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

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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
WHO (1987) WHO (1992) Strict (1986) % Normal 50 30 14 Head length (m) Head width (m) W/L

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
LH FSH Testosterone Diagnosis Hypothalamic or pituitary failure Gonadal failure N Germinal compartment failure N or  Androgen resistence Idiopathic

16 Causes of female infertility

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

18 Cyclical changes in female reproductive hormones

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

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

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 211

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. 212

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?) 213

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?

51 Use of tumor markers Screen for disease
Diagnosis of symptomatic patients Staging Prognostic indicators Detect recurrence of disease Monitoring response to therapy Radioimmunolocalization

52 Prostate-specific antigen
A serine protease in the kallikrein family Produced exclusively by epithelial cells in the prostate Forms complexes with 1-antichymotrypisin (ACT) and 2-macroglobulin Most immunoassays measure both free PSA and PSA-ACT, but not PSA-AMG

53 Prostate cancer 2nd most common cancer (19%), and 2nd leading cause of cancer death, in men Sensitivity of PSA (at 4.0 g/L) is 78%; specificity is approximately 33%. PSA concentration correlates with clinical stage of cancer PSA is used to monitor therapy

54 Free PSA Measurement of uncomplexed (free) PSA can improve the specificity Reported as %fPSA Prostate cancer is associated with higher concentrations of PSA-ACT BPH is associated with higher free PSA concentrations

55 hCG Glycoprotein secreted by the syncytiotropoblastic cells of the placenta  subunit is shared with LH, FSH, TSH  subunit is specific to hCG Assays can measure intact (sandwich) or both intact and  subunit Cancer patients produce both intact hCG and  subunit

56 Use of hCG Pregnancy Elevated with virtually all trophoblastic tumors
C/P Hyatidiform mole () Choriocarcinoma Elevated in 70% of nonseminomatous testicular tumors

57 Alpha-Fetoprotein Major fetal protein (70 kd glycoprotein)
Synthesized in the yolk sac, fetal liver, GI tract, kidney Structurally related to albumin Used as a marker for neural tube defects Moderate elevations in liver disease (hepatitis/cirrhosis) Concentrations >1000 g/L are associated with hepatocellular carcinoma Lower cutoff is used for screening

58 Combined AFP/hCG Useful for differentiating germ cell tumors AFP hCG
Yolk sac tumors nl Choriocarcinoma Embryonal carcinoma Seminomas Teratoma Nonseminomatous testicular tumor or

59 Carcinoembryonic antigen
Family of up to 36 large, cell-surface glycoproteins Elevated in . . . 70% of colorectal cancers 45% of lung cancers 50% of gastric cancers 40% of breast cancers 55% of pancreatic cancers 25% of ovarian cancers 40% of uterine cancers

60 Use of CEA Elevated in non-malignant conditions:
Cirrhosis, emphysema, rectal polyps, benign breast disease, ulcerative colitis Most useful in staging and monitoring recurrence of disease

61 Carbohydrate Antigens
Glycoproteins expressed by tumor cells (surface or excretory) High molecular weight mucins (mucopolysaccaride protein)

62 Breast cancer Most common malignancy in U.S. women (7% of women develop breast cancer by age 70) Episialin is expressed by mammary epithelium CA 15-3, CA 549, and CA are three distinct epitopes on episialin

63 Specificity of episialin markers
Sensitivity Specificity CA 15-3 69% Pancreatic, lung, ovarian, colorectal, liver CA 549 77% Ovarian, prostate, lung CA 27.29 58% 98% (FDA –approved for monitoring recurrence)

64 CA 125 High MW glycoprotein recognized by mAb OC125
Isolated from a serous ovarian tumor Elevated in 50% of stage I ovarian cancer Elevated in 90%+ of stage II, III, and IV Overall, sensitivity 95%; specificity 82%; PPV 78%; NPV 91%.

65 DU-PAN-2 100-500 kd mucin (80% carbohydrate)
Found mainly in pancreatic and biliary epithelium Also in breast, bronchi, salivary glands, stomach, colon, intestine 60% sensitivity for pancreatic cancer 45% sensitivity for biliary tract cancer 44% sensitivity for hepatocellular carcinoma

66 Blood group antigens CA 19-9 CA 19-5 CA 50 CA 72-4 CA 242
Sensitivity 80% for pancreatic cancer;, 30% for colorectal cancer CA 19-5 GI, pancreatic, ovarian cancer CA 50 Sensitivity 90% for pancreatic cancer; as high as 73% for Duke’s stage C or D colon cancer. Also elevated in esophageal, liver, gastric cancer CA 72-4 Sensitivity 40% in GI cancer, 40% in lung cancer, 36% in ovarian cancer CA 242 Sensitivity 75% for pancreatic cancer, 70% for colorectal cancer, 44% for gastric cancer

67 Other tumor markers Oncogenes Suppressor genes Receptors
ras, HER-2/neu, bcl-2, c-myc Suppressor genes Retinoblastoma, p53, BRCA1 and 2 Receptors ER/PR

68 Oncogene associations
N-ras AML, neuroblastoma K-ras Leukemia, lymphoma c-myc B, T-cell lymphoma, small cell lung cancer HER-2/neu Breast, ovarian, GI cancer bcl-2

69 Pancreatic cancer Fourth most common cause of cancer deaths in men (fifth in women) Incidence is increasing worldwide 2:1 male preference Early diagnosis is unusual Epigastric pain and significant weight loss are the most common presenting signs 1 year survival is <10%; 5 year is 2%.

70 Cancer incidence and mortality in the United States (cases per 100,000)
Source: Cancer 2002;94 (1999 data)

71 Incidence and mortality of GI/pancreatic tumors (1999 data)
Source: SEER Cancer Statistics Review

72 Laboratory values in pancreatic cancer
Serum amylase is usually elevated, but only after significant progression of the disease Does not distinguish between pancreatitis and carcinoma At least half of pancreatic adenocarcinomas are ductal and mucin-producing CA19-9 is the best marker (80-90% sensitivity) 5% are endocrine (islet cells) and may be hormone secreting Insulinoma (β-islet cells), glucagonoma (-islet cells), somatostatin, calcitonin, ACTH

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