Fundamentals of Laboratory Medicine-part 2 W3:L2

Tubes are described by:

Width x Height in mm

To Deliver pipette:

Calibrated to deliver stated amount of fluid that is
drained from the pipette through gravity
(self-draining)

-Do NOT expel small amount of fluid at the end of the tip

UNLESS: Blow-Out: has an etched (frosted) ring or 2 thin
etched or coloured rings on the stem. Use a bulb to expel fluid in the tip!

To Contain pipette:

Calibrated to contain the stated amount of fluid.
Correct volume is NOT delivered through draining
-Fluid remaining inside the pipet wall is significant and must be drained too

When do we use a TC pipette?

whole blood samples

How do we ensure an accurate amount is dispensed?

wash with diluent/reagent

What happens if the room temp is not the same as the calibration temp?

seeing variances in the amount of volume we are delivering or containg iniside the pipette, most of labs are room temp

Volumetric pipet:

calibrated to deliver a fixed volume of liquid by drainage
-Calibration Mark etched on stem, “bulb” in middle,
gradual taper at tip
-To be used when accuracy is required to deliver an
exact amount
-For ex: a 5 mL Volumetric pipette has a tolerance of 0.01 m

Ostwald (Ostwald-Folin) pipet:

similar to volumetric but with bulb closer to the
tip. Used for viscous fluids (ex. serum0

Graduated pipet:

calibrated to deliver a volume of liquid between 2 calibration marks
-Can use to transfer various volumes
-To be used when speed is required
Less accurate than volumetric pipettes (tolerance of 0.02ml)

There are two types:
-Mohr and serologic

When should a Graduated pipette be used in the clinical lab?

when we need to make aliquots of a chemical (we don't need the exact amount)
the

Mohr pipet:

Calibration marks do NOT go to
the tip of the pipette

-cannot be “BLOW OUT” pipettes

Serologic pipet:

-calibration marks go down to the tip
-has wider tip opening

Reading the Meniscus:

Bring the pipette up to your eye level
-Don’t bend down to the pipette level

Micropipettors:

Used for < 1.0 mL
-Various volumes
-Technique dependent

Transfering solutions:

Pasteur Pipette (glass)
Transfer Pipette (plastic)

When do we use tranfer pipettes?

-blood bank
-tranfer fluid and doesn't have to be exact

Sedimentation principle:

centripetal acceleration causes more dense substances to separate out along the radial direction while the lighter objects move to the top

-separate substances (heave at the bottom, lighter at top)

Centrifugation:

process by which centrifugal force is used to separate lighter portions of a solution from the heavier portions (or solids from liquids)

Precipitate (pellet):

the packed particles (solid) at the bottom of a centrifuged tube

Supernatant:

the liquid portion at the top of a centrifuged tube

Decantation: *

separation of the supernatant from the pellet

Centrifuge:

apparatus used to separate substances of different density or particle size, when suspended in a fluid, by spinning them about an axis in a suitable container

Rotor and carriers attached to the vertical shaft of a motor and enclosed in a metal covering

Rotor (centrifuge head):

primary component of a centrifuge which holds the material to be centrifuged and which is rotated by the drive system

Types of Centrifuges:

-Benchtop vs. floor
-Refrigerated or not refrigerated
-Rotor head type (fixed, hematocrit,
swinging-bucket, angled)
-Maximum speed attainable

Common applications:

-Separating serum or plasma from blood cells
-Separating supernatant from a precipitate in analytic reaction
-Separating two immiscible liquids or expelling air (oil and water)

Normal separation of serum(liquid) from clot (blood cell):

min of 1000g (force) for 10 mins, can completely separate

What speed should you set the centrifuge to?

cannot set centrifuge at a sepcific g-force, you have to set it at an RPM/speed

Relative Centrifugal Force (RCF):

the force required to separate 2 phases in a centrifuge. Based upon 3 variables:
Mass, speed, and radius

RCF = 1.118 x 10-5 x r x rpm2

Where:
r = radius (cm) from center of rotation to bottom
of tube in the rotor cavity

rpm = speed of rotation of the rotor in
revolutions per minute

Rotating tip radius:

The distance measured from the rotor axis to the tip of the liquid inside the tubes at the greatest horizantal distance from the rotor axis is the rotating tip raius

Types of Centrifuges (based on Rotor
Head):

1. Horizontal-head (swinging-
bucket): bucket moves to allow tubes to spin in a horizontal position and rest in a vertical position
-flat surface
-particles travel constantly along the tube

2. Fixed-angle (angle-head):

tubes are held in a fixed position (25 – 40 angle) during centrifugation
-particles are driven outward
-at an angle

Miscellaneous centrifuges:

Microfuge: bench-top, used for rapid
separation of small samples (microtubes) Rpm: up to 15,000
-Rcf: up to 12,000 x g

Ultracentrifuge: high-speed centrifuge (fixed angle rotor) –used to clear lipids (chylomicrons)
Rcf: up to 1,000,000 x g
-Rotor chamber covered w/steel armor plate

Balancing a Centrifuge:

Must be balanced by position, tube type
And by volume

What to do if NO balance tube and odd
number of tubes?

always start with a triangle (3 spaces in btwn)
then places the remaining two tubes opposite from each other
NEVER PLACES TUBES IN OPP. POSIT. OF THE TRIANGLE

Balancing a Fixed Angle Centrifuge:

Sample number is even:
-load pairs of tubes in opposite positions

Sample number is odd:
-load three tubes at equal intervals in the rotor, forming an equilateral triangle
-load the remaining tubes in pairs in opposite positions

Weight = Mass x Gravity

Mass: the quantity of matter
Weight is a function of mass under the influence of gravity as per:

Types of Balances:

Gravimetric Analysis: technique through which
the amount of an analyte can be determined
through the measurement of mass

Weighing: comparison of an unknown to a known
mass as determined through the use of a balance

Classified by:
-# of Pans: single vs. double pan
-Mechanical vs. Electronic

Types
-Analytical Balance
-Top-Loading or Pan Balance

Mechanical Balance Principle: *

balance unknown mass with known mass

Electronic Balance Principle: *

-Uses electromagnetic force compensation
-Pan connected to a coil placed in a magnetic field

Capacity:

maximum load that can be weighed

Linearity:

the smallest to largest load that can be weighed accurately

Resolution:

the smallest increment in weight that can be discernible