Immunology and Serology Lec

[MIDTERMS] LESSON 9
Transcribed by: ANGELICA AGUILAR
DO NOT REDISTRIBUTE WITHOUT PERMISSION!
PRINCIPLES OF SEROLOGICAL TESTING B. GRADUATED
- marked to allow for varying amounts
TABLE OF CONTENTS  Serological
1 Blood Specimen Preparation and Measuring  Blowout
2 Dilution
3 Simple dilutions C. MICROPIPETTES
4 Compound dilutions - deliver small volumes
5 Testing parameters  Maximum capacity of 1mL or 1000uL

BLOOD SPECIMEN PREPARATIONG AND

MEASURING
SEROLOGY
-study of fluid components in the blood, especially
ANTIBODIES
 Immunology = reaction of blood in vivo (inside)
 Serology = reaction of
SERUM
-liquid portion of blood minus coagulation factors
 Coagulated blood
o Fibrinogen  fibrin  clot formation
 serum
 Appearance: Clear yellow
o Due to loss of fibrinogen

-most frequently encountered specimen in immunologic

testing

-separated from other components of a blood specimen

via CENTRIFUGE Use mechanical suction
 Due to difference in SPECIFIC GRAVITY  Before dipping pipette, always press bulb
o Top (lighter) = serum  Place on top of serological pipette
o Bottom (heavier) o Snuggly fit
1
 WBCs,  Dip the pipette inside the tube
 RBCs  Release the bulb to aspirate desired
 Platelet volume
 Place gloved finger on top
-ideally, use fresh serum that has not been heated Wipe off outside of pipette with gauze
 Heating 2  Do not wipe the tip
o inactivates factors o Gauze/ tissue may absorb the fluid
o denatures proteins 3 Adjust the meniscus
 Ex. Antibody Drain into receiving vessel
4
 After setting the volume to “0”
-for certain tests, complement may need to be
inactivated
 COMPLEMENT = proteins that help fight off
antigen
o Goal: cell lysis
 Complement may interfere with tests results.
 Heat sample to 56 C for 30 minutes
o 30 mins start when the temp of water
bath reaches 56 C
 When temp is reached, put the
sample inside
o Alternative: 65 C for 10 mins
STORAGE
-if testing is delayed
 Bet. 2C – 8C for up to 72 hours
 Frozen at -20C or below
o For >3 days
PIPETTE - Look at the pipette at eye level when setting the pipette
A. VOLUMETERIC to “0”
- deliver only 1 volume  Clear sample = lower meniscus
 Colored = upper meniscus
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 Immunology and Serology Lec
[MIDTERMS] LESSON 9
Transcribed by: ANGELICA AGUILAR
DO NOT REDISTRIBUTE WITHOUT PERMISSION!

DILUTION
- Demonstration of visible end point (
 If the relative proportions of antigen and antibody
present are not similar, the reaction cannot be
detected
o Too much Antibody = end point may
not be reached
 Serum w/ antibody must be
diluted

- SOLUTE = material being diluted -Ex. SERIAL DILUTON

 Serum = most common  same amount of diluent in each tube, w/ dye
 antibiotic  every dilution made, amount of dye is cut in
 RBC & WBC half in each successive tubes
 final volume of each tube should be the same
- DILUENT = medium making up the rest of the solution  color becomes lighter
 Normal Saline Solution (NSS) = most
common - STEPS
 1st tube = 1mL solute and 1mL solvent = ½
- indication of relative concentration (expression of  2nd tube = 1mL of 1st tube and 1mL solvent = ½ x
concentration) 2=¼
 3rd tube = 1mL of 2nd tube and 1 mL solvent = ¼
- relationship between the two is express as fraction or x 2 = 1/8
ratio STEPS
 Ex. 1:2 = 1 part serum: 2 part of NSS Plan the number and size of simple dilution
1
necessary to reach the desired end point
-Equations are used to determine Calculate each simple dilution, keeping the dilution
2
 Total volume of solution factor the same at each step
 Amount of solute needed 3 Set up the series of tube
 Amount of diluent needed Obtain the final dilution
 Count the number of tube used
4  Set up a multiplication series in w/c the
-3 WAYS original dilution factor is raised to a
power equal to the number of tubes
 Serial dilution
*TITER
 Compound Dilution
-the last tube in w/c a positive reaction is visible
 Simple dilution

SIMPLE DILUTIONS -an indicator of an antibody’s strength

-  ↑ titer = ↑ analyte of interest
o Ex. Titer 32 vs 1028
1 amount of solute  1028 = higher (more
= antibodies)
dilution total volume
FORMULA -Ex.

or
1 1 1 1 1
1 amount of solute Dilution:
= 2 4 8 16 32
dilution−1 amount of diluent 1
64
Rxn: (+) (+) (+) (+) (+) (-)
COMPOUND DILUTIONS Titer: 32
-made when large dilutions are needed
 Ex. 1:500, 1:600 TEST PARAMETERS
A. SENSITIVITY
-requires several steps of making several smaller - proportion of people who have a disease or condition
dilutions (SERIAL DILUTION) and who have a positive test

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 Immunology and Serology Lec
[MIDTERMS] LESSON 9
Transcribed by: ANGELICA AGUILAR
DO NOT REDISTRIBUTE WITHOUT PERMISSION!

-indicates how small an amount can be and still -SERIAL DILUTION involves making a several dilutions in
produce a positive test result which the dilution factor is the same

true positives - Serial dilutions are used to determine the TITER, or

%= × 100 strength, of an antibody.
t rue positives+ false negatives
B. SPECIFICITY  The last tube in which a visible reaction is
seen is considered the END POINT.
- proportion of people who have DO NOT HAVE a
disease or condition and who have a negative test
- SENSITIVITY is defined as the proportion of people who
have a specific disease or condition and have a positive
- measure the substance that is designed to measure,
test for that disease or condition.
not interfering substances

true negatives - SPECIFICITY refers to the proportion of people who do

%= × 100
t rue negatives+ false positives
C. POSITIVE PREDICTIVE VALUE
- The probability that a person with a positive screening
test actually has the disease
true positives
%= ×100
t rue positives+ false postives
D. NEGATIVE PREDICTIVE VALUE
- The probability that a person with a negative screening
test does not have the disease
true negative
%= ×100
t rue negative+ false negative
SUMMARY
- SERUM for serological testing is obtained by allowing a
sterile tube to clot at either room temperature or 4°C
and then carefully removing the serum from the clot
after centrifugation
 Minimum time for clotting = 30 mins
o Thrombin tube = 5 minutes
 Minimum centrifuge time = 15 minutes
not have the disease or condition and who have a
negative test for that disease or condition
- If a test is HIGHLY SENSITIVE and HIGHLY SPECIFIC,
it is a good indicator that a patient has the disease or
condition if positive results are obtained.
- The POSITIVE PREDICTIVE VALUE is the likelihood
that a person with a positive screening test actually
has the disease
- The NEGATIVE PREDICTIVE VALUE is the probability
that a person with a negative screening test does not
have the disease
- POSITIVE and NEGATIVE PREDICTIVE VALUES help
the clinician to determine whether a positive or a
negative test is likely to be a true result based on a
specific test population.

-TYPE OF PIPETTES
 Volumetric
o hold a specified amount of liquid
o To deliver
 Graduated
 Serological
o Calibrated all the way to the bottom
o Blownout
 Micropipettes

- A DILUTION is the addition of a liquid to make a

weaker solution of either a reagent or a patient
specimen.
 Done to see a visible end point in an antigen-
antibody reaction

- Patient serum, the SOLUTE, is made weaker by

adding diluent so that the antibody present is not as
concentrated.

- The relationship between the serum and the total

volume can be expressed as a:
 Ratio = Ex. 1:20,
 Fraction = Ex. 1/ 20.
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