s elbow in the outstretched hand;
–– use their other arm to reach across the child and grasp its wrist in a palm-up position
(reaching across the child anchors the child’s shoulder, and thus prevents twisting or
rocking movements; also, a firm grasp on the wrist effectively provides the phlebotomist
with a “tourniquet”).
If necessary, take the following steps to improve the ease of venepuncture.
• Ask the parent to rhythmically tighten and release the child’s wrist, to ensure that there
is an adequate flow of blood.
• Keep the child warm, which may increase the rate of blood flow by as much as
sevenfold, by removing as few of the child’s clothes as possible and, in the case of an
infant, by:
–– swaddling in a blanket; and
–– having the parent or caregiver hold the infant, leaving only the extremity of the site of
venepuncture exposed.
• Warm the area of puncture with warm cloths to help dilate the blood vessels.
• Use a transilluminator or pocket pen light to display the dorsal hand veins and the veins
of the antecubital fossa.
Finger and heel-prick
See Section 7.2, which describes the steps for both finger and heel-pricks, for paediatric
and neonatal patients, and for adults. Select the proper lancet length for the area of
puncture, as described in Section 7.2.
4.3 Illustrations for paediatric and neonatal blood sampling

47
5.0 Personal Involvement with the wash-up and separation Division
While with the wash-up and separation division I was practically involved in:
1. Separation of serum/plasma from whole blood.
2. Performing urinalysis; interpretation and documentation of results.
3. Performing pregnancy test.
4. Standard laboratory procedures of washing test tubes and other recyclable instruments
Urinalysis
I practically performed urinalysis using the DIRUI H10 urinalysis strip following
standard operating procedure (SOP) given below:
Note appearance(clear, turbid or cloudy) and color of urine(amber, yellowish, pale
yellowish or colorless)
Dip the strip into urine up to the test area for no more than two (2) seconds.
Draw the edge of the strip along the brim of the vessel to remove excess urine.
Turn the strip on its side and tap twice to remove any excess urine.
Hold the strip horizontally and prevent possible mixing of chemicals when excessive
urine is present
Compare the colors of the reagent pads exactly after sixty (60) seconds with the color
chart on the vial label under good light.
Result interpretation of the chemical analysis of urine (urinalysis)
To perform the chemical examination, most clinical laboratories use commercially
prepared test strips. These are narrow plastic strips that hold small squares of paper called
test pads arranged in a row. The test pads have chemicals impregnated into them. When a
strip is briefly but completely dipped into urine, the test pads absorb the urine and a
chemical reaction changes the color of the pad within seconds to minute.
The degree of color change on a test pad can also give an approximation of the amount of
substance present. For example a slight color change in the test pad for protein may
indicate a small amount of protein present in the urine whereas a deep color change may
indicate a large amount.

48
Fig 3.6: urinalysis colour chart

49
The most frequently performed chemical tests using reagent strips are:
Specific gravity
pH
Protein
Glucose
Ketones
Blood
Leukocyte esterase
Nitrite
Bilirubin
Urobilinogen
Specific gravity
The first test, specific gravity is actually a physical characteristic of the urine, a measure
of urine concentration that can be determined using a chemical test. There are no
“abnormal” specific gravity values. This test simply indicates how concentrated the urine
is. SG measurements are actually are actually a comparison of the amounts of solutes
(substances dissolved) in urine as compared to pure water. Since all urine have some
solutes present, a urine SG of 1.000 is not possible. If a person drinks excessive quantities
of water in a short period of time or gets intravenous (IV) fusion of large volumes of
fluid, then the urine SG may be as low as 1.002. The upper limit of the test pad, an SG of
1.035 indicates concentrated urine, one with many solutes in a limited amount of water.
Knowing urine concentration helps health care providers decide if the urine specimen
they are evaluating is the best one to detect a particular substance. For example, if they
are looking for a very small amount of protein, a concentrated morning urine specimen
would be the best sample.
pH
As with specific gravity, there are typical but not abnormal pH values. The kidneys play
an important role in maintaining the acid-base balance of the body. Therefore, any

50
conditions that produce acids or bases in the body such as acidosis or alkalosis, or the
ingestion of acidic or basic foods can directly affect urine pH.
Diet can be used to modify urine pH. A high protein diet or consuming cranberries will
make the urine more acidic. A vegetarian diet, a low carbohydrate diet or the ingestion of
citrus fruits will tend to make the urine more alkaline.
Some of the substances dissolved in urine will precipitate out to form crystals when the
urine is acidic; others will form crystals when the urine is basic. If crystals form while the
urine is being produced in the kidney, a kidney stone or calculus can develop. By
modifying urine pH through diet or medications, the formation of these crystals can be
reduced or eliminated.
Protein
The protein test pad measures the amount of albumin in the urine. Normally there will not
be detectable quantities. When urine protein is elevated, a person has a condition called
proteinuria; this can be an early sign of kidney disease. Albumin is smaller than most
other proteins and is typically the first protein that is seen in the urine when kidney
dysfunction begins to develop. Other proteins are not detected by the test pad but may be
measured with a separate urine protein test.
Glucose
Glucose is normally not present in urine. When glucose is present, the condition is called
glucosuria. It results from either
1. An excessively high glucose concentration in the blood, such as may be seen with the
people who have uncontrolled diabetes mellitus.
2. A reduction in the “renal threshold”. When blood glucose levels reach a certain
concentration, the kidneys begin to excrete protein into the urine to decrease blood
concentration. Sometimes the threshold concentration is reduced and glucose enters the
urine sooner, at a lower blood glucose concentrations.
Some other conditions that can cause glucosuria include hormonal disorders, liver
disease, medications and pregnancy. When glucosuria occurs, other tests such as blood
glucose is usually performed to further identify the specific cause.

51
Ketones
Ketones are not normally found in the urine. They are intermediate products of fat
metabolism. They can form when a person does not eat enough carbohydrates (for
example, in cases of starvation or high protein diets) or when a person’s body cannot use
carbohydrates properly. When carbohydrates are not available, the body metabolizes fat
instead to get the energy it needs to keep functioning. Ketones in urine can give an early
indication of insufficient insulin in a person who has diabetes, exposure to cold and less
of carbohydrate such as with frequent vomiting can also increase fat metabolism resulting
in ketonuria.
Blood (hemoglobin)
This test is used to detect hemoglobin in the urine (haemoglubinuria). Haemoglobin is an
oxygen-transporting protein found inside red blood cells (RBC). It’s presence in the urine
indicates blood in the urine (hematuria). The small number of RBC’s normally present
in the urine usually results in a negative test. However, when the number of RBC’s
increase, they are detected as a positive result. Even small increase in the amount of RBC
in urine can be significant. Numerous diseases of the kidney and urinary tract, as well as
trauma, medications, smoking, or strenuous exercise can cause haematuria or
haemaglobinuria. Sometimes a chemical test for blood in the urine is negative but the
microscopic examination shows increased numbers of RBC’s, when this happens the
laboratorian may test the sample for ascorbic acid (vitamin C) because vitamin C has
been known to interfere with the accuracy of urine blood test results, causing them to be
falsely low or falsely negative.
Leukocyte esterase
Leukocyte esterase is an enzyme present in most white blood cells (W.B.C’s). Normally,
a few WBCs are present in urine and this test is negative. When the number of WBC’s
increases significantly, this screening test will become positive.

52
When WBC count in urine is high it means that there is inflammation of the urinary tract
or kidneys. The most common cause for WBC’s in urine (leukocyturia) is a bacterial
urinary tract infection (UTI), such as bladder or kidney infection.
Nitrite
This test detects nitrite and is based upon the fact that many bacteria can convert nitrate
to nitrite in the urine. Normally, the urinary tract and urine are free of bacteria. When
bacteria find their way into the urinary tract, they can cause a urinary tract infection
(UTI). A positive nitrite test result can indicate a UTI. However, since not all bacteria are
capable of converting nitrate to nitrite, some can still have a UTI despite a negative nitrite
test.
Bilirubin
Bilirubin is not present in the urine of normal, healthy individuals. Bilirubin is a waste
product that is produced by the liver from the haemoglobin of RBC’s that are removed
from circulation. It becomes a component of bile, a fluid that is secreted into the
intestines to aid in food digestions.
In certain liver diseases such as biliary obstruction or hepatitis, bilirubin leaks back into
the blood stream and is excreted in urine. The presence of bilirubin in the urine is an early
indication of liver disease and can occur before clinical symptoms such as jaundice
develop.
Urobilinogen
Urobilinogen is normally present in urine in low concentrations. It is formed in the
intestine from bilirubin and a portion of it is absorbed back into the blood stream.
Positive test result help detect liver diseases such as hepatitis and cirrhosis and conditions
associated with increased RBC destruction (haemolytic anemia). When urine
urobilinogen is low or absent in a person with urine bilinogen and/or signs of liver
dysfunction, it can indicate the presence of hepatic or biliary obstruction.

53
NOTE: Urine samples that have stayed 24hours after collection should not be analyzed
as they must have been acted upon by bacteria and some unstable biological components
degraded by simple metabolic processes e.g. glucose. Analyzing such samples will give
wrong results.
Pregnancy test
This test is used to detect pregnancy in women using urine or blood plasma. Pregnancy
tests in this unit are often conducted using blood plasma already separated from whole
blood by dipping the pregnancy test strip in it. The strip is then allowed to stay for 3-10
minutes.
Interpretation of results
Fig 3.7: pregnancy test strip result indicator
Negative: Only one color band appears on the control region. No apparent band on the
test region. This indicates that no pregnancy has been detected.
Positive: Distinct color bands appear on the control and test regions. Presence of both test
line and control line indicate that you are pregnant. The color intensity of the test bands

54
may vary since different stages of pregnancy have different concentrations of HCG
hormone.
NOTE: A positive test line will appear directly below the control line on the same
test surface (or 'result window' area). Any line or accumulation of color/dye that
appears at the juncture between test components should not be mistaken for "test line"
(this is only the source of the test reagent & dye).
Invalid: No visible band at all. The control band will not appear if an insufficient volume
of specimen is added into the test kit. Proper procedures may not have been followed in
performing the test. Repeat with a new test kit. Please consult above instructions and
follow precisely
A pregnancy test works by checking the bloodstream for a hormone called human
chorionic gonadotropin, or hCG. This is only produced when a fertilized egg is implanted
in the uterus, and it’s produced from about six days after the egg is fertilized in most
women. In around ten percent, however, implantation occurs a little later, which can
cause a false negative if a pregnancy test is taken early. In these cases pregnancy won’t
be detectable until the first period is missed.
There are two ways to test for pregnancy – checking for the presence of hCG in blood
(plasma) or in urine. Blood tests, which test for the presence and concentration of hCG in
the blood, are more reliable, and can detect pregnancy as early as 7-10 days after
contraception, whereas that of urine, which test for the presence of hCG in urine, cannot
be relied upon to give a define yes or no until the mother to be has missed her first period.
If hCG is present in the urine or blood plasma, it will soak into the strip along with the
urine. This then binds to an antibody bonded to a pigment molecule embedded in a line
on the indicator strip; it binds to the antibody molecules, causing the dye to become
visible. If there is no hCG the line does not appear. Most tests will also have a test line
which will change colour to indicate that the test has been used correctly. This allows one
to read the results very quickly, usually within ten minutes.
It’s important to wait for ten minutes to obtain the most accurate result. Time is also a
factor – the later you do a test the more hCG there will be and the more accurate it will

55
be. This is also why some tests in the chemical pathology laboratory are done first thing
in the morning to boost accuracy.
Notes on Pregnancy Tests / Test Limitations and Terms of Use
- The Urine hCG Pregnancy Test is for in vitro diagnostic use only (for external use
only).
- As with all diagnostic tests, a final clinical diagnosis should not be based on the results
of a single test, but should only be made by a physician.
- A negative result obtained from a urine specimen collected from a mother in very early
pregnancy may be due to an extremely low concentration of hCG. In such cases, the test
should be repeated on a fresh specimen obtained two days later.
- If a urine sample is too diluted, it may not contain a representative urinary hCG
concentration. If a negative result is obtained with a low specific gravity specimen and
pregnancy is still suspected, obtain a first morning urine specimen and retest.
- Test instructions must be followed precisely to insure accurate results. Please read
instructions and follow exact test procedure. Please check with your doctor to verify
diagnostic results – or if you receive unexpected or inconsistent test results.
COLLECTION OF LABORATORY SAMPLES
Today’s technologies allow testing on an impressively wide variety of samples
(sometimes called specimens) that are collected from the human body. Most commonly
these are blood, urine, saliva, sputum, faeces, semen and other bodily fluids, as well as
tissue.
Urine, faeces, sputum & semen
Some samples such as urine, faeces, sputum, and semen can be easily collected by the
patient. For many people, collecting the samples themselves is often the preferred way
because of greater privacy but sometimes professional help is needed. These types of
samples can be collected at home and brought to a medical practitioner's rooms or clinic
or a pathology collection centre. When samples are collected professionally there is
always someone on hand to help and to provide instructions. Experienced health

56
professionals are well aware of the potential for embarrassment and often there are
printed instructions on how to obtain samples – contaminated or badly collected samples
can be unable to be tested in the laboratory.
Here are some examples of types of samples typically collected by the patient. It is very
important that all instructions for sample collection are carefully followed. Requirements
can vary between laboratories, hospitals and doctors.
Semen — Patients ejaculate into a specimen container. Usually, they need to refrain from
ejaculating for 3 to 4 days before collecting the specimen. The specimen must be kept
warm and brought to the lab within the time period specified.
Sputum — Patients are instructed to cough up sputum from as far down in the lungs as
possible. (Some doctors prefer people to do this under professional guidance in the
surgery.)
Stool — People usually collect this sample themselves during toileting. Instructions
usually emphasize the importance of preventing the sample from becoming contaminated
from other material in the toilet bowl and to wash their hands well after handling the
sample. They may also be told to avoid certain foods during the test period. Depending
on the test, they may be instructed to collect the sample into a container, scoop a small
portion into a vial, or smear a small amount on special test paper.
Urine — Most urine specimens are collected by urinating into a container or receptacle.
To keep the sample from becoming contaminated by materials outside the urinary tract,
instructions are provided on how to clean the area. For some tests, a first-void urine
sample is required. This is when the urine is at its most concentrated. Other tests require a
mid-stream urine sample – after a small amount of urine has been passed. Collecting the
urine specimen can be awkward. For certain tests, 24-hour urine samples are collected at
home and may need to be refrigerated. It’s important to wash hands well after collecting
the sample.

57
Saliva — This type of sample may be collected using a swab or, if a larger volume is
needed for testing, by expectorating into a container.
Blood — Samples can be collected from blood vessels (capillaries, veins, and sometimes
arteries) by trained phlebotomists (usually pathology collectors or nurses). A fine needle
is used and blood is withdrawn into a special collection tube. The procedure takes just a
few minutes and can sting just a bit, typically when the needle is inserted or withdrawn.
See
Swabs – Many samples are collected by running a swab over an affected area.
Procedures of this type can be collected by the patient or by a professional. The sample
can be sent to a laboratory for analysis, although a few tests can provide immediate
results in the doctor’s rooms. Throat, nasal, vaginal, and superficial wound cultures are
obtained in this way. The procedures, while they occasionally can be uncomfortable, are
generally quick, relatively painless and have no after-effects.
Samples from open wounds and sores — If a wound or sore is located in the outer layer
of skin, the specimen is typically collected on a swab by brushing it over the area and
gathering a sample of fluid or pus. Touching the open wound area may be temporarily
painful since the wound is likely to be tender and sore. If a wound or infection is deep,
however, a needle and syringe may be used to aspirate a sample of fluid or pus from the
site.
Secretions and fluids from the nose or throat — Samples are collected by running a
swab over the area. People typically respond to swabbing of their throat with a
momentary gag reflex. If the throat is sore, the sample collection, brief as it is, can be
uncomfortable. Similarly, a nasal swab may be a bit uncomfortable as the swab is
inserted and reaches areas inside the nose that are typically never touched.
Secretions and tissues from the female reproductive system — Samples of vaginal
secretions are obtained by running a cotton swab over the walls of the vagina. Also,

58
cervical cells for a Pap smear are obtained using a special spatula or a tiny brush.
Endometrial tissue samples are obtained by inserting a thin, flexible, hollow tube into the
uterus, during which a slight pinch or brief cramping might be felt. It’s common for
people to feel embarrassed and vulnerable because of how these samples are collected. A
sensitive approach by the health care professional contributes greatly to a person’s
emotional comfort. If you are physically uncomfortable, try asking for what you need
(such as a smaller speculum). Also, if you would be more at ease if a woman performs
these procedures ask your clinic for a female practitioner.
Biopsies, needle aspirations, surgery, CSF & bone marrow
Some samples can only be obtained by more invasive procedures conducted by specially-
trained doctors, pathologists and other medical personnel.
Because of the nature of these collections techniques, some discomfort may be involved.
Knowing what the procedure involves can help alleviate anxiety. Some common
collections of this type include:
Tissue biopsy — Samples of bodily tissue can be obtained from various places in the
body such as breast, lung or skin and, depending on the site, may involve varying degrees
of invasiveness and pain or discomfort. The time required to perform the procedure and
for recovery can also vary greatly. These procedures are conducted by medical
practitioners, pathologists and occasionally other medical personnel who have specialist
training. Biopsies can be collected using procedures such as:
• Needle biopsy or aspiration — A needle is inserted into the site and cells or fluids
are withdrawn using a syringe. A slight pinch may be felt at the site of needle
insertion. Usually no recovery time is required, and slight discomfort may be
experienced afterwards.
• An open biopsy is a minor surgical procedure in which an incision is made and a
portion of tissue is cut from the site. A closed biopsy is a procedure in which an
incision is made (usually smaller than an open biopsy) and an instrument is
inserted to help guide the surgeon to the appropriate site and to obtain the sample.
These biopsies are usually performed in a doctor’s procedure room or hospital

59
operating room. A local or general anaesthetic is used, depending on the
procedure, to ensure the patient remains comfortable. Very occasionally, a general
anaesthetic is needed.
• Surgery – samples are also obtained during surgical procedures. These are called
‘frozen sections’ after the technique used: tissue is removed and frozen for
dissection to allow immediate investigation. Samples taken this way can be used
to give the surgeon preliminary diagnosis while the surgery is in progress.
Samples are also removed and sent to the lab for more detailed analysis.
Cerebrospinal fluid (CSF) — A sample of cerebrospinal fluid is obtained by lumbar
puncture, sometimes called a spinal tap. This is a specialized but relatively routine
procedure. It is usually performed while the person is lying on his or her side in a curled
up fetal position but may sometimes be performed in a sitting position. The back is
cleaned with an antiseptic and a local anaesthetic is injected under the skin. A special
needle is inserted through the skin, between two vertebrae, and into the spinal canal. The
doctor collects a small amount of CSF in multiple sterile vials. Then the needle is
withdrawn and a sterile dressing and pressure are applied to the puncture site. The patient
will then be asked to lie quietly in a flat position, without lifting their head, for one or
more hours to avoid a potential post-test spinal headache. The lumbar puncture procedure
usually takes less than half an hour. For most people, it is a moderately uncomfortable to
somewhat painful procedure. The most common sensation is a feeling of pressure when
the needle is introduced.
Other body fluids such as synovial fluid, peritoneal fluid, pleural fluid and pericardial
fluid are collected using procedures similar to that used for CSF in that they require
aspiration of a sample of the fluid through a needle into a collection vessel, such as a
syringe or specimen container. They often require some patient preparation, use of a local
anaesthetic, and a resting period following sample collection.

60
Separation of plasma/serum from whole blood (Blood fractionation)
Blood fractionation is the process of fractionating whole blood, or separating it into its
component parts. This is typically done by centrifuging the blood. The resulting
components are:
• a clear solution of blood plasma in the upper phase (which can be separated into
its own fractions, see Blood plasma fractionation),
• the Buffy coat, which is a thin layer of leukocytes (white blood cells) mixed with
platelets in the middle, and
• Erythrocytes (red blood cells) at the bottom of the centrifuge tube.
Cryogen vials are the tubes used in the wash up/separation unit containing a silicone gel;
when centrifuged the silicone gel forms a layer on top of the buffy coat, allowing the
blood serum to be removed more effectively for testing and related purposes.
Draw 12 mL of whole blood for each 5 mL of serum or plasma needed. Collect in an
appropriate collection tube. Centrifuge for at least 5 minutes at 1500-2500 RPM. Pipette
the serum or plasma into a clean plastic screw-cap cryogen vial and attach the label.
Serum, plasma or whole blood collection
Draw blood in the color-coded Vacutainer tube indicated in the alphabetical test listing.
For serum or plasma, draw approximately 2 1/2 times the requested volume. For serum,
allow the blood to clot sixty minutes and separate by centrifugation. For plasma and
whole blood, completely fill the Vacutainer whenever possible to eliminate dilution from
the anticoagulant or preservative and immediately mix the blood by gently and
thoroughly inverting the tube five to ten times. Separate plasma by centrifugation.
Transfer the serum, plasma or whole blood to a plastic transport tube (see Pediatric
Specimen Tubes below). To prevent injury and exposure to potentially infectious
material, do not ship frozen serum, plasma, or whole blood received in glass tubes or SST
(glass or plastic).

61
The color-coded Vacutainer tubes on the inside cover are recommended unless otherwise
indicated in the alphabetical test listing. Color-coded pediatric Vacutainer tubes are
provided to facilitate special handling.
Most blood specimens can be obtained using routine phlebotomy techniques; however,
there are some exceptions. The use of a tourniquet can cause stress and is not
recommended in some cases. The patient’s posture either sitting, standing or lying down,
or the time of day relative to the patient’s sleep cycle can be important factors in some
tests. Whenever specific issues of this nature are important, they will be listed as part of
the specimen requirements or patient preparation for the individual tests in the General
Test Listing section.
Whole blood
Collect whole blood according to instructions provided for the individual test.
Thoroughly mix the blood with the additives by gently inverting the tube eight times
(four times in the case of light blue-top (sodium citrate) tubes. Maintain the specimen at
room temperature or on cool packs before submitting to our laboratory unless instructed
otherwise by the specimen requirements. Never freeze whole blood unless specifically
instructed in the specimen requirements.
If you store cool packs in the freezer, be sure to allow enough time for them to warm to
refrigerator temperature before placing whole blood specimens near them. To minimize
the risk of hemolysis, do not place whole blood specimens in direct contact with cool
packs.
Plasma
Plasma contains fibrinogen and other clotting factors when separated from the red blood
cells. Evacuated tubes used to collect plasma specimens contain anticoagulant and,
frequently, a preservative. The additive in each tube is specified on the label and tube
stoppers are color coded according to the additive present. Consult the individual test

62
specimen requirements to determine the correct additive/tube to use. Indicate that the
specimen is plasma on the plastic screw-cap vial for transport and test requisition.
Centrifugation: When plasma is required, or when not using a serum gel separator tube,
follow these instructions:
1. Draw 12 mL of whole blood for each 5 mL of serum or plasma needed. Collect in
an appropriate collection tube.
2. Centrifuge for at least 15 minutes at 2200-2500 RPM.
3. Pipette the serum or plasma into a clean plastic screw-cap vial and attach the
label. Do not transfer red cells to the vial. Screw cap on firmly to prevent
leakage.
Serum
Please check individual specimen requirements for restrictions. When using a serum
separator tube, follow these instructions:
1. Perform venipuncture as with any other blood collection device.
2. Invert the tube gently no more than eight times. Further inversion may cause
alterations in sample integrity.
3. Do not remove the stopper at any time. Do not centrifuge immediately after
drawing blood. Allow the blood to clot in an upright position for at least 30
minutes but not longer than 1 hour before centrifugation.
4. Perform venipuncture as with any other blood collection device.
5. Centrifuge for at least 5 minutes at 1500-2500 RPM within one hour of collection.
6. Transfer the serum to a plastic screw-cap cryogen vial for transport to the
laboratory.
Note: Do not use serum separator tubes for therapeutic drug monitoring or toxicological
analysis. The plastic serum separator material extracts lipophilic substances (most drugs),
resulting in a falsely low drug concentration result. Instead, collect the specimen in a
plain red-top tube containing no anticoagulants or preservatives. Transfer the serum with

63
a pipette to a plastic vial for transport to the laboratory. Serum should be clear and free
from all red cells.
Frozen serum or plasma specimens
Serum or plasma specimens need to be frozen only if specifically stated in the specimen
requirement. However, in these cases, it is essential to freeze the specimen as soon as it is
separated from the cells. Always freeze specimens in plastic tubes unless specifically
instructed otherwise.
Lay the tube at a 45° angle to avoid tube breakage caused by expansion during freezing.
Do not freeze plastic Serum Separator Tubes. An exception is a specimen submitted in a
PPT tube; the plasma can be frozen and transported in the original tube.
Extreme cold may cause ordinary plastic labels to become brittle and detach from the
specimen tube. Use clear tape to secure label to specimen transport tube.
Note: If more than one test is requested on a frozen specimen, split the sample prior to
freezing. Use a separate test requisition when submitting a frozen specimen; frozen and
non-frozen specimens must not be submitted on the same test requisition. Indicate on
specimen container and test requisition if specimen is plasma or serum.
If more than one test is ordered on a single frozen sample, we will call you to choose
which test you want performed before testing can proceed.
Fig 3.8: Blood components after centrifugation.

64
Fig 3.9: cryogen vials Fig 4.0: swab stick
Common causes of unacceptable serum or plasma specimens and inaccurate test
results:
Haemolysis
Hemolysis occurs when the membrane surrounding red blood cells is disrupted and
hemoglobin and other intracellular components escape into the serum or plasma.
Hemolyzed serum or plasma varies in color from faint pink to bright red, rather than the
normal straw color. Grossly or moderately hemolyzed specimens may be rejected and
even slight hemolysis may alter certain test results.
Hyperbilirubinemia
Icteric serum or plasma varies in color from dark to bright yellow, rather than the normal
straw color. Icterus may affect certain determinations. Upon receipt of such specimens, a
new sample may be requested to assure results of diagnostic value.
Turbidity (lipemia)
Turbid, cloudy or milky serum (lipemic serum) may be produced by the presence of fatty
substances (lipids) in the blood. Bacterial contamination may also cause cloudy serum.
Moderately or grossly lipemic specimens may alter certain test results.

65
This separation is done by collecting blood in a lithium heparin anticoagulated vacutainer
tube after which it is spun at a revolution of 1500RPM for five minutes. After
centrifuging, the blood separates into it’s components with the plasma appearing at the
top (supernatant) and red blood cells at the bottom with the white blood cells in-between.
With the aid of a Pasteur pipette, the plasma is then transferred into a cryogen vial and
then labeled with the patient’s information (name, laboratory number, intended test(s)
etc).
3.3 Personal Involvement with the Chemical Pathology Main Laboratory
Liver function test (LFT), Fasting Lipid Profile (FLP), Fasting Blood Glucose/sugar
(FBG/FBS), Random Blood Glucose/Sugar (RBG/RBS), Electrolytes (Na+
, K+
, Ca2+
,
PO4
2-
,), Urea, Creatinine, protein test, Oral Glucose Tolerance Test (OGTT), Two hours
post prandia glucose (2HrPP), cerebrospinal fluid biochemistry are the tests conducted in
this laboratory following standard scientific procedures.
GLUCOSE
Clinical significance
Glucose is a major carbohydrate present in the blood & serves as a primary source of
energy. It is usually obtained from ingested starch & sugar. The glucose concentration is
normally maintained at constant level. Excessive glucose is stored as a inactive glycogen
mainly in the liver & little in the muscles.
Elevated blood glucose levels are found in diabetes mellitus, hyperthyroidism,
hyperadrenalism & certain liver diseases.
Decreased levels are found in Insulinoma, hypothyroidism, hypopituitarism.
Principle
Enzymatic colorimetric determination of glucose according to the following reaction.
Glucose Oxidase
Glucose+ O2 + H2O -----------------------> Gluconic acid + H2O2
Peroxidase
2H2O2+phenol + 4-Aminoantipyrine -------------------> Quinonimine + 4H2O

66
Reagent composition
GLUCOSE (S.L) R1 5 x 100 mL / 1 x 1000 mL
Tris Buffer, (pH 7.40) 92 mmol/L
Phenol 0.3 mmol/L
Glucose Oxidase 15000 U/L
4- Aminophenazone 2.6 mmol/L
GLUCOSE STANDARD 1 x 4 mL
Glucose standard concentration 100 mg/dL
Storage and stability
The sealed reagents are stable up to the expiry date stated on the label, when stored at 2 -
80C.
Linearity
This reagent is linear up to 600 mg/dL
If the concentration is greater than linearity (600 mg/dL), dilute the sample with normal
saline and repeat the assay. Multiply the result with dilution factor.
Normal range
It is recommended that each laboratory establish its own reference values.
The following value may be used as guide line.
Serum / Plasma: 70-105 mg/dL
C S F: 50 -70 mg/dL
Preparation and stability of working reagent
The Reagent is ready to use.
Precaution
To avoid contamination, use clean laboratory wares.
Avoid direct exposure of reagent to light.
Sample
Serum / plasma (free of haemolysis) / Cerebrospinal fluid
Laboratory procedure
Arrange three test tubes (this may depend on the number of intended test samples at
hand) in the order of blank, standard and sample and pipette the specified volume of
reagents as shown in the table below into each. The blank serve as to clear the memory of
the spectrophotometer, the standard is a known concentration of which the test sample
concentration is compared.
Blank Standard Sample
Working reagent 1000µL 1000µL 1000µL
Standard - 10µL -
Sample - - 10µL

67
Mix and incubate for 1minute at 37˚c. Measure the absorbance of sample and standard
against reagent blank spectrophotometrically at wavelength of 630nm
Table 2.5 creatinine test procedure
Calculation
Absorbance of Sample
Glucose Conc. (mg/dL) = ------------------------------ x 100
Absorbance of standard
ALBUMIN
Albumin which is synthesized in the liver constitutes a major part of the total proteins in
the body, the other part being globulin; they form the major portion of the dissolved
substances in the plasma. Functions of albumin includes distribution of extracellular
fluids, regulation of osmotic pressure, acts as transport agent for a wide variety of
substances such as hormone, lipids, vitamins etc.
Increased levels are seen in dehydration.
Decreased levels are seen in liver diseases (hepatitis, cirrhosis), malnutrition, kidney
disorders, and increased fluid loss during extensive burns and malabsorbtion.
Principle
The reaction between albumin from serum or plasma and the dye bromocresol-green
produces a change in colour that is proportional to the albumin concentration.
Reagent composition
Albumin reagent 4˟ 50Ml, 1 * 500Ml, 1 * 1000Ml
Succinate buffer (pH 4.20) 75mmol/L
Bromocresol green 0.14g/L
Albumin standard 1*3Ml
Albumin standard concentration 3g/dL
The sealed reagents are stable up to the expiry date stated on the label, when stored at
room temperature and standard at 2-8˚c.
Linearity
This reagent is linear up to 6g/dL
If the concentration is greater than linearity (6g/dL) dilute the sample with normal saline
and repeat the assay. Multiply the result with dilution factor.

68
Normal range
It is recommended that each laboratory establish its own reference values. The following
may be used as guideline.
Serum/plasma 3.5-5.5g/dL
Sample
Serum/plasma (free of haemolysis)
Precaution
Arrange three test tubes (this may depend on the number of intended test samples at
hand) in the order of blank, standard and sample and pipette the specified volume of
reagents as shown in the table below into each. The blank serve as to clear the memory of
the spectrophotometer, the standard is a known concentration of which the test sample
concentration is compared.
Standard - 10µL -
Sample - - 10µL
Table 2.6 creatinine test procedure
Calculation
Cholesterol concentration (g/dL) = absorbance of sample/absorbance of standard * 3
ALKALINE PHOSPHATASE
Alkaline phosphatase (ALP) is widely distributed throughout the body, but clinically
important one for diagnostic reasons are in bone, liver, placenta and intestine. Growing
bone associated with the release of ALP and so in childhood the level of ALP is around
three times that of adult. During pregnancy in 2nd
and 3rd
trimester the enzyme rises
considerably due to placenta releasing ALP. It can be used to examine placental function.
Elevated levels are seen in bone diseases, e.g. Pagets disease, rickets, osteoblastic
metastatic and in obstructive disease of biliary tract.
Decreased levels are rarely seen, e.g. in vitamin A resistant rickets.

69
Principle
Kinetic determination of ALP according to the following reaction
Para-nitrophenyl phosphate +H20--------ALP------> p-nitrophenol + inorganic phosphate
ALP= Alkaline phosphatase.
Reagent composition
Alkaline Phosphatase R1 2*24ml/2*40ml/2*100ml/4*100ml
Diethanolamine buffer (pH 10.2) 125mmol/L
Magnesium chloride 0.625mmol/L
Alkaline Phosphatase R2 2*6ml/2*10ml/2*25ml/4*25ml
P-nitrophenyl phosphate 50mmol/L
The sealed reagents are stable up to the expiry date stated on the label, when stored at 2-
8˚C.
Linearity
This reagent is linear up to 200U/L
If the concentration is greater than linearity (200U/L), dilute the sample with normal
Normal Range
may be used as guideline
Women 64-306U/L
Men 80-306U/L
Children 160-1200U/L
Mix 4 volume of reagent 1 (R1) with 1 volume of reagent 2 (R2). The working reagent is
stable for 30 days at 2-8˚c.
NOTE: Discard the working reagent if the absorbance exceeds 1.00 at 405nm

70
Sample
Precaution
Laboratory Procedure
working reagent 1000µL
Sample 20µL
Mix and incubate at 37˚c for 1minute. Measure the change in absorbance per minute
( OD/min) during 3 minutes at 405nm wavelength.
Table 2.7 Alkaline phosphatase test procedure
Calculation
ALP activity (U/L) = ( OD/min) * 2750
CHOLESTEROL
Clinical Significance
Cholesterol is the main lipid found in the blood, bile and brain tissues. It is also one of the
most important steroids of the body and is a precursor of many steroid hormones. Two
thirds of the steroid present in the blood is esterified. The liver metabolizes the
cholesterol and it is transported in the blood stream by lipoproteins.
Increased levels are found in hypercholesterolemia, hyperlipidemia, hypothyroidism,
uncontrolled diabetes, nephritic syndrome and cirrhosis.
Decreased levels are found in malabsorption, malnutrition, hyperthyroidism, anaemia and
liver diseases.
Test Principle
Enzymatic colorimetric determination of total cholesterol is according to the following
reactions
Cholesterol ester + H2O------------------------------------------> cholesterol + fatty acids
Cholesterol + O2-----------------------------------------------> 4-cholesten-3-one + H2O2

71
2H2O2 + phenol + 4-Aminoantipyrine------------------------------------> red quinine +
4H2O
Reagent Composition
Cholesterol R1 4 * 50Ml
Pipes buffer (pH 6.90) 50mmol/L
Phenol 24mmol/L
Sodium cholate 0.5mmol/L
Cholesterol R2 4*50Ml
Cholesterol esterase >200U/L
Cholesterol oxidase >250U/L
Peroxidase >1000U/L
4-Aminoantipyrine 0.5mmol/L
Cholesterol Standard 1 * 4Ml
Cholesterol standard reagent 200mg/dL
8˚C
Linearity
This reagent is linear up to 500mg/dL
If the concentration is greater than linearity (500mg/dL), dilute the sample with normal
Normal Range
may be used as guideline.
Serum/plasma 150-220mg/dL
Dissolve contents of reagent 2 (R2) with the amount of reagent 1 (R1) indicated on the
vial label.
The working reagent is stable for 90 days at 2-8˚c.
NOTE: Discard the working reagent if the blank absorbance exceeds 0.08
Sample
Precaution

72
Standard - 10µL -
Sample - - 10µL
Mix and incubate for 5 minutes at 37˚c. Measure the absorbance of sample and standard
against reagent blank spectrophotometrically at wavelength of 505 (492-540) nm.
Table 2.8 Cholesterol test procedure
Calculation
Cholesterol concentration (mg/dL) = absorbance of sample/absorbance of standard * 200
SERUM GLUTAMATE PYRUVATE AMINOTRANSFERASE (SGPT)/ALANINE
AMINOTRANSFERASE (ALT)
It is found in most of the tissues, but mainly found in the liver. Increase levels are found
in hepatitis, cirrhosis, obstructive jaundice and other hepatic disease. SGPT activity is
markedly elevated even before clinical signs of jaundice become apparent in disease
associated with hepatic necrosis. Slight elevations are also found in myocardial
infraction.
Principle
Kinetic determination of Alanine Aminotransferase (ALT) is based upon the following
reactions.
L-Alanine + alpha-ketoglutarate------ALT------------------> pyruvate + L-glutamate
Pyruvate + NADH + H+---------------LDH---------------> L-Lactate + NAD+
ALT= Alanine aminotransferase
LDH= Lactate dehydrogenase
Reagent Composition
SGPT R1 4*50Ml
Tris buffer (pH 7.5) 110mmol/L
L-Alanine 550mmol/L
SGPT R2 4*50Ml
LDH >200U/L
NADH 0.20mmol/L
Alpha-ketoglutarate 16mmol/L
Storage and Stability
8˚C.
Linearity

73
Reference range
Serum -up to 49U/L
Reconstitute the reagent 2 (R2) with the volume of reagent 1 (R1) mentioned on the vial
label. The reconstituted reagent is stable for 50 days at 2-8˚c.
NOTE: Discard the working reagent if the blank absorbance is less than 1.00 at 340nm
Sample
Precaution
Sample 100µL
Table 2.9 SGPT test procedure
Calculation
SGPT activity (U/L) = ( OD/min) * 1768
SERUM GLUTAMATE OXALATE TRANSFERASE (SGOT)/ASPARTATE
AMINOTRANSFERASE (AST)
SGOT is present in most of the tissues. Especially in cardiac muscle, liver cells, skeletal
muscle and kidneys. Injury to these tissues results in the release of the enzymes in blood
stream.
Increased levels are found in myocardial infarction. The duration and extent of increase is
related to the infract. SGOT determination is of considerable value to differentiate
myocardial infraction from other cardiac disorders.
Increased levels are also found in various types of liver disease, skeletal muscle trauma
and in renal diseases.
Decreased levels may be found in pregnancy, Beriberi and diabetic ketoacidosis.
Principle

74
Kinetic determination of Aspartate Aminotransferase (AST) is based upon the following
reaction.
L-Aspartate + alpha-ketoglutarate-----AST/SGOT----------> oxaloacetate + L-glutamate
Oxaloacetate + NADH + H+---------MDH------------>L-malate + NAD+
AST= Aspartate aminotransferase
MDH= Malate dehydrogenase
Reagent Composition
SGOT R1 4*50Ml
Tris buffer (pH 7.8) 88mmol/L
L-Aspartate 260mmol/L
SGOT R2 4*50Ml
MDH >600U/L
LDH >900U/L
NADH 0.20mmol/L
Alpha-ketoglutarate 12mmol/L
8˚C
Linearity
Reference range
Serum -up to 46U/L
Reconstitute the reagent 2 (R2) with the volume of reagent 1 (R1) mentioned on the vial
label. The reconstituted reagent is stable for 30 days at 2-8˚c.
NOTE: Discard the working reagent if the blank absorbance is less than 1.00 at 340nm
Sample
Precaution
Sample 100µL

75
Table 3.0: SGOT test procedure
Calculation
SGOT activity (U/L) = ( OD/min) * 1768
TRIGLYCERIDES
Triglycerides are simple lipids, formed in the liver by glycerol and fatty acids. They are
transported by VLDL, LDL and constitute about 95% of fat, stored as a source of energy
in the tissue and plasma.
Increased levels are found in hyperlipidemia, diabetes, nephrotic syndrome and
hypothyroidism. Increased levels are risk factor for arteriosclerotic coronary disease,
peripheral vascular disease, acute pancreatic and hyperlipoproteinaemia. Decreased levels
are found in malnutrition and hyperthyroidism.
Principle
Enzymatic determination of triglyceride is based on the following equations:
TGL+H2O--------------> Glycerol + fatty acid
Glycerol + ATP------------------>Glycerol-3-phosphate + ADP
Glycerol-3-phosphate + O2------------>Dihydroxyacetone phosphate + H2O2
2H2O2 + 4-Aminoantipyrine + TOPS---------->Violet coloured complex
GPO= Glycerol-3-phosphate oxidase
LPL= lipoprotein lipase
GK= glycerol kinase
Reagent Composition
Triglycerides Reagent 5*25ml/4*50ml/5*100ml
Pipes-buffer (pH 7.00) 5mmol/L
TOPS 5.3mmol/L
Potassium ferrocyanate 10mmol/L
Magnesium salt 17mmol/L
4-aminoantipyrine 0.9mmol/L
ATP 3.15mmol/L
Lipoprotein lipase >1800U/L
Glycerol kinase >450U/L
Glycerol-3-phosphate oxidase >3500U/L
Peroxidase >450U/L
Triglycerides Standard 1 * 4Ml
Triglycerides standard concentration 200mg/dL

76
8oC
Linearity
This reagent is linear up to 1000mg/dL
If the concentration is greater than linearity (1000mg/dL), dilute the sample with normal
Normal range
Male 60-165mg/dL
Female 40-140mg/dL
Sample
Precaution
Standard - 10µL -
Sample - - 10µL
Table 3.1: Triglycerides test procedure
Calculation
Cholesterol concentration (mg/dL) = absorbance of sample/absorbance of standard * 200
HDL CHOLESTEROL
Lipoproteins are the proteins which mainly transport lipids in the blood stream. They are:
high density lipoproteins (HDL), low density lipoprotein (LDL), Very low density
lipoprotein (VLDL) and chylomicrons. LDL carries cholesterol to the peripheral tissues
where it can be deposited and increase the risk of atherosclerotic heart and peripheral
vascular disease. Hence high levels of LDL are artherogenic. HDL transports cholesterol
from peripheral tissues to the live and then for excretion, hence HDL has a protective
effect. Hence the determination of serum HDL cholesterol is a useful tool to identify
patients at risk of developing coronary heart disease.

Students' Industrial Training Report on Clinical Chemistry Tests

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