Roger L. Bertholf, Ph.D. Associate Professor of Pathology

1. Interpretation of Laboratory Tests:A Case-Oriented Review of Clinical Laboratory Diagnosis (Part 2) Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida Health Science Center/Jacksonville Mark A. Bowman, MT(ASCP), Ph.D. Associate Professor of Clinical Pathology Clinical Laboratory Sciences Program Director University of Iowa College of Medicine

2. Case 1: Failure to Conceive

3. Case History A couple visits their family doctor, complaining that the wife had been unable to become pregnant. What questions should you ask?

4. Infertility • Definition: One year of unprotected intercourse without pregnancy • 1°: No previous pregnancies • 2°: Previous pregnancy (not necessarily live birth) • Fecundability: Probability of achieving pregnancy within a menstrual cycle • 20-25% for normally fertile couples • 90% of couples should conceive within one year • 10-15% of couples experience infertility

5. Probabilities of failure to conceive 5 months 50 Nulliparous 2.7 months Parous

6. Requirements for conception • Male must produce adequate numbers of normal, motile spermatozoa • Male must be capable of ejaculating the sperm through a patent ductal system • The sperm must be able to traverse an unobstructed female reproductive tract • The female must ovulate and release an ovum • The sperm must be able to fertilize the ovum • The fertilized ovum must be capable of developing and implanting in appropriately prepared endometrium

7. Sperm Morphology • % normal spermatozoa • Head, acrosomal region • Vacuoles • Midpiece abnormalities • Tail defects

8. Comparison of Criteria

9. Evaluation of semen • 2-3 days abstinence prior to collection • Gelation/liquefaction (macroscopic) • Color/volume/consistency/pH

10. Sperm morphology

11. Sperm motility

12. The Endocrine System Hypothalamus/Pituitary/Pineal Thyroid/Parathyroid Thymus Adrenal Pancreas Kidney Ovary Testis

13. Evaluation of male infertility H&P N A Semen analysis Follow-up N A PCT Antisperm antibodies Sperm mucuous penetration Repeat N A LH, FSH, Testosterone

14. Male Hypothalamic-Pituitary-Gonadal Axis GnRH Testosterone Inhibin LH, FSH FSH acts on Sertoli cells LH acts on Leydig cells

15. Male reproductive endocrinology

16. Causes of female infertility

17. Female Hypothalamic-Pituitary-Gonadal Axis GnRH Estradiol Progesterone LH, FSH FSH stimulates follicular growth LH stimulates ovulation

18. Ovulation Cyclical changes in female reproductive hormones

19. Vaginal Uterine Ovarian Adrenal disorders Thyroid disorders Pituitary/hypothalamic disorders Pregnancy/lactation Uterine Ovarian Adrenal disorders Thyroid disorders Pituitary disorders Hypothalamic disorders Iatrogenic Evaluation of amenorrhea Primary causes Secondary causes

20. Primary amenorrhea • 40% due to Turner’s syndrome or pure gonadal dysgenesis • Turner’s syndrome: 45X karyotype • Pure GD: 46XX or XY karyotype • Müllerian duct agenesis or dysgenesis • Testicular feminization • Androgen receptor deficiency in XY karyotype

21. Secondary amenorrhea • Pregnancy is the most common cause • Prolactin elevation • Tumor • Iatrogenic • Thyroid disease • Effects on the metabolism of estrogens and androgens

22. Regulation of thyroid hormones TRH TSH T4 (T3) T3 (rT3)

23. Thyroid disease and infertility • Hypothyroidism • Pre-pubertal • Delayed sexual maturation, or rarely, precocious puberty • Post-pubertal • TSH may have leuteotropic effect • Hyperthyroidism • Amenorrhea

24. Endometriosis • Appearance of endometrial tissue elsewhere in the pelvic cavity. • Origin is uncertain • One of the most common diseases of menstruating women • Involved in 20-50% of infertility cases

25. Causes of infertility

26. Unexplained infertility • Exclusionary diagnosis, after all diagnostic tests are normal • Most studies report a 15-25% incidence • Conservative protocol: • Semen analysis, mid-luteal phase progesterone, tubal patency • Liberal protocol: • Above, plus cervical mucous evaluation, endometrial maturation, immunology studies

27. Immunological causes of infertility • Male or female? • Source • Vaginal fluid (IgA, IgE) • Fallopian tubes (IgA) • Variations throughout cycle • Experimental induction of infertility • Baskin, 1932 • Animal studies

28. Anti-sperm autoantibodies • 1955: Rumke and Hellinga demonstrate association between humoral autoantibodies to sperm and unexplained infertility • Results were controversial, and hampered by inadequate analytical techniques • Humoral antibodies do not effect fertility unless they exist in the reproductive tract • Antibodies must be demonstrated on the sperm surface

29. Effect of sperm autoantibodies • Spontantous agglutination • Motility/penetration • Binding to tail • Disruption • IgG mediated complement fixation (tail) • Seminal fluid contains complement inhibitors, so membrane attack occurs in the female reproductive tract

30. Anti-sperm antibodies in the female • Clinically significant only in high titers (in serum) • Anti-sperm antibodies may exist in vaginal secretions or cervical mucus even when humoral antibodies are not detected

31. Diagnosis of immune-related infertility • Post-coital test • Evaluates sperm viability in the cervical mucus • Humoral antibodies • Not diagnostic • Demonstration of antibodies on the sperm surface

32. Case 3: Unexplained Weight Loss

33. Case History A 62 year old man visited his family doctor because of weight loss from 185 lbs. to 163 lbs. The patient was not obese prior to his weight loss, and he described his appetite as “normal.” He had occasional indigestion. The patient was afebrile, and vital signs were normal. The patient had normal bowel movements. What other questions would you ask this patient?

34. Pre-test • What are “tumor markers”? • What are desirable characteristics of a tumor marker? • In what ways are tumor markers used?

35. Leading causes of death in the United States Source: National Vital Statistics Report (1999 data)

36. Types of tumor markers • Enzymes and isoenzymes • Hormones • Oncofetal antigens • Carbohydrate antigens • Receptors • Oncogene products • Genetic markers

37. Desirable characteristics of tumor markers • Easy to measure • Specific for tumor • Always present with tumor

38. Sensitivity vs. Specificity • Sensitivity and specificity are inversely related.

39. Marker concentration - + Disease

40. Sensitivity vs. Specificity • Sensitivity and specificity are inversely related. • How do we determine the best compromise between sensitivity and specificity?

41. True positive rate (sensitivity) False positive rate 1-specificity Receiver Operating Characteristic

42. Evaluating the clinical performance of laboratory tests • The sensitivity of a test indicates the likelihood that it will be positive when disease is present • The specificity of a test indicates the likelihood that it will be negative when disease is absent • The predictive value of a test indicates the probability that the test result, positive or negative, correctly classifies a patient

43. Predictive Value The predictive value of a clinical laboratory test takes into account the prevalence of a certain disease, to quantify the probability that a positive test is associated with the disease in a randomly-selected individual, or alternatively, that a negative test is associated with health.

44. Illustration • Suppose you have a new marker for liver cancer • The test correctly identified 98 of 100 patients with confirmed liver cancer (What is the sensitivity?) • The test was positive in 15 of 100 patients with no evidence of liver cancer (What is the specificity?)

45. Test performance • The sensitivity is 98.0% • The specificity is 85% • Liver cancer has an incidence of 1.5:100,000 • What happens if we screen 1 million people?

46. Analysis • In 1 million people, there will be 15 cases of liver cancer. • Our test will (most likely) identify all of these cases (TP) • Of the 999,985 healthy subjects, the test will be positive in 15%, or about 150,000 (FP).

47. Predictive value of the positive test The predictive value is the % of all positives that are true positives:

48. What about the negative predictive value? • TN = 849,985 • FN = 0

49. Summary of predictive value Predictive value describes the usefulness of a clinical laboratory test in the real world. Or does it?

50. Lessons about predictive value • Even when you have a very good test, it is generally not cost effective to screen for diseases which have low incidence in the general population. Exception? • The higher the clinical suspicion, the better the predictive value of the test. Why?