Procedures Manual

The procedures manual must be a complete document of current techniques and approved policies that is available at all times in the immediate bench area of laboratory personnel. It is extremely important that all personnel review this manual periodically. The manual should comply with the CLSI format for a procedure (see Box 7-1). The procedural format found in this text generally follows these guidelines.

Alternate techniques can be included with each procedure if more than one technique is acceptable. New pages must be dated and initialed when inserted and removed pages must be retained for 5 years, with the date of removal and the reason for removal indicated. It may be legally necessary to identify the procedure followed for a particular reason.

Procedures used in immunology apply many techniques common to other scientific disciplines, such as chemistry. In the field of immunology, different serologic techniques are used to detect the interaction of antigens with antibodies. These methods are suitable for the detection and quantitation of antibodies to infectious agents, as well as microbial antigens and nonmicrobial antigens (see Part III of this text).

Blood Specimen Preparation

After blood has been obtained from a patient, in a plain evacuated tube, without anticoagulant, it should be allowed to clot and the serum should be promptly removed for testing. Clotting and clot retraction should take place at room temperature or in the refrigerator, depending on the protocol for the specific procedure. Complete clot retraction normally takes about 1 hour. After clot retraction, the clot should be loosened from the sides of the test tube with an applicator stick and centrifuged for 10 minutes at a moderate speed.

After centrifugation, serum can be transferred to a labeled tube with a Pasteur pipette and rubber bulb. If the serum is contaminated with erythrocytes, it should be recentrifuged. The serum-containing tube should be sealed.

Excessive heat and bacterial contamination are avoided. Heat coagulates the proteins and bacterial growth alters protein molecules. If the test cannot be performed immediately, the serum should be refrigerated. In most cases, if the testing cannot be done within 72 hours, a serum specimen must be frozen at −20° C. Standard Precautions must be followed when blood specimens are handled.

For some testing, the serum complement must first be inactivated (see following discussion). If the protein complement is not inactivated, it will promote lysis of the red blood cells and other types of cells and can produce invalid results. Complement is also known to interfere with certain tests for syphilis.

Types of Specimens Tested

Most immunology tests are done on serum, although body fluids may also be tested. Lipemia, hemolysis, or any bacterial contamination can make the specimen unacceptable. Icteric or turbid serum may yield valid results for some tests but may interfere with others. Blood specimens should be collected before a meal to avoid the presence of chyle, an emulsion of fat globules that often appears in serum after eating, during digestion. Contamination with alkali or acid must be avoided because these substances have a denaturing effect on serum proteins and make the specimens useless for serologic testing.

Other specimens include urine for pregnancy tests and tests for urinary tract infection. It is important that the urine specimen be collected after thorough cleaning of the external genitalia to prevent contamination of microbiological assays. Urine for the hCG assay (pregnancy test) must be collected at a suitable time interval after fertilization to allow the concentration of the hCG hormone to rise to a significantly detectable level.

Any specimen must be collected into a suitable container to prevent in vitro changes that could affect the assay results. Proper handling and storage of the specimen until testing are essential. Immunologic assays are also done on cerebrospinal fluid (CSF), other body fluids, and swabs of various body exudates and discharges. The established protocol for each specific assay must be followed in terms of specimen collection requirements and conditions for the assay itself.

Inactivation of Complement

Some procedures require the use of inactivated serum. Inactivation is the process that destroys complement activity. Complement is known to interfere with the reactions of certain syphilis tests and complement components (e.g., C1q). It can agglutinate latex particles and cause a false-positive reaction in latex passive agglutination assays. Complement may also cause lysis of the indicator cells in hemagglutination assays.

Complement in body fluids can be inactivated by heating to 56° C for 30 minutes. When more than 4 hours has elapsed since inactivation, a specimen can be reinactivated by heating it to 56° C for 10 minutes.

Pipettes

Pipettes are used in the immunology-serology laboratory for the quantitative transfer of reagents and the preparations of serial dilutions of specimens such as serum (Fig. 8-1). Although semiautomated micropipettes have replaced traditional glass pipettes in the laboratory, traditional methods may still be needed at times.

With practice, it is important to develop a good technique for handling pipettes (Fig. 8-2). The same general steps apply to pipetting with all manual pipettes (Box 8-1), with few exceptions.

Laboratory accidents frequently result from improper pipetting techniques. The greatest potential hazard is when mouth pipetting is done instead of mechanical suction. Mouth pipetting is never acceptable in the clinical laboratory.

After the pipette has been filled above the top graduation mark, removed from the vessel, and held in a vertical position, the meniscus must be adjusted. The meniscus is the curvature in the top surface of a liquid (Fig. 8-3). The pipette should be held so that the calibration mark is at eye level. All readings must be made with the eye at the level of the meniscus. The delivery tip is touched to the inside wall of the original vessel, not the liquid, and the meniscus of the liquid in the pipette is eased, or adjusted, down to the calibration mark.

Before the measured liquid in the pipette is allowed to drain into the receiving vessel, any liquid adhering to the outside of the pipette must be wiped off with a clean piece of gauze or tissue. If this is not done, any drops present on the outside of the pipette might drain into the receiving vessel along with the measured volume. This would make the volume more than that specified and an error would result.