Basic Serologic Laboratory Techniques



Basic Serologic Laboratory Techniques



Learning Objectives


At the conclusion of this chapter, the reader should be able to:


• Identify and explain the parts of a procedure.


• Describe the preparation of blood specimens for testing.


• Provide examples of the types of specimens that can be tested using immunologic procedures.


• Explain how complement is inactivated in a serum sample.


• Compare the differences between the two types of pipettes typically used in the immunology-serology laboratory.


• Describe and demonstrate pipetting techniques using manual and automatic pipettes.


• Define the term dilution.


• Calculate the concentration of a substance using the dilution factor.


• Calculate the concentration of a single dilution.


• Compare the characteristics of the acute and chronic phases of illness.


• Define the term antibody titer.


• Analyze a case study with the interpretation of the assay results.


• Correctly answer case study related multiple choice questions.


• Be prepared to participate in a discussion of critical thinking questions.


• Correctly answer end of chapter review questions.


• Explain and prepare a serial dilution.



Key Terms



Serologic testing has long been an important part of diagnostic tests in the clinical laboratory for viral and bacterial diseases. Immunologic testing is done in many areas of the clinical laboratory—microbiology, chemistry, toxicology, immunology, hematology, surgical pathology, cytopathology, immunohematology (blood banking)—and a great variety of specimens are tested. Rapid testing is typically used in the laboratory as well as in home-testing kits.


The advent of monoclonal antibody (MAb) technology has led to the development of highly specific and sensitive immunoassays. Common serologic and immunologic tests include pregnancy tests for human chorionic gonadotropin (hCG) and tests for infectious mononucleosis and syphilis.



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.



Box 8-1   Pipetting With Manual Pipettes




1. Check the pipette to ascertain its correct size, being careful also to check for broken delivery or suction tips.


2. Wearing protective gloves, hold the pipette lightly between the thumb and the last three fingers, leaving the index finger free.


3. Place the tip of the pipette well below the surface of the liquid to be pipetted.


4. Using mechanical suction or an aspirator bulb, carefully draw the liquid up into the pipette until the level of liquid is well above the calibration mark.


5. Quickly cover the suction opening at the top of the pipette with the index finger.


6. Wipe the outside of the pipette dry with a piece of KimWipe tissue to remove excess fluid.


7. Hold the pipette in a vertical position with the delivery tip against the inside of the original vessel. Carefully allow the liquid in the pipette to drain by gravity until the bottom of the meniscus is exactly at the calibration mark. To do this, do not entirely remove the index finger from the suction hole end of the pipette; rather, by rolling the finger slightly over the opening, allow slow drainage to take place.


8. While still holding the pipette in a vertical position, touch the tip of the pipette to the inside wall of the receiving vessel. Remove the index finger from the top of the pipette to permit free drainage. Remember to keep the pipette in a vertical position for correct drainage. In TD (to deliver) pipettes, a small amount of fluid will remain in the delivery tip.


9. To be certain that the drainage is as complete as possible, touch the delivery tip of the pipette to another area on the inside wall of the receiving vessel.


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.




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.


image
Figure 8-1 Types of manual pipettes.
TD, To deliver. (From Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby.)


Graduated Pipettes


A method for delivering a particular amount of liquid is to deliver the amount of liquid contained between two calibration marks on a cylindrical tube, or pipette. Such a pipette is called a graduated pipette, or measuring pipette. It has several graduation, or calibration, marks. Graduated pipettes are used when great accuracy is not required, although these pipettes should not be used with any less care than volumetric pipettes. Graduated pipettes are used primarily for measuring reagents but are not calibrated with sufficient tolerance for measuring standard or control solutions, unknown specimens, or filtrates.


A graduated pipette is a straight piece of glass tubing with a tapered end and graduation marks on the stem separating it into parts. Depending on the size used, graduated pipettes can be used to measure parts of a milliliter or many milliliters. These pipettes come in various sizes, or capacities, including 0.1, 0.2, 1.0, 2.0, 5.0, 10, and 25 mL. If 4 mL of deionized water is to be measured into a test tube, a 5-mL graduated pipette would be the best choice.


Because graduated pipettes require draining between two marks, they introduce one more source of error compared with volumetric pipettes, which have only one calibration mark. This makes measurements with the graduated pipette less precise. Because of this relatively poor precision, the graduated pipette is used when speed is more important than precision (e.g., measurement of reagents) and is generally not considered accurate enough for measuring samples and standard solutions.



Serologic Pipettes


Another pipette used in the laboratory, the serologic pipette, looks similar to the graduated pipette. However, the orifice, or tip opening, is larger in the serologic pipette than in other pipettes. The rate of fall of liquid is much too fast for great accuracy or precision.


The serologic pipette is recognized by a frosted ring at the noncalibrated end, with calibrations extending to the tip. The letters TD (to deliver) appear on the pipette and, for quick recognition, each size of pipette has an imprinted, color-coded band that indicates the volume. The serologic pipette is usually allowed to empty by gravity. Depending on the calibration, the remaining drop needs to be expelled to deliver the full volume.


Each serologic pipette is marked with identifying numerals (e.g., 10 mL in 110image). The first of these numbers represents the total capacity of the pipette. The second number represents the smallest gradation into which the pipette is divided. In the example cited, therefore, the total pipette volume is 10 mL. Markings then divide it into 1-mL sections and each milliliter is further divided into tenths. Sizes of serologic pipettes most frequently used are 10 mL in 110image, 5 mL in 110image, 2 mL in 110image, 2 mL in 1100image, 1 mL in 110image, and 1 mL in 1100image. For greatest accuracy, the smallest pipette that will hold the desired volume should be used.




Pipetting Techniques


Manual Pipettes


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.


image
Figure 8-2 Pipetting technique. (From Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby.)

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.


image
Figure 8-3 Reading the meniscus. (From Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby.)

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.



Automatic Pipettes


Automatic pipettes allow fast, repetitive measurement and delivery of solutions of equal volumes. The sampling type measures the substance in question. The sampling-diluting type measures the substance and then adds the desired diluent. The sampling type of automatic pipette is mechanically operated and uses a piston-operated plunger. These are adjustable so that varying amounts of reagent or sample can be delivered with the same device. Disposable and exchangeable tips are available for these pipettes. Automatic pipettes and micropipettors must be calibrated before use.



Micropipettors


Automatic micropipetting devices allow rapid repetitive measurements and delivery of predetermined volumes of reagents or specimens. The most common type of micropipette used in many laboratories is one that is automatic or semiautomatic, called a micropipettor. These are piston-operated devices that allow repeated, accurate, reproducible delivery of specimens, reagents, and other liquids requiring measurement in small amounts. Many micropipettors are continuously adjustable so that variable volumes of liquids can be dispensed with the same device. Delivery volume is selected by adjusting the settings. Different types or models are available, which allow volume delivery ranging, for example, from 0.5 to 5000 µL. The calibration of these micropipettes should be checked periodically.


The piston, usually in the form of a thumb plunger, is depressed to a stop position on the pipetting device. The tip is placed in the liquid to be measured, and then the plunger is slowly allowed to rise back to the original position (Fig. 8-4

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Jun 12, 2016 | Posted by in IMMUNOLOGY | Comments Off on Basic Serologic Laboratory Techniques

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