Presentation on theme: "Roger L. Bertholf, Ph.D. Associate Professor of Pathology"— Presentation transcript:
1 Interpretation of Laboratory Tests: A Case-Oriented Review of Clinical Laboratory Diagnosis (Part 2) Roger L. Bertholf, Ph.D.Associate Professor of PathologyUniversity of Florida Health Science Center/JacksonvilleMark A. Bowman, MT(ASCP), Ph.D.Associate Professor of Clinical PathologyClinical Laboratory Sciences Program DirectorUniversity of Iowa College of Medicine
3 Case HistoryA couple visits their family doctor, complaining that the wife had been unable to become pregnant.What questions should you ask?
4 InfertilityDefinition: One year of unprotected intercourse without pregnancy1°: No previous pregnancies2°: Previous pregnancy (not necessarily live birth)Fecundability: Probability of achieving pregnancy within a menstrual cycle20-25% for normally fertile couples90% of couples should conceive within one year10-15% of couples experience infertility
5 Probabilities of failure to conceive 5 months50Nulliparous2.7 monthsParous
6 Requirements for conception Male must produce adequate numbers of normal, motile spermatozoaMale must be capable of ejaculating the sperm through a patent ductal systemThe sperm must be able to traverse an unobstructed female reproductive tractThe female must ovulate and release an ovumThe sperm must be able to fertilize the ovumThe fertilized ovum must be capable of developing and implanting in appropriately prepared endometrium
7 Sperm Morphology % normal spermatozoa Head, acrosomal region Vacuoles Midpiece abnormalitiesTail defects
8 Comparison of Criteria WHO (1987)WHO (1992)Strict (1986)% Normal503014Head 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
20 Primary amenorrhea40% due to Turner’s syndrome or pure gonadal dysgenesisTurner’s syndrome: 45X karyotypePure GD: 46XX or XY karyotypeMüllerian duct agenesis or dysgenesisTesticular feminizationAndrogen receptor deficiency in XY karyotype
21 Secondary amenorrhea Pregnancy is the most common cause Prolactin elevationTumorIatrogenicThyroid diseaseEffects on the metabolism of estrogens and androgens
22 Regulation of thyroid hormones TRHTSHT4(T3)T3(rT3)
23 Thyroid disease and infertility HypothyroidismPre-pubertalDelayed sexual maturation, or rarely, precocious pubertyPost-pubertalTSH may have leuteotropic effectHyperthyroidismAmenorrhea
24 EndometriosisAppearance of endometrial tissue elsewhere in the pelvic cavity.Origin is uncertainOne of the most common diseases of menstruating womenInvolved in 20-50% of infertility cases
26 Unexplained infertility Exclusionary diagnosis, after all diagnostic tests are normalMost studies report a 15-25% incidenceConservative protocol:Semen analysis, mid-luteal phase progesterone, tubal patencyLiberal protocol:Above, plus cervical mucous evaluation, endometrial maturation, immunology studies
27 Immunological causes of infertility Male or female?SourceVaginal fluid (IgA, IgE)Fallopian tubes (IgA)Variations throughout cycleExperimental induction of infertilityBaskin, 1932Animal studies
28 Anti-sperm autoantibodies 1955: Rumke and Hellinga demonstrate association between humoral autoantibodies to sperm and unexplained infertilityResults were controversial, and hampered by inadequate analytical techniquesHumoral antibodies do not effect fertility unless they exist in the reproductive tractAntibodies must be demonstrated on the sperm surface
29 Effect of sperm autoantibodies Spontantous agglutinationMotility/penetrationBinding to tailDisruptionIgG 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 testEvaluates sperm viability in the cervical mucusHumoral antibodiesNot diagnosticDemonstration of antibodies on the sperm surface
33 Case HistoryA 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 antigensCarbohydrate antigensReceptorsOncogene productsGenetic markers
37 Desirable characteristics of tumor markers Easy to measureSpecific for tumorAlways present with tumor
38 Sensitivity vs. Specificity Sensitivity and specificity are inversely related.
42 Evaluating the clinical performance of laboratory tests The sensitivity of a test indicates the likelihood that it will be positive when disease is presentThe specificity of a test indicates the likelihood that it will be negative when disease is absentThe predictive value of a test indicates the probability that the test result, positive or negative, correctly classifies a patient211
43 Predictive ValueThe 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 1.5:100,000What 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 = 849,985FN = 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 patientsStagingPrognostic indicatorsDetect recurrence of diseaseMonitoring response to therapyRadioimmunolocalization
52 Prostate-specific antigen A serine protease in the kallikrein familyProduced exclusively by epithelial cells in the prostateForms complexes with 1-antichymotrypisin (ACT) and 2-macroglobulinMost immunoassays measure both free PSA and PSA-ACT, but not PSA-AMG
53 Prostate cancer2nd most common cancer (19%), and 2nd leading cause of cancer death, in menSensitivity of PSA (at 4.0 g/L) is 78%; specificity is approximately 33%.PSA concentration correlates with clinical stage of cancerPSA is used to monitor therapy
54 Free PSAMeasurement of uncomplexed (free) PSA can improve the specificityReported as %fPSAProstate cancer is associated with higher concentrations of PSA-ACTBPH is associated with higher free PSA concentrations
55 hCGGlycoprotein secreted by the syncytiotropoblastic cells of the placenta subunit is shared with LH, FSH, TSH subunit is specific to hCGAssays can measure intact (sandwich) or both intact and subunitCancer patients produce both intact hCG and subunit
56 Use of hCG Pregnancy Elevated with virtually all trophoblastic tumors C/P Hyatidiform mole ()ChoriocarcinomaElevated in 70% of nonseminomatous testicular tumors
57 Alpha-Fetoprotein Major fetal protein (70 kd glycoprotein) Synthesized in the yolk sac, fetal liver, GI tract, kidneyStructurally related to albuminUsed as a marker for neural tube defectsModerate elevations in liver disease (hepatitis/cirrhosis)Concentrations >1000 g/L are associated with hepatocellular carcinomaLower cutoff is used for screening
59 Carcinoembryonic antigen Family of up to 36 large, cell-surface glycoproteinsElevated in . . .70% of colorectal cancers45% of lung cancers50% of gastric cancers40% of breast cancers55% of pancreatic cancers25% of ovarian cancers40% of uterine cancers
60 Use of CEA Elevated in non-malignant conditions: Cirrhosis, emphysema, rectal polyps, benign breast disease, ulcerative colitisMost 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 cancerMost common malignancy in U.S. women (7% of women develop breast cancer by age 70)Episialin is expressed by mammary epitheliumCA 15-3, CA 549, and CA are three distinct epitopes on episialin
63 Specificity of episialin markers SensitivitySpecificityCA 15-369%Pancreatic, lung, ovarian, colorectal, liverCA 54977%Ovarian, prostate, lungCA 27.2958%98% (FDA –approved for monitoring recurrence)
64 CA 125 High MW glycoprotein recognized by mAb OC125 Isolated from a serous ovarian tumorElevated in 50% of stage I ovarian cancerElevated in 90%+ of stage II, III, and IVOverall, sensitivity 95%; specificity 82%; PPV 78%; NPV 91%.
65 DU-PAN-2 100-500 kd mucin (80% carbohydrate) Found mainly in pancreatic and biliary epitheliumAlso in breast, bronchi, salivary glands, stomach, colon, intestine60% sensitivity for pancreatic cancer45% sensitivity for biliary tract cancer44% 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 cancerCA 19-5GI, pancreatic, ovarian cancerCA 50Sensitivity 90% for pancreatic cancer; as high as 73% for Duke’s stage C or D colon cancer. Also elevated in esophageal, liver, gastric cancerCA 72-4Sensitivity 40% in GI cancer, 40% in lung cancer, 36% in ovarian cancerCA 242Sensitivity 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-mycSuppressor genesRetinoblastoma, p53, BRCA1 and 2ReceptorsER/PR
68 Oncogene associations N-rasAML, neuroblastomaK-rasLeukemia, lymphomac-mycB, T-cell lymphoma, small cell lung cancerHER-2/neuBreast, ovarian, GI cancerbcl-2
69 Pancreatic cancerFourth most common cause of cancer deaths in men (fifth in women)Incidence is increasing worldwide2:1 male preferenceEarly diagnosis is unusualEpigastric pain and significant weight loss are the most common presenting signs1 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 diseaseDoes not distinguish between pancreatitis and carcinomaAt least half of pancreatic adenocarcinomas are ductal and mucin-producingCA19-9 is the best marker (80-90% sensitivity)5% are endocrine (islet cells) and may be hormone secretingInsulinoma (β-islet cells), glucagonoma (-islet cells), somatostatin, calcitonin, ACTH