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LESSON 4

CLINICAL CHEMISTRY SUPPLIES &

— google.com REAGENTS

= A medical technologist manipulates a number of equipment, encounters different

glassware, and prepares and uses reagents in clinical chemistry laboratory in order
to
perform different procedures on biological specimens. Some of these supplies maybe
familiar to you, however, there is a need to carefully consider the proper
operation or
usage to avoid laboratory- acquired injuries, damage to property, and to produce
the
desired clinical chemistry results. Injuries and damage to property can lessen
laboratory
productivity and entails additional expenses on the part of the injured
laboratorian. On the
other hand, deviation from the true value can greatly affect the diagnosis and can
lead to
improper patient management and treatment.

I. SUPPLIES
The current chemistry laboratory has shifted mostly to automation. Some of these
supplies may not anymore used, but it is still important that a medical
technologist to be
knowledgeable of their operation and use.

A. Laboratory Balance
A properly operating balance is vital in producing high-quality reagents. Its use
has
minimized because most chemistry laboratories are now purchasing commercially
prepared reagents.

o General rules in using balances:

1. Balances must be placed in area where temperature is constant, free of

corrosive fumes and dust, absence of strong vibrations.

2. Keep balances clean, wipe up any spills promptly.

3. Avoid jarring the instrument. Position in on a sturdy table not close to a

source of heat and must not be exposed to sunlight

4. Zero point must be rechecked before and after weighing

5. Forceps should always be used to handle weights.

6. Calibrate balances regularly through the use of test weights from appropriate
ANSI/ASTM classes 1 to 4.

o Balances are categorized according to design, number of pans ( single or

double) , and whether they are mechanical or electronic or classified by
operating ranges.

o The most popularly used balances are analytic and electronic balances:

a) Analytic balance is for the preparation of primary standard. It has a single

pan enclosed by a transparent sliding door. The weight range is from 0.01
mg to 160 g.

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MLS 111A, Lesson 4

b) Electronic balance is also a single pan balance that uses electromagnetic

force to counterbalance the weighed sample’s mass. Its measurements
equal the accuracy and precision of any available mechanical balance, with
the advantage of a fast response time ( less than 10 seconds).

B. pH meters

pH is an expression of the degree of acidity or alkalinity of a solution. The value

does not actually indicate the true hydrogen ion concentration but rather hydrogen
ion
activity. pH values can be digitally displayed or on galvanometer type scale.

o Hydrogen electrode is the heart of the pH meter. It generates an electrical

potential once in contact with hydrogen ion in solution. The 2 types:

a. glass electrode: inside is a wire, usually composed of solver coated with

silver chloride. It does not have any opening to the solution whose pH we
wish to measure.

b. Reference electrode: a more complicated devise. There is an opening to

the solution with which it is immersed.

C. Thermometers

A lot of procedures in the chemistry laboratory require monitoring of

accurate temperature so a chemical reaction can proceed. Reactions that are
temperature dependent use some type of heating, cooling cell, heating/ cooling
block,
or water/ice bath to provide the correct temperature for the reaction environment.

o Laboratory refrigerators and current clinical chemistry analyzers have built-in

thermometers for temperature maintenance.
o Types of thermometers are: liquid-in-glass and electronic or thermistor probe.
a. Liquid-in-glass thermometers use colored liquid; encased in plastic or glass
material; measure between 20°C and 400°C; calibrated periodically against
NiST-certified or NIST-traceable thermometer for critical laboratory
applications.
o NIST has an SRM thermometer with various calibration points ( 0°C,
25°C 30°C 37°C)
b. Electronic (thermistor) thermometers are more accurate and provide faster
reading; calibrated periodically against an SRM thermometer.

D. Centrifuge

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

mass and densities. A centrifuge is made up of a head or rotor, carriers, or
shields that
are attached to the vertical shaft of a motor or air compressor and enclosed in a
metal
covering. Rotor is the part of the centrifuge that holds the tubes and rotates
during the
operation of the centrifuge. Modern centrifuges have lid for safety operation,
brake or a
built-in tachometer; some are refrigerated.

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MLS 111A, Lesson 4
o Centrifugal force depends on 3 factors: mass, speed, radius. Speed is
expressed in revolutions per minute (rpm). The faster the speed and the
longer the radius, the better is the quality of the filtrate obtained
o RCF means relative centrifugal force

Formula: RCF=1.118x10° x rx (rpm)?

where:
1.118x 10° =aconstant, determined from angular velocity
r = radius in cm, measured from the center of the

centrifuge axis to the bottom of the bucket

o Important aspects to observe during centrifugation:

1. Principle of balance.
¥ tubes and carriers must be of equal weight, shape, size, and must
be placed in opposing positions in the centrifuge head.
¥ The principle of balance is necessary to avoid noise, vibration, or
shaking of centrifuge.
v¥ An unbalanced load also decreases the speed of centrifuge leading
to improper separation of phases.
2. Always operate centrifuges with lids closed to avoid aerosol
contamination.
3. Allow the rotor to stop spinning before opening the centrifuge lid.
4. Use only tubes that are specified as appropriate for that particular
centrifuge
5. Tachometer (Speed indicator) should be used as a guide for reproducible
centrifugation.
6. Clean any spills or debris ( glass or blood )

o Classification of centrifuge depends on the model type and use.

Table 1. Types of centrifuge

Types of Centrifuge Description

Clinical centrifuge 1. For urinalysis or serum separation

2. speed capacity is 0-3,000 rpm

3. holds tube ranging from 5-50 mL sizes

Serofuge Small centrifuge used in blood banking and

serology to spin serological tubes with 2-3
mL sizes

Microcentrifuge Used to spin microtubes with capacity of 0.5-

15 mL sizes at high speed usually up to

12,000-14,000 rpm

Refrigerated centrifuge Rotate speed at 0-20,000 rpm

Ultracentrifuge Rotate speeds over 50,000 rpm,
eee

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MLS 111A, Lesson 4

oThere are also traditional classification of

laboratory determinations. These are:

centrifuges used in routine

1. horizontal-head centrifuge: the cups holding the tubes of material to be

centrifuged
occupy a vertical position when the centrifuge is at rest, but assume a horizontal
position when the centrifuge res9olves. The horizontal-head is also called swinging
bucket

2. fixed-angle-head centrifuge: the cups are held in a rigid position at a fixed

angle.
This position makes the process of centrifuging more rapid than with the
horizontal-
based centrifuge. There is a less chance that the sediment will be disturbed when
the centrifuge stops.

E. Glassware and plasticware

Laboratory supply consisted of glass is termed as glassware. There are different
types of glass materials. The different categories are shown in table 2.

Nowadays, plastics are becoming more popular and eventually have replaced some
glassware; there are certain characteristics of plasticware that could be better
than
glassware. Table 3 shows the comparison between plastic and glassware.

Regardless of type, the supplies must satisfy tolerances of accuracy and must
belong into 2 classes of precision tolerances ( class A or class B) given by the
American Society for Testing and Materials (ASTM).

Table 2. Types of glasses

Thermal-resistant Alumina-silicate | Acid-resistant Low-actinic Flint glass

(Borosilicate) Glass | glass and alkali- (amber-colored)
resistant glass glass
Low alkali High silica Boron —free Amber or red | Also called
content content which color to soda-lime
makes it reduce the glass.
comparable to amount of
fused quartz light through
in heat the
resistance, substance
thermal inside the
stability, and glassware
electrical
characteristics
Resistant to Strengthened | Referred as Used for Composed of
heat, corrosion chemically “soft glass” substances a mixture of
and thermal rather than because its that are the oxides ,
shock thermally thermal particularly calcium, and
resistance is | sensitive to sodium
much less light, like
than that of bilirubin and
borosilicate VitA

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MLS 111A, Lesson 4

Commercial Commercial Most less

names: Pyrex; brand: Corex expensive
kimax
Balanced Used for high Less resistant
mechanical, precision to high-
thermal, and analytical temperature
chemical work in and sudden
strengths radiation change in
resistant and temperature
used for
optical
reflection and
mirrors
Chemical
resistance is
fair
Can cause
contamination
because it is
releases alkali
into solutions

Table 3. Comparison of plasticware and glassware

Plastic Glass

Advantage Cheaper Chemical stability

Durable
Unbreakable Clarity

Store alkaline solutions

Disadvantage

-Leaching of surface-
bound constituents
-Permeability water vapor
-Evaporation through
breathing of plastic
-Absorption of dyes,
stains, or proteins

o Whenever in contact with biohazardous material, plasticware or glassware

must be decontaminated according to appropriate protocols.

oContainers and Receivers are

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of high quality material; they are not

calibrated to hold an exact volume. These include beakers, test tubes,
Erlenmeyer flask, and reagent bottles.
MLS 111A, Lesson 4

e Beakers are wide, straight-sided cylindrical vessels and are available in

many sizes and on several forms. The most common form used in the
laboratory is known as the “ Griffin low form’. They are made of glass that is
chemical and heat resistant.

e Separating funnel is used for simple extraction procedures

e Flasks have flat bottom and sloping sides that gradually narrow in diameter
so that the top opening is bottle-like. They are to hold liquids, mix solutions
on non-critical values. There are kinds of flasks:

a. Erlenmeyer flasks have a flat bottom and sloping sides that gradually
narrow in diameter. They are often used for preparing reagents and for
titration procedures. They also come in various sizes: 10,mL-4,000 mL.

b. Florence flask has a flat bottom and rounded sides that give rise to a long
cylindrical neck. The only marking seen is the total capacity in mL. Size
range: 50-2,000 mL

c. Volumetric flasks have round bulb at the bottom. This tapers to a long
neck, on which the calibration mark is found. They are calibrated “to
contain” (TC) the exact volume but are not calibrated “to deliver” (TD) the
exact volume. For each use of volumetric flask, there are certain
allowable limits within which its volume must lie. This is called the
tolerance of the flask.

e Test tubes come in various sizes. Tubes without lips are most satisfactory
because there is less chance of chipping and breakage.

e Graduated cylinder is commonly called “graduates”. It is a long, straight-

sided, cylindrical glassware with calibrated marking. It is used to measure
volumes of liquids when a high degree of accuracy in not essential. It is not
caliabrated as accurately as volumetric flasks.

e Reagent bottles can be made of glass or other material. They come in

various sizes that suit the intended use.

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MLS 111A, Lesson 4

Figure 1. Types of laboratory glassware

Erlenmeyer laboratory
flask bottle

measuring ee

cylinder

laboratory glassware
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o Pipettes are types of volumetric plasticware or glassware used commonly in

the laboratory. They are used to transfer or measure aliquots of a liquid.
They come in different sizes and transfer different volumes of liquid less than
20 mL. Bigger volumes need automated pipetting device.

Pipettes can be classified according to designs: to contain (TC) and to

deliver (TD)

a. TC pipette holds a particular volume but does not dispense that exact
volume.

b. TD pipette dispense the volume indicated

Other Pipette classifications

a. drainage characteristics
o blowout
o self-draining
b. type
o Measuring
¥ Serologic
¥ Mohr
¥ Bacteriologic
v Ball, Kolmer, or Kahn
¥ Micropipette

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MLS 111A, Lesson 4

o Transfer
¥ .volumetric
¥ ostwald-folin
v Pasteur
¥ Automatic macropipettes or micropipettes

A blowout pipette has continuous etched ring or two small, close,

continuous rings located near the mouth or top of the pipette. This indicates that
the
remaining liquid at the tip must be expelled into the receiving container. Without
the
markings, pipette is classified as self-draining where the fluid is allowed to
drain by
gravity. With the exception of Mohr pipette, the tip must remain in contact with
the
vessel with the side of the receiving container for some second after the liquid
has
drained.

Serological and Mohr pipets are also called graduated/measuring pipets.

They are graduated pipets that can measure different amounts of liquid. They can
be purchased as reusable or disposable. Both pipets are NOT used to measure
calibrators or controls.

Serological pipet is known as a “to deliver(TD)/blowout pipet ”. Calibration is

until
the tip.

Mohr pipet has the same measuring technique as the serological pipet. It is
however, a TD/"no blowout” pipet. The end-calibration line for measuring the full
volume of the pipet is before the tip of the pipet. The pipet is held vertical and
the
liquid is allowed to drain from the beginning calibration line to the end
calibration
line to measure the full volume. The receiving receptacle is tilted so that the
pipet
tip touches the inside wall of the container. Mohr is more accurate than the
serological.

Volumetric pipet has a bulb in the middle of the pipet and two slender pieces of
glass on either side. It is not graduated. It measures only one volume. It is a
TD/’no
blowout” pipet. It is more accurate than serological and is used to add diluent to
a
lyophilized control or measure standards and reagents.

Ostwald-Folin Pipet is a TD/blowout” pipet. It’s use for measuring viscous

solutions such as whole blood, but is not used much in laboratory anymore. The
same pipetting technique applies.

Micropipettes are pipets that measure small amount of a liquid, less than 1 mL.
They can be deigned either as Mohr or a serologic but they are usually “to contain”
(TC) pipets, To deliver the contained volume, the pipet contents are usually pushed
out by a small pipetting bulb and the inner walls are rinsed out with the diluent
in the
receiving receptacle using an aspiration technique and a small aspirating bulb.
This
pipet is more precise if used properly.

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MLS 111A, Lesson 4

Unopette is a special micropipette used in Hematology laboratory. It is self-

filling
pipet.

Capillary pipets (TC) are other disposable micropipette. It is inexpensive and is

made of capillary tubing with a calibration line marking a specified volume.

Pasteur and Disposable Transfer pipets are used for transferring liquid from one
receptacle to another. Disposable pipets are made of plastic, while, Pasteur
usually
have a reusable suction top and disposable glass pipet bottom.

Figure 2
TYPES OF PIPETTES

lI

a
SSS Se
°-Volumetric
-Measuring
-Mohr
Serological google.com,

Pipetting with manual pipets

. Check the pipet to ascertain its correct size, being careful also to check for
broken
delivery or suction tips. Volume indication and type of pipette can be seen near
the
mouth of the pipette.

. Wearing protective gloves, hold the pipette lightly between the thumb and the
last
three fingers.

. Place the tip of the pipet well below the surface of the liquid to be pipetted.
. Using mechanical suction or an aspirator bulb, carefully draw the liquid up into
the
pipette until the level of the liquid is well above the calibration mark.

. Quickly cover the suction opening at the top of the pipet with the index finger

. Wipe the outside of the pipet dry with a piece of gauze or tissue to remove
excess
fluid.

. Held the pipette in a vertical position with the delivery tip against the inside
of the
original vessel. Carefully allow the liquid in the pipet to drain by gravity until
the
bottom of the meniscus is exactly at the calibration mark. (The meniscus is the
concave or convex surface of a column of liquid as seen in a laboratory pipette,
buret, or other measuring devise). To do this, do not entirely remove the index
finger from the suction-hole end of the pipette; rather , by rolling the finger
slightly
over the opening allow slow drainage to take place.

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MLS 111A, Lesson 4

8. While still holding the pipette in a vertical position, touch the tip of the
pipette to the
inside wall of the receiving vessel. Remove the index finger from the top of the
pipette to permit free drainage. Remember to keep the pipette in a vertical
position
for correct drainage. In TD pipettes, a small amount of fluid will remain in the
delivery tip.

9. To be certain that the drainage is as complete as possible, touch the delivery

tip of
the pipette to another area on the inside wall of the receiving vessel.

10.Remove the pipette from the receiving vessel, and place it in the appropriate
place
for washing

Figure 3. Correct manners of pipetting

A. C.
Serologic/Mohr The effect of the pipetting position
(e.g. using a 2-10 ml pipette)
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Volumetric/Ostwald-Folin i_o 7 Peal f
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MLS 111A, Lesson 4

B.

D.

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How to “take” pipette.

Do this. Not do this.

Bo

Figure 4. Reading fluids using a pipette (e051e.com)

A. Meniscus level

google.com

C. Read meniscus at eye level

Reading the meniscus

Read the volume at the dottow

ofthe meniscus

Read here...

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Meniscus

Eye level
MLS 111A, Lesson 4

B. Differentiating a convex and concave meniscus

Convex

Concave

D. Effects of meniscus misreading

Ca

Jj ¥ Low reading

Show | ener eee ee D Correct position of eye

By

Meniscus

“D High reading

B. Semiautomated Pipets offer more convenience and efficacy to pipetting. No

pipetting bulb is needed. It makes use of plastic tips which are disposable and
autoclavable. Plastic tips retain less inner surface film than the glass tips .

The pipets can be single or multichannel (8-.12). A plunger or trigger is used to

aspirate the liquid into the pipet. These pipets use air or positive displacement
to draw up
the fluid. The air displacement technique uses suction to draw up fluid. Positive
displacement uses a mechanical devise such as piston to displace the liquid to be
drawn
up. It operates much like the movement of the barrel in a hypodermic syringe.
Positive
displacement pipetting can be used for very dense liquids.

Figure 5. Single channel pipette

Figure 6. Multichannel pipette

Push button
(Operating button)
(PA)

Tip Ejector
(PA)
Push button rod
(ABS)

Display cover a
(P.

A)

Handle
(ASAIPC)

Tip Ejector Collar

(PA)

Tip cone Cc E e@.

2.5-1000u1(PVDF) ree
Sa

2000-10000yu1(PPS) '

—.. . e

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MLS 111A, Lesson 4

Steps in using semi-automated micropipettors:

1.
2.
3.

4.

Attach the proper tip to the pipettor, and set the delivery volume.

Depress the piston to a stop position on the pipettor.

Place the tip into the solution, and allow the piston to rise slowly back to its
original
position.

Some tips are wiped with a dry gauze at this step, and some are not wiped. Follow
the manufacturer’s directions.

Place the tip on the wall of the receiving vessel, and depress the piston, first to
a
stop position where the liquid is allowed to drain, then to a second stop position
where the full dispensing of the liquid takes place.

Dispose of the tip in the waste disposal receptacle. Some pipettors automatically
eject the used tips, thus minimizing biohazard exposure.

Figure 7. Semi-automated Pipette

Method 1:
Forward pipetting

Upper stop
ees oot Tt
Second stop

mm
Electronic pipetters or automatic micropipettors provide a variety of programmable
application parameters. The applications include the pipetting mode, the fixed-
volume
mode, and the dispensing mode. The pipetting mode allows the user to create
programs
for reverse pipetting, a method used for highly viscous fluid. The pipetting mode
includes:

e blowout, which provides manual control when dispensing residual liquid;

e manual pipetting for manual-like control of aspiration and dispensing;
e rinsing, which is used for mixing applications

The fixed volume allows the user to program a number of the most frequently used
volumes for quick recall. The dispensing mode provides repetitive dispensing of a
constant volume.

REAGENTS
Reagent is defined as any substance employed to produce a chemical reaction.
With automation, most instruments use their own ready-to-use form or “kit”.

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MLS 111A, Lesson 4

A. Chemicals
A.1. Analytic chemicals are in varying grades of purity.

e Analytic reagent (AR) grade are of a high grade of purity and are used often in
the preparation of reagents.

e ultrapure

e Chemically Pure (CP) Grade indicate that the impurity limitations are not stated
and that the preparation of these chemicals is not uniform. Clinical laboratories
cannot use these chemicals in reagent preparation unless with reagent blank.
However, the CP designation does not reveal the limits of impurities that are
tolerated.

e United Sates Pharmacopeia (USP) and National Formulary (NF) grade are
chemicals that are generally less pure that CP-grade chemicals, because the
tolerances specified are such that USP and NF chemicals are not injurious to

health, rather than chemically pure.

e Technical or commercial grade are chemicals used only for industrial purposes
and are generally not used in the preparation of reagents for clinical laboratory.

A.2. Reference Materials

Clinical chemistry analyses involved measurement of biochemical

products which makes purification of material with exact composition impossible.
This explains why traditional standards are not applicable to clinical chemistry.

Primary standard is a highly purified chemical that can be measured directly

to produce a substance of exact known concentration of purity. National Institute
of Standards and Technology (NIST) develops reference certified reference
materials for clinical chemistry use.

B. Reagent Water
Water is the most frequently used reagent in the laboratory.

1. levels of water purity

type 7 reagent water is the most pure and should be used for procedures that

require maximum water purity. It must be used immediately after it is produced and
not to be stored.

Type Ii reagent water is used for qualitative chemistry procedures and for most
procedures done in Hematology, immunology, microbiology, and other clinical test
area.

Type Iii reagent water can be used for some qualitative laboratory tests, such as
done in general urinalysis. It is used as a source for type 1 or type II water. It
is also
used for washing and rinsing glassware.

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MLS 111A, Lesson 4

2. Purification of water process

Tap water has a lot of impurities that disqualifies it for analyses. There are
purification processes to qualify water for laboratory use,
Distilled water : is made by boiling water and the resulting steam is cooled.
Distilled water meets the specifications of type 1 and II water.

Double-distilled water is water without substances still found in distilled water.

These substances are removed through redistillation process. These are carbon
dioxide, chlorine and ammonia.

Deionized water is obtained from passing the water through a resin column
containing positively and negatively charged particles.

Combination of deionization and distillation. Water of higher purity is also

produced by special distillation units which the water is first deionized and then
distilled; this eliminates the need for double distillation.

Reverse Osmosis process water under pressure through semipermeable

membrane made of cellulose acetate or other materials. This removes
approximately 90% of dissolved solids, 98% of organic impurities, insoluble
matter, and microbiological organisms. It does not remove dissolved gases and
only about 10% of ionized particles.

C. Buffers are weak acids or bases and their related salts that, as a result of
their dissociation characteristics, minimize changes in the hydrogen ion
concentration.

Hydrogen ion concentration — is often expressed as pH. The pH scale

ranges from 0-14.

Henderson-Hasselbalch equation:

pH = -log [H]

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