Clinical Chemistry Laboratory Equipment

CLINICAL CHEMISTRY 1
Instructor: Prof. Kimberly Ann Pulga, RMT
LAB 2

Outline ❖ Must be held vertically and the tip placed against the side of the
At the end of the session, the student must be able to learn: container and must not touch the liquid in it.
Basic Laboratory Equipment and Supplies ❖ A small amount of fluid will remain in the tip of the Pipette
Pipets ❖ Meet requirements of transfer pipets
Laboratory Vessels A. Ex: mohr, serologic, volumetric transfer pipets
Classification of Pipets
Base on design
Base on Drainage
Base on use
Types of Automatic Pipet
Air Displacement
Positive Displacement
Dispenser and Dilutor/Dispenser
Procedures on using the pipet
Meniscus

Classification – Drainage Characteristics

1. Blow out
BASIC LABORATORY EQUIPMENTS AND SUPPLIES
❖ Has a continuous etched ring or two small continuous rings located
near the top of the pipet.
A. Pipets ❖ Last drop should be expelled into receiving vessel.
A. Ex: Serologic, Ostwald-Folin
❖ The Pipettes are glass or plastic tubes, usually open at both ends, 2. Self-Draining
which are used to transfer specific amounts of liquid from one ❖ Allows the content to drain by gravity.
container to another. ❖ The tip of the pipet should not be in contact with
❖ They are usually used for volumes between 1 and 100 milliliters. ❖ The accumulating fluid in the receiving vessels during drainage
- Usually in lab, 10 or 15 ml ang pinakamaraming makikita sa stock room
except mohr pipet.
A. Ex. Volumetric, Mohr
Classification of Pipets
Blow out Pipet
❖ Depends on the amount of liquid needed to wet the interior surface
of the ware and the amount of any residual liquid left in the pipet
❖ The frosted band should not be confused with thicker colored rings
tip:
or colored dots, which are a manufacturer’s code for the maximum
A. I. Base on design
volume of the pipette.
▪ To contain (TC)
❖ Remember, only blow out a serological pipette if it has a frosted
▪ To deliver (TD)
band or two thin rings.
B. II. Base on drainage:
▪ Blow out
▪ Self-draining

C. Base on use
▪ Measuring/Graduated
▪ Volumetric/Transfer

1. TC – To Contain Pipet
❖ Contains a particular volume but does not dispense the exact Classification Based on Use
volume.
❖ Rinse out pipet (diluting fluid)
1. Measuring or Graduated
❖ A small amount of fluid will cling to the inside wall of the pipet
A. Example: if you pipet 10, usually that is 9 or 8
B. Example: Sahli Hemoglobin, Lang Levy ❖ Deliver the amount of liquid contained between two calibration
marks not calibrated with sufficient tolerance to use in measuring
standard or control solutions.

1. Serologic
2. Mohr
3. Bacteriologic
4. Ball, Kolmer, or Kahn
5. Micropipet

2. Transfer
Sahli-Hemoglobin Lang-Levy

1. Volumetric
❖ 2. TD – To Deliver Pipet 2. Ostwald-Folin
❖ Can dispense the amount of volume indicated 3. Pasteur Pipets
❖ Designed to be drain by gravity 4. Automatic Macropipets or Micropipets
 Measuring Pipets Automatic Pipet

❖ Measuring pipettes are divided into: ❖ Automatic pipet is usually use in the laboratory
A. Mohr Pipettes ❖ Most use pipet because they are safe, stable and more
▪ They are calibrated between two marks on the stem
precise
▪ The volume of the space between last calibration mark
and the delivery is not known ❖ fix volume automatic pipet meaning only one volume pipet
▪ The space cannot be use for measuring ❖ Variable volume automatic pipet meaning they can be able
▪ The graduation on these always end before the tip. to select different volumes
❖ Micropipette which dispenses less than 1ml
❖ Macropipette which dispenses more than 1ml

Types of Automatic Pipet

B. Serological Pipettes
1. Air Displacement
▪ Generally, a blowout pipet
▪ They are long tubes with uniform diameters ❖ Relies on piston for suction creation to draw the sample into a
▪ Graduated or they have graduation marks to the delivery disposable tip that must be changed for use.
tip of the pipet ❖ The piston does not come in contact with the liquid
▪ Has larger orifice or mouth than mohr pipet
▪ The distance from last calibration mark up to the tip is 2. Positive-Displacement
known.
▪ The graduation marks continue to the tip. ❖ Operates by moving the piston in the pipet tip.
❖ Like syringe.
❖ Doesn’t require different tips
❖ Rinsing and blotting between samples maybe required
3. Dispenser and Dilutor/Dispenser

Transferring Pipet ❖ Obtain the liquid from common reservoir and dispense it
repeatedly.
Ostwald-Folin Pipet (Transfer Pipet) 1. Bottle Top
2. Motorized
No graduation marks and only exact for: 3. Handheld
4. Attached to a Dilutor
❖ For viscous fluids.
❖ Blow out
❖ Has bulb near the tip
Legend

(A & C) air displacement automatic pipet

(B & D) positive displacement automatic

pipet

Volumetric Pipet (Transfer Pipet)

❖ Has used to deliver a single specific volume of liquid, usually
between 1 and 100 ml.
❖ The bulb is located at the middle of the pipet DISPENSER AND DILUTOR (AUTOMATIC PIPET)
❖ Shaped like rolling pins with a large belly, one blunt end, the neck, ❖ The dilutor often combines sampling and dispensing functions
and one tapering end, the tip.

Pasteur Pipet (Transfer Pipet)

❖ Do not have calibration marks

❖ Use to transfer solutions without consideration of a specific volume

Pipet Bulbs

❖ Has a pipette bulb is used to draw liquid up into the pipette. There
are many types of pipette bulbs:

1. Rubber Bulb
2. Pipet Filler
3. Pipet Aid
4. Pipet Pumper
 10. Touch the tip to the inside of the tilted container, lift your finger off
the end and allow the liquid to drain out of the pipette

-if that is a blow out pipet you have to expel the remaining
liquid inside the walls of the pipet

11. Hold the pipette in this position for a few seconds after it stops
draining wipe the pipet again before disposal or cleaning the pipet

Meniscus
Rubber Bulb Pipet Pumper ❖ Curvature in the top surface of the liquid. Pipette should be held
that the calibration mark is at the eye level.
B. Laboratory Vessels
A. Lower Meniscus
▪ Clear Solutions
B. Upper Meniscus
▪ Colored or Viscous Solutions

Laboratory Vessels

❖ Calibration – Glassware or other apparatus used in quantitative

measurement is checked to determine its exact volume.
❖ National Bureau of Standards – Calibrated by weight using
Pipet Pumper Pipet Aid distilled water and analytic balance.

Procedure on using the Pipet Volumetric Flasks

1. Hold the pipette about 8 cm below the mouthpiece with one hand then
with your other hand squeeze the bulb and touch the opening to the
mouth of the pipette.

2. Insert no more than one half cm of the pipette into the bulb
❖ Calibrated to hold one exact volume of liquid (TC)
3. Place the tip into the colored liquid and slowly release the pressure ❖ This is a to contain
on the bulb

-once you release the pressure on the bulb, you will see that Erlenmeyer Flasks
the liquid is going up

4. The liquid will be drawn up into the pipette and will form a curved
surface against the glass

-Curved surface is known as meniscus

5. This surface is called the meniscus pull the bottom of the meniscus
up about 1 cm past the desired level ❖ Designed to hold different volumes rather than one exact amount.

-Read at the bottom of the meniscus for colorless liquid (lower Beaker
meniscus) and upper meniscus for colored liquid
-Eye level when releasing the volume

6. Then quickly, but carefully, remove the bulb as you slip your free index
finger over the tip of the mouthpiece hole

*Never use your thumb your index finger will allow better
control and will also enable you to hold other items with your
free fingers when necessary. ❖ Hold different volumes rather than one exact amount
❖ Griffin beakers
7. Then with your finger still on the end of the pipette, gently lift the ❖ Berzelius beakers
pipette out of the solution
Graduated Cylinder
8. Then raise your finger just enough to allow the bottom of the meniscus
to line up with the desired graduation mark. You should observe the
meniscus at eye level while doing this

* When the meniscus is at the desired level, touch the tip of

the pipette to the inside of the container holding the liquid, to
remove any drops of liquid on the end of the pipette. Now,
there is precisely aspirated liquid in your pipette. ❖ Used to measure liquid when high degree of accuracy is not
essential
9. keeping your finger on the end of the pipette, wipe the sides of the ❖ Calibrated to deliver
pipet with tissue paper & gently move it to the waste container ❖ Can be used to measure specified volume of Liquid
 THERMOMETER

1. Liquid-in-Glass

❖ Use color liquid or mercury encased in plastic or glass material with

bulb at the end and a graduated stem.
A. Not use in laboratory or in hospitals
B. has side effects if a person is expose to mercury
C. Usually measures temperature between 20 degrees to 400
degrees celsius.

❖ Three Types of Liquid-in-Glass Thermometer

A. Partial Immersion used for measuring temp in units such as
heating blocks and water bath. Should be immersed to the
proper height as indicated by the continuous line etched in the
thermometer

B. Total Immersion used for refrigeration application

C. Surface Thermometers for flat surface such as oven or

incubator.

2. Electronic Thermometer/ Thermistor Probe

❖ Advantage: size and millisecond response time

❖ Disadvantage: expensive

3. Digital Thermometer

Additional infos:
TD - To Deliver Pipet
- When you are transferring the aspirated solution, it must not touch the liquid present in the receiving vessel.
Classification Drainage Characteristics
1. Blow out – last drop should be expelled into the receiving vessel. You have to squeeze the aspirator and then release to aspirate
the solution into your pipet. Lahat ng laman sa loob should be expelled, kasi kasama siya sa bilang or volume
2. Self-Draining - after you aspirate, you transfer it to the receiving vessel, you just have to remove your index finger. After you
transfer - release index finger, dire deretso ng magfoflow or dispense yung naaspirate na solution
Blow Out Pipet
- Frosted bands should not be confused with the thick color rings in the pics.
- Frosted bands are manufacturer's code for the maximum volume of the pipet.
- Two rings - blow out pipet, to deliver pipet. Maximum capacity - 10 ml. Per marking 1/10.
Classification Based on the use
1. Measuring or Graduated - merong graduation markings
2. Transfer - walang markings, we only have 1 because we could transfer 1 volume only.
Measuring Pipets
Mohr Pipet - We don’t know what is the volume from the tip to below the line. The volume of the space between the last calibration
marking up to the tip is not know, it can’t be used in measuring. Self-draining, kahit may matira sa
inner walls ng pipet okay lang kasi most likely yung tip or remaining or excess
nagcocontribute diyan.
Serological Pipet - 10 ml kasi from 9 to the tip is 1 ml volume. That’s why tese pipette are blow out pipet because you need to
expel this because it is still part of the measurement. You have to expel everything.
Ostwald- Folin Pipet - A blow out pipet. Bulb for oswald- folin is near the tip, and can also be used in single volume (5 ml)
Volumetric Pipet (Transfer Pipet) - In volumetric, the bulb is in the middle
Pasteur Pipet (Transfer Pipet) - 2 drops/ 5 drops. Innacurate
Types of Automatic Pipet
1. Air displacement - used in our lab in school, most routinely used. Safe, stable, and most precise. We have fixed volume pipette (1
volume), Variable volume - we can able to select different volume or set different volumes. Micropipet - dispenses less than 1 ml, Macropipet
- greater than 1ml.
2. Positive displacement - parang syringe class, you will pull the plunger-like that can serve as your aspirator. When you pull it, you are
already aspirating the solution from the original container or source vessel.
3. Dispenser and Dilutor/Dispenser - You can see this in the chemical analyzer or auto analyzer.
B, Laboratory vessels
Volumetric flasks (To contain)
- Bawal magtantya ng volume
Graduated cylinder
- Can hold different volumes, but if high degree accuracy hindi pwede gamitin.
Thermometer
1. Liquid in glass - mercury containing thermometers bawal na kasi may have adverse effects.
1 CCHM321 LAB – LESSSON: Venipuncture Equipment’s
VENIPUNCTURE EQUIPMENTS • Needle size varies by both length and gauge
(diameter).
- 16 gauge daw sabi ni ma’am
• For routine venipuncture, 1-inch and 1.5-inch
lengths are used.
- It gives better control and is less frightening to
patient
- If the seal is broken, needle should not be
used
- 21g, 22g, 23g will be used in CCHM,

- Hub is usually colored, depending on the

gauge
- Venipuncture – puncuring if vein for us to - Evacuated tube needle is a mutisample needle
collect or withdraw a blood sample or two-way needle. The other needle is
- Three methods: needle and syringe, ets, and covered with rubber sleeve. We are not
butterfly/ winged infusion set. allowed to use needle with hub.
- The phlebotomy drawing station can also be • Needle gauge refers to the diameter of the
reclined. In hospital, they have this. needle bore.
EVACUATED TUBE SYSTEM • Needles vary from large (16-gauge – blood
collection) needles used to collect units of
blood for transfusion to much smaller (23-
gauge) needles used for very small veins.
• The smaller the gauge number the bigger
the diameter of the needle.
• Multisample needles have the stopper
puncturing needle covered by a rubber
sheath that is pushed back when a tube is
attached and returns to full needle coverage
when the tube is removed. This prevents
leakage of blood when tubes are being
changed.
- Safet shield is no longer required - The needle in the rubber sheath isused to
- Tube holder/ adapter used for the multisample puncture the rubber In evacuated tube.
needle (used in ats mehod) or two-way needle
NEEDLE HOLDERS
- 23: blue
Made of rigid plastic and may be designed to act as a
NEEDLES
safety shield for the used needle.
• Multisample needles
- Also known as tube adapter. Actually
- You are not allowed to use needles with hub in
disposable and can be used once only, pero
retdem.
we can disinfect nalang para makatipid, also
• Hypodermic needles (needle and syringe)
the torniquet.
• Winged blood collection needles
- You can have smaller needles because you
will be using it to collect blood from hand.
Gauge depends: uaually 23,25.
• Sterile, disposable, and are used only once.
- 1 for every patient, no reusing
2 CCHM321 LAB – LESSSON: Venipuncture Equipment’s

SYRINGES

• Advantage

– Phlebotomist is able to control the suction pressure

on the vein by slowly withdrawing the syringe plunger
- Tube advancement mark: this is where you will
insert the tube only – Blood will appear in the hub of the needle when the
- You will know that you hit the vein if the blood vein has been successfully entered.
appeared in the tube. Pag hindi na hit, remove
the tube first, and the kapa, align, tusok. Kasi
wala tayong hub, since ito yung nag iindicate if
you already hit the vein. Since we don’t have
that kukunin natin yung tube then itutusok.
- If walang blood: Remove the tube first, adjust
the needle, kapa, align tusok, and then ibalik
yung tube
• Blood drawn in a syringe is immediately
transferred to appropriate evacuated tubes to
prevent the formation of clots.
• It is not acceptable to puncture the rubber
stopper with the syringe needle and allow the
blood to be drawn into the tube.

Blood Transfer Device

• Provides a safe means for blood transfer

without using the syringe needle or removing
the tube stopper
• It is an evacuated tube holder with a rubber-
sheathed needle inside.
• After blood collection, the syringe tip is
inserted into the hub of the device and
evacuated tubes are filled by pushing them
onto the rubber-sheathed needle in the holder
NEEDLE DISPOSAL SYSTEMS as in an evacuated tube system

Rigid, puncture-resistant, leak-proof disposable

“sharps” containers labeled BIOHAZARD that are
easily sealed and locked when full.

COLLECTION TUBES WINGED BLOOD COLLECTION SETS

• Evacuated tubes • “Butterflies”

• Vacutainers • Used for the infusion of IV fluids and for
performing venipuncture from very small or
very fragile veins often seen in children and in
the geriatric population.
3 CCHM321 LAB – LESSSON: Venipuncture Equipment’s
• Usually 21 or 23 gauge with lengths of ½ to
3/4 inch.

TOURNIQUETS

Used during venipuncture to make it easier to locate

patients’ veins.

- Blood pressure cuffs can be used as

alternative, you should just adjust the cuff to
40-60 mmhg

PUNCTURE SITE PROTECTION SUPPLIES

• 70% isopropyl alcohol - primary antiseptic

used for cleansing the skin in routine
phlebotomy
• 2x2-inch gauze pads - used for applying
pressure to the puncture site after the needle
has been removed.

– It is not recommended to use cotton balls to apply

pressure because the cotton ball fibers can stick to the
venipuncture site and may cause bleeding to begin
again when the cotton is removed.

– Video: Venipuncture Equipments

1 CCHM321 LAB – LESSON: Venipuncture using a syringe
EQUIPMENT:

• Requisition form
• Gloves
• Tourniquet
• 70 percent isopropyl alcohol pad
• Syringe needle
• Evacuated tubes
• 2 × 2 gauze
• Sharps container Step 4.
• Indelible pen
Verify if the patient has fasted, has allergies to latex,
• Bandage or has had previous problems with venipuncture.
• Biohazard bag
- If the test is fasting blood sugar, the patient
PROCEDURE: must have fasted 8 to 10 hours, lipid profile 10
Step 1. to 12 hours. Overfast (repeat fasting) or
underfast (wait further) you should not collect
Obtain and examine the requisition form. sample.

Step 5.

Select correct tubes and equipment for the procedure.

Have extra tubes available.

- You cannot collect a sample without the

request from the physician

Step 2.

Greet and reassure the patient and explain the

procedure to be performed.

Step 6.

Wash hands and apply gloves.

- Introduce yourself. Patient must understand

everything before you start.

Step 3.
- If not visibly soiled, no need. Kahit hand
Identify the patient verbally by having him or her state sanitizer.
both the first name and last name and compare the
Step 7.
information on the patient’s ID band with the requisition
form. Position the patient’s arm slightly bent in a downward
- Bawal medtech magtatanong position so that the tubes fill from the bottom up. Do
- If the patient cannot talk or is unconscious, you not let the patient hyperextend the arm. Ask the patient
can ask the relatives or guardian available. to make a fist.
2 CCHM321 LAB – LESSON: Venipuncture using a syringe
- Make sure it’s slightly bent so that you can - Push it forward to remove any air in the
easily palpate the vein. Vein palpating can be syringe.
horizontal or vertical direction. Assess the vein
kung anong gauge gagamitin. Step 11.

Reapply the tourniquet, remove the needle cap, and

inspect the needle.

- You only have 2 times in applying torniquet

Step 12.

Ask the patient to remake a fist, and anchor the vein

by placing the thumb of the nondominant hand 1 to 2
inches below the site and pulling the skin taut.

Step 8. Step 13.

Apply the tourniquet 3 to 4 inches above the Hold the syringe in the dominant hand with the thumb
antecubital fossa. Palpate the area in a vertical and on top near the hub and the other fingers underneath.
horizontal direction to locate a large vein and to
Smoothly insert the needle into the vein at a 15- to 30-
determine the depth, direction, and size. The median
degree angle with the bevel up until you feel a
cubital is the vein of choice followed by the cephalic
lessening of resistance. A flash of blood will appear in
vein.
the syringe hub when the vein has been entered.
The basilic vein should be avoided if possible. Remove
Brace the fingers against the arm to prevent
the tourniquet and have the patient open his or her fist.
movement of the needle when pulling back on the
- Index finger for palpating. Not allowed to use plunger.
basillic vein.
- Deeper vein, increase the angle, kung
mababaw lang decrease the angle. Same with
gauge pag makapal or malaki, decrease the
gauge, pag manipis taasan gauge.

Step 14.

Pull back the syringe plunger slowly using the

nondominant hand to collect the appropriate amount of
blood.

Step 9. Step 15.

Clean the site with 70 percent isopropyl alcohol in Release the tourniquet and have the patient open the
concentric circles moving outward and allow it to air fist.
dry.
- Release torniquet first before withdrawing
- Inner outer or back and forth needle.

Step 16

Cover the puncture site with gauze, remove the needle

smoothly, and apply pressure.

Step 17.

Remove the needle from the syringe and discard it in

the sharps container.
Step 10.
Step 18.
Assemble the equipment as the alcohol is drying.
Fill tubes in the correct order. Mix anticoagulated tubes
Attach the hypodermic needle to the syringe.
as soon as they are removed from the transfer device.
Pull the plunger back to ensure that it moves freely
Step 19.
and then push it forward to remove any air in the
syringe. After tubes are filled, the entire syringe are discarded
into a sharps container.
3 CCHM321 LAB – LESSON: Venipuncture using a syringe
- Do not puncture the rubber of your tubes

Step 20.

Label the tubes and confirm identification with the

patient.

Step 21.

Examine the puncture site and apply a bandage.

Step 22.

Remove gloves and wash hands.

1 CCHM321 LAB – LESSON: Other Venipuncture Procedures
OTHER VENIPUNCTURE PROCEDURES Step 10. Assemble the equipment while the alcohol is
VENIPUNCTURE USING AN EVACUATED TUBE drying.
SYSTEM • Attach the multisample needle to the holder.
EVACUATED TUBE SYSTEM Step 11. Insert the tube into the holder up to the tube
• Preferred, because it is direct, efficient, relatively safe advancement mark.
for the patient and the blood drawer, Step 12. Reapply the tourniquet. Do not touch the
and allows multiple tubes to be easily collected puncture site with an unclean finger. Ask the patient to
- Unlike with the needle and syringe, kung 5ml remake a fist.
barrel mo as is nayon, pag may pahabol wala - Bawal may hub sa multisample needle. You hit
ng madadagdag lalo na kung ongoing the vein if blood appears when tube is
collection. In ets, you can have as many tubes inserted.
as you can. Step 13. Remove the plastic needle cap and examine
the needle for defects such as nonpointed or barbed
PURPOSE: ends.
To obtain a blood specimen for patient diagnostic or Step 14. Anchor the vein by placing the thumb of the
monitoring purposes from an antecubital vein using nondominant hand 1 to 2 inches below the site and
the evacuated tube system (ETS) pulling the skin taut.
Step 15. Grasp the assembled needle and tube
EQUIPMENT: holder using your dominant hand with the thumb on
• Tourniquet the top near the hub and your other fingers beneath.
• Gloves • Smoothly insert the needle into the vein at a
• antiseptic prep pad 15- to 30-degree angle with the bevel up until
• ETS needle you feel a lessening of resistance.
• tube holder and tubes • Brace the fingers against the arm to prevent
• gauze pads movement of the needle when changing tubes.
• sharps container Step 16. Using the thumb, advance the tube onto the
• permanent ink pen evacuated tube needle, while the index and middle
• bandage fingers grasp the flared ends of the holder.
Step 17. When blood flows into the tube, release the
PROCEDURE: tourniquet, and ask the patient to open the fist.
Step 1. Review and accession test request. Step 18. Gently remove the tube when the blood stops
Step 2. Greet and reassure the patient and explain the flowing into it.
procedure to be performed. • Gently invert anticoagulated tubes promptly.
Step 3. Identify the patient verbally by having him or • Insert the next tube using the correct order of
her state both the first name draw.
and last name and compare the information on the • Fill tubes completely.
patient’s ID band with the requisition form. Step 19. Remove the last tube collected from the
Step 4. Verify if the patient has fasted, has allergies to holder and gently invert.
latex, or has had previous Step 20. Cover the puncture site with clean gauze.
problems with venipuncture. • Remove the needle smoothly and apply
Step 5. Select correct tubes and equipment for the pressure or ask the patient to apply pressure.
procedure. Have extra tubes - Remove the tube first before you withdraw the
available. needle. Pwede irelease sa 2nd tube yung
- First, you have to know the different tests torniquet, okay lang din kung sa 3rd tube na
requested by the physician. Purple/ lavender basta within 1 minute.
top – cbc, platelet count, wbc count, rbc count. Step 21. Activate the safety device.
Blue top/ citrate – coagulation. Red top – - Kahit walang safety shield
clinincal chemistry: fasting blood sugar, BUN, Step 22. Dispose the needle/holder assembly with the
creatine, bilirubin enznymes: ast, alt, acp. safety device activated into the sharps container.
Step 6. Wash hands and apply gloves. Step 23. Label the tubes before leaving the patient
Step 7. Position the patient’s arm slightly bent in a and verify identification with the patient ID band or
downward position so that the tubes fill from the verbally with an outpatient.
bottom up. • Observe any special handling procedures.
- Vein selection okay lang walang gloves Complete paperwork.
Step 8. Apply the tourniquet 3 to 4 inches above • - let him state his full name, date of birth, age
the antecubital fossa. and gender, date and time of collection, and
• Palpate the area in a vertical and horizontal initials as phlebotmoist
direction to locate a large vein and to Step 24. Examine the puncture site and apply
determine the depth, direction, and size. bandage.
• Remove the tourniquet and have the patient • Place bandage over folded gauze for
open his or her fist additional pressure.
Step 9. Clean the site with 70 percent isopropyl Step 25. Prepare sample and requisition for
alcohol in concentric circles moving outward and allow transportation to the laboratory.
it to air dry. • Dispose of used supplies.
2 CCHM321 LAB – LESSON: Other Venipuncture Procedures
Step 26. Thank the patient, remove gloves, and wash - Mabilis magfoflow yung blood, half of the
hands rubber tubing ay malalagyan niya na agad.
Pag may dugo na sa rubber tubing tyaka na
VENIPUNCTURE USING A WINGED BLOOD ipull plunger. Pag wala pang blood sa rubber
COLLECTION SET tubing wag muna hilahin plunger.
EQUIPMENT: Step 15
• Requisition form • Pull back on the plunger of the syringe slowly and
• Gloves smoothly with the nondominant hand to collect blood.
• Tourniquet • Do not pull back on the syringe plunger if a blood
• 70 percent isopropyl alcohol pad flash does not appear.
• Winged blood collection set • Invert anticoagulated tubes immediately
• Blood transfer device Step 16
• Evacuated tubes • Release the tourniquet.
• 2 × 2 gauze • Cover the puncture site with gauze, remove the
• Sharps container needle smoothly or activate the safety device on
• Indelible pen needles designed to be retracted while the needle is in
• Bandage the vein.
• Biohazard bag Step 17. Remove the winged blood collection set from
the syringe and discard it in the
PROCEDURE: sharps container.
Steps 1-6: Similar with other venipuncture procedures Step 18. Fill the evacuated tubes in the correct order.
Step 7: Support the hand on the bed or drawing chair Step 19. After tubes are filled, the syringe and blood
armrest and have the patient make a fist. transfer device are discarded into a
Step 8: Apply the tourniquet 3 to 4 inches above the sharps container.
wrist bone. Step 20. Label the tubes and confirm identification with
Step 9. Palpate the top of the hand or wrist. Select a the patient.
vein that is large and straight and that can be easily Step 21. Examine the puncture site and apply a
anchored. bandage.
- We perform this on patient with extreme age, Step 22. Remove gloves and wash hands.
geriatricts, or pediatricts, or obese patients pag
di makuhanan. Disadvantage is you can only
get small amount of sample.
Step 10. Release the tourniquet, have the patient relax
the fist, and clean the site with 70 percent isopropyl
alcohol in concentric circles and allow to air dry.
- Collect 3ml for butterfly method
Step 11.
• Assemble the equipment as the alcohol is drying.
• Attach the winged blood collection set to the
evacuated tube holder or the syringe.
• Stretch out the coiled tubing.
• Pull the plunger back to ensure that it moves
freely and then push it forward to remove any air
in the syringe.
- Choose non branching vein or staight, mahirap
pag yung branching
Step 12
• Reapply the tourniquet, remove the needle cap, and
inspect the needle.
• Lay the syringe and tubing next to the patient’s hand.
Step 13
• Anchor the vein by placing the thumb of the non-
dominant hand below the knuckles and pulling the skin
taut.
• Having the patient make a fist may be helpful.
Step 14
• Grasp the needle between the thumb and index
finger by holding the back of the needle or by folding
the wings together.
• Smoothly insert the needle into the vein at a shallow
10- to 15-degree angle with the bevel up.
• Thread the needle into the lumen of the vein until the
bevel is firmly “seated” in the vein.
• A flash of blood will appear in the tubing when the
needle has entered the vein.
 GLUCOSE
DETERMINATION

KIMBERLY ANN M. PULGA, RMT, MPH

COLLEGE OF MEDICAL LABORATORY SCIENCE
OUR LADY OF FATIMA UNIVERSITY
VALENZUELA CAMPUS
UNIT OUTCOMES

To achieve this unit a learner must:

1. Rationalize the consideration in patient preparation, specimen

collection, transport and processing and handling.
2. Describe the specimen of choice, principle involved, advantages
of the different laboratory methods of glucose determination.
3. Be able to properly perform fasting blood sugar determination.
4. Be able to evaluate FBS results correctly.
PRE-ANALYTICAL PHASE

Method: Quantitative Enzymatic-Colorimetric Determination of

Glucose in Serum or Plasma

Principle:
PRE-ANALYTICAL PHASE

Method: Quantitative Enzymatic-Colorimetric Determination of

Glucose in Serum or Plasma

Principle:
GO
Glucose + O2 + H2O à Gluconic acid + H2O2

PO
2H2O2 + 4-aminoantipyrine + Phenol à Quinone complex
PRE-ANALYTICAL PHASE

Specimen Collection and Preparation:

Remove from the clot within 30 minutes of collection in order to

prevent glycolysis.

Plasma: an anticoagulant containing fluoride is recommended.

ANALYTICAL PHASE

Materials:
Spectrophotometer
Accurate pipetting devices
Heating block or water bath (100 degrees Celsius)
Cuvettes
Vortex mixer
Interval timer
ANALYTICAL PHASE

Reagents:
Glucose liquicolor reagent Non-reactive ingredients & preservatives
4-aminoantipyrine preservatives
Glucose oxidase Glucose standard (100 mg/dL)
Peroxidase Glucose in aqueous benzoic acid
Phenol

Storage and stability: Stable at 2-8 degrees Celsius

Do not freeze
ANALYTICAL PHASE

Manual Procedure:
1. Pipet into tubes the following REAGENT STANDARD (S) SAMPLE (U)
volumes (mL) and mix well: BLANK (RB)

REAGENT 1.5 1.5 1.5

2. Incubate all tubes at 100
degrees Celsius for 3 minutes. STANDARD - 0.050 -

SAMPLE - - 0.050

3. Immerse test tubes in room

temperature water for 2
minutes to cool. Read S and U
vs. RB at 600 nm.
 ANALYTICAL PHASE

Glucose Determination

Glucose Determination 2
POST-ANALYTICAL PHASE

Formula for glucose concentration:

C (mg/dL) = __Abs sample___ x value of standard (100mg/dL)

Abs standard

C (mmol/L) = __Abs sample___ x value of standard (100mg/dL) x 0.0056

Abs standard
POST-ANALYTICAL PHASE

Normal values (Profame):

Serum/Plasma (fasting): 65-110mg/dL
Spinal fluid: 40-70 mg/dL
 LIPID
DETERMINATION
(CHOLESTEROL, HDL AND TRIGLYCERIDE)

KIMBERLY ANN M. PULGA, RMT, MPH

COLLEGE OF MEDICAL LABORATORY SCIENCE
OUR LADY OF FATIMA UNIVERSITY
 CHOLESTEROL
DETERMINATION
 CHOLESTEROL DETERMINATION

UNIT OUTCOMES:
To achieve this unit, a learner must:

1. Prepare accurately sample for cholesterol

determination.

2. Perform correctly total cholesterol determination.

 CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:
METHOD: Quantitative Enzymatic-Colorimetric Determination of Total
Cholesterol in Serum or Plasma

PRINCIPLE:
 CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:

Specimen collection and preparation:

Blood should be collected following a 12-hour fast. Specimen may be

serum, or plasma collected with EDTA as an anticoagulant. Avoid
hemolysis.
 CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:

Materials:
Spectrophotometer
Accurate pipetting devices
Heating block or water bath (37 degrees Celsius)
Cuvets
Interval timer
 CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:

Reagents:
4-aminophenazone Phenol
Peroxidase Cholesterol esterase
Cholesterol oxidase Buffers and stabilizers
Cholesterol standard (200 mg/dL)
-Buffered aqueous solution of cholesterol with stabilizers,
surfactants and preservative
 CHOLESTEROL DETERMINATION

ANALYTICAL PHASE:
Manual Procedure:
1. Pipette into cuvets the following volumes (mL) and mix well:

Reagent Blank (RB) Standard (S) Sample (U)

Reagent 1.0 1.0 1.0

Standard - 0.010 -
Sample - - 0.010
 CHOLESTEROL DETERMINATION

ANALYTICAL PHASE:
Manual Procedure:

2. Incubate all cuvets at 37 degrees Celsius for 5 minutes.

3. Read S and U vs. RB at 500nm within 60 minutes.

Cholesterol Determination
 CHOLESTEROL DETERMINATION

POST-ANALYTICAL PHASE:
Formula for cholesterol concentration:

C (mg/dL) = Abs sample x value of standard (200 mg/dL)

Abs standard

Reference range:
- Adult: 140-310 mg/dL
- Children: 120-200 mg/dL
 CHOLESTEROL DETERMINATION

POST-ANALYTICAL PHASE:
Reference range:
- Adult: 140-310 mg/dL
- Children: 120-200 mg/dL
HDL CHOLESTEROL
DETERMINATION
 HDL CHOLESTEROL DETERMINATION

UNIT OUTCOMES:
To achieve this unit, a learner must:

1. Perform correctly the laboratory methods of HDL

cholesterol determination.
2. Carry our calculations involved in HDL cholesterol
determination.
3. Evaluate the HDL cholesterol result.
4. Correlate the HDL cholesterol result with health status
of the patient.
 HDL CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:
METHOD: Quantitative Enzymatic-Colorimetric Determination of High
Density Lipoprotein (HDL) Serum or Plasma

PRINCIPLE:
LDL cholesterol and VLDL cholesterol fractions are precipitated from
serum or plasma by means of a magnesium chloride/dextran sulfate reagent,
according to Finley et al. HDL cholesterol is the determined in the supernatant
fluid using a Cholesterol reagent and the derived dilution factor in the
calculation.
 HDL CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:

Specimen collection and preparation:

Blood should be collected following a 12-hour fast. Specimen may be

serum, or plasma collected with EDTA as an anticoagulant. Avoid
hemolysis.
 HDL CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:

Materials:
Spectrophotometer
Accurate pipetting devices
Heating block or water bath (37 degrees Celsius)
Cuvets
Vortex mixer
Interval timer
 HDL CHOLESTEROL DETERMINATION

PRE-ANALYTICAL PHASE:
Reagents:
HLD cholesterol precipitating reagent:
-Magnesium sulfate
-Dextran sulfate
-Stabilizers and preservatives
HDL cholesterol standard (50mg/dL)
-Buffered aqueous solution of cholesterol with stabilizers,
surfactants and preservative
 HDL CHOLESTEROL DETERMINATION

HDL SEPARATION PROCEDURE:

1. To 0.5 ml (500ul) serum or plasma in a test tube, add 0.05 ml (50 ul)
HDL precipitating agent.
2. Mix well (vortex) and allow to stand 5 minutes.
3. Centrifuge for 10 minutes at high speed (1000xg).
 HDL CHOLESTEROL DETERMINATION

ANALYTICAL PHASE:
Manual Procedure:
1. Pipette into cuvets the following volumes (mL) and mix well:

Reagent Blank (RB) Standard (S) Sample (U)

Reagent 1.0 1.0 1.0

Standard - 0.025 -
Sample - - 0.025
 HDL CHOLESTEROL DETERMINATION

ANALYTICAL PHASE:
Manual Procedure:

2. Incubate all cuvets at 37 degrees Celsius for 5 minutes.

3. Read S and U vs. RB at 500nm within 60 minutes.

HDL Cholesterol Determination

 HDL CHOLESTEROL DETERMINATION

POST-ANALYTICAL PHASE:
Formula for HDL cholesterol concentration:

C (mg/dL) = ∆AbsHDL sample x value of standard (55 mg/dL)

∆ Abs standard

Note: 50 mg/dL is the concentration of the standard (mg/dL) multiplied by the

dilution factor of 1.1.

HDL cholesterol standard, 50 mg/dL x 1.1 = 55 mg/dL

 HDL CHOLESTEROL DETERMINATION

POST-ANALYTICAL PHASE:
Reference range:
 TRIGLYCERIDE
DETERMINATION
 TRIGLYCERIDE DETERMINATION

UNIT OUTCOMES:
To achieve this unit, a learner must:

1. Perform correctly the laboratory methods of lipid

determination.
2. Carry our calculations involved in lipid determination.
3. Evaluate the lipid profile result.
4. Correlate the lipid profile result with health status of
the patient.
 TRIGLYCERIDE DETERMINATION

PRE-ANALYTICAL PHASE:
METHOD: Colorimetric Determination of Triglycerides in Serum or Plasma

PRINCIPLE:
 TRIGLYCERIDE DETERMINATION

PRE-ANALYTICAL PHASE:

PRINCIPLE:
 TRIGLYCERIDE DETERMINATION

PRE-ANALYTICAL PHASE:

Specimen collection and preparation:

Sample stability: triglycerides are reportedly stable for at least 10 days at 2

– 8 degrees Celsius. Do not store samples at 15 – 25 degree Celsius as
phospholipids may hydrolyze, releasing free glycerol and falsely elevating
triglyceride values.
 TRIGLYCERIDE DETERMINATION

PRE-ANALYTICAL PHASE:

Materials:
Spectrophotometer
Accurate pipetting devices
Heating block or water bath (37 degrees Celsius)
Cuvets
Vortex mixer
Interval timer
 TRIGLYCERIDE DETERMINATION

PRE-ANALYTICAL PHASE:
Reagents:
-Enzymatic triglyceride reagent
-Triglyceride activator
-Triglyceride standard

Preparation:
- Add 9 drops of triglyceride activator to one bottle of triglyceride reagent or add 50
μL of activator for every 5.0 mL of reagent.
- Invert gently 3 – 4 times. •Before use allow to stand for at least 15minutes at room
temperature.
 TRIGLYCERIDE DETERMINATION

ANALYTICAL PHASE:
Manual Procedure:
1. Pipette into cuvets the following volumes (mL) and mix well:

Reagent Blank (RB) Standard (S) Sample (U)

Reagent 1.0 1.0 1.0

Standard - 0.010 -
Sample - - 0.010
 TRIGLYCERIDE DETERMINATION

ANALYTICAL PHASE:
Manual Procedure:

2. Incubate all cuvets at 37 degrees Celsius for 5 minutes.

3. Read S and U vs. RB at 500nm.

TAG Determination
 TRIGLYCERIDE DETERMINATION

POST-ANALYTICAL PHASE:
Formula for triglyceride cholesterol concentration:

C (mg/dL) = Abs sample x value of standard (200 mg/dL)

Abs standard

Reference range:
30-150 mg/dl
 Thanks!
Do you have any questions?
Email: kmpulga@[Link]

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