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  • Oncogenes

    Oncogenes are genes that function abnormally and help cause cancer. Oncogenes are inherited in a nononcogene form known as a protooncogene and require a triggering event to start abnormal activity. This event could be a mutation that occurs in the protooncogene itself within a single cell during mitosis. It could also be due to a more complicated chromosome abnormality occurring during mitosis in which the protooncogene is relocated in some area that promotes the oncogenic potential of the protooncogene (e.g., the abl protooncogene on chromosome 9 that is translocated to chromosome 22 and helps form the Philadelphia chromosome oncogene of CML. Another possibility is cell injury from a variety of causes such as radiation. Most of the oncogenes, when active, increase cell proliferation and thereby the number of cells with the oncogene (oncogenic “amplification”) increasing oncogene products (“overexpression”) leading to or causing carcinogenesis. Some oncogenes are actually oncogene suppressors before becoming oncogenes. If the suppressor protooncogene is deleted, damaged, or mutated on one or on both chromosomes (depending on the particular gene), the nonsuppressor oncogene (if activated as described above) is released from inhibition. The abnormal suppressor gene may even produce abnormal gene products that are synergistic to the other oncogene’s effect (e.g., the p53 suppressor protooncogene). There are many protooncogenes and oncogenes, and more are discovered every year. Some of the current most important are listed in Table 33-5. Of these, the Rb, p53, FAP, DCC, wt, and nf-1 genes are suppressors.

    Some currently important oncogenes

    Table 33-5 Some currently important oncogenes

  • Chromosome Abnormalities in Malignancy

    Certain malignancies have characteristic chromosome abnormalities. These can be chromosome deletions (the whole chromosome is absent or only a portion of a chromosome); additions (e.g., trisomy, when a third chromosome is present in a group that normally would consist of two); translocation, either single (where part of one chromosome breaks off and attaches to another) or reciprocal (where two chromosomes exchange a portion of each chromosome); or gene rearrangement (on the same chromosome). The most famous chromosome abnormality is the Philadelphia chromosome of chronic myelogenous leukemia (CML), present by standard chromosome analysis in about 85% of cases and by nucleic acid probe for gene rearrangement in about 95%–97% of cases (also present in about 25% of adult ALL, 5% of childhood ALL, and 1%–2% of adult acute myelogenous leukemia [AML]. This is a reciprocal translocation in which a portion of the long arm of chromosome 22 breaks off at an area known as the breakpoint cluster region (BCR or ph1 oncogene) and attaches to the long arm of chromosome 9, while the distal portion of the long arm of chromosome 9 (known as the abl oncogene) breaks off and replaces the missing part of chromosome 22. Chromosome 22 becomes shorter but finishes with part of the BCR oncogene still in place plus the addition of the ab1 oncogene, creating a very abnormal chromosome. In genetic terminology, the various changes involved in the Philadelphia chromosome abnormality are summarized by t(9;22) (q34;q11); t is the translocation; (9;22) are the chromosomes involved, with the lowest chromosome number placed first; (q34;q11) is the location of the changes on the chromosomes (q = long arm of the chromosome, p = short arm; the first number refers to a region; the second is a band within the region [chromosome quinocrine banding method]; a decimal followed by a number is a subband). Other symbols are del (deletion), inv (inversion), qh+ or – (long arm increased or shortened), ph+ or – (short arm increased or shortened), I (isochromosome [mirror-image chromosome composed of 2 long arms or 2 short arms]).

  • Immunohistochemical Tumor Differentiation

    Following microscopic diagnosis of malignancy, several questions immediately arise: Is it carcinoma, sarcoma, or lymphoma? If carcinoma, is it squamous or glandular (adenocarcinoma)? If sarcoma, what is the tissue of origin? Is it primary or metastatic? If metastatic, what is the primary site (site of origin)?

    In the majority of cases, the pathologist can differentiate carcinoma, sarcoma, and lymphoma and determine if a carcinoma is squamous or glandular. However, some tumors are poorly differentiated or the biopsy may be small or obtained in a tumor area that does not have unequivocal distinguishing features. In these cases, special tissue stains using antibodies against various cell antigens can often be of assistance. The majority of these antibodies are against some component of tissue intermediate filaments. Intermediate filaments are one of four major filamentous proteins that constitute the skeleton of cells. Intermediate filaments comprise most of the intracellular matrix and are intermediate in diameter compared to the other three filamentous structural proteins. Intermediate filaments contain five protein components: cytokeratin, vimentin, desmin, glial fibrillary acidic protein, and neurofilaments. A different one of these five predominates in each of the five histologic types of mammalian tissues (epithelial, mesenchymal, muscle, neuronal, and glial). This relationship is shown in Table 33-1. It was not long before investigators found so many tumor categories in each of the five intermediary filament subgroups (Table 33-2) that antibodies specific to individual tumor types and even subgroups were required. Since that time there has been a steady stream of new antibodies from several manufacturers that attempt to fill this need. Usually the new antibody is introduced as specific (or at least, “relatively specific”) for some tumor or tissue. Usually, over time, it is found that a certain number of patient neoplasms in tumor categories not expected to be reactive with the antibody were in fact reactive to greater or lesser degree (Table 33-3). Then the antibody is promoted as part of a “cocktail” or panel of antibodies rather than as a single clear-cut diagnostic reagent. The reader is cautioned that technical details (type of tissue fixation, correct technique, and experience with the procedure), selection of the most sensitive and strongest-reacting antibodies, and experience in interpreting the result all play a major role in final results. Antibodies from different manufacturers frequently do not behave exactly the same for various reasons, in some cases because the antibody recognizes a different antigen (epitope) within a group of antigens or reacts with more than one antigen. This may sometimes become a problem when one defines a tumor on the basis of reactivity with a single antibody, either alone or as part of a panel. In addition, although immunohistologic stains are able to solve (or at least partially solve) many diagnostic problems, there is well-documented variation of results, both positive and negative, between laboratories and different investigators, as well as some individual tumors that do not produce a recognizable antibody pattern or that produce one that does not fit the clinical or microscopic picture. Finally, the multiplicity of antibodies and manufacturer’s trade names for these antibodies is confusing to nonexperts attempting to understand consultation reports.

    Original concepts of diagnosis by intermediate filament antibodies

    Table 33-1 Original concepts of diagnosis by intermediate filament antibodies

    Current diagnosis by intermediate filament antibodie

    Table 33-2 Current diagnosis by intermediate filament antibodies

    Some antibodies useful in tumor identification

    Table 33-3 Some antibodies useful in tumor identification

  • Cell Proliferation Markers

    These tests measure the quantity of various antigens associated with cell proliferation, not the actual rate of proliferation. Except for FCM, measurement is done by applying immunohistologic stains on microscopic tissue sections; either fresh tissue or with some methods, preserved and paraffin-embedded tissue. An antigen-antibody reaction is seen under the microscope by a color reaction in nuclei that contains the proliferation marker antigen. There are four types of cell proliferation marker tests:

    1.FCM S-phase measurement: This is discussed in the section on FCM. Technical problems with S-phase measurement have led to research for other proliferation markers that are more easily and universally employed and do not require special equipment. However, it is still the reference method for proliferation markers.
    2.Nuclear mitotic count (or index): The number of mitoses per microscope high-power field (usually 400Ч magnification) is the first of the cell proliferation markers, since the number of mitoses roughly correlates with tumor cell replication and with degree of tumor differentiation. The greater the number of mitoses, the more likely, in general, that the tumor will be less differentiated and more aggressive. However, this does not hold true in every tumor type nor is there a linear relationship with metastases or prognosis. Also, mitotic counts may differ in different areas of the same tumor and even in the same area are not as reproducible as would be desirable. This technique is used more often in soft tissue sarcomas.
    3.Ki-67: This is a monoclonal antibody that detects a protein in cell nuclei that appears only in the growth phase of the cell proliferation cycle (G1, S, G2, and M phases). Detection begins in mid-G1 phase and lasts throughout the remainder of the proliferation phase. This is a measure of total tumor growth fraction. It correlated well with FCM S-phase measurements. This method requires fresh tissue and is performed on cryostat frozen tissue sections.
    4.PCNA (Cyclin): This is a stable protein produced mostly during the proliferative phase of the cell cycle. It correlates directly with cell proliferation rate. In general, there is good correlation with flow cytometry S-phase measurements, but some discrepancies have been reported. Different commercial antibodies do not react with the same PCNA epitopes. The original method required cryostat-frozen fresh tissue sections, but at least one commercial kit will react with antigen in paraffin-embedded, formalin-fixed tissue. There is some evidence that PCNA production is greatest in the S-phase of the cell cycle.

  • Flow Cytometry in Cancer

    FCM has until recently been predominantly used to phenotype leukemias and lymphomas and to aid in prognosis of nonhematologic tumors.

    Nonhematologic tumors

    In nonhematologic tumors, predominately aneuploid neoplasms (especially if the S-phase value is increased) generally are more aggressive and have shorter survival time than tumors that are predominantly diploid and have normal S-phase values. However, this varies considerably between different tumor types, and there is often variation between tumors in different patients with the same tumor type. Added to this are various technical problems, such as mixtures of diploid and aneuploid tumor cells, mixtures of normal cells and tumor cells, differences in degree of tumor anaplasia in different areas, whether the tissue is fresh or formalin-fixed, proper adjustment of the instrumentation, and experience in avoiding or interpreting variant DNA peaks. S-phase work in nonhematologic tumors is more difficult than standard ploidy determination and sometimes cannot be done adequately.

    The most intensively studied (or reported) nonhematologic malignancies have been breast, colorectal, prostate, urinary bladder, ovary, and uterus.

    In breast carcinoma, there has been considerable disagreement between various studies, but overall suggestion that DNA ploidy is not a reliable independent factor in predicting likelihood of lymph node metastasis or length of survival. S-phase analysis is much more difficult to perform adequately but appears to have some predictive value regarding lymph node metastasis, degree of tumor differentiation, and presence of estrogen receptors. In colorectal cancer, the majority of studies have found that aneuploid tumors usually have shorter survival than diploid tumors and there is some correlation with probable Duke’s tumor stage (except for stage D) and therefore overall survival. In early superficial (noninvasive) transitional cell carcinoma of the urinary bladder, degree of aneuploidy has predictive value for invasiveness and tumor grade. FCM on bladder washings has additive value to biopsy of early superficial lesions. If the DNA index (measuring aneuploidy) of the biopsy differs from that of bladder washings before treatment, this suggests higher risk of tumor invasion. If the bladder washing DNA index is aneuploid and that of the biopsy is diploid, this suggests carcinoma in situ. However, if both are aneuploid, there is no prognostic assistance. Bladder washing FCM is considered the best test to follow up a patient after tumor surgery. If intravesical chemotherapy is given, it is necessary to wait 6 months after the last chemotherapy dose to resume bladder washing surveillance (because of chemotherapy-induced abnormalities in normal epithelial cells). Fresh urine specimens are much better than preserved specimens; the fresh specimen should be refrigerated immediately and analyzed as soon as possible, but no more than 12 hours later. If that is not possible, appropriate fixative must be added. In prostate carcinoma, DNA diploid tumors tend to be better differentiated (lower grade), respond better to radiation therapy, and have longer survival time; aneuploid tumors tend to be less differentiated (higher grade) with a worse prognosis and usually have less response to estrogen therapy. In ovarian carcinoma, diploid carcinomas in stage III or less have a much better prognosis than aneuploid carcinomas. In melanoma and in renal, endometrial, bone and cervix carcinomas, diploid state has some chance of a better prognosis.

    Hematopoietic malignancies

    In hematopoietic malignancies, malignant lymphomas that are diploid are usually lower grade and less aggressive, with the opposite generally true for aneuploid lymphomas. However, not all reports agree. S-phase analysis is also said to have prognostic value. Burkitt’s lymphoma has an interesting FCM profile, since it is usually diploid but has a high S-phase value and behaves like a typical aneuploid tumor. Childhood acute lymphocytic leukemia (ALL) that is aneuploid has a better response to chemotherapy. This has not been shown with adult ALL or with acute myelogenous leukemia.

  • Image Analysis Cytometry (IAC)

    Image analysis cytometry (IAC) combines some aspects of traditional visual morphology of cell nuclei with nuclear DNA analysis as done in FCM but using nonfluorescent visible nuclear stains. The instrument’s operator finds cancer cells on a tissue slide or smear and instructs the equipment’s computer to search for a certain number of tumor nuclei using instructions on what the nuclei should look like in terms of size, shape, chromatin density or distribution, and nucleoli. This helps differentiate tumor nuclei from nontumor nuclei and permits the instrument to search later on its own. The slide can then be stained with a nuclear material stain such as Feulgen (similar to flow cytometry nonspecific nuclear staining but with a different type of stain). The instrument then finds nuclei that fit the parameters given to it and analyzes the intensity of the nuclear staining reaction (again, similar to FCM). The instrument uses the sum of the nuclear density readings to calculate the average amount of DNA and displays this information as a bar-graph histogram (as in FCM) showing nuclear density composition compared to normal diploid control cell nuclei. IAC is most often performed on smears made from fresh cellular material but can be done on smears prepared from fixed tissue or even on regular formalin-fixed paraffin-embedded microscopic slides. However, tissue slide sections (rather than smears) present more difficulty due to overlapping nuclei and tissue background.

    The major advantages of IAC over FCM is that the cell selection process is more likely to analyze tumor cells only. The major advantage of FCM is less variation in the DNA peak composition (height and width) due to better counting statistics generated from thousands of cell nuclei rather than the 100-200 cells (range, 50-250 cells) usually counted in IAC. Therefore, differences in diploid-aneuploid results between FC and IAC have ranges from 9%–24%. This most commonly occurs when the number of tumor cells is very small, such as often occurs in effusions or (to a lesser extent) in fine-needle aspirate smears. Under these circumstances, IAC tends to detect malignant cells somewhat (but not always) more often than FCM. On the other hand, the relatively small numbers of cells analyzed in IAC make S-phase peak analysis very difficult or impossible, necessitating other ways to obtain cell proliferation activity information (such as monoclonal antibody stain for proliferating cell nuclear antigen or for Ki-67 antigen).

  • Flow Cytometry (FCM)

    Considerably simplified, flow cytometry (FCM) counts and analyzes certain aspects of cells using instrumentation similar in principal to many current hematology cell-counting machines. If the cells to be analyzed come from solid tissue, the cells or cell nuclei must first be extracted from the tissue and suspended in fluid. Next, the cell nuclei are stained with a fluorescent dye that stains nucleic acids in order to assay the amount of nuclear deoxyribonucleic acid (DNA); in addition, antibodies with a fluorescent molecule attached can be reacted with cell antigens in order to identify or characterize (phenotype) the cells. The cells or cell nuclei are first suspended in fluid and then forced through an adjustable hole (aperture) whose size permits only one cell at a time to pass. As the cells pass through the aperture each cell also passes through a light beam (usually produced by a laser) that activates (“excites”) the fluorescent molecules and also strikes the cell, resulting in light scatter. A system of mirrors and phototubes detects the pattern of light scatter and the fluorescent wavelengths produced (if a fluorescent dye is used), then records and quantitates the information. The pattern of light scatter can reveal cell size, shape, cytoplasm granules (in complete cells), and other cell characteristics. The electronic equipment can sort and analyze the different pattern. This information is most often collected as a bar-graph histogram, which is then displayed visually as a densitometer tracing of the bar graph; the concentration of cells in each bar appears as a separate peak for each cell category, with a peak height proportional to the number of cells in each bar of the bar graph. If all cells had similar characteristics (according to the parameters set into and recorded by the detection equipment) there would be a single narrow spikelike peak. One great advantage of cell counting by FCM rather than counting manually is that many thousand (typically, 10,000) cells are counted in FCM compared to 100 (possibly 200) in manual counts. Therefore, FCM greatly reduces the statistical error that is a part of manual cell counts.

    At present, flow cytometry is most often used for the following functions:

    1. Cell identification and phenotyping. Use of fluorescent-tagged antibodies, especially monoclonal antibodies specific for a single antigen, helps to identify various normal and abnormal cells and also subgroups of the same cell type. For example, lymphocytes can be separated into B- and T-cell categories; the T-cells can be further phenotyped as helper/ inducer, suppressor/cytoxic, or natural killer cell types. This technique may also be used to identify subgroups of certain malignancies, most often of hematologic origin (leukemias and lymphomas; see also the discussion of cell lineage studies in Chapter 7).
    2. Analysis of nuclear DNA content. Cell nuclear chromatin is stained (usually with a reagent that also contains a fluorescent molecule), and the fluorescence corresponding to total nuclear chromatin content is measured by the flow cytometer. The major determinant of total nuclear chromatin content when cells are in their resting stage is chromosome material, which in turn is related to the number of chromosomes (“ploidy”). Normally, there are 2 sets of chromosomes (“diploid”). As described previously, the FCM produces a visual representation of the DNA content of various cell groups present that in diploid cells from a resting state would be displayed as a single homogeneous spikelike peak appearing in a certain expected location of the FCM densitometer graph. A sufficient number of cell nuclei with more or less DNA than normal (an arbitrary cutoff point most often set at 5 percent deviance from diploid state) is called aneuploidy and often is seen as a separate peak from the usual diploid peak. Another way of demonstrating this is to obtain a DNA index in which the patient DNA content is compared to DNA of a (normal) control cell specimen. Ordinarily, the patient and control DNA patterns would be identical, providing a DNA index of 1.0. A DNA index sufficiently more than or less than 1.0 indicates aneuploidy. Aneuploidy is most often associated with malignancy, and if found in certain tumors may predict a more aggressive behavior. However, it should be noted that aneuploid DNA can be found in some nonneoplastic cells as part of the reaction to or regeneration after inflammation or tissue destruction and has also been reported in some benign tumors.
    3. Cell proliferation status. In standard FCM, this is done by determination of the S-phase fraction (percent S-phase; percent S; SPF). A normal cell activity cycle consists of five time periods. The cell normally spends most of its time in the G0 (“gap zero”) resting phase. It may be stimulated to enter the G1 (gap 1) stage in which ribonucleic acid (RNA) and some protein is synthesized. Next comes the S (synthesis) phase when DNA content increases to twice the resting amount in preparation for mitosis. Then comes the G2 (gap 2) period when additional RNA is produced and a very short M (mitosis) period, which is difficult to separate from G2, when mitosis takes place. After this the cell returns to G0. The S-phase (proliferation phase) features doubling of the nuclear DNA content, which is detected on the FCM histogram. The number of cells in the S-phase, if considerably increased, is shown as a small peak or elevation midway between the G0/G1 and the G2M peaks in the FCM histogram. Increase in the S-phase fraction (SPF) area suggests an unusual degree of cell proliferative activity. Since tumor cells tend to replicate more readily than normal cells, increased SPF activity can therefore raise the question of malignancy. In a considerable number of tumors, the degree of SPF activity correlates roughly to the degree of aggressiveness in tumor spread, which has prognostic significance. SPF has become one of the better and more reliable overall tumor prognostic markers (indicators). However, there are certain problems. Not all tumors with increased SPF are malignant; not all malignant tumors with increased SPF metastasize; and not all malignant tumors with relatively small SPF fail to metastasize. In addition, the S-phase peak is usually not large, even when considerable S-activity is occurring. The S-phase peak can be interfered with by cell debris or by poor separation of the G0/G1 and G2/M peaks. SPF can be falsely increased to variable degrees by reactive normal tissue cells (e.g., fibroblasts, endothelial cells, inflammatory cells, or residual areas of nontumor epithelial cells) intermixed with or included with tumor cells.
    4. To provide helpful information in patients with cancer. This includes use of information from the previous two categories (analysis of nuclear DNA content and cell proliferation status) to help determine prognosis; to help diagnose cancer in effusions, urine, or other fluids in which cancer cells may be few or mixed with benign cells such as activated histiocytes or mesothelial cells; to detect metastases in lymph nodes or bone marrow when only small numbers of tumor cells are present; or to supplement cytologic examination in fine-needle aspirates.

  • Biochemical Tests for Congenital Anomalies

    Besides giving information on fetal well-being, amniocentesis makes it possible to test for various congenital anomalies via biochemical analysis of amniotic fluid and tissue culture chromosome studies of fetal cells (see Chapter 34). In addition, certain substances of fetal origin may appear in maternal serum. In some cases it is possible to detect certain fetal malformations by screening tests in maternal serum.

    Maternal serum alpha-fetoprotein

    One of the most widely publicized tests for congenital anomalies is the alpha-fetoprotein (AFP) test in maternal serum for detection of open neural tube defects. Although neural tube defects are much more common in infants born to families in which a previous child had such an abnormality, about 90% occur in families with no previous history of malformation. AFP is an alpha-1 glycoprotein with a molecular weight of about 70,000. It is first produced by the fetal yolk sac and then mostly by the fetal liver. It becomes the predominant fetal serum protein by the 12th or 13th week of gestation but then declines to about 1% of peak levels by delivery. It is excreted via fetal urine into amniotic fluid and from there reaches maternal blood. After the 13th week both fetal serum and amniotic fluid AFP levels decline in parallel, the fetal blood level being about 200 times the amniotic fluid level. In contrast, maternal serum levels become detectable at about the 12th to 14th week and reach a peak between the 26th and 32nd week of gestation. Although maternal serum screening could be done between the 15th and 20th weeks, the majority of investigators have decided that the interval between the 16th and 18th weeks is optimal, since the amniotic fluid AFP level is still relatively high and the fetus is still relatively early in gestation. Maternal AFP normal levels differ for each week of gestation and ideally should be determined for each laboratory. Results are reported as multiples (e.g., 1.5Ч, 2.3Ч) of the normal population mean value for gestational age. In any patients with abnormal AFP values it is essential to confirm fetal gestational age by ultrasound, since 50% of abnormal AFP results are found to be normal due to ultrasound findings that result in a change being made in a previously estimated gestational date. Some reports suggest that maternal weight is also a factor, with heavier women tending to have lower serum AFP values (one group of investigators does not agree that maternal AFP values should be corrected for maternal weight). There are also some reports that AFP values are affected by race, at least when comparing values from Europeans and African Americans.

    Maternal AFP levels reportedly detect about 85%-90% (literature range, 67%-97%) of open neural tube defects; about one half are anencephaly and about one half are open or closed spinabifida. There is an incidence of about 1-2 per 1,000 live births. The test also detects a lesser (but currently unknown) percentage of certain other abnormalities, such as fetal ventral wall defects, Turner’s syndrome, pilonidal sinus, hydrocephalus, duodenal atresia, multiple hypospadias, congenital nephrosis, and cystic hygroma. In addition, some cases of recent fetal death, threatened abortion, and Rh erythroblastosis produce elevated maternal AFP levels, as well as some cases of maternal chronic liver disease and some maternal serum specimens obtained soon after amniocentesis. A theoretical but unlikely consideration is AFP-producing tumors such as hepatoma. More important, twin pregnancies cause maternal values that are elevated in terms of the reference range established on single-fetus pregnancies. A large minority of elevated maternal AFP levels represent artifact due to incorrect estimation of fetal gestational age, which, in turn, would result in comparing maternal values to the wrong reference range. There is also the possibility of laboratory error. Most authorities recommend a repeat serum AFP test 1 week later to confirm an abnormal result. If results of the second specimen are abnormal, ultrasound is usually suggested to date the age of gestation more accurately, to examine the fetus for anencephaly, and to exclude twin pregnancy. However, even ultrasonic measurements may vary from true gestational age by as much as 5-7 days. Some perform ultrasonic evaluation if the first AFP test result is abnormal; if ultrasound confirms fetal abnormality, a second AFP specimen would be unnecessary. In some medical centers, about 40%-59% of elevated maternal AFP levels can be explained on the basis of technical error, incorrect fetal gestation date, and multiple pregnancy.

    Some conditions produce abnormal decrease in maternal serum AFP values. The most important is Down’s syndrome (discussed later). Other conditions that are associated with decreased maternal serum AFP levels include overestimation of fetal age and absence of pregnancy (including missed abortion).

    Amniotic fluid alpha-fetoprotein

    Amniocentesis is another technique that can be used to detect open neural tube defects. It is generally considered the next step after elevated maternal AFP levels are detected and confirmed and the age of gestation is accurately determined. As mentioned previously, amniocentesis for this purpose is generally considered to be optimal at 16-18 weeks of gestation. Assay of amniotic fluid AFP is said to be about 95% sensitive for open neural tube defects (literature range, 80%-98%), with a false positive rate in specialized centers less than 1%. Most false positive results are due to contamination by fetal blood, so a test for fetal red blood cells or hemoglobin is recommended when the amniotic fluid AFP level is elevated. Amniotic fluid AFP normal values are age related, similar to maternal serum values.

    Screening for Down’s syndrome

    While maternal serum AFP screening was being done to detect neural tube defects, it was noticed that decreased AFP levels appeared to be associated with Down’s syndrome (trisomy 21, the most common multiple malformation congenital syndrome). Previously, it had been established that women over age 35 had a higher incidence of Down’s syndrome pregnancies. In fact, although these women represent only 5%-8% of pregnancies, they account for 20%-25% (range, 14%-30%) of congenital Down’s syndrome. Since it was discovered that mothers carrying a Down’s syndrome fetus had AFP values averaging 25% below average values in normal pregnancy, it became possible to detect about 20% of all Down’s syndrome fetuses in pregnant women less than age 35 years in the second trimester. Combined with approximately 20% of all Down’s syndrome fetuses detected by amniocentesis on all possible women over age 35, the addition of AFP screening to maternal age criteria potentially detected about 40% of all Down’s syndrome pregnancies. Later, it was found that serum unconjugated estriol (uE3) was decreased about 25% below average values seen in normal pregnancies, and hCG values were increased at least 200% above average normal levels; both were independent of maternal age. Addition of hCG and uE3 to AFP screening raised the total detection rate of all Down’s syndrome patients to about 60%. Later, there was controversy whether including uE3 was cost effective. Even more recently it was found that substituting beta-hCG for total hCG increased the total Down’s syndrome detection rate to 80%-86%. Also, it was found that screening could be done in the first trimester as well as the second trimester (although AFP was less often abnormal). Finally, it was found that if AFP, uE3, and beta-hCG were all three decreased (beta-hCG decreased rather than elevated), about 60% of fetal trisomy 18 could be detected. Trisomy 18 (Edward’s syndrome) is the second most common congenital trisomy. Decreased AFP can also be caused by hydatidiform mole, insulin-dependent diabetes, and incorrect gestational age estimation.

    Amniotic fluid acetylcholinesterase

    Acetylcholinesterase (ACE) assay in amniotic fluid has been advocated as another way to detect open neural tube defects and to help eliminate diagnostic errors caused by false positive AFP results. Acetylcholinesterase is a major enzyme in spinal fluid. Results from a limited number of studies in the late 1970s and early 1980s suggest that the test has 98%-99% sensitivity for open neural tube defects. Acetylcholinesterase assay has the further advantage that it is not as dependent as AFP on gestational age. It is not specific for open neural tube defects; amniotic fluid elevations have been reported in some patients with exomphalos (protrusion of viscera outside the body due to a ventral wall defect) and certain other serious congenital anomalies and in some patients who eventually miscarry. Not enough data are available to properly evaluate risk of abnormal ACE results in normal pregnancies, with reports in the literature ranging from 0%-6%. There is also disagreement as to how much fetal blood contamination affects ACE assay. The test is less affected than AFP assay, but substantial contamination seems capable of producing falsely elevated results.

    Chromosome analysis (cytogenetic karyotyping) on fetal amniotic cells obtained by amniocentesis is the standard way for early prenatal diagnosis of fetal trisomies and other congenital abnormalities. However, standard karyotyping is very time-consuming, requires a certain minimum number of fetal cells that need culturing, and usually takes several days to complete. A new method called fluorescent in situ hybridization (FISH) uses nucleic acid (deoxyribonucleic acid, DNA) probes to detect certain fetal cell chromosomes such as 13, 18, 21, X, and Y, with identification accomplished by a fluorescent dye coupled to the probe molecules. Correlation with traditional cytogenetics has generally been over 95%, with results in 24 hours or less. FISH modifications have made it possible to detect fetal cells in maternal blood and subject them to the same chromosome analysis. One company has a combined-reagent procedure that can be completed in 1 hour. Disadvantages of FISH include inability to detect abnormalities in chromosomes other than the ones specifically targeted by the probes and inability to detect mosaic abnormalities or translocations, thereby missing an estimated 35% of chromosome defects that would have been identified by standard karyotyping methods.

    Preterm labor and placental infection

    It has been estimated that about 7% of deliveries involve mothers who develop preterm labor. It has also been reported that chorioamnionitis is frequently associated with this problem (about 30%; range, 16%-82%). Less than 20% of infected patients are symptomatic. Diagnosis of infection has been attempted by amniotic fluid analysis. Amniotic fluid culture is reported to be positive in about 20% of cases (range, 4%-38%). Mycoplasmas are the most frequent organisms cultured. Amniotic fluid Gram stain is positive in about 20% of patients (range, 12%-64%). Amniotic fluid white blood cell count was reported to be elevated in 57%-64% of cases. However, there was great overlap between patients with or without infection and also between those with proven infection. In three reports, the most sensitive amniotic fluid test for infection was amniotic fluid interleukin-6 (IL-6) assay (81%-100%). However, at present most hospitals would have to obtain IL-6 assay from large reference laboratories.

  • Fetal Maturity Tests

    Tests for monitoring fetal maturity via amniocentesis are also available. Bilirubin levels in erythroblastosis are discussed in chapter 11. Amniotic creatinine assay, amniotic epithelial cell stain with Nile blue sulfate, fat droplet evaluation, osmolality, and the Clemens shake test, alone or in combination, have been tried with varying and not entirely satisfactory results. Most current tests measure one or more components of alveolar surfactant. Surfactant is a substance composed predominantly of phospholipids; it is found in lung alveoli, lowers the surface tension of the alveolar lining, stabilizes the alveoli in expiration, and helps prevent atelectasis. Surfactant deficiency causes neonatal respiratory distress syndrome (RDS), formerly called “hyaline membrane disease.” The major phospholipid components of surfactant are phosphatidylcholine (lecithin, about 80%; range, 73%-88%), phosphatidylglycerol (PG, about 3%; range, 1.8%-4.2%), and sphingomyelin (about 1.6%). The current most widely used tests are the lecithin/sphingomyelin (L/S) ratio, assay of phosphatidylglycerol (PG), the foam stability index (FSI), and TDx fluorescent polarization.

    Lecithin/Sphingomyelin (L/S) ratio. The L/S ratio has been the most widely accepted fetal maturity procedure. Lecithin (phosphatidylcholine), a phospholipid, is the major component of alveolar surfactant. There is a 60% or greater chance of RDS in uncomplicated pregnancies when the fetus is less than 29 weeks old; about 8%-23% at 34 weeks; 0%-2% at 36 weeks; and less than 1% after 37 weeks. In amniotic fluid, the phospholipid known as sphingomyelin normally exceeds lecithin before the 26th week; thereafter, lecithin concentration is slightly predominant until approximately the 34th week, when the lecithin level swiftly rises and the sphingomyelin level slowly decreases so that lecithin levels in the 35th or 36th week become more than twice sphingomyelin levels. After that happens it was originally reported (not entirely correctly), that there was no longer any danger of neonatal RDS. The L/S ratio thus became a test for fetal lung and overall maturity. Certain precautions must be taken. Presence of blood or meconium in the amniotic fluid or contamination by maternal vaginal secretions may cause a false increase in lecithin and sphingomyelin levels, so that “mature” L/S ratios are decreased and “immature” L/S ratios are increased. The amniotic fluid specimen must be cooled immediately, centrifuged to eliminate epithelial cells and other debris, and kept frozen if not tested promptly to prevent destruction of lecithin by certain enzymes in the amniotic fluid.

    Evaluations in unselected amniocentesis patients have revealed that about 55% of neonates with immature L/S ratios using the 2.0 ratio cutoff point do not develop RDS and about 5% (literature range, 0%-17%) of neonates with a mature L/S ratio (ratio >2.0) develop RDS. Some have attempted to eliminate the falsely mature cases by changing the cutoff point to a ratio of 2.5, but this correspondingly increases the number of falsely immature results. In clinically normal pregnancies, only about 3% of neonates with a mature L/S ratio, using proper technique, develop RDS. In complicated pregnancies, especially those with maternal type I insulin-dependent diabetes, hypertension, or premature rupture of the amniotic membrane, about 15% (literature range, 3%-28%) of neonates with mature L/S ratios are reported to develop RDS. In other words, RDS can develop at higher L/S ratios in a relatively small number of infants. The wide range in the literature reflects differences in opinion among investigators as to the effect of diabetes on neonatal L/S ratios. Also, the L/S ratio can produce falsely mature results if contaminated by blood or meconium. It takes experience and careful attention to technical details to obtain consistently accurate L/S results.

    Phosphatidylglycerol (PG). A number of other tests have been developed in search of a procedure that is more accurate in predicting or excluding RDS and that is also technically easy to perform. PG is a relatively minor component (about 10%) of lung surfactant phospholipids. However, PG is almost entirely synthesized by mature lung alveolar cells and therefore is a good indicator of lung maturity. In normal pregnancies PG levels begin to increase after about 30 weeks’ gestation and continue to increase until birth. It normally becomes detectable about the 36th week. In conditions that produce severe fetal stress, such as maternal insulin-dependent diabetes, hypertension, and premature membrane rupture, PG levels may become detectable as early as 30 weeks’ gestation. Most of the limited studies to date indicate that the presence of PG in more than trace amounts strongly suggests that RDS will not develop, whether the pregnancy is normal or complicated. Overall incidence of RDS when PG is present seems to be about 2% (range 0%-10%). It is considered to be a more reliable indicator of fetal lung maturity than the L/S ratio in complicated pregnancy. It may be absent in some patients with clearly normal L/S ratios and occasionally may be present when the L/S ratio is less than 2.0. PG assay is not significantly affected by usual amounts of contamination by blood or meconium.

    PG can be assayed in several ways, including gas chromatography, thin-layer chromatography (TLC), enzymatically, and immunologically. The TLC technique is roughly similar to that of the L/S ratio. Some report the visual presence or absence of PG, with or without some comment as to how much appears to be present (trace or definite). Some report a PG/sphingomyelin (PG/S) ratio. A PG/S ratio of 2.0 or more is considered mature. A commercially available enzymatic PG method (“PG-Numeric”) separates phospholipids from the other components of amniotic fluid (using column chromatography or other means), followed by enzymatic assay of glycerol in the phospholipid fraction. After several years there is still an insufficient number of published evaluations of this technique. Immunological methods are still restricted to a slide agglutination kit called Amniostat FLM-Ultra (improved second-generation test). Current small number of evaluations indicate that Amniostat FLM-Ultra detects about 85%-90% of patients who are positive for PG on TLC. The risk of RDS is about 1%-2% if the test is reactive (positive).

    Foam stability index (FSI). The FSI is a surfactant function test based on the ability of surfactant to lower surface tension sufficiently to permit stabilized foaming when the amniotic fluid is shaken. This depends on the amount and functional capability of surfactant as challenged by certain amounts of the antifoaming agent ethanol. It is thought that the phospholipid dipalmitoyl lecithin is the most important stabilizing agent. The FSI is actually a modification of the Clemens shake test, which used a final amniotic fluid-ethanol mixture of 47.5% ethanol. The FSI consists of a series of seven tubes containing amniotic fluid with increasing percentages of ethanol in 1% increments from 44%-50%. The endpoint is the tube with the highest percentage of ethanol that maintains foam after shaking. An endpoint of the 47% tube predicts about a 4% chance of RDS and an endpoint in the 48% tube predicts less than 1% chance. Because even tiny inaccuracies or fluctuations of ethanol concentration can influence results considerably, and also the tendency of absolute ethanol to adsorb water, some problems were encountered in laboratories making their own reagents. To solve these problems a commercial version of the FSI called Lumidex was introduced featuring sealed tubes containing the 1% increments of ethanol to which aliquots of amniotic fluid are added through the rubber caps that seal the tubes. The FSI (or Lumidex) has been reported to be more reliable than the L/S ratio in predicting fetal lung maturity. At least two reports indicate that the FSI correctly demonstrates fetal lung maturity much more frequently than the L/S ratio in fetuses who are small for their gestational age. Drawbacks of the FSI method in general are interference (false positive) by blood, meconium, vaginal secretions, obstetrical creams, and mineral oil. A major drawback of the current Lumidex kit is a shelf-life of only 3 weeks without refrigeration. Although the shelf life is 3 months with refrigeration, it is necessary to stabilize the tubes at room temperature for at least 3 hours before the test is performed.

    TDx-FLM fluorescent polarization. The TDx is a commercial instrument using fluorescent polarization to assay drug levels and other substances. It has been adapted to assay surfactant quantity indirectly by staining surfactant in amniotic fluid with a fluorescent dye and assaying surfactant (in mg/gm of albumin content) using the molecular viscosity of the fluid as an indicator of surfactant content. The assay in general produces results similar to the L/S ratio and a little better than the FSI. There is some difference in results depending on whether a single cutoff value is used, what that value is, and whether multiple cutoff values are applied depending on the situation. Test technical time is about 30 minutes. Specimens contaminated with meconium, blood, or urine (in vaginal pool material) interfere with the test.

    Lamellar body number density. Surfactant is produced by alveolar type II pneumocytes in the form of a concentrically wrapped small structure about 3 microns in size that on cross-section looks like an onion and is called a lamellar body. It is possible to count the lamellar bodies using some hematology platelet counting machines, with the result calculated in units of particle density per microliter of amniotic fluid. In the very few evaluations published to date, results were comparable to those of the L/S ratio and FSI.

    Amniocentesis laboratory problems. Occasionally, amniotic puncture may enter the maternal bladder instead of the amniotic sac. Some advocate determining glucose and protein levels, which are high in amniotic fluid and low in normal urine. To prevent confusion in diabetics with glucosuria, it has been suggested that urea and potassium levels be measured instead; these are relatively high in urine and low in amniotic fluid. Another potential danger area is the use of spectrophotometric measurement of amniotic fluid pigment as an estimate of amniotic fluid bilirubin content. Before 25 weeks’ gestation, normal pigment levels may be greater than those usually associated with abnormality.

  • Fetal or Placental Function

    Urine estriol or total estrogens. Estriol is an estrogenic compound produced by the placenta from precursors derived from fetal adrenal cortex and fetal liver. Newly synthesized estriol is unconjugated; therefore, unconjugated estriol represents a product of the entire fetoplacental unit. The unconjugated estriol reaches maternal serum (where it has a half-life of about 20 minutes) and is taken to the maternal liver, where about 90% is conjugated with a glucuronide molecule. The conjugated form of estriol is excreted in maternal urine. A lesser amount of conjugated estriol is produced by the maternal liver from nonestriol estrogens synthesized by the placenta from maternal adrenal precursors. Serum estriol can be measured either as total estriol or as unconjugated estriol. It usually is measured as unconjugated estriol to exclude maternal contribution to the conjugated fraction. Urine estriol can be measured as total estriol or as total estrogens, since estriol normally constitutes 90%-95% of urine total estrogens.

    Historically, urine total estrogen was the first test used, since total estrogen can be assayed by standard chemical techniques. However, urine glucose falsely increases results (which is a problem in diabetics, who form a large segment of the obstetrical high-risk group), and certain other substances such as urobilinogen also may interfere. In addition, urine total estrogen results are a little more variable than urine estriol patterns. Eventually, other biochemical procedures that were more specific for urine estriol (in some cases, however, the “estriol” being measured using biochemical methods is actually total estrogen) were devised. These procedures also have certain drawbacks, some of which are shared by the total estrogen methods. Both urine total estrogens and urine estriol have a significant degree of between-day variation in the same patient, which averages about 10%-15% but which can be as high as 50%. Both are dependent on renal excretion, and, therefore, on maternal renal function. Both have a maternal component as well as the fetal component. Urine total estrogen necessitates a 24-hour collection. The standard method for urine estriol also is a 24-hour specimen. There is substantial difficulty collecting accurate 24-hour specimens, especially in outpatients. Also, there is a 1- to 2-day time lag before results are available. Some have proposed a single voided specimen based on the estriol/creatinine ratio. However, there is controversy whether the single-voided specimen method (reported in terms of either estriol per gram creatinine or estriol/creatinine ratio) provides results as clinically accurate as the 24-hour specimen.

    Estriol can be detected by immunoassay as early as the ninth week of gestation. Thereafter, estriol values slowly but steadily increase until the last trimester, when there is a more pronounced increase. Clinical use of estriol measurement is based on the fact that severe acute abnormality of the fetoplacental unit (i.e., a dead or dying placenta or fetus) is manifested either by failure of the estriol level to continue rising or by a sudden marked and sustained decrease in the estriol level. Urine specimens are usually obtained weekly in the earlier part of pregnancy, twice weekly in the last trimester, and daily for several days if a problem develops.

    In general, urine estriol or estrogen excretion correlates reasonably well with fetal health. However, there are important exceptions. Only severe fetal or placental distress produces urine estrogen decrease of sufficient magnitude, sufficient duration, and sufficiently often to be reliable (i.e., mild disorder may not be detected). There is sufficient daily variation in excretion so that only a very substantial and sustained decrease in excretion, such as 40%-50% of the mean value of several previous results, is considered reliable. Some consider an estriol value less than 4 mg/24 hours (after 32 weeks’ gestation) strongly suggestive of fetal distress and a value more than 12 mg/24 hours as indicative of fetal well-being. Maternal hypertension, preeclampsia, severe anemia, and impaired renal function can decrease urine estrogen or estriol excretion considerably. Decrease may also occur to variable degree in variable numbers of fetuses with severe congenital anomalies. Certain drugs such as ampicillin and cortisol may affect urine estriol or estrogen values by effects on production, and other substances such as mandelamine or glucose can alter results from biochemical interference with some test methods. Some investigators have reported a decrease shortly before delivery in a substantial minority of normal patients. Maternal Rh-immune disease may produce a false increase in urine estriol levels. Continued bed rest has been reported to increase estriol excretion values an average of 20% over levels from ambulatory persons, with this increase occurring in about 90% of patients in the third trimester.

    The literature contains widely differing opinions regarding clinical usefulness of estrogen excretion assay in pregnancy. In general, investigators have found that urinary estrogen or estriol levels are decreased in about 60%-70% of cases in which fetal distress occurs (literature range, 33%-80%). The more severe the fetal or placental disorder, the more likely that urine estrogen or estriol levels will be low. The percentage of falsely low values is also said to be substantial, but numerical data are not as readily available.

    Serum unconjugated estriol. Plasma or serum unconjugated estriol, measured by immunoassay, has been used as a replacement for urine hormone excretion. Advantages include ease of specimen collection (avoidance of 24-hour urine collection problems), increased specificity for fetoplacental dysfunction (no maternal hormone contribution), no 24-hour wait for a specimen, closer observation of fetoplacental health (due to the short unconjugated estriol half-life), little technical interference by substances such as glucose, and less dependence on maternal kidney function. Drawbacks include the majority of those drawbacks previously described for urine hormone excretion (effect of bed rest, hypertension, and other conditions, and medications affecting estrogen production). Also similar to urine excretion, there is substantial between-day variation, averaging about 15% (reported maximum variation up to 49%). However, there are also considerable within-day fluctuations, which average about 15% (with maximum variation reported as high as 51%). Thus, a single value is even more difficult to interpret than a urine value. Some believe that 24-hour urine measurements may thus have some advantage, since within-day fluctuations are averaged out. Also similar to urine values, the current trend of interpretation is to require a sustained decrease of 40%-50% from the average of several previous serum values to be considered a significant abnormality. The serum specimens should preferably be drawn at the same time of day in the same patient position and assayed by the same laboratory. Although frequency of sampling is not uniform among investigators, many obtain one or two specimens per week during the earlier part of pregnancy and one per day if there is clinical suggestion of abnormality or one serum value becomes significantly decreased. Although there is some disagreement, the majority of investigators indicate that serum unconjugated estriol has a little better correlation with clinical results than does urine hormone excretion.

    Because of the problems associated with collection of urine or serum estriol specimens and interpretation of the values, as well as the disturbing number of false positive and false negative test results, many clinicians depend more on other procedures (e.g., the nonstress test, which correlates the rate of fetal heartbeat to fetal movement) than on estrogen values to monitor fetal well-being.

    Placental lactogen. HPL is a hormone produced only by the placenta, with metabolic activity similar in some degree to that of prolactin and GH. Values correlate roughly with the weight of the placenta and rise steadily in maternal serum during the first and second trimesters before entering a relative plateau in the third. Serum levels of hPL are higher than those attained by any other peptide hormone. Serum half-life is about 30 minutes. Although hPL cross-reacts with GH in most radioimmunoassay (RIA) systems, the high level of hPL relative to GH at the time of pregnancy when hPL levels are measured prevents clinical problems with GH interference. Serum hPL has been evaluated by many investigators as a test of placental function in the third trimester. Its short half-life is thought to make it a more sensitive indicator of placental failure than measurements of other hormones, especially urine measurements. Since hPL levels normally can fluctuate somewhat, serial measurements are more accurate than a single determination. Estriol, which reflects combined fetal and placental function, still seems to be used more than hPL.