INTRODUCTION TO CLINICAL CHEMISTRY

Clinical chemistry, also known as chemical pathology, clinical biochemistry, or diagnostic

chemistry is a branch of pathology in medical laboratory sciences that focuses on qualitative and
quantitative tests on analytes or markers in body fluids and tissues using analytical techniques
and specialized instruments for diagnosis and treatment of diseases. The function of clinical
chemistry laboratory therefore is to perform qualitative and quantitative analysis on body fluids
and excretions such as blood, urine, cerebrospinal fluid (CSF) as well as faeces or stool, calculi,
sweat and other material.

Clinical chemistry discipline in medical laboratory sciences is concerned with the diagnosis of
diseases that influence metabolic processes of the body such as hormones, enzyme disturbances,
toxiclogical investigations and with monitoring of treatment, screening of apperentntly healthy
populations for pre-clinical diseases. It also involves interpretation of test results and ensuring
total laboratory quality management.

COLLECTION OF SPECIMEN

All clinical specimens are potentially pathogenic and therefore it is important that all necessary
precautions must be observed when collecting specimen. In addition, proper identification of the
patient must be made before collecting specimen. A properly filled request form must accompany
each specimen. Specimen collection is the process of acquiring body fluids or tissue for
laboratory analysis

Types of specimens

1. Blood specimens – specimen containers used for blood for biochemical analysis must be
leak proof and chemically clesn. Syringes and needlesfor collecting the blood samples
must also be chemically clean, dry and sterile. Blood specimen may be obtained from
veins, capillaries and artries.
 Venous blood- the median cubical vein in the anti-cubical fossa or crook of the
elbow is the preffered site for collecting venous blood in the adults since the vein
is both large and close to the surface of thw skin. Veins in the back of the hand or
the ankle can also be used.

Method:

 Select the vein by palpitation, clean the intended site with 70% alcohol and allow
the skin to dry.
 Apply the tourniquet 4-6 inches above the intended puncture site to obstruct the
return of venous blood to the heart
 Ast the patient to close his/her fist
 Insert the needle and draw the predetermined amount of blood

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  Untie the tourniquet, remove the needle from the vein, place a dry absorbent
cotton wool on the puncture site and ask the patient to apply a little pressure to
stop the bleeding.
 Remove the needle from the syringe and dispense the blood into the appropriate
container. Vein puncture is used when large quantity of blood, serum or plasma is
required.

Capillary Blood – this is used when small volume of blood is required. The tip of a finger, ear
lobe are site of collection in an adult or grown child, while in an infant less than one year old, the
lateral or medial planta surface of the foot should be used.

Method:

 Clean the site with cotton wool soaked in 70% alcohol and allow to dry
 Prick the site with a lancet making an insertion less than 2.5 mm deep
 Wipe off the first drop of blood and collect the subsequent blood into appropriate
container be gentle contact
NB: milking or squeezing or massaging of the site should be avoided because it causes
the flow of debris and tissue fluids that have a different composition from that of plasma.

Arterial blood collection – arterial samples can be obtained either through a catheter placed in an
artery or by using a needle and syringe to puncture an artery. The syringes are pre-heparinized
and handled to minimize air exposure that will alter the blood gas values. Several sites can be
used for arterial blood sampling in both adult and pediatric patients, however the preferred site is
the radial artery. Arterial blood is used for arterial blood gas analysis

TYPES OF BLOOD SPECIMEN

1) Whole blood – consists of red blood cells, white blood cells and platelets suspended in a
protective yellow fluid known as plasma. Whole blood transports oxygen and nutrients to
the lungs and tissues.
2) Plasma – plasma is the liquid portion of blood. it serves as a transport medium for
nutrients to the cells and organs and transport medium for waste products ro the kidneys,
liver and lungs for excretion. It plays an important role in maintenance of blood pressure,
distribution of heat throughout the body and maintenance of acid-base balance in the
blood and body. Plasma is derived when all the blood cells are separated from whole
blood through the use of an anticoagulant.
3) Serum – serum is the fluid component of blood without the blood cells and clothing
factors, but it contains all proteins not used in clothing factors, all electrolytes, antibodies,
antigens, hormones and any exogenous substances. Serum is used in many diagnostic
tests as well as blood typing. To obtain serum, a blood sample is allowed to clot, the
sample is then centrifuged to remove the clot and blood cells, the resulting liquid
supernatant is serum.

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2. URINE COLLECTION

The type of urine to be collected depend on the test to be performed. Collection and
transportation of urine specimens to the clinical laboratory are important because variables
such as collection method, container, transportation, and storage affect the analysis outcome
and consequently diagnostic and therapeutic decisions based on the results. Clinical
laboratory staff are responsible for patient instruction, collection and labeling of urine
specimens and timely transportation of specimens to the Laboratory when necessary. Urine
samples can be collected in the following ways in the laboratory.

1. Early morning urine specimen – The first urine voided in waking up in the morning is
refferd to as early morning specimen. This specimen usually contains the highest
concentration of substances than others passed later in the day. It is therefore suitable for
easy identification of abnormalities.
2. Random Urine Specimen - For chemical and microscopic examination, a random urine
specimen is usually more suitable. This is a urine specimen collected at any time of the
day. A randomly collected specimen may be collected at unspecified times
and is often more convenient for the patient. A random specimen is suitable for
most screening purposes e.g for the detection of glucose, blood or bilirubin in urine.
3. Midstream “clean catch” specimen.
This specimen provides a safer, less traumatic method for obtaining urine for
bacterial culture. It also offers a more representative and less contaminated
specimen for microscopic analysis than the random specimen. Mid stream urine is
collected by passing the first part of urine into the toilet bowl during urination and the
collect the middle part into a clean sterile container, the remaining urine flow is passed
into the toilet bowl. A cleansing material can be used to collect mid-stream urine.
4. Timed urine specimen – This is a type of urine collected at a specified period of time or a
predetermined interval of time. Examples of timed urine include: 24 hour urine
collection, 2hr post-prandial urine collection or 30 minutes/1 hour interval urine
collection for glucose tolerance test. Before beginning a timed urine collection, a patient
should be given instruction on the methods of collection, factors that can affect the
validity of the test result like drug ingestion, diet or other activities that can interfere with
the analytical procedures.
24 hour urine collection – This is a timed urine collected for a period of 24 hours for the
quantitative analysis of substances such as hormones, phosphates, calcium, and protein. It
necessary to collect a 24 hour urine because the concentration of some substances in
urine varies from sample to sample in a person and so a pooled specimen for a period of
24 hours will give a more accurate quantity of a substance in a patient. The successful
collection of a 24 hour urine depends on explaining the procedure fully to the patient.
This procedure is as follows:

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 
Instruct the patient at a specific time, usually 8am to empty his/her bladder,
discard the urine , this should not be part of the collection.
 Give the patient a large (2-3 liter) wide mouth well container containing the
appropriate preservative
 Instruct the patient to collect into the container all the urine he/she passes in the
next 24 hours upto and including the urine passed at 8.am the following morning.
 The urine should reach the laboratory as soon as possible after collection. If a
delay is unavoidable, the specimen must be refrigerated at 2-8 oC until it can be
delivered to the laboratory.
 The container must be well labeled with the patient’s name, hospital/lab number,
the date and time of collection and completion.
3. COLLECTION OF FAECES

Small aliquots of stool are frequently analyzed in clinical chemistry laboratory to detect the
presence of occult blood as an effective clue to the presence of bleeding ulcer or malignant
disease in the gastro intestinal tract (GIT). The patient is given a stool sample container to collect
a little quantity of stool into the container for the analysis. Random specimen of stool are
collected for occult blood test.

A 72 hour stool specimen can also be collected in an adult for the measurement of feacal
nitrogen and faecal fat. This is done to assess the severity of malabsorption.

4. COLLECTION OF CEREBROSPINAL FLUID

Cerebrospinal fluid (csf) is normally obtained from the lumber region. The sample is collected
into sterile tubes or containers to avoid contamination. The chemical analysis or investigations
commonly required are protein, glucose and electrolytes. Rapid processing of specimen is a
clinical requirement for the test on spinal fluid. The specimen must be handled with utmost care
to avoid repeat of collection.

SEPARATION AND STORAGE OF SPECIMEN

Plasma or serum should be separated from cells as soon as possible within two hours of blood
collection.

1) Separation of plasma
 Tube with an anti coagulant eg: Edta (lavender top)sodium heparin (green top), sodium

citrate (blue top) are used for separating Plasma. Separation should be done within one

hour of receiving the specimen.

 Collect whole blood into tube with the appropriate anticoagulant

 Mix the blood with the anticoagulant gently or with roller mixer

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  Centrifuge at 200rpm for five minutes
 After centrifugation, separate the plasma from the blood cells gently with clean Pasteur
pipette into a dry clean specimen tube.

Separation of serum

 Whole blood is collected into a plain dry tube or into a serum separator tube using
standard procedures
 Allow sample to clot for one hour at room temperature
 Centrifuge for 10 minutes at 2000rpm
 Using a clean pipette transfer the serum gently into a clean dry specimen tube

STORAGE OF SPECIMEN

Serum or plasma samples are stored in capped tubes and kept at room temperature if they are to
be analyzed immediately. If tests on serum or plasma samples are not analyzed immediately
within eight hours of collection, they are stored at temperatures of +2 oC to +8oC (refrigerator).
Tests should be carried out within seven days if samples are stored at +2 to +8 oC. samples are
stored at -15oC to -20oC when analysis are delayed for two weeks to one month. This is to
maintain stability of the specimen and to reduce evaporation. If analysis is delayed for more than
a month or above, the samples should be stored at -70oC to -80oC.

Note:
Universal Precautions must be observed when working with blood. Use of personnel
Protective equipment is mandatory.

ANTICOAGULANTS
Anticoagulants are chemical substances which when added to whole blood prevents clotting
either by removal or inactivation of one of the steps in coagulation. Blood cells clots within 5 to

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10 minutes outside the human body, so if whole blood or plasma is needed for laboratory
analysis an anticoagulant must be added to the specimen immediately it is withdrawn in other to
inactivate or remove one of the steps involved in the coagulation process.

WORKING PRINCIPLE OF ANTICOAGULANT

Most of the anticoagulant commonly used acts by removing the calcium ions present in the blood
which is required for coagulation process. The anticoagulant binds with the calcium and thus
prevents blood from clotting. Also some anticoagulants are used for specific assays due to their
unique properties such as fluoride which inhibits the activities of glycolytic enzymes responsible
for the breakdown of glucose in the blood and provide precise results for blood sugar estimation.

TYPES OF ANTICOAGULANT
There are different types of anticoagulant and the type of investigation must be considered in
choosing the anticoagulant to be used since it can eliminate, inactivate or interfere with the
substance being estimated. Every anticoagulant is added in fixed proportion to blood. The
following are the most commonly used anticoagulant in the laboratory for routine analysis:
1. Heparin – This is a natural or physiological anticoagulant which cannot be prepared in
the laboratory. It is obtained from leech. Heparin is present in most of the body tissues,
but in concentration less than that required to prevent clotting. It is available as sodium,
potassium, lithium and ammonium salts. It is the most widely used anticoagulant in
clinical chemistry. It is used in 0.1 to 0.2mg/ml of blood. The heparinized blood is used to
determine blood gas analysis. ESR, PCV, osmotic fragility test etc. it is a very costly
anticoagulant.
Mode of action: It interferes with the activity of thrombin and inhibits the thromboplastin
formation and destroys the thrombin (anti-thrombin activity) and hence disrupts the clothing
mechanism.
2. ETHYLENE DIAMMINE TETRA ACETIC ACID (EDTA) – it is a widely used
anticoagulant in the laboratory especial haematology laboratory. EDTA acts as a chelating
agent that binds to calcium ions. There are two forms of EDTA namely tri-potassium
(K3EDTA), Di-sodium (Na2EDTA). It is used at a concentration of 0.5 to 2.0 mg/ml of
blood. It is mainly used for blood cell counts. It gives better preservation of cellular
morphology of blood cells and inhibits clumping of platelets.
Mode of Action: it acts by chelating calcium forming insoluble calcium salt thereby preventing
blood clotting.
3. TRI-SODIUM CITRATE

It was first used as an anticoagulant in blood transfusion in earlier 20 th century. Its usefulness
continues till today in blood collection tubes for ESR estimation by Westergren method and
coagulation studies as well as for the preservation of blood in blood banks. It has little
application on clinical chemistry laboratory. 3.8gm/dl is used in a ratio of 0.5ml to 2ml of blood.

Mode of Action – the action of citrate ions is to form calcium citrate complexes which disrupt
the blood clotting mechanism by chelating or biding with the calcium ion in the blood and
inhibits the coagulation of blood.

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 4. OXALATES – This can be available as sodium, potassium, and ammonium oxalate. It
can be used as single oxalates but are commonly used as double oxalates like potassium
oxalate and ammonium oxalate. This is because when used alone can cause shrinkage or
swelling of red cells. 0.5ml of dried double Oxalates to 5ml of blood. This anticoagulant
can be used for blood chemistry, PCV, ESR, white blood cell count.

Mechanism of Action – it acts as a chelating agent and binds with the calcium ions present in
the blood and forms insoluble precipitates of calcium oxalates.

5. SODIUM FLUORIDE – this is the anticoagulant of choice for the estimation of blood
glucose. It stabilizes glucose in plasma. 6mg/ml of blood is used.

Mode of Action – It binds with calcium ions present in the blood and forms calcium fluoride.
Also it is commonly used whenever blood glucose estimation is required because sodium
fluoride is the competitive inhibitor of phosphorylase enzymes and prevents glycolysis by
blocking its activity.

6. IODOACETATE – this is used as sodium iodoacetate. It inhibits glucose -3-phosphate

dehydrogenase activity in the sample thereby preventing glycolysis. It can be substituted
with sodium fluoride. 0.2mg/ml of blood is used.

PRESERVATIVES

Preservatives are chemicals which have the ability to retain the chemical integrity of specimen
by reducing bacterial growth or chemical decomposition. Also it decreases atmospheric oxidation
of unstable compounds in the specimen and solubilize constituents that might precipitate out of
solution.

Some preservatives commonly used are:

1. Refrigeration – This is generally used for preservation of serum or plasma samples, urine
samples and stool samples. When there is a delay in examining urine or stool, refrigerate
at 2-8oC. Urine can be freezed at -20 to -16 oC. Refrigeration of urine does not interfere
with the urine chemical tests and it prevents bacterial growth.
2. Hydrochloric acid – 6N HCl is used to preserve a 24hr urine for quantitative analysis of
calcium, creatinine etc
3. Boric acid – used for preservation of 24hr urine especially for protein quantitation.
5g/30ml of urine
4. Formalin (formaldehyde) useful for urine sediments
5. Sodium fluoride – acts as a preservation in blood glucose estimation
6. Phenol – for urine preservative
7. Toluene
8. Thymol

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 9. Sodium carbonate
10. 10% aqueous formalin – for stool preservative
11. Polyvinyl-alcohol for stool

SOLUTIONS

A solution is a homogenous mixture of one or more solutes dissolved in a solvent. Solutions are
prepared by dissolving a solute in a solvent.

Types of Solutions

1. Standard solution – a standard solution is a solution containing a precisely known

concentration of a substance. Standard solutions are integral part of every qualitative
analysis in the clinical chemistry laboratory and are used during sample analysis to
determine the concentration of substances in the sample or specimen. The
concentrations of standard solutions are expressed in mol/L, mmol/L, mg/dl etc. There
two types of standard solution namely:
 Primary standard solution – This is a solution of accurately known concentration
prepared from chemicals of high purity, which can be weighed out directly for the
preparation of solutions of selected concentration or for the standardization of solutions
of unknown concentration. These chemicals must be stable substances of definite
composition which will not change its composition during weighing and they must not be
hydroscopic. Example sodium chloride (NaCl), sodium carbonate (Na2CO3) etc.
 Secondary standard solution – These are solutions whose concentration cannot be
prepared directly by weighing the solute and dissolving a known amount into a volume of
the solvent. The actual concentration is obtained by standardization of an aliquot with a
primary standard mainly by titration. E.g NaOH, HCl etc.

TYPES OF SOLUTIONS

1. Molar solution – this is the molecular weight of a substance in gram dissolved in 1 liter of
a solvent. It is written as Mol/L or M. example 1M of NaCl is prepared by dissolving
58.5 (molecular weight) of NaCl salt in 1 liter of distilled water.
2. Millimoles per liter (Mmol/L) – This expresses the concentration of solutions in terms
of the molecular weight in milligrams (mg) per liter of solvent. Example the molecular
weight of NaCl is 58.5, therefore a solution of 1mmol/L of NaCl contains 58.5mg of
NaCl in 1 liter of solvent.
3. Percentage solutions – a percentage solution is a type of solution that expresses the
amount of solute dissolved in a given amount of solvent as a percentage of the total
solution. It is used in the laboratory to represent the concentration of a solution.
Percentage solutions can be prepared in various ways as follows:

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  Weight
/volume (W/V%) – contains Xg of solute dissolved and made upto100ml of
solvent. E.g. 20% of Na2CO3 is made by dissolving 20g of solid Na 2CO3 in
distilled water and making up the final volume to 100ml with distilled water.
 Volume/Volume (V/V%) solution – this contains Xml of solvent that is made up to 100ml
with distilled water or another solvent. E,g, 30% alcohol is prepared by
measuring 30ml of alcohol and making up the volume to 100ml with distilled
water.
 Weight /weight (W/W%) – this contain Xg of solute in 100g of solvent
4. Normal solution (N) – this is a solution containing equivalent mass of the solute in 1
liter of solvent. One normal solution (1N) is Gram molecular weight divided by the
valence per liter of solvent. Gram molecular weight/valence per liter of solvent. E.g to pre pare 1N
solution of SO42- = Molecular weight of SO4 = (S=32+, O=16x4=64)=96, valence =2
therefore 1N of SO4 = 96/2 = 48g of SO42- dissolved in 1 liter of solvent.
5. Isotonic solution – this is a solution that exert equal osmotic pressure as blood. It is any
external solution that has the same solute concentration or osmolarity and water
concentration compared to body fluids. An example of isotonic solution is normal saline
or physiological saline which is prepared by dissolving 8.5 g NaCl in 1 liter of distilled
water.

COLORIMETER AND SPECTROPHOTOMETER – principle, component and

application

A colorimeter is an instrument used to measure the absorbance of light of a coloured solution.

It is widely used method for finding the concentration of biochemical compounds or solutes
in a coloured solution. It measures absorbance of light between 400 to 700nm (nanometer) i.e
visible spectrum of light.

Some basic definitions

1. Wavelength – light travels in form of waves. Wavelength is the distance between two
wave peaks measured in nanometers (nm).

The wavelength of light determines the colour of the light seen by the naked eye.
Wavelength between 400 to 700nm forms the visible spectrum which is visible to the
human eye. When this visible white light passes through a prism, they are separated into a
spectrum of colours: red, orange, yellow, green, blue, indigo and violet. Red colour has
the longest wavelength of 700nm while violet has the shortest wavelength of 400nm.
Wavelengths less than 400nm (<400nm) are not visible to the naked eye and are referred
to as ultraviolet e.g. sunlight, fluorescent light. Wavelengths above 700nm (>700nm) are
also not visible to the naked eye, they are referred to as infra-red e.g. microwave, remote
control, electric heaters etc.

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 2. Absorption or optical density – formally the amount of light absorbed by a coloured
solution is referred to as optical density (OD). The OD is measured using a
colorimeter or spectrophotometer. The measurement of OD is based on the Beer-
Lambert’s law. OD measurement is applied to calculating the concentration of a
substance in an unknown test solution by using the formula:

Optical density of test

X concentration of standard

Optical density of standard

PRINCIPLE OF COLORIMETER

The principle of colorimeter is based on the theories proposed by two scientists Beer and
Lambert.

 Beer’s law – This law states that the amount of light absorbed by a coloured solution is
directly proportional to the concentration of solute in the solution.
 Lambert’s law – This law states that the amount of light absorbed by a coloured solution
is directly proportional to the length and thickness of the solution under analysis.

The working principle of colorimeter and spectrophotometer is based on the combination of

Beer’s law and Lambert’s law which states as follows:

Beer-Lambert’s law – States that the amount of light absorbed by a coloured solution is directly
proportional to the concentration of the solution and the length of the light path through the
solution.

Mathematically expressed: A=ƐLC

Where:

A= absorbance or optical density

Ɛ = molar absorption coefficient which is specific and constant to every chemical and
wavelength used.
L= light Path
C = concentration
If the same light path is used for all measurement by using the same cuvette to hold all solutions
to be measured then
A=C
Therefore AT = CT (Absorbance of Test = Concentration of Test)
And AS = CS (Absorbance of Standard = Concentration of Standard)

So AT x CS = AS x CT
CT = AT x CS
AS

Where: CT = concentration of Test

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 AT = Absorbance of Test
CS = Concentration of Standard
AS = Absorbance of Standard
The above is the basic equation for the determination of concentration of analytes in clinical
chemistry laboratory.

Application

 Used in determination of the concentration of analytes in body fluids like blood,

urine, serum etc. example blood glucose, urea, cholesterol, creatinine.
 Used by the food industry and by manufacturers of paints and textiles
 For the determination of course of reaction by measuring the rate of formation
and disappearance of light absorbing compounds.

SPECTROPHOTOMETER

A spectrophotometer is an instrument that measures the intensity of light absorbed after it passes
through sample solution. With the spectrophotometer, the concentrations of a known chemical
substance can be determined by measuring the intensity of light detected. The spectrophotometer
is based on the Beer-Lambert Law which states that the amount of light absorbed in a solution is
directly proportional to the concentration of the solute in the solution and thickness of the
solution under analysis. Spectrophotometry is most commonly applied to ultraviolet, visible,
and infrared radiation. Spectrophotometry is an important technique used in many biochemical
experiments that involve DNA, RNA, and protein isolation, enzyme kinetics and biochemical
analyses.

Differences between colorimeter and spectrophotometer

colorimeter spectrophotometer
1. It uses wavelength in the visible range It uses wavelengths in the ultraviolet, visible and
(400 – 700nm) infra-red range (200-800nm)
2. Wavelength is selected using a coloured Wavelength is selected by using a monochromator
filter
3. It consists of a sensor and data It consists of a sensor, data processor and a
processor computer
4. Does not use prism or gratings It uses prisms or gratings for monochromatic light
5. It is less expensive and less complex It is more expensive and complex
6. It is less sensitive, less precise and less It s more sensitive, precise and specific
specific

ESSENTIAL PARTS OF A COLORIMETER/SPECTROPHOTOMETER

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 1. Light source – this is a simple tungsten lamp/bulb that provides light energy of the
required intensity in the visible and non visible range of the spectrum. This light energy is
separated into the proper wavelength by the monochromator or filter, which then falls on
the solution to be measured.
2. Slit system – they confine the light energy beam to a narrow path and help exclude
energy from stray light
3. Monochromator /filters – this isolates specific wavelength of light emitted by the light
source. This is achieved by a prism or grating in spectrophotometer or by filters in
colorimeters. The filters are made of glass or dyed gelatin
4. Cell or cuvette – this is used to hold the solution to be measured. There are two types, the
cylindrical or the rectangular type
5. Photocell detector – this measures the light intensity. It helps in converting the resulting
transmitted light rays once it passes the sample solution into an electrical signal which is
proportional to the light intensity. There are three types namely selenium photocell,
photomultiplier tube and silicon photocell.
6. Galvanometer – it measures the electric signal generated by the photocell detectors and
displays the value in the display area. The display area can be analogue or digital.

FLAME EMMISSION PHOTOMETER

Flame emission photometer is most commonly used for the quantitative measurement of sodium
potassium and lithium in body fluids.

Principle – When an element in its atomic state is placed in a flame, the atoms increase in
energy, become exited, and emit energy in the form of light in order to return to their original
energy level. The light emitted is specific in wavelength for each atom (sodium emits light of
wavelength 589nm (yellow), while potassium emits light of wavelength 767nm (violet). The
intensity of the emission is directly proportional to the number of atoms returning to their
original energy level. The amount of light emitted is in turn directly proportional to the
concentration of sodium and potassium present in the sample.

COMPONENTS OF A FLAME PHOTOMETER

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 1. Flame – this is provided by a compressed gas. High pressure tubing must be used to lead
the gas to the flame. The temperature of the flame is one of the critical factors in flame
photometry
2. Atomizer/nebuliser – this is used to spray the sample into the flame as fine droplets. The
atomizer or nebulizer provides a means of drawing the sample through the aspirator and
converting it into a fine mist which then enters the flame.
3. Optical system – this consists of convex mirror and convex lens. The convex mirror
transmits the light emitted from the atoms and also helps to focus the emissions to the
lens. The lens helps to focus the light in the slit
4. filters – the reflections from the mirror pass through the slit and reach the filters. Filters
isolates the wavelength to be measured
5. photo detector – the intensity of radiation emitted by the flame is measured by the photo
detector. The emitted radiation is converted to an electrical signal with the help of the
photo detector. These electrical signals are directly proportional to the intensity of light.
6. Galvanometer - it measures the electric signal generated by the photocell detectors and
displays the value in the display area.

Procedure

1. The sample is diluted in de-ionized water which is converted to an aerosol by the aid of
the atomizer/nebulizer.
2. The aerosol is mixed with gas in the burner

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 3. The mixture is then ignited in the flame, the heat from the flame releases free atoms from
the aerosol and increases their energy level
4. As the atoms return to their ground state, they emit energy in form of light
5. The emitted light is then measured by the detector which is proportional to the
concentration of the ions (Na+ and K+) in the sample

PROTEIN FREE FILTERATE

A protein free filtrate is a sample of blood, serum or plasma from which all proteins have been
removed by chemical or physical, dialysis, ultra filtration or solvent extraction methods. Proteins
interfere with some blood tests and may alter the results of some investigations, therefore the
need to remove proteins or make protein free filtrate before performing these laboratory
investigations. Some blood tests that require protein free filtrate include:

 Estimation of blood urea

 Estimation of glucose level in serum or plasma
 Estimation of serum creatinine
 Estimation of uric acid level in serum

Preparation of protein free filtrate (PFF)

PFF is prepared by the precipitation of protein. There are two methods used

1. Precipitation by heavy metals – proteins acts as an anion when its PH shifts towards
alkalinity. Therefore they can be precipitated using heavy metals like copper, silver etc
2. Precipitation by large anions – proteins can be precipitated using large anions such as
tungstic acid, tri-chloro acetic acid (TCA), phosphortungstic acid etc. this method is
based on Folin-Wu method for PFF.

Precipitation by large anions (Folin Wu Method)

Principle – proteins are precipitated by large anions like tungstic acid or tri-chloro acetic acid
(TCA)

Method:

 Collect 5ml of blood and obtain the serum or plasma

 In a 50ml centrifuge tube pipette 4.5ml of 5% TCA or 2.5ml of tungstic acid
 Add 0.5ml of serum or plasma
 Cover and mix vigorously by rotating the tube until precipitate forms
 Filter or centrifuge for 5-10 minutes at 2,000 rpm
 The supernatant should be clear and colorless

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 Separate the clear colorless supernatant fluid from the sediment into a clean and
sterile sample tube using a Pasteur pipette
 This is a PFF, label and store at 2-8oC

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