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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 Case 1: Failure to Conceive Case History A couple visits their family doctor, complaining that the wife had been unable to become pregnant. What questions should you ask? 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 Probabilities of failure to conceive 100 Percent failed 5 months 50 Nulliparous 2.7 months Parous 10 0 2 4 6 8 10 Months of unprotected intercourse 12 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 Sperm Morphology • • • • • % normal spermatozoa Head, acrosomal region Vacuoles Midpiece abnormalities Tail defects Comparison of Criteria % Normal WHO (1987) 50 WHO (1992) 30 Strict (1986) 14 Head length (m) 3.0-5.0 4.0-5.5 5.0-6.0 Head width (m) 2.0-3.0 2.5-3.5 2.5-3.5 W/L 1.5-2.0 1.5-1.75 1.0-1.67 Evaluation of semen • 2-3 days abstinence prior to collection • Gelation/liquefaction (macroscopic) • Color/volume/consistency/pH Sperm morphology Sperm motility The Endocrine System Hypothalamus/Pituitary/Pineal Thyroid/Parathyroid Thymus Adrenal Pancreas Kidney Ovary Testis Evaluation of male infertility H&P N Semen analysis N A Follow-up A PCT Repeat Antisperm antibodies N A Sperm mucuous penetration LH, FSH, Testosterone Male Hypothalamic-PituitaryGonadal Axis GnRH Testosterone Inhibin LH, FSH FSH acts on Sertoli cells LH acts on Leydig cells Male reproductive endocrinology LH FSH Testosterone Diagnosis N N N N or N N N Hypothalamic or pituitary failure Gonadal failure Germinal compartment failure Androgen resistence Idiopathic Causes of female infertility Other Immunologic 15% factors 5% Pelvic factors 50% Ovulatory disorders 30% Female Hypothalamic-PituitaryGonadal Axis GnRH Estradiol Progesterone LH, FSH FSH stimulates follicular growth LH stimulates ovulation Cyclical changes in female reproductive hormones LH Estradiol Progesterone Ovulation FSH 1 4 8 12 16 Days since onset of menses 20 24 28 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 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 Secondary amenorrhea • Pregnancy is the most common cause • Prolactin elevation – Tumor – Iatrogenic • Thyroid disease – Effects on the metabolism of estrogens and androgens Regulation of thyroid hormones TRH TSH T4 (T3) T3 (rT3) Thyroid disease and infertility • Hypothyroidism – Pre-pubertal • Delayed sexual maturation, or rarely, precocious puberty – Post-pubertal • TSH may have leuteotropic effect • Hyperthyroidism – Amenorrhea 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 Causes of infertility Seminal Defect 25% Tubal Defect 22% Ovulation Defect 27% Endometriosis 5% Other 4% Unexplained 17% 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 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 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 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 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 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 Case 3: Unexplained Weight Loss 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? Pre-test • What are “tumor markers”? • What are desirable characteristics of a tumor marker? • In what ways are tumor markers used? Leading causes of death in the United States Total Deaths Percent of total 2.391,399 100 Cardiovascular disease 725,192 30.3 Cancer 539,838 23.0 Cerebrovascular 167,366 7.0 COPD 124,181 5.2 Accidents 97,860 4.1 All causes Source: National Vital Statistics Report (1999 data) Types of tumor markers • • • • • • • Enzymes and isoenzymes Hormones Oncofetal antigens Carbohydrate antigens Receptors Oncogene products Genetic markers Desirable characteristics of tumor markers • Easy to measure • Specific for tumor • Always present with tumor Sensitivity vs. Specificity • Sensitivity and specificity are inversely related. Disease + Marker concentration Sensitivity vs. Specificity • Sensitivity and specificity are inversely related. • How do we determine the best compromise between sensitivity and specificity? True positive rate (sensitivity) Receiver Operating Characteristic False positive rate 1-specificity 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 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. 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?) 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? 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). Predictive value of the positive test The predictive value is the % of all positives that are true positives: TP PV 100 TP FP 15 100 15 150,000 0.01% What about the negative predictive value? • TN = 849,985 • FN = 0 TN PV 100 TN FN 849,985 100 849,985 0 100% Summary of predictive value Predictive value describes the usefulness of a clinical laboratory test in the real world. Or does it? 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? Use of tumor markers • • • • • • • Screen for disease Diagnosis of symptomatic patients Staging Prognostic indicators Detect recurrence of disease Monitoring response to therapy Radioimmunolocalization 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 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 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 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 Use of hCG • Pregnancy • Elevated with virtually all trophoblastic tumors – C/P Hyatidiform mole () – Choriocarcinoma • Elevated in 70% of nonseminomatous testicular tumors 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 Combined AFP/hCG • Useful for differentiating germ cell tumors Yolk sac tumors Choriocarcinoma Embryonal carcinoma Seminomas Teratoma Nonseminomatous testicular tumor AFP nl nl nl or hCG nl nl 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 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 Carbohydrate Antigens • Glycoproteins expressed by tumor cells (surface or excretory) • High molecular weight mucins (mucopolysaccaride protein) 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 27.29 are three distinct epitopes on episialin Specificity of episialin markers Sensitivity Specificity CA 15-3 69% Pancreatic, lung, ovarian, colorectal, liver CA 549 77% Ovarian, prostate, lung 58% 98% (FDA –approved for monitoring recurrence) CA 27.29 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%. 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 Blood group antigens CA 19-9 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 Other tumor markers • Oncogenes – ras, HER-2/neu, bcl-2, c-myc • Suppressor genes – Retinoblastoma, p53, BRCA1 and 2 • Receptors – ER/PR 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 Leukemia, lymphoma 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%. Cancer incidence and mortality in the United States (cases per 100,000) Females Males Site Incidence Deaths Incidence Deaths Lung 91.5 (2) 57.7 (1) 52.0 (2) 41.0 (1) Prostate 160.6 (1) 33.9 (2) 1.3 0.3 131.9 (1) 28.8 (2) 67.4 (3) 26.3 (3) 49.0 (3) 18.5 (3) 12.1 12.2 (4) 9.5 9.3 (4) Breast Colon/rectum Pancreas Source: Cancer 2002;94 (1999 data) Incidence and mortality of GI/pancreatic tumors (1999 data) Site New Cases (est.) Deaths (actual) Colon/rectum 129,400 57,155 Pancreas 28,600 29,081 Stomach 21,900 12,711 Esophageal 12,500 11,917 Source: SEER Cancer Statistics Review 1973-1999 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