Alanine

Anti-sperm antibodies

Q I recently read about anti-sperm antibodies and infertility. How often is infertility caused by these antibodies? How does one perform this test?

A Scientific studies have shown considerable evidence that antibodies on the sperm surface and in cervical mucus are associated with interference in sperm motility and gamete interaction.[1,2] Thus, sperm antibodies detected on the sperm surface or in the cervical mucus play a definite role in fertility.[1] The prevalence of sperm-bound antibodies among infertile men is about 10%.[3,4]

The role of sperm antibodies in the inhibition of sperm penetration into cervical mucus has been clearly defined. Moghissi, et al,[5] found 25% of women from 172 infertile couples had sperm antibodies in the cervical mucus and 13% of the women had sperm antibodies in serum.

In one study, 64% of 66 couples without significant sperm surface anti-sperm antibody had adequate sperm motility in cervical mucus, compared with only 26% of 122 couples where sperm-associated anti-sperm antibodies were detected.[6]

The presence of sperm antibodies in serum is not closely correlated with the presence of sperm antibodies in cervical mucus in a given individual.[7-10] The clinical significance of serum sperm antibodies in both men and women is controversial.[1,2]

There are numerous methods for determining sperm antibodies. These include tray slide sperm agglutination, mixed agglutination reaction, sperm immobilization, sperm cytotoxicity, ELISA, indirect immunofluorescence, radiolabeled antiglobulin, immunobead binding, and flow cytometric sperm antibody.[1,11,12]

Anti-sperm antibodies may be determined in the blood or semen of men.[12,13] The optimal method for anti-sperm antibody detection should quantitatively measure isotype-specific, sperm-surface antibody on living sperm cells.[1]

The assay employed in our laboratory, developed by Bronson, et al, is an immunobead test that quantitates sperm surface antibody on living cells. Defining the immunoglobulin (Ig) isotype further determines specificity and loci of binding. The immunobead test identifies antibody located on the sperm surface by incubation with polyacrylamide beads coated with an antibody specifically directed against the specific IgG, IgA, and IgM human isotypes.[1,14,15] The attachment of polyacrylamide beads to regional sites on motile sperm can be quantitatively measured under light microscopy:

* A weakly positive response is present if 20%-49% of sperm are bound by immunobeads.

* An intermediate response is present when 50%-89% of sperm bind to immunobeads.

* A high response is present when 90%-100% binding is present.[16]

This method allows identification of the region of the sperm surface bound by antibodies. Head-directed antibodies can interfere with sperm penetration into cervical mucus and with attachment and fusion with oocyte membrane and structures.[12,16] Tail-principal, piece-directed antibodies affect penetration into and through cervical mucus. Tail-tip-directed IgG and IgA antibodies have negligible impact, but tail-tip-directed IgM antibodies may interfere with progression through cervical mucus.[16]

Immunoglobulin-specific immunobead assays can be used to measure antibodies attached to the sperm after they are incubated with the female's serum or cervical mucus.[12] In further studies of relevant sperm antibodies involved in human infertility, the relevant sperm cell antigens should be identified and characterized. Also, reliable assays of anti-sperm antibody activity reactive with the specific sperm antigens should be developed.[1]

- Robert M. Nakamura

References

1. Marshburn PB, Kutteh WH. The role of anti-sperm antibodies in infertility. Fertil Steril. 1994; 61: 799-811.

2. Naz RK, Menge AC. Anti-sperm antibodies: Origin, regulation, and sperm reactivity in human infertility. Fertil Steril. 1994; 61: 1001-1013.

3. Pattinson HA, Mortimer D. Prevalence of sperm surface antibodies in the male partners of infertile couples as determined by immunobead screening. Fertil Steril. 1987; 48: 466-469.

4. Eggert-Kruse W, Hofsab A, Hauru E, Tilgen W, Gerhard I, Runnebaum B. Relationship between local anti-sperm antibodies and sperm-mucus interaction in vitro and in vivo. Hum Reprod. 1991; 6: 267-276.

5. Moghissi KS, Sacco AG, Borin K. Immunologic infertility. 1. Cervical mucus antibodies and postcoital test. Am J Obstet Gynecol. 1980; 136: 941-950.

6. Mathur S, Williamson HO, Baker ME, Rust PF, Holtz GL, Fudenberg HH. Sperm motility on postcoital testing correlates with male autoimmunity to sperm. Fertil Steril. 1984; 41: 81-87.

7. Haas GG Jr. The inhibitory effect of sperm-associated immunoglobulins on cervical mucus penetration. Fertil Steril. 1986; 46: 334-337.

8. Bronson RA, Cooper GW, Rosenfeld DL. Autoimmunity to spermatozoa: Effect on sperm penetration of cervical mucus as reflected by postcoital testing. Fertil Steril. 1984; 41: 609-614.

9. Menge AC, Medley NE, Mangione CM, Dietrich JW. The incidence and influence of anti-sperm antibodies in infertile human couples on sperm-cervical mucus interactions and subsequent fertility. Fertil Steril. 1982; 38: 439-446.

10. Shai S, Bar-Yoseph N, Peer E, Naot Y. A reverse (antibody capture) enzyme-linked immunosorbent assay for detection of anti-sperm antibodies in sera and genital tract secretions. Fertil Steril. 1990; 54: 894-901.

11. Rasanen M, Lahteenmaki A, Saarikoski S, Agrawal Y. Comparison of flow cytometric measurement of seminal anti-sperm antibodies with the mixed antiglobulin reaction and the serum tray agglutination test. Fertil Steril. 1994; 61: 143-150.

12. Smith S. Use of the laboratory in infertility and recurrent spontaneous miscarriage. Clin Lab Med. 1992; 18: 393-409.

13. Clarke GN, Elliot P J, Smaila C. Detection of sperm antibodies in semen using the immunobead test: A survey of 813 consecutive patients. Am J Reprod Immunol Microbiol. 1985; 7:118-123.

14. Bronson RA, Cooper GW, Rosenfeld DL. Correlation between regional specificity of anti-sperm antibodies to the spermatozoan surface and complement-mediated sperm immobilization. Am J Reprod Immunol. 1982; 2: 222-224.

15. Bronson RA, Cooper GW, Rosenfeld DL. Membrane-bound sperm-specific antibodies: Their role in infertility. In: Jagiello G, Vogel H, eds. Bioregulators of Reproduction. New York, NY: Academic Press; 1981; 521-527.

16. Bronson RA, Cooper GW, Rosenfeld DL Sperm antibodies: Their role in infertility. Fertil Steril. 1984; 42: 171.

ALT and blood donors

Q I read that alanine aminotransferase (ALT) tests are no longer necessary for blood donors. Is that true, and if so, who made the decision?

A In January 1995, the National Institutes of Health Consensus Statement, "Infectious Disease Testing for Blood Transfusions," was published.[1] The objective of the study was to provide physicians and other transfusion medicine professionals with a consensus on the matter. The consensus panel consisted of 12 experts in hematology, cardiology, transfusion medicine, infectious disease, and epidemiology. They concluded ALT should be discontinued as a surrogate marker for blood donors. Their logic was based on two points:

* There are more specific tests than ALT to identify hepatitis C carriers.

* Too many donors were being eliminated from the blood donor pool because of elevated ALT values.

In the past there was no specific test to measure the presence of the hepatitis C carrier state. In fact, in those days it was referred to as non-A, non-B hepatitis. In 1986 and 1987, ALT was introduced to identify donors at risk of transmitting post-transfusion non-A, non-B hepatitis. This was based on two major studies in the late 1970s that indicated the likelihood of non-A, non-B hepatitis in transfusion recipients was higher in those receiving units from donors with elevated ALT values.

The numbers were dramatic. About 45% of recipients receiving blood from donors with high ALT (60-284 U/L) developed non-A, non-B hepatitis; only 5% of recipients of blood from donors with low ALT (1-14 U/L) were so affected. Based on these results as well as on the use of anti-hepatitis B core antigen testing, up to 50% of the non-A, non-B post-transfusion hepatitis could be eliminated.

Enter the new generation of tests for anti-HCV, which are more sensitive and much more specific. They have made the ALT donor test redundant and inappropriate. So why continue to use a test of lesser diagnostic utility when a superior test is available?

There is one additional problem with ALT testing: Increased ALT values may occur due to many non-pathological reasons. In simpler terms, some healthy people who never had hepatitis have ALT values above 60 U/L. This phenomenon caused the discarding of up to 200,000 good units of donated blood each year. The NIH-consensus statement concludes: "ALT testing of volunteer blood donors should be discontinued. Persons previously deferred for an isolated elevation in ALT may now be reevaluated for donor eligibility."[1]

- Bernard E. Statland

Reference

1. NIH Consensus Group. Infectious Disease Testing for Blood Transfusions. Washington, DC: NIH; Jan. 9, 1995; 13(1).

Arterial line draws

Q What are the recommended procedures for using arterial lines, Grosshong catheters, and Port-caths to collect blood samples? How much blood should be discarded with each device used? Should the discard be returned to the patient to avoid hospital-induced anemias? Can the discard specimen be used for any testing? Can coagulation be drawn from any of these lines? Should a saline rinse be administered before blood collection? We've used arterial lines to collect blood samples for years, but we feel we need to rethink our procedures, considering all the new products being used. And we're concerned we may be compromising the quality of care of our patients.

A Arterial and venous catheters usually contain a flush solution with a small amount of heparin - enough to invalidate coagulation tests unless the solution is thoroughly removed from the line before obtaining a sample for coagulation tests. The line is cleansed by withdrawing blood. Typically intravenous and arterial lines have a dead-space volume of 0.7-2.0 mL. We and many others who have studied this problem recommended that about six times the dead-space volume be withdrawn before obtaining a sample for coagulation tests and that one to two times the volume be withdrawn before obtaining blood for noncoagulation testing.

When obtaining a sample from an indwelling catheter, the procedure should be performed only by personnel who are familiar with sterile techniques to avoid infecting the patient. Following the withdrawal of the specimen, it is necessary to flush the catheter with a dilute heparin solution to prevent clotting in the line. In many hospitals, this only is done by a registered nurse.

The first fluid withdrawn from the catheter contains flushing solution. This cannot be returned to the patient. I've heard of giving a patient some of the withdrawn blood, but I would not do so in my lab.

We require that 2 mL of blood be discarded if noncoagulation tests are to be drawn and 6 mL if coagulation testing is scheduled. If the line is unusually long, a larger amount must be discarded. To reduce the amount of blood withdrawn, and to use as much as possible for testing, I recommend that blood for chemistry tests be obtained after the 2-mL discarded sample has been removed. This should be sufficient to rinse the line for coagulation tests. Several papers in critical care nursing literature have discussed this issue in detail.

- Daniel M. Baer

Suggested reading

Clapham MCC, Willis N, Mapleson WW. Minimum volume of discard for valid blood sampling from indwelling arterial cannulae. Br J Anaesth. 1987; 59: 232-235.

Molyneaux RD, Papciak B, Rorem DA. Coagulation studies and the indwelling heparinized catheter. Heart Lung. 1987; 16: 20-23.

Rudisill PT, Moore LA. Relationship between arterial and venous activated partial thromboplastin time values in patients after percutaneous transluminal coronary angio-plasty. Heart Lung. 1989; 18: 514-519.

Edited by Bernard E. Statland, M.D., Ph.D., Statland Laboratory Consulting, Nashville, Tenn.

Panelists (name following each answer indicates respondent panelist):

Daniel M. Baer, M.D., Chief of Pathology, Veterans Affairs Medical Center, and professor of pathology, Oregon Health Sciences University, Portland, Ore. Kathleen G. Beavis, M.D., assistant director, microbiology laboratory, Thomas Jefferson University Hospital, Philadelphia, Pa. John A. Koepke, M.D., professor emeritus of pathology, Duke University Medical Center, Durham, N.C. Byron A. Myhre, M.D., Ph.D., professor of pathology and chief of clinical pathology, UCLA School of Medicine, Harbor General Hospital Campus, Torrance, Calif. Robert M. Nakamura, M.D., senior consultant and chairman emeritus, department of pathology, Scripps Clinic and Research Foundation, La Jolla, Calif.

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