PREFACE

This laboratory manual was designed for the third-year medical technology students
involved in clinical chemistry. The emphasis is on the proper collection of specimens,
method to be used, sources of error, reference ranges, clinical significance and quality
control system. Since laboratory procedures vary among institutions, the principle of the
procedure is included to guide the students.
It is my hope that this manual will help students understand the importance of
laboratory testing to prepare them better for their role in the clinical laboratory.

Nenita SJ Eustaquio
 TABLE OF CONTENTS

Activity Title Rating

number
1 Basic Equipment, Glassware’s and Reagents

2 Preparation of Concentration of Solutions

3 Collection, Preparation and Handling of Specimens

4 Preparation of Protein Free Filtrate

5 Colorimetric Technique

6 Quality Control System

7 Blood Glucose Determination

8 Cholesterol Determination

9 Blood Urea Nitrogen Determination

10 Uric Acid Determination

11 Creatinine Determination

12 Total Protein Determination

 LABORATORY SAFETY PRACTICES

"All blood and body Fluids are considered potentially infected with blood-borne pathogens"

1. Smoking, eating, and application of cosmetics are prohibited to prevent the spread of
hand to mouth infectious agents or toxic chemicals.
2. To prevent contact with or spread of infectious agents, personal protective equipment
such as laboratory gown and gloves must be used
3. Contact lens should be discouraged since these will allow substances to remain on the
cornea longer.
4. Goggles or face shields are recommended if contact lenses are worn, especially if there
is a high risk of fumes, aerosols, or splashes.
5. Long hair must be secured with a tie and avoid wearing dangling jewelry since this may
pose safety hazard by coming in contact with specimens.
6. Mouth pipetting is strictly prohibited.
7. During biological spill cleanup always wear gloves and a lab gown, disposable towels
should be placed over the spill and disinfectant poured on the towel. Recommended
disinfectant is 10% bleach.
8. If a chemical is splashed in the eye, go to the nearest eye wash and wash eye for 5
minutes. Then seek medical attention
9. If a chemical is splashed on the person or clothing, go to the nearest body shower and
rinse for 15 minutes. Then seek medical attention
10. Infectious waste should be segregated from the regular trash and placed in infectious
waste containers clearly marked with biohazard label
11. Needles and sharps should be place in rigid, containers that can be sealed with a tight-
fitting lid.
12. Infectious waste can be treated by incineration, autoclaving, or discharge into the
sanitary sewer system.
13. Flammable liquids such as xylene, ether, peroxides mercury and other heavy metals
should not be flushed down the drain. These should flow to wastewater treatment plant
with at least 100-fold excess of water to dilute the chemical.
14. Chemicals should be stored in an uncluttered area that is properly ventilated and away
from a heat source. They should not be stored above eye level.
15. All chemical containers should be clearly labeled with the name of the chemical and any
precautions for handling. Separate chemicals according to flammable solvents,
Corrosives and Acids.
16. Fume hoods are used when chemical reagents may produce a hazardous fume.
17. Report any small shocks; unplug and tag equipment until serviced. If a severely shocked
person cannot let go of instrument, unplug it or knock person loose with nonconductive
material, such as wood.
18. Replace all frayed wires and plugs
19. Dispose of all radioactive material in appropriate labeled container
20. In case of fire, close doors to contain the fire, Pull the nearest fire alarm, find the nearest
fire extinguisher and attempt to put off the fire
Name: Nurhaliza P. Halid Section: BSMT-3B Date: Oct. 11, 2023

Activity 1

Basic Laboratory Equipment, Glassware's and Chemicals

Objective: At the end of the activity, the learner should be able to;

1. Identify the different equipment, glassware, and chemicals used in clinical chemistry.
2. Explain the operating principle and safety measures of each piece of equipment.
3. Describe the different reagents used in laboratory analysis.

A. Chemicals Grade:

Chemicals exist in varying degrees of purity. For quantitative measurements and

preparation of accurate standard solutions, it is essential to use pure chemicals and identify
exact amounts of contaminants. For the most highly purified chemicals, the American Chemical
Society (ACS) established the specifications of chemicals in the "reagent grade," "analytical
reagent grade" category. The United States Pharmacopoeia (USP) and National Formulary
(N.F.) represent other purity grades and mean that these chemicals meet the stipulations listed
in the United States. Although, at the same time, they are adequate for human consumption,
they may not be pure enough for specific chemical applications.

B. Standards:

A Primary Standard (P.S.) is a chemical for the highest purity and can be measured
directly to produce a substance of exactly known concentration. The International Union of Pure
and Applied Chemistry (IUPAC) requires that P.S. be at least 99.98% pure and that the Working
Standards be 99.95%.

C. Water Specifications:

Reagent Grade Water (RGW) is water suitable for Reagent and standard preparation. Most
procedures use distilled or deionized water.

Type I - is used for procedures that require maximum water purity, such as;
● preparation of standard solutions

● ultra-microchemical analysis

● measurement of nanogram or sub nanogram concentration

● tissue or cell culture

Type II - used in most laboratory determinations that do not require Reagent grade 1 water.
For chemistry, hematology, microbiology, immunology, and other chemical laboratory areas.

Type III - used in most qualitative measurements/examinations, in urinalysis, parasitology

and histology, washing glassware.
D. Glassware’s:

● Types of Glassware’s as to the texture

a) Borosilicate glass – the most common type encountered in volume measurements. They
have a high degree of thermal resistance and have low alkali content. Ex. Pyrex and Kimax

b) Alumina-silicate – they are strengthened chemically rather than thermally. They are six
times stronger than borosilicate glass. Ex. Corex

c) Corning Boron Free Glass - is soft glass, poor heat resistance but highly resistant to alkali.
d) Low Actinic glassware – are of high thermal resistance with a red color, protecting light-
sensitive materials.

1. Pipets - in general, pipets fall into two classes:

a) Volumetric or transfer pipets – calibrated for one specified volume measurement; either "to
deliver" (T.D.) or "to contain (T.C.), they are easily distinguished by the bulb-like
enlargement partway up the stem. Contents of T.D. Pipets are drained freely or blown out.
Blow-out pipets are indicated by the opaque ring near the mouthpiece of the pipet. ex.
Volumetric pipet, Ostwald Folin pipet, Pasteur pipet, Automatic macro/micropipettes

b) Graduated or measuring pipets – are long, cylindrical tubes drawn out to a tip and are
calibrated in uniform fractional volume measurement. They are either Mohr or serologic.
The Mohr pipet is calibrated between two marks on the stem while the serologic pipet is
calibrated up to the tip.

c) Automatic pipetting devices permit rapid, repetitive measurement and delivery of equal
volumes.

2. Volumetric glassware

a) A volumetric flask is calibrated to hold one exact volume of liquid.

b) Erlenmeyer flasks graduated cylinders, and burets are designed to hold a variety of
volumes

E. Balances

a) Single-pan analytical balance operates on the principle of weighing by substitution.

b) Single pan top-loading balance always operates by the principle of weighing by

substitution.

c) Electronic balances operate on the principle of electromagnetic force compensation.

F. Centrifuge – is a machine that uses centrifugal force to separate phases of different

 densities.

G. Dry Oven Sterilizer – used to dry glassware.

I. Single tube mixers – a vortex mixer is capable of a variable speed oscillation which results in
a swirling motion to liquid contents of a test tube or other container.

J. Thermometer

K. Refrigerator

L. Needles and Syringes

K. Test tubes/vacutainer tubes

Drawing: Sketch and Label the different equipment’s and glassware used in Clinical Chemistry

MICROSCOPE BLOOD ANALYZERS

___________________________________ __________________________________
PIPPETTE VOLUMETRIC FLASK

GRADUATED CYLINDER VACUTAINER TUBES

SYRINGE AND NEEDLE TEST TUBE

DRY OVEN STERILIZER CENTRIFUGE

THERMOMETER TEST TUBE MIXER

Name: Nurhaliza P. Halid Section: BSMT-3B Date: Oct. 11, 2023

Activity 2

Laboratory Calculations

Objective: At the end of the activity, the learner should be able to;

1. Prepare different concentrations of solutions.

A Solution is a homogenous molecular mixture of 2 or more substances. Solutions may be

classified as; Dilute, Concentrated, Saturated, Supersaturated and Standard.

Percent Solutions are defined as the amount in grams (solid) or volume in millimeters (liquid)
of solute per 100 ml of solution. There are 3 types of % solutions expressed in physical unit
these are weight per weight (% w/w), volume per volume (% v/v), and weight per volume (%
w/v)

Concentration of solutions may also be expressed in chemical unit namely Normal solutions
which contains one-gram equivalent weight of solute in one liter of solution, and Molar solutions
which contain one mole or I gram molecular weight of solute per liter of solution.

Dilution of Solution – involves making a weaker solution from a stronger one. It is usually
expressed as 1 unit of the original solution to the total number of units of final solution.
Therefore a 1:10 dilution calls for 1 unit of concentrated solution to be diluted to a total volume
of 10 units.

Standardization – may be prepared directly by dissolving a weighed amount of a pure, dry

chemical and diluting the solution to an exactly known value.

Procedure:

1. Prepare 10% w/v solution of NaCl

2. Prepare 10% v/v solution of HCL

3. Prepare 10% w/w NaCl aqueous solution

4. How much 95% alcohol is required to make 200 ml of 5% alcohol?

5. Prepare 500 ml of 0.5 N H2SO4

6. A 5N solution is diluted 1:5, rediluted 2:10 and rediluted once more 3:15. Give the resultant
concentration.
Name: Nurhaliza P. Halid Section: BSMT-3B Date: Oct. 11, 2023

Activity 3

COLLECTION, PREPARATION, AND HANDLING OF LABORATORY SPECIMENS

Objective: At the end of the activity, the learner should be able to:

1. Perform the different methods for blood collection.

2. Prepare different blood sample specimen

In general, blood for clinical analysis should be drawn in the fasting state. Therefore, an
overnight fast of 8 to 12 hours is ideal. The types of samples are whole blood (collected with
anticoagulant), plasma, and serum.

The number of samples are as follows:

1. macro method – 1 ml and above

2. micro method – 0.1 ml – 0.9 ml (100 ul – 900 ul)
3. ultramicro method – 0.01 – 0.09 ml ( 10 ul – 90 ul )
4. nanoliter method – 0.001 – 0.009 ml ( 1 ul – 9 ul )

Collection of samples may be through:

a. Capillary Method or Prick method for micro, ultramicro, and nanoliter technique from
the finger tip, earlobe, or the toe and heel plantar surface.

b. The venipuncture Method for the macro method is taken from the basilic and
cephalic veins of the antecubital forearm, femoral veins, ulnar and radial veins at the
dorsum of hand and foot.

c. Arterial Method - macro method from the radial artery.

Procedure:

With a partner, perform the capillary and venipuncture methods of collecting a blood
sample. Make an illustration and describe the steps in blood sample collection.
Questions:

1. Draw and label the essential parts/components of a syringe and vacutainer set (adaptor
with needle)

2. Why is serum preferred over plasma and whole blood in blood chemistry analysis?

This is because our chemistry reference intervals are based on serum, not plasma. In general,
there is little difference between serum and plasma, except for certain analytes.

3. What are the different biologic factors that will affect the result of blood chemistry?

The most common may affect the result of blood due to the diet, hunger, exercise, posture, daily
and seasonal fluctuations, menstrual cycle, and pregnancy are the most prevalent of these
elements.

5. What are some changes in the blood that may occur upon long-standing?
Additionally, moving from lying down to standing increased haemoglobin, hematocrit, red blood
cell, leukocyte, neutrophil, lymphocyte, basophil, and platelet counts as well as mean platelet
volume clinically significantly. Moving from sitting to standing also increased haemoglobin
clinically significantly.

5. Give some common causes of hemolysis

In order to prevent cross-contamination between the various additives present in blood

collection tubes, the procedure for drawing blood is now postulated according to international
guidelines. The order is as follows: blood culture bottle, non-additive tube, coagulation tube, clot
activator, serum separator tube, sodium heparin, plasma separator tube,
ethylenediaminetetraacetic acid (EDTA), blood tube, and oxalate/fluoride.

6. What is the correct "order of draw"? Enumerate the different anticoagulants and respective
actions.
In order to prevent cross-contamination between the various additives present in blood
collection tubes, the procedure for drawing blood is now postulated according to international
guidelines. The order is as follows: blood culture bottle, non-additive tube, coagulation tube, clot
activator, serum separator tube, sodium heparin, plasma separator tube,
ethylenediaminetetraacetic acid (EDTA), blood tube, and oxalate/fluoride.

7. What are the sites to be avoided when performing venipuncture

-obvious skin infection (including newly tattooed areas)

-extensive scarring
-don't use same side as mastectomy
-avoid previous hematoma
-avoid arm w/ fistula/cannula -edamtous extremity