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Table of Contents
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OBJECTIVES
By the end of this session the reader should be able to:
- Describe the time course of serum human chorionic gonadotropin during pregnancy
- Describe the relative sensitivity of urine home pregnancy test kits in detecting pregnancy (day of the first missed period)
- Describe the relative sensitivity of laboratory qualitative urine pregnancy tests in detecting pregnancy (7-10 days post-conception)
- Discuss the use of the serum β-hCG test (5-8 days post-conception) in diagnosing and monitoring ectopic pregnancy
- Identify the characteristics of normal semen
- Discuss the most common semen findings in infertility (increased or decreased volume, reduced motility, increased abnormal forms, low counts, high counts)
- Discuss the lecithin: sphingomyelin ratio in diagnosing fetal lung maturity
- Discuss the microviscosity test by fluorescence polarization for determining fetal lung maturity
- Discuss the use of the amniotic fluid "delta O.D." in diagnosing and monitoring hemolytic disease of the newborn
KEY TERMS
Amniotic fluid - Substance that protects the developing fetus; derived mostly from fetal urine.
Ectopic Pregnancy - A pregnancy in which the embryo develops in the fallopian tube or abdomen.
Embryo - A developing infant that has not yet finished organ development (less than 10 weeks gestation).
Erythroblastosis fetalis - A fetal disease caused by maternal antibody-mediated fetal erythrocyte destruction.
Fetus - A developing infant that has finished organ development (more than 10 weeks gestation).
Gestation - Length of pregnancy measured in weeks from the first day of the last menstrual period.
Hydatidiform mole - Abnormal pregnancy resulting from pathologic ovum.
Oligohydramnios - The presence of less amniotic fluid than is usual for a given gestational age.
Polyhydramnios - The accumulation of an excessive amount of amniotic fluid.
Respiratory Distress Syndrome - A disease of premature newborns caused by a deficiency of lung surfactant.
Zygote - The cell resulting from the union of male and female gamete.
BACKGROUND/SIGNIFICANCE
It is advantageous to diagnose a pregnancy as promptly as possible. The diagnosis of pregnancy is initially made by measuring human chorionic gonadotropin (hCG) in urine or blood. Since this test is available over the counter in most pharmacies and since positive results can result from causes other than a viable pregnancy it is important that pharmacists understand how to interpret these tests.
DIAGNOSIS OF PREGNANCY
The tests used in the clinical laboratory for the diagnosis of pregnancy are based on immunological techniques to detect the presence of hCG in urine or blood. hCG is a glycoprotein of 40,000 MW consisting of an alpha and a beta subunit. The alpha subunits of luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and hCG are identical while their beta subunits are unique for each hormone and are responsible for their biological action. Antibodies formed against the whole hCG molecule cross-react extensively with LH, FSH, TSH and this has been a source of bothersome interference with the specificity of pregnancy tests. A major improvement occurred with the development of specific antibodies against the beta subunit of hCG, such that the test is not affected, for instance, by the mid-cycle or menopause elevations of LH. Unfortunately the hopes for complete specificity of this test have not been borne out in practice. Traces of hCG have been demonstrated in the blood of normal, healthy men and nonpregnant females. The sources of this hCG appear to be the testes and the pituitary. Furthermore, anti-hCG-beta-subunit antibodies do cross-react to a small extent with other hormones, particularly with LH. Thus, values below 5 mIU/mL of serum are interpreted as negative and values over 30 mIU/mL as positive. With the most sensitive methods, values indicating pregnancy are usually reached after the first week post-conception; i.e., well before the first missed menstrual period. Quantitation of hCG in blood also is used in the diagnosis and monitoring of therapeutic response in trophoblastic neoplasms and non-seminomatous germ-cell tumors.
The serum values related to pregnancy are:
| Nonpregnant | 0-5 mIU/mL |
| Indeterminate | 6-20 mIU/mL |
| First week post-conception | 20-30 mIU/mL |
| Second week post-conception | 30-100 mIU/mL |
| Third week post-conception | 100-1000 mIU/mL |
| First trimester peak (7-12 weeks) | 10,000-160,000 mIU/mL |
| Second trimester | 6,000-30,000 mIU/mL |
| Third trimester | 400-15,000 mIU/mL |
The measurement of β-hCG in serum is also useful in the diagnosis of ectopic pregnancy which may produce an “acute abdomen.” In this situation the measurement is extraordinarily important in the workup of the etiology of the abdominal pain and is carried out with the quantitative serum test. If positive, life saving surgical intervention may be indicated. In ectopic pregnancy, β-hCG decreases in serum over time.
Principally, three analyses are now used to detect pregnancy:
Urine Home Pregnancy Test Kits
Drugstores now carry urine pregnancy testing products that are easy to use, 99% specific and sensitive (when used as directed) and relatively inexpensive. The detection limit for pregnancy is the day of the first missed period. If negative they recommend retesting in 5-7 days if menses has not occurred. Specimens collected from pregnant women less than twelve days from conception may contain concentrations of hCG below the limit of detection of the test. In these instances the test should be repeated two to three days later since hCG approximately doubles every two days during early pregnancy. This allows the increase in concentration of hCG to reach the detection limit of the assay.
Urine Qualitative Pregnancy Test - Clinical Laboratory
The rapid and sensitive qualitative Color Immunochromatographic Assay (Quidel, San Diego, CA) utilizes a monoclonal/polyclonal antibody method capable of detecting the intact molecule of hCG in urine. This test unit consists of a plastic container with a membrane strip which provides the solid support for the immunochromatographic assay. One end of the membrane provides contact with the urine well, which contains an absorbent pad which provides an even flow of the urine along the membrane. In the test, 220 uL of urine is added to the “Add Urine” well, saturating the absorbent pad which then transports the urine to the attached membrane strip. As the urine moves to the first zone of the membrane, it mobilizes the mouse anti-B-hCG monoclonal antibody conjugated with red latex beads. The urine continues to move the antibody-red latex conjugate across the membrane to the immobilized anti-B-hCG zone, a rabbit polyclonal antibody which is immobilized on the vertical line. If hCG is present in the urine specimen, a “sandwich” of solid phase/hCG/redtex is formed. The vertical line will appear, resulting in a positive sign (+) visible in the “Read Results” window, indicating the presence of hCG. If hCG is not detected, the “Read Results” window will only contain the pre-printed blue horizontal line, indicating a negative (-) result. As urine continues to move across the membrane, it comes in contact with the reagent in the “Test Complete” window. A blue line will appear, indicating the test is complete. See Figure.
Sensitivity: 30 mIU/mL
In normal pregnancy, hCG concentrations can reach this level as early as 7 to 10 days post conception.
- Specimen
- For optimal results it is best to test the first urine voided in the morning, because it contains the greatest concentration of hCG. However, urine collected anytime during the day can also be used.
Limitations
- Positive results from very early pregnancy may later prove negative due to natural termination of pregnancy. This is estimated to occur in up to 50% of all conceptions. It is recommended that weak positive results be retested with a fresh urine sample 48 hours later.
- A negative result obtained with a urine specimen collected from a very early pregnancy (or a very dilute urine) should be retested on a fresh specimen after two days.
- Patients with trophoblastic and nontrophoblastic disease may have elevated hCG levels; therefore, the possibility of hCG-secreting neoplasms should be ruled out prior to the diagnosis of pregnancy
Serum Quantitative Pregnancy Test - Clinical Laboratory
Serum should be used in the quantitative B-hCG assays. The assay range is from 1.9 mIU/mL to 500 mIU/mL and may be extended by dilution of the specimen by the laboratory. This detection limit enables the laboratory to detect pregnancy about 2 days before the urine qualitative method gives a positive result. This test should be used in the detection of ectopic pregnancy and tumors.
SEMEN ANALYSIS
In examining a semen specimen for investigation of infertility the following items are important:
Samples:
A minimum of two specimens collected 7 days apart are tested in order to compensate for the considerable variation in sperm output.
Interval from last ejaculation:
The recommended interval is three to five days. Shorter periods often result in reduced numbers and volume. Longer periods result in an increase in immotile forms.
Time of collection:
The collection time is best in the morning or during the day because it is difficult for the laboratory to offer such special services during the evening and night hours. The time elapsed between collection and examination is important for assessing the percentage of motile sperm as well as liquefaction. Testing should be scheduled in advance with the laboratory.
Technique of collection:
No differences are known in the quality of the semen specimen whether it has been obtained through masturbation or coitus with a condom. In the latter case, the condom must be free of any chemicals or powders.
Sample handling:
Collect specimen in wide-mouthed jar or condom. Transport to the laboratory without any additions, without delay. Keep sample near body temperature. Prevent temperature extremes, drying, or freezing. The time of collection should be indicated.
Time of analysis is recorded by the laboratory to assess the time elapsed from collection.
Macroscopic Examination
Volume:
Average 3-5 mL; however, fertility may be seen with specimens of 1 mL up to 15 mL. Below
1 mL, fertility is reduced. However, infertile males can also have increased semen volume.
Appearance:
Translucent, whitish-gray or opalescent.
Mucolysis: (liquefaction)
Normal semen coagulates a few seconds after ejaculation, then undergoes liquefaction within 30 minutes. Coagulation is due to formation of a fibrin clot by the action of the prostatic clotting enzyme from the seminal vesicles. Liquefaction is caused by the action of fibrinolytic enzymes of prostatic origin. Liquefaction is defective or absent in the semen of males with lesions or absence of the vasa deferentia and seminal vesicles.
+MICROSCOPIC EVALUATION OF SPERMATOZOA
Sperm count (number/mL)
Counting technique: The thoroughly mixed sample (after liquefaction) is diluted with a special diluent in a leukocyte pipet, and counting is performed in a hemocytometer chamber.
Normal values: 60-150 million/mL
Counts less than 20 million/mL are distinctly abnormal, but no sharp cutoff exists where infertility begins.
Counts over 300 million/mL are associated with increased incidence of infertility.
The "sperm count" is frequently the only parameter utilized for evaluation of the "completeness" of a vasectomy. Precision of this technique is poor.
Motility
Technique: A drop of liquefied semen is placed on a slide, covered with a coverslip and at least 200 spermatozoa are observed for motility. The microscope stage be maintained at body temperature (37o C).
Normal:
Nonmotile: <20% (Motile: > 80% initially)
Sluggish: <20%
Progressive motility: >60% within three hours of collection.
Motile forms decrease approximately 5% with every hour elapsed from collection.
Morphology
Technique: Examine stained smear (Papanicolau) and perform differential count of normal and abnormal forms.
Normal: > 70% normal forms.
Abnormal forms are characterized as: large, small, tapering, amorphous, bicephalic, double-tailed, constricted head, short tails, and/or immature.
Formed elements other than spermatozoa: crystals, leukocytes, macrophages, erythrocytes, bacteria, trichomonas, usually signify inflammation.
The most frequently encountered change associated with infertility is reduced motility and an increase in abnormal forms. Variation in volume and numbers seems less important.
LABORATORY EXAMINATION OF AMNIOTIC FLUID
The mechanism of the production of amniotic fluid is poorly understood. Amniotic water is in dynamic equilibrium with both fetal and maternal blood. Near term 500 mL of water in the amniotic fluid is exchanged per hour. The solutes are exchanged at a slower rate. At midterm, amniotic fluid contains from 0.5 to 1.0 g of protein/dL, and as compared to plasma levels, 1/3 less calcium and magnesium, 90% less copper and iron and almost the same amount of sodium and potassium. Bilirubin in amniotic fluid is considerably lower than the plasma levels, consisting almost exclusively of the unconjugated type, which is protein bound, even if the fetus is capable of conjugating bilirubin.
Towards term the concentration of most solutes decreases, the protein content falling below 100 mg/dL, but uric acid, creatinine and lecithin increase. Uric acid and creatinine determinations had previously been used to assess fetal renal maturity.
Tests of clinical significance on amniotic fluid are directed toward 1) assessment of fetal lung maturity; 2) the presence and severity of erythroblastosis fetalis; and 3) genetic abnormalities.
Tests for fetal maturity
Lecithin-sphingomyelin ratio (L:S ratio)
The rapid assessment of fetal lung maturity (FLM) is critical for managing high-risk obstetric patients. The question that the physician is asking, when FLM is evaluated, is "what is the probability of the infant developing respiratory distress syndrome (RDS) if I induce delivery at this time?"
- Basic physiology
- During the first two trimesters of development, there is little pulmonary surfactant in the amniotic fluid. The type II pneumocytes begin secreting large quantities of surfactants during the last trimester. Initially in the third trimester there are equivalent quantities of lecithin (L) and sphingomyelin (S). Lesser quantities of other phospholipids, such as phosphatidylinositol (PI) and phosphatidylglycerol (PG) are present. At 33 to 34 weeks gestation there is a rapid increase in the L content of secreted surfactant, while S content begins to slowly decrease. This results in a rapid increase in the L/S ratio, the basis of the first widely accepted test for FLM. At 35 to 36 weeks there is a very rapid increase in PG content.
Biochemical Methods
- L/S ratio
- This was the first broadly accepted test for FLM assessment. The assay is based upon the isolation of phospholipids from the amniotic fluid and subsequent separation by thin-layer chromatography (TLC). Charring allows visualization of the L and S content and reflectance densitometry allows determination of the relative quantities. A ratio of 2 or more is indicative of a mature fetal lung and is not associated with RDS. The problem is that the assay is among the most labor and skill-intensive methods employed in a clinical laboratory and requires approximately 3 hours to complete. In addition, lecithin and sphingomyelin exist in all cell membranes. Whole blood has an L/S ratio of 1.5 and its presence (e.g., after a traumatic tap) may artifactually lower the ratio. Diabetic patients cannot be assessed with this method as high L/S values (well in excess of 2.0) are often present in diabetics who may develop severe RDS. In these cases PG is measured.
- PG content
- The increase in PG content in amniotic fluid is a relatively late event, and when PG content represents 3% (or more) of total phospholipid the fetal lungs are mature. Initially, the assessment of PG content required the use of two-dimensional TLC. Currently commercial kits are available which allow detection by either latex agglutination (antibody based) or an enzymatic approach. The latex agglutination assay is more prevalent due to its simplicity and rapidity. The test is reported as either "PG detected" or "PG absent." When PG is detected, the risk of RDS is minimal.
Biophysical Methods
The role of pulmonary surfactant is to reduce surface tension and prevent alveolar collapse. Therefore, a variety of approaches involves measuring directly or indirectly the surface tension of the amniotic fluid. Among the many methods are (1) optical absorbance at 650 nm, (2) surface tension (falling drop) (3) foam stability, and (4) microviscosity. The first three are no longer commonly used.
- Microviscosity by fluorescence polarization
- This fluorescence polarization assay (FPA) is based on the principle that, upon addition of an exogenous fluorophore to amniotic fluid, the degree of fluorescence depolarization produced by the amniotic fluid correlates with FLM. This type of FLM assay (often called the TDx FLM after the first commercial kit) is relatively inexpensive, fast, accurate, highly reproducible, and correlates well with the L/S ratio and PG content. In addition, this assay can be used with diabetic high-risk obstetric patients, although a different cutoff value for FLM is required. For these reasons, this approach has become the preferred approach for initial assessment of FLM at many medical centers. A major problem with the assay, however, is that the presence of blood or meconium interferes with the test.
Approach to FLM determination
The initial approach is to determine if there is blood or meconium present. If they are present, then the AF is sent for L/S testing by two-dimensional thin-layer chromatography.
- If no meconium or blood is present, then the AF is sent for the FPA.
- If the patient is diabetic, a value above 90 (mg surfactant/g albumin) is considered "mature."
- If the patient is not diabetic, a value above 55 (mg surfactant/g albumin) is considered "mature."
AF for which an "immature" value is obtained by FPA is then tested for PG content, as a small percentage of AF samples will have detectable PG (indicating FLM), but low FPA or L/S values. If PG is detected, then the fetal lung is considered "mature," and, if it is not detected, then it is considered "immature."
Diagnosis and Monitoring of Erythroblastosis Fetalis: Amniotic Fluid Absorbance
In normal pregnancy a small concentration of unconjugated (indirect) bilirubin is present in the amniotic fluid, and this concentration falls as term is approached. In erythroblastosis fetalis (e.g., due to Rh-incompatibility), however, the bilirubin concentration increases with time as the hemoglobin liberated by the hemolytic process is converted to bilirubin. Due to fetal hepatic immaturity all bilirubin is generally of the unconjugated type and is bound to albumin. When the bilirubin concentration is so high that the binding capacity is exceeded, the free unconjugated bilirubin can penetrate the brain and produce kernicterus. Hence, the assessment of the presence and severity of erythroblastosis fetalis relies heavily on the monitoring of bilirubin in the amniotic fluid.
Sensitization of pregnant women is due to exposure to Rh-positive fetal blood which can gain access to the maternal circulation. Although this antigenic challenge is generally small, it may be sufficient in some women to provoke an antibody response. A much larger antigenic exposure may result from disruption of the integrity of the fetal compartment during spontaneous or induced abortion, ectopic pregnancy, or delivery.
When a sensitized woman has another Rh-positive pregnancy, the antibodies (usually of the IgG class) can readily cross the placenta and can cause destruction of fetal erythrocytes leading to production of bilirubin. Repeated exposure during pregnancy or delivery leads to an augmented response and to more severely affected later pregnancies.
Sensitization can usually be prevented by intramuscular administration of anti-Rho (D) immune globulin (RhIg, RhoGAM, Ortho Diagnostic systems) which inactivates the potentially stimulating immunogens. A small number of sensitized pregnancies, however, continues to occur despite immunization, and in pregnancies in which immunization has not been performed.
- Determination of Bilirubin
- Although bilirubin in serum is measured colorimetrically by diazotization methods, bilirubin in amniotic fluid is monitored by direct visible scanning spectrophotometry of a centrifuged specimen.
Direct spectrophotometry is utilized for the following reasons:
- It is much more rapid than “wet” chemical methods;
- The presence of substances other than bilirubin (e.g., hemoglobin) can be inferred from the shape of the visible spectrum (Figure 1).
- There is analytical interference in chemical methods by amniotic fluid
Bilirubin in serum is not determined by scanning spectrophotometry due to the presence of other interfering compounds in serum.
The bilirubin content obtained by visible spectrophotometry is expressed as absorbance. The absorbance (formerly optical density or “O.D.”) of bilirubin at its wavelength of maximum absorption (450 nm) is measured and corrected for background absorbance by subtraction of a constructed tangent baseline (Figure 1). Hence the term “delta A” (or “delta O.D.”).
Bilirubin is highly photosensitive, and this is particularly true in amniotic fluid, in which concentrations are generally low and in which few cells are present to protect the bilirubin from light. Hence amniotic fluid for bilirubin measurement must be protected from light by collecting it into an amber-colored tube or (preferably) by wrapping the tube with aluminum foil or masking tape.
Frequently hemoglobin may be present in amniotic fluid concurrently with bilirubin due to the ongoing hemolytic process (hemoglobin of fetal origin) and/or to repeated amniocentesis (hemoglobin of maternal origin). Since the so-called “Soret band” of hemoglobin (410 nm) is near the absorbance band of bilirubin (450 nm) (Figure 1), substantial positive interference can occur in the determination of absorbance. Therefore, whenever hemoglobin is noted in the initial scan of the fluid, the fluid is extracted with an equal volume of chloroform. Since the bilirubin is highly soluble (100% recovery) in chloroform and hemoglobin is insoluble in chloroform, the chloroform extract will contain only bilirubin. Since a volume of chloroform equal to that of the amniotic fluid is used, the absorbance of bilirubin in the chloroform extract will be equal to that in the original amniotic fluid, free from interference by hemoglobin (Figure 1).
Amniotic fluid absorbance values are interpreted by plotting them semilogarithmically on a “Liley-Prognostication Chart”, which contains weeks of gestation on the abscissa (linear axis) and absorbance on the ordinate (logarithmic axis). The chart is divided into three zones: “zone C - very severely affected babies” (zone of severe hemolysis), “zone B - indeterminate zone,” and “zone A - unaffected babies” (no hemolysis). Persistence of values in the severely affected zone may induce the clinician to perform intrauterine transfusion or (if the fetus is determined to be viable by the tests previously described) to induce labor. Frequently exchange transfusion is performed at birth in such situations. A plot of serial amniotic fluid absorbance is shown in Figure 2.
- Diagnosis of Genetic Abnormalities
- Sufficient cells can be recovered from amniotic fluid to permit culture and subsequent karyotyping for such abnormalities as Down syndrome (trisomy 21). In addition, sex of the fetus can be determined (important in certain X-linked disorders when one or both parents are affected).
Figure 1: Determination of Amniotic Fluid Absorbance (30 weeks gestation)
Upper = Visible spectrum of unextracted amniotic fluid (Note the absorption band of bilirubin at 450 nm and the interfering Soret band of hemoglobin at 410 nm)
Lower = Visible spectrum of chloroform extract of same amniotic fluid (Note the single absorption band of bilirubin at 450 nm, free from hemoglobin interference)
Absorbance = 0.11 (“indeterminate zone” of Liley chart)
Figure 2: Liley Amniotic Fluid Prognostication Chart
CASE STUDY
Case 1
(BrighamRAD Teaching Case Database, August 19, 1994)
An 18-year-old woman at 11.5 weeks gestation presented with an elevated serum beta-subunit human chorionic gonadotropin (b-hCG) concentration of 285,730 mIU/ml. Transabdominal ultrasound images show complex solid and cystic areas filling the endometrial cavity of the uterus.
- What is the differential diagnosis in this case?
- What drugs should be used to treat this patient?
volum .2ml co
volume. 2ml color -greyish opalascent viscosity - normal ph-8.0 fructose-present total sperm cout -140ml motility -45% total motility at the 1hr-45% actively motile 35% sluggishly motile -10% abnormal forms-20% pus cells-2-4/hpf rbc-s-absent epithelial cells-absent
wow, very detailed analysis
Hi, want for a seminal test the count is 1.6ml, whitish, opaletscent fluid. want to know if is normal or not.
thank you.