Transcript Case 1: Failure to Conceive

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