Guidelines for human andrology laboratories The American Fertility Society Birmingham, Alabama
I. Organization of the Laboratory and Definition of Services A. General Laboratory 1. The institutional affiliation, history, definition of services, and the purpose of the laboratory should be clearly defined. 2. The laboratory must be in compliance with any state or federal licensing requirements. Any current licenses, permits, and certification by any other groups or agencies should be listed. 3. The laboratory must satisfy any Institutional Review Board (or equivalent Human and/or Animal Investigation Committee) requirements for any provided service or procedure, if applicable. B. Specific Laboratory Procedures It is recognized that a single standardized protocol is inappropriate or unavailable for many andrology laboratory procedures. In the absence of a widely accepted, standardized protocol, each laboratory must develop its own protocol for that particular procedure with appropriate controls and methodology to assure reliable, acceptable results. Andrology laboratories perform some or all of the following procedures: 1. Semen analysis and procedures: the standards for semen analysis are detailed in the World Health Organization (WHO) Laboratory Manual (1) for the Examination of Human Semen and Semen-Cervical Mucus Interac-
Drafted and revised by a committee composed of William Byrd, Ph.D., Chairperson; Jeffrey P. Boldt, Ph.D.; and Don P. Wolf, Ph.D.; and approved by the Board of Directors of The American Fertility Society, June 1992. A. F. S. staff assistance was provided by Robert D. Visscher, M.D.; and Joyce Zeitz. Vol. 58, No.4, October 1992
tion. In addition to the standard semen analysis, semen may be tested for fructose, adenosine triphosphate (ATP), or other biochemical markers. Assays may include tests for sperm survival, sperm viability, and sperm membrane integrity. Sperm may be tested for their ability to penetrate human or bovine cervical mucus in either a cross-match test or in capillary tubes (2, 3). 2. Sperm antibody testing: the sperm antibody assays used must be able to measure the presence of sperm antibodies on the sperm as well as in the serum, cervical mucus, follicular fluid, seminal plasma, or other fluids. These assays may work either directly or indirectly on the sperm and fluids. Both positive and negative controls must be used to validate the assay. The mixed antiglobulin reaction and the immunobead test are described in the WHO Manual (1). Other protocols are also available (4). 3. Sperm penetration assay or the zonafree hamster oocyte test: human sperm fertility potential is measured by the ability of sperm to penetrate zona-free hamster eggs. Positive controls must be utilized to validate test results. A discussion of the zona-free hamster oocyte assay is found in the WH 0 Manual (1). Other descriptions of the assay are available (5). 4. Sperm cryopreservation: sperm cryopreservation involves the freezing and storage of human sperm for future use. Sperm for freezing may be obtained from either patients or donors. Guidelines for Semen Donor Insemination Supplement 1
have been established by The American Fertility Society (6-8). 5. Preparation of sperm for intrauterine insemination with husband or donor sperm: fresh and frozen sperm may be processed for intrauterine and intracervical insemination. Refer to the Guidelines for Embryology Laboratories for information regarding quality control (10) and necessary equipment (11) for washing sperm in preparation for intrauterine insemination. When frozen donor sperm is used for insemination, the Guidelines for Semen Donor Insemination of The American Fertility Society must be followed (6-8). 6. Computer assisted semen analysis (CASA): laboratories must have a protocol that, on a periodic basis, validates that their assisted semen analysis equipment is functioning correctly. II. Laboratory Personnel A. Personnel Qualifications and Responsibilities There should be sufficient personnel to provide andrology services as required in a timely fashion with a mechanism in place to provide back-up for laboratory personnel as required. There are several categories of personnel. Staffing levels should be appropriate for the size and volume of the program; a minimum of two qualified persons are required who are capable of performing all technical services. 1. Laboratory Director: a. Qualifications: The individual must fulfill both of the following requirements: Hold an earned doctorate degree (Ph.D.) from an accredited institution in a chemical, physical, or biological science as the major subject, or be a M.D. or D.O. licensed in the state where the laboratory is located, or be serving as a laboratory director and qualified or could have qualified on or before January 1, 1992, and must have expertise in biochemistry, biology, and the physiology of reproduction, with experience in experimental design, statistics, and problem solving. 12S
Have two years documented experience in a laboratory performing andrology procedures. This experience should include familiarity with laboratory quality control, quality assurance, inspection, accreditation and licensing procedures, and detailed knowledge of all of the technical andrology procedures performed in the laboratory. b. Responsibilities: These include the overall operation and administration of the laboratory including hiring competent personnel, formulating laboratory policies and protocols, and communicating regularly with the medical director regarding patient progress and patient protocols as they affect laboratory aspects of treatment. The laboratory director must be accessible to the laboratory to provide on site, telephone, or electronic consultations as needed. c. If the medical director is also the laboratory director, there should be a qualified, designated laboratory supervisor. 2. Laboratory Supervisor: a. Qualifications: The individual must fulfill both of the following requirements: Possess a current state license from the state where the laboratory is located, if so required by the state, and have a master's degree in sci.ence or clinical laboratory science or medical technology with at least two years of training and experience in a clinical laboratory, or have a bachelor's degree in science or medical technology and have at least four years of training or experience in a clinical laboratory. Have at least two years of laboratory training or experience in andrology, reproductive physiology, tissue culture, and sterile techniques, and be able to demonstrate competence in the technical procedures performed in the andrology laboratory. b. Responsibilities: These include providing technical supervision for Fertility and Sterility
each of the procedures performed in the laboratory. The supervisor is not required to be on site at all times procedures are performed, but must be available as needed and also accessible to provide on site, telephone, or electronic consultation. 3. Auxiliary Personnel (Technologist/ Technician): a. Qualifications: An andrology laboratory technician must fulfill both of the following requirements: Hold an earned bachelor's degree from an accredited institution with a biological, chemical, or physical science as the major subject. Individuals without this educational requirement may be qualified provided they meet all other requirements prior to January 1, 1992. Have documented experience in tissue culture, sterile techniques, and demonstrated competence in the technical procedures performed in the andrology laboratory. b. Responsibilities: These include processing specimens, being able to independently perform all of the routine technological procedures carried out in the andrology laboratory under the supervision of a laboratory director or supervisor, and reporting test results. B. Personnel Records There must be written documentation of compliance with the section described above. This should include the following items: 1. List all personnel, the capacity in which they work, and their shifts, if applicable. 2. List the education, training, and qualifications of all laboratory personnel (consider the use of Laboratory Personnel Qualification Appraisal Form OMB No. 0938-0049 or its equivalent). 3. Document the chain of command so that a responsible individual can always be identified. A qualified individual must be on duty or on call at all times. 4. Itemize personnel participation in training courses, educational proVol. 58, No.4, October 1992
grams, and/or technical meetings. . Documentation to verify education and training including any new courses must be maintained. 5. Conduct performance reviews for personnel. 6. Document the training of personnel for each specific laboratory test offered. Definitive training programs for all procedures should be established. 7. Document the health status, physicals, routine scans for isotopes, or laboratory tests on personnel whenever required. III. Laboratory Space and Design The andrology laboratory should have adequate space and a design that is appropriate for the volume and type of procedures performed and that ensures safe and comfortable working conditions. A. The andrology laboratory may share space physically with other laboratory activity. However, any activity requiring sterile · technique (i.e., sperm preparation for intrauterine insemination) should be physically separated from other activities. B. Adequate space should be provided for record keeping, data entry, and related administrative functions. C. Material for laboratory construction, ventilation ofthe area, and cleanliness should be appropriate to the laboratory work. The use of carpet in tissue culture or work areas is prohibited.
IV. Laboratory Procedure Manuals There should be a manual(s) in the laboratory describing all procedures in sufficient detail to assure reproducibility and competence in handling of mammalian gametes. The National Committee for Clinical Laboratory Standards (NCCLS) has a specified format for all laboratory procedure manuals. This format is described in NCCLS publication GP-2A. Procedure manuals should include, but are not limited to the following: A. Patient instructions for proper collection, labeling, and delivery of specimens. Each patient should have a unique identification number. A requisition slip or a form designating the patient's name, unique identification number, assay(s) to be perSupplement I
formed, and the referring physician's. name should accompany the specimen. B. Procedure sheets for the tests performed by the laboratory including the principles of the test, preparation of any standards or controls, the methodology used, references, and criteria for unacceptable results, if appropriate. If specimens are to be rejected, the criteria for rejection and procedure for safe disposal of the specimen must be established. C. All material in the laboratory manuals should be reviewed and revised annually by the director and signed. Auxilliary personnel should be updated and trained in any revised procedures. D. Every laboratory should have some effective mechanism by which the specimen is logged in with the appropriate information on the specimen and patient. For example, given a date, the laboratory should be able to document whether or not a specimen was tested, the results of the test, the referring physician, and some unique code that identifies the patient. E. The location of all patient records must be recorded in a manual. The records should identify the person performing the work, the test results, as well as results interpretation. The results should be reviewed by the laboratory director or supervisor and signed. These test results should be kept for a minimum of two years. F. Maintenance manuals for all laboratory equipment must be kept in the laboratory. This manual should include any records of equipment performance and any maintenance. G. Policy manuals should be maintained in the laboratory. These policies should include, but are not limited to, procedures for record keeping, result reporting, laboratory communication, and consent procedures (if required by the institution). V. Laboratory Equipment and Supplies (9) A. Laboratory Equipment/Facilities Laboratories are required to maintain or have access to all equipment necessary to perform those services outlined in section LB. that they perform. Since each labo14S
ratory may perform or offer different services, it is the responsibility of the laboratory director to ensure that the proper equipment is in place to perform the necessary assays. 1. Larger laboratory equipment (i.e., laminar flow hoods, biohazard lab hoods, balances) must be certified by a qualified agency on an annual basis. Certifications must be maintained on file for review. 2. There should be a regular laboratory program for checking and calibrating laboratory equipment such as pipettors, thermometers, pH meters, centrifuges, and refrigerators. Manufacturer supplied manuals or maintenance manuals for all laboratory equipment must be maintained in the laboratory. 3. There must be a mechanism for the safe handling and disposal of biohazardous waste material in the laboratory. 4. All material that comes in contact with sperm that is being prepared for cryopreservation or intrauterine insemination must be tested for toxicity. This testing requires the use of an appropriate bioassay or animal model system. 5. All laboratory chemicals and reagents must be labeled with the date received and date opened and should be stored as recommended by the supplier/manufacturer. B. Culture Medium Preparation and Quality Control Testing 1. Culture medium formulated de novo should utilize dedicated reagents, glassware, and tissue-culture-grade water (or its equivalent) in its preparation. Quality control testing utilizing an appropriate bioassay system to evaluate the medium is required. More extensive discussion for the procedures and documentation necessary to prepare medium can be found in the Guidelines for Human Embryology Laboratories, Section IV.C.l.c. 2. Quality control testing is optional when commercial media is purchased and used within its labeled expiration period. However, documentation of quality control testing using an approFertility and Sterility
priate bioassay system must be supplied by the manufacturer. It is strongly suggested that each batch of culture medium be tested using an appropriate animal or cell culture model, since pretesting by the manufacturer may not reflect medium suitability when in actual use in the laboratory. C. The use of human serum or human albumin from any source (with the exception of maternal serum being used in the serum donor) will require documentation or testing to establish the absence of sexually transmitted diseases such as Hepatitis B, Hepatitis C, and human immunodeficiency virus (HIV). D. Cryopreservation medium for sperm freezing: each new lot of medium utilized for cryopreservation must be tested for postthaw survival of sperm. An acceptable survival of donor sperm is 50 percent of the initial motile cell population. VI. Laboratory Safety and Infection Control Laboratory procedures and policies on lab safety must be available to all laboratory personnel and should be reviewed annually by the laboratory director. Protocols should be available for fire and electrical safety and internal and external disaster preparedness. In addition, the following guidelines are recommended: A. The laboratory must undergo a safety inspection on a regular basis (at least annually) by personnel trained in this task. The results of this inspection must be kept on file for reference. This includes fire inspection, storage of hazardous materials, infection control, and storage of volatile or hazardous materials. Adequate fire and safety precautions must be posted. The facility should be well lighted and ventilated. B. All body fluids should be handled as if they were contaminated. Safety precautions are to be taken when handling and disposing of any biological specimens. This includes wearing gloves and laboratory coats and avoiding accidental wounds from sharp instruments. Hands must be washed after removing gloves and immediately if they become contaminated with biological material. Disposable laboratory supplies should be used whenever possible. Vol. 58, No.4, October 1992
C. All procedures involving body fluids should minimize the creation of aerosols or droplets. The use of appropriate hoods should be considered when procedures are conducted that have a high potential for creating aerosols such as centrifugation. Alternatively, centrifuges designed to contain aerosols can be utilized. Capped tubes must be used during centrifugation. D. All laboratory personnel should be offered vaccinations for Hepatitis B and testing for sexually transmitted diseases. A copy of these results or a statement of declination should be included in the laboratory records. E. Containers must be labeled as to their contents and dated. F. Adequate facilities should be available for safe disposal of hazardous or biological waste. All biohazardous waste should be placed in a container marked BIOLOGICAL HAZARD and disposed of accord-· ingly. G. There should be no smoking, eating, drinking, or application of makeup in the laboratory. H. Mechanical pipetting devices must be used for manipulation of liquids in the laboratory. Mouth pipetting is not permitted. I. All work surfaces must be decontaminated with a disinfectant immediately after any spills occur and after the end of each shift.
J. There must be sufficient space available for working. The room temperature, ventilation, noise level, and fume removal should be adequate. Utilities, communication equipment, and housekeeping should be adequate. K. Radioisotopes or biohazardous chemicals cannot be used in the same room where sperm are prepared for intrauterine insemination or cryopreservation. L. Laboratory personnel should periodically review their safety guidelines, for instance "Safety Guidelines for the Andrology Laboratory (10)." VII. Quality Control/Assurance (11) A. Quality Control To assure reliable results, the following recommendations must be followed: Supplement I
1. Document all new protocols, review
and place in appropriate manuals. Protocols should include a description of the assay, standards, controls, calibration, tolerances, and limits, where applicable. New procedures should be evaluated by parallel testing (when possible) during evaluation. The National Committee for Clinical Laboratory Standards (NCCLS) publication GP-2A specifies the format for laboratory protocols. 2. The laboratory director and/or supervisor will review and update all procedures on at least a yearly basis. Copies of old protocols and updated procedures must be kept for at least two years. Record effective dates of all changes in protocols. 3. Maintain and calibrate equipment against National Safety Board (NSB) Standard Reference Materials, when possible, on a regular basis. This ineludes a record of instrument calibration, functional checks of equipment, when possible, evidence of an active review of records, and documentation of corrective action taken when instruments malfunction. 4. Date all reagents, media, or chemicals. All outdated materials· should be discarded in an appropriate manner. 5. Use positive and negative controls when performing sperm antibody testing and positive controls when performing the sperm penetration assay. 6. Infection control: use only hepatitis and HIV -screened materials. Use sterile techniques, appropriate disease screens, and relevant laboratory procedures. 7. Conduct daily monitoring of temperature, gases, and humidity (when appropriate) for all equipment used on a daily basis. B. Quality Assurance The quality assurance program should include a mechanism to review and analyze
data in order to identify problems related to the quality of care provided by the laboratory. This should include, but is not limited to, the following: 1. Mechanisms to detect clerical, transcriptional, or analytical mistakes. 2. Data from the laboratory should be gathered and analyzed on a regular basis and the information gathered should be used to identify and resolve problems. A copy of this report should be kept for review. Quality assurance should include the turnaround time for reports and consistency of service as well as statistical analysis of data. 3. Infection control: Use HIV and Hepatitis B and C screened serum products. 4. An adverse reaction file should be maintained. REFERENCES 1. World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1987:1-67. 2. Blasco L. Clinical tests of sperm fertilizing ability. Fertil Steril 1984;41:177-92. 3. Moghissi KR. The function of the cervix in human repro· duction. Curr Probl Obstet Gynecol1984;7:1-58. 4. Bronson R, Cooper G, Rosenfeld D. Sperm antibodies: their role in infertility. Fertil Steril1984;42:171-83. 5. Wolf DP, Sokoloski JE. Characterization of the sperm penetration bioassay. J Androl 1982;3:445-51. 6. The American Fertility Society. New guidelines for the use of semen donor insemination: 1986. Fertil Steril1986;46(Supp 2):95S-110S. 7. The American Fertility Society. Revised new guidelines for the use of semen donor insemination. Fertil Steril 1986;49: 211. 8. The American Fertility Society. New guidelines for the use of semen donor insemination: 1990. Fertil Steril1990;53(Supp 1):1S-13S. 9. Gerrity M. Selection and use of equipment. In: Wolf DP, editor; Bavister BD, Gerrity M, Kopf GS, assoc. editors. In vitro fertilization and embryo transfer: a manual of basic techniques. New York: Plenum Press, 1988:7-24. 10. Schrader SM. Safety guidelines for the andrology laboratory. Fertil Steril1989;51:387-9. 11. Gerrity M. Quality control and laboratory monitoring. In: Wolf DP, editor; Bavister BD, Gerrity M, Kopf GS, Assoc. editors. In vitro fertilization and embryo transfer: a manual of basic techniques. New York: Plenum Press, 1988:25-45.
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