Basic Serologic Laboratory Techniques
ANTIBODY TITER
A central concept of serologic testing is the manifestation of a rise in titer, or concentration, of an antibody. The antibody titer is defined as the reciprocal of the highest dilution of the patient’s serum in which the antibody is still detectable. That is, the titer is read at the highest dilution of serum that gives a positive reaction with the antigen. If a serum sample has been diluted 1:64 and reacts positively with the antigen suspension used in the testing process, and if the next highest dilution of 1:128 does not give a positive reaction, the titer is read as 64. A high titer indicates that there is a relatively high concentration of the antibody present in the serum.
Determination of the concentration of antibody (titer) for a specific antigen involves the following two steps:
1. Preparing a serial dilution of the antibody-containing solution (e.g., serum)
2. Adding an equal volume of antigen suspension to each dilution
A high titer indicates that a considerable amount of anti-body is present in the serum. For most pathogenic infections, an increase in the patient’s titer of two doubling dilutions, or from a positive result of 1:8 to a positive result of 1:32 over several weeks, is an indication of a current infection. This is known as a fourfold rise in the antibody titer.
Serial Dilution Principle
Serial dilutions are a method for determining the concentra-tion of a substance (e.g., antibody). The greatest dilution of the CASE STUDY
JJ, a 9-year-old boy, was taken to the emergency depart-ment with a sore throat. On examination, he had redness of the throat and slightly swollen glands. The physician assistant ordered a throat culture and blood drawn for an antistreptolysin-O antibody (ASO). An antibiotic was prescribed for a 10-day period. His mother was told to make an appointment with his pediatrician for a follow-up.
At the follow-up visit 2 weeks later, the results of the laboratory test revealed a throat culture with a few colonies of β-streptococci. The qualitative ASO test result was reported as positive. The acute serum was frozen at the
time of testing. The pediatrician ordered a convalescent specimen to be tested semiquantitatively in parallel with the acute specimen for an ASO titer.
The acute and convalescent specimens were prepared as twofold serial dilutions of each specimen (see table).
Tube
1 2 3 4 5 6
Saline (µL) — 50 50 50 50 50
Serum (µL) 50 50 50 (1:2) 50 (1:4) 50 50 Mix and
transfer to next tube
50 50 50 50 50
Dilution/titer 1:1 1:2 1:4 1:8 1:16 1:32
IU/mL 200 4008 800 1600 3200 6400
The results of the parallel testing of the acute and convalescent specimens revealed the following:
• Acute specimen positive, 1:1 dilution/titer (IU/mL • Convalescent specimen positive, 1:4 dilution/titer 200)
(IU/mL 800) Questions
1. The convalescent specimen demonstrated:
a. No evidence of streptococci infection b. A possibility of streptococci infection
c. Significant evidence of streptococci infection d. Evidence of a chronic streptococci infection 2. Comparing acute and chronic patient
speci-mens can:
a. Distinguish acute from chronic infection.
b. Diagnose the cause of the infection.
c. Demonstrate at least a two-fold dilution rise that is significant for an acute infection.
d. Demonstrate at least a two-fold dilution rise that is significant for a chronic infection.
See Appendix A for the answers to these questions.
Critical Thinking Group Discussion Questions 1. Is the difference between the acute and
conva-lescent titers significant?
2. What does a rise in titer mean?
See instructor site for the discussion of the answers to these questions.
sample that yields a positive result is the end point. This end (e.g., hemagglutination). Colorimetric reactions can be per- formed (e.g., enzyme immunoassay) and quantitated spectropho-tometrically with specialized instruments for microtiter plates.
Interpretation of Results
In clinical immunology, the titer of an antibody in an individ-ual’s serum can have clinical significance, depending on the antibody in question. Antibody titers are sometimes used to evaluate a person’s immune status. Titers may be obtained over time, as with acute and convalescent specimens for infectious diseases or monitoring a mother’s titer for blood group anti-bodies during pregnancy.
CHAPTER HIGHLIGHTS
• Traditional serologic tests have been done for viral and bacterial diseases. Other common tests include pregnancy tests for human chorionic gonadotropin (hCG) and immunologic tests for infectious mononucleosis and syphilis.
• The procedures manual describes current techniques (in CLSI format) and approved policies and is always available to laboratory personnel.
• After a blood sample has clotted, serum should be promptly removed for testing or frozen at −20° C. Standard Precau-tions must be followed when blood specimens are handled.
• Lipemia, hemolysis, and bacterial contamination can make the specimen unacceptable. Icteric or turbid serum may give valid results or may interfere. Blood specimens should be collected before a meal to avoid chyle. Contamination with alkali or acid must be avoided.
• Some procedures require inactivated serum. Complement can be inactivated by heating to 56° C for 30 minutes or, after 4 hours, reinactivated by heating for 10 minutes.
• A graduated pipette delivers the liquid between two calibration marks. A serologic pipette resembles the gradu-ated pipette, but has a frosted ring and enlarged tip opening.
• Automatic pipettes allow fast repetitive measurement and delivery of solutions of equal volumes.
• All dilutions are a ratio. Dilution is an indication of relative concentration.
• A dilution factor is used to correct for having used a diluted sample in a determination rather than the undiluted sample. The result (answer) using the dilution must be multiplied by the reciprocal of the dilution made.
• When the concentration is too high or less specimen is available for analysis, the original specimen may be diluted or the initial dilution (or filtrate) further diluted. These
single dilutions are usually expressed as a ratio (1:2, 1:5, 1:10) or a fraction (½, 1⁄5, 1⁄10).
• A dilution is the volume or number of parts of the sub-stance to be diluted in the total volume, or parts, of the final solution. A dilution is an expression of concentration, the relative amount of substance in solution. Dilutions can be made singly or in series.
• In a dilution series, all dilutions, including or following the first one, are the same, called serial dilutions.
• A complete dilution series usually contains 5 or 10 tubes, although any single dilution may be made directly from an undiluted specimen or substance.
• When testing antibody levels for a specific infectious organism, blood should be drawn during both the acute and convalescent phases.
• A difference in the amount of antibody present, or the antibody titer, may be noted when two different samples are tested concurrently. A rise in titer is central to serologic testing.
• The antibody titer is defined as the reciprocal of the highest dilution of the patient’s serum in which the antibody is still detectable.
REVIEW QUESTIONS
1. A written procedural protocol should contain the following information, in the correct order: ____, ____, ____, ____. Choose from (A) to (D).
A. Specimen collection and storage B. Reference values
C. Reagents, supplies, and equipment D. Procedural method
a. A, B, C, D b. B, C, A, D c. A, C, D, B d. D, C, B, A
2. Factors that can denature, coagulate, or alter protein molecules include:
a. Heat
b. Strong acid solution c. Strong alkali solution d. All of the above
3. If testing cannot be done within _____ hours of collec-tion, a serum specimen should be frozen at −20° C.
a. 24 b. 48 c. 72 d. 96
4. Complement can be inactivated in human serum by heating to _____° C.
a. 25 b. 37 c. 45 d. 56
5. A specimen should be reinactivated when more than ______ hour(s) has (have) elapsed since inactivation.
a. 1 b. 2 c. 4 d. 8
6. A graduated pipette can be used when:
a. Extreme accuracy is not needed.
b. Very precise accuracy is needed.
c. Precision is more important than speed.
d. Precision and speed are important.
7. A meniscus is the:
a. Curvature in the top surface of a liquid b. Zero mark on a pipette
c. Last marking on a serologic pipette d. Flat line of liquid in a pipette 8. Automatic pipettes have the advantage of:
a. Being fast
b. Allowing repetitive measurement of solutions c. Delivering equal volumes of solutions d. All the above
9. A dilution is a(n):
a. Ratio of volume or number of parts of the substance to be diluted in the total volume, or parts, of the final solution
b. Indication of relative concentration
c. Frequently used measure in serologic testing d. All the above
10. If a serial dilution is prepared in 1:2 dilutions, the final dilution in tube 6 is:
a. 1:25 b. 1:32 c. 1:64 d. 1:256
11. and 12. To prepare 10 mL of a diluted serum specimen 1:10, 11. ____ part of serum is needed.
a. 1.0 b. 0.75 c. 0.50 d. 0.20 and
12. ____ parts of distilled water is (are) needed to reach the total volume.
a. 10 b. 9 c. 4.5 d. 0.1
13. Serum for detection of antibodies should be drawn during the:
a. Acute phase of illness only
b. Acute and convalescent phases of illness c. Convalescent phase of illness only
d. Acute and convalescent phases, as well as 6 months after an illness
14. A central concept of serologic testing is:
a. Antigen-antibody interaction
b. Determination of antibody composition c. Quantitation of antigen titer
d. Manifestation of a rise in antibody titer BIBLIOGRAPHY
Bishop ML, Fody EP, Schoeff L: Clinical chemistry: principles, procedures, correlations, ed 6, Philadelphia, 2010, Lippincott Williams & Wilkins.
Burtis CA, Ashwood ER, Bruns DB, editors: Tietz fundamentals of clinical chemistry, ed 6, St Louis, 2008, Saunders.
Campbell JM, Campbell JB: Laboratory mathematics: medical and biological applications, ed 5, St Louis, 1997, Mosby.
Kaplan LA, Pesce AJ: Clinical chemistry: theory, analysis, correlation, ed 5, St Louis, 2010, Mosby.
Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby.
135 ectopic pregnancy
follicle-stimulating hormone (FSH) human chorionic gonadotropin
(hCG)
immunochromatographic luteinizing hormone monoclonal
nontrophoblastic neoplasms