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Serial Dilutions

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Serial Dilutions &
Antibody Titers
CLS 240
May 21, 2014

LABORATORY EXERCISE – WEEK 2 Serial Dilutions
Principle
A set of dilutions where the dilution factor is exactly the same at each step, is known as a serial dilution. Serial dilutions are commonly used in the clinical laboratory to determine the strength or titer of a particular antibody in patient serum. This information can be used to help diagnose the disease state of the patient, which will help the provider develop an effective treatment plan. Serial dilutions are created with a known quantity of both serum and diluent. When making serial dilutions it is import to ensure the zone of equivalence is maintained. The zone of equivalence is where there are an equal amount of antigens and antibodies within the dilution. If the zone of equivalence is not obtained and there are too many antibodies in the dilution it is known as prozone, whereas too antigens it is known as postzone.
Hemagglutination studies use the concentration of antibodies to help diagnose patients. When a patient first becomes ill his/her titer will be low, which is known as the acute phase. In the acute phase the titer is low because the patient has recently become ill and the patient’s body has not had enough time to develop antibodies. It is estimated within 2 weeks the patient will enter the convalescent phase where the titer will become increased, suggesting the body has developed antibodies to fight the infection. A higher titer also suggests the patient is recovering from the illness.
Quality Control
I documented the lot numbers as well as the expiration date for the A1 cells.
Sources of error * Inaccurate pipetting technique. * The use of expired reagents. * Not centrifuging for the recommended time. * Errors with the transfer process * Skipping a tube when making the serial dilutions.

Results
Patient ID:__Tyler Davis___ Tech Name/Date:__Regina O’Neal / May 14, 2014___ Tube | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Agglutination(IS) | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | Aliquot (ml)Anti-A (1:10) | .25 ml | .25 ml | .25 ml | .25 ml | .25 ml | .25 ml | .25 ml | .25 ml | Diluent (ml)saline | ----- NA | .25 ml | .25 ml | .25 ml | .25 ml | .25ml | .25ml | .25ml | Total volume | .25 ml | .50ml | .50 ml | .50ml | .50ml | .50ml | .50ml | .50ml | Dilution*Use for RBC | 1:10original | 1:20 | 1:40 | 1:80 | 1:160 | 1:320 | 1:640 | 1:1280 | | | | | | | | | | RBCs (ml) | .25ml | .25ml | .25ml | .25ml | .25ml | .25ml | .25ml | .25ml | Volume-newRBCs+ dilution | .50 ml | .50 ml | .50ml | .50ml | .50ml | .50ml | .50ml | .50ml | *Dilution factor (RBCs) | 1:20 | 1:40 | 1:80 | 1:160 | 1:320 | 1:640 | 1:1280 | 1:2560 | Titer reported | 20/1= 20 | 40 | 80 | 160 | 320 | 640 | 1280 | 2560 |
*****Dilution factor= aliquot volume/total volume (which is the same as) aliquot volume/ aliquot volume +diluent volume
Remember tube # 1 no saline added. Tube 2-8 you removed .25ml from every tube!
*You do this the same way you determine any dilution factor. 0.25/.25+.25 or .25/.50 or .5 or 50/100 or 1:2
Interpretation
Following the completion of the serial dilution I identified agglutination in all 8 tubes. Due to the positive agglutination I have determined that the antibody titer cannot be reported out. The next step would be to continue the serial dilution until I get a negative reaction. The last tube with positive agglutination will be reported out as the titer. I would conclude the patient is in the convalescent phase due to the elevated antibody titers, which suggest the patient’s body has effectively developed antibodies to fight the infection.
References
M.L. Turgeon. (2009). Immunology and Serology in Laboratory Medicine. Fourth Edition.

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