LAB # 5
SERIAL DILUTION
Dilution
For many tests, a measured amount of a serum
sample is used directly for detection of
antibodies.
However, in order for a visible end point to
occur in a serological reaction, the relative
proportions of antigen and antibody present are 2-folds Dilution
important. - SOLUTE, TOTAL VOLUME, DILUENT
Zone of equivalence: equal amount of antigen **If in this instance, an endpoint (Agglutination) was
and antibody for better reaction reached at tube number five, the actual titer would be
High concentration Diluted 32.
Serial dilution: determine the titer of antibody;
Clinical Chemistry TITER
A dilution involves two entities: spread of antibody; reciprocal of final dilution
Solute – serum sample; being diluted where the last positive reaction occurs
Diluent – Normal Saline Solution Serial dilutions do not always have to be
doubling dilutions. (5-folds, 10- folds
Solute dilution, etc…)
DILUTION= 1:5→1:25→1:125→1:625→1:3125
Total Volume
SOLUTE=Dilution ×Total Volume Pre-Requisite:
Principle of Serial Dilution
Solute Blood extraction (Venipuncture)
TOTALVOLUME=
Dilution Centrifugation➔ Serum Sample (Red top – no
anticoagulant)
DILUENT =Total Volume−Solute Pipetting technique using Automatic pipette
ABO antibodies
SIMPLE DILUTION
Only one test tube/ container is used ABO Antibodies
Once you mixed the solute and the diluent ➔ BLOOD GROUP ANTIBODY
Desired Dilution PRODUCED
A Anti-B
COMPOUND DILUTION B Anti-A
Two or more test tubes are used. AB No antibody
The amount of diluent on each test tubes may O Anti-A, Anti- B, Anti-
vary. AB
The last test tube has the desired dilution and
will be used in the procedure. - By Karl Landsteiner
- ABO Antibodies – naturally occurring (at
SERIAL DILUTION birth)
If in each step of the dilution, the dilution - If you do not have the antigen, you have
factor is exactly the same. the antibody against that missing antigen
A series of test tubes is set up with exactly the - EX: You are Blood type A; you have Anti
same amount of diluent on each test tube. B on your plasma or serum
EX: All 10 test tube has NSS
Avoid formation of bubbles PROCEDURE: 2-folds dilution
Doubling dilution – most common; initial Prepare a serum sample from a Blood Type A
dilution for each test tube is 1:2 dilution and Blood type O patient. (both have Anti-B)
Final dilution – to get the titer Prepare 2 sets of test tubes, labeled with A1-
Initial dilution x final dilution of A10 and B1-B10.
previous test tube
Add 0.5mL of NSS (Diluent) to test tubes A1- REMEMBER, THIS PATIENT IS BLOOD TYPE
A10 and B1-B10. O WITH ANTI-B ON ITS SERUM SAMPLE
On A1 test tube, Add 0.5mL of serum from
Blood type A patient and mixed properly Next step is to test each dilutions.
(aspirate, dispense, aspirate, dispense…) Prepare 2 new sets of test tubes, labeled A1-
AND On B1 test tube, Add 0.5mL of serum A10 and B1-B10.
from Blood type O patient and mixed Using sterile dropper/Pasteur pipette, add 1
properly (aspirate, dispense, aspirate, drop of B-cell Reagent (has B-antigen) on
dispense…). Each test tubes. A1-A10 and B1-B10
Aspirate 0.5mL from A1 test tube and transfer Add 2 drops of A1 dilution to A1 test tube
it to A2 test tube and mixed properly containing 1 drop of B-cell reagent and
(aspirate, dispense, aspirate, dispense…). mixed the test tube gently.
REPEAT THE PROCEDURE UP TO THE Add 2 drops of A2 dilution to A2 test tube
LAST TEST TUBE (A10). containing 1 drop of B-cell reagent and mixed
On the last test tube (A10), Aspirate the test tube gently.
0.5mL and then discard. Repeat the procedure up to the last dilution.
Aspirate 0.5mL from B1 test tube and transfer Add 2 drops of B1 dilution to B1 test tube
it to B2 test tube and mixed properly containing 1 drop of B-cell reagent and mixed
(aspirate, dispense, aspirate, dispense…). the test tube gently.
REPEAT THE PROCEDURE UP TO THE Add 2 drops of B2 dilution to B2 test tube
LAST TEST TUBE (B10). containing 1 drop of B-cell reagent and mixed
On the last test tube (B10), Aspirate the test tube gently.
0.5mL and then discard. Repeat the procedure up to the last
SAME PIPPETE WITH SAME SAMPLE. dilution.
**USE DIFFERENT PASTEUR PIPETTES
AFTER PERFORMING THESE STEPS: WHAT **Can be performed on a glass slide
DO WE HAVE NOW?
REMEMBER, THIS PATIENT IS BLOOD TYPE AFTER PERFORMING THESE STEPS: WHAT
A WITH ANTI-B ON ITS SERUM SAMPLE DO WE HAVE NOW?
REMEMBER, THESE TEST TUBES
CONTAINS A MIXTURE OF 2 DROPS FROM
THE DILUTION AND 1 DROP OF B-CELL
REAGENT.
AFTER PERFORMING THESE STEPS: WHAT
DO WE HAVE NOW?
AFTER PERFORMING THESE STEPS: WHAT
DO WE HAVE NOW?
REMEMBER, THESE TEST TUBES
CONTAINS A MIXTURE OF 2 DROPS FROM
THE DILUTION AND 1 DROP OF B-CELL
REAGENT.
Centrifuge all the mixtures for 15 seconds at
3400 rpm. (Add parafilm/cover to mix
properly)
Avoid over centrifugation false-positive
Dislodge the mixture after centrifugation
(gentle mixing/tapping)
Do not read the result without mixing gently
after centrifugation false-positive
INTERPRET THE RESULT
POSITIVE REACTION: WITH
AGGLUTINATION; hemolysis
(seldom)
NEGATIVE REACTION:
WITHOUT AGGLUTINATION
REPORT THE TITER:
“TITER is the reciprocal of the final
dilution of the last test tube with
positive reaction”
Compare
- higher titer, higher concentration of Anti-
B
