Articles on Medical Diseases and Conditions

Month: December 2009

  • Congenital Diseases of Skeletal Muscle

    Several well-known disorders affecting skeletal muscle either are not congenital or do not yet have any conspicuously useful laboratory test. Among these are disorders whose primary defect is located in the central nervous system or peripheral nervous system rather than in skeletal muscle itself. In this group are various neurologic diseases that secondarily result in symptoms of muscle weakness. The following discussion involves inherited muscle disorders. Some can be diagnosed in the first trimester of pregnancy by means of amniotic villus biopsy.

    Muscular dystrophies. The muscular dystrophies can be divided into several subgroups. The most common is Duchenne’s (pseudohypertrophic) dystrophy. Duchenne’s muscular dystrophy and the closely related Becker’s muscular dystrophy is transmitted as a familial sex-linked recessive disorder in 60%-65% of cases and is said to be the most common lethal sex-linked genetic disease. As in all sex-linked genetic diseases, the X chromosome carriers the abnormal gene. In Duchenne’s dystrophy this gene controls production of dystrophin, a protein found in skeletal, cardiac, and smooth muscle at the muscle fiber outer membrane, where it apparently helps provide strength and elasticity to the muscle fiber. Although both males and females may have the defective gene, females rarely develop clinical symptoms. About one third of cases are sporadic gene mutations. The male patient is clinically normal for the first few months of life; symptoms develop most often between ages 1 and 6 years. The most frequent symptoms are lower extremity and pelvic muscle weakness. There is spotty but progressive muscle fiber dissolution, with excessive replacement by fat and fibrous tissue. The latter process leads to the most characteristic physical finding of the disease, pseudohypertrophy of the calf muscles.

    Laboratory tests. Screening tests are based on the fact that certain enzymes are found in relatively high amounts in normal skeletal muscle. These include creatine phosphokinase, aldolase, aspartate aminotransferase (AST), and lactic dehydrogenase (LDH). Despite external pseudohypertrophy, the dystrophic muscles actually undergo individual fiber dissolution and loss of skeletal muscle substance, accompanied by release of muscle enzymes into the bloodstream. In many tissues AST, LDH, and aldolase are found together. Pulmonary infarction, myocardial infarction, and acute liver cell damage among other conditions cause elevated serum levels of these enzymes. Aldolase follows a pattern similar to AST in liver disease and to LDH otherwise. Creatine Kinase (previously creatine phosphokinase) or CK is found in significant concentration only in brain, heart muscle, and skeletal muscle.

    The two most helpful tests in Duchenne’s muscular dystrophy are CK and aldolase assays. Aldolase and CK values are elevated very early in the disease, well before clinical symptoms become manifest, and the elevations usually are more than 10 times normal, at least for CK. This marked elevation persists as symptoms develop. Eventually, after replacement of muscle substance has become chronic and extensive, the aldolase level often becomes normal and the CK level may be either normal or only mildly elevated (less than 5 times normal). In the hereditary type of Duchenne’s dystrophy, most males with the abnormal gene have elevated CK values. In females with the abnormal gene, about 50%-60% have elevated CK. Aldolase values are much less frequently abnormal; AST and LDH values tend to parallel CK and aldolase values but at a much lower level. Therefore, other than CK, these enzymes are not of much use in detecting carriers. Even with CK, a normal result does not exclude carrier status.

    The CK isoenzyme pattern in Duchenne’s dystrophy may show an increased MB isoenzyme as well as MM fraction, especially in the earlier phases of the illness.

    In fascioscapulohumeral dystrophy and limbgirdle dystrophy, conditions that resemble Duchenne’s dystrophy in many respects, CK and aldolase levels are variable but frequently are normal.

    Other muscular disorders in which the serum enzyme levels may be elevated are trauma, dermatomyositis, and polymyositis. The levels of elevation are said to be considerably below those seen in early cases of Duchenne’s dystrophy. Neurologic disease is usually not associated with elevated levels, even when there is marked secondary muscular atrophy.

    Definitive diagnosis. Diagnosis of the muscular dystrophies may sometimes be made on the basis of the clinical picture and enzyme values. A more definitive diagnosis can be made with the addition of muscle biopsy. This becomes essential when the findings are not clear cut. The biceps or quadriceps muscles are the preferred biopsy location. The biopsy is best done at a pediatric or congenital disease research center where special studies (e.g., histochemical staining or electron microscopy) can be performed and the proper specimen secured for this purpose (these special studies provide additional information and are essential when biopsy results are atypical or yield unexpected findings). Biopsy specimens show greatly decreased dystrophin on assay or essentially absent dystrophin on tissue sections stained with antidystrophin antibody. In Becker’s dystrophy, dystrophin is present in tissue sections but considerably reduced. Another diagnostic method is DNA probe. About two thirds of Duchenne’s and Becker’s cases are due to partial deletion from the dystrophin gene. These cases can be diagnosed by standard DNA probe. The 35% without detectable deletion can be tested for with the restriction length polymorphism DNA probe method, which is less accurate than gene deletion DNA methods. Diagnosis in the first trimester of pregnancy can be done using DNA probe techniques on a chorionic villus biopsy specimen.

    Malignant hyperpyrexia (MH) Malignant hyperpyrexia (MH) is a rare complication of anesthesia triggered by various conduction and inhalation agents (most commonly succinylcholine) that produces a marked increase in both aerobic and anaerobic skeletal muscle metabolism. This results in greatly increased production of carbon dioxide, lactic acid, and heat. Such overproduction, in turn, is clinically manifested by a marked increase in body temperature, tachycardia, muscle rigidity, tachypnea, and finally shock. The first clinical sign is said to be muscle rigidity, which occurs in about 70%-75% of patients. The next clinical evidence of developing MH is tachycardia or cardiac ventricular multifocal arrhythmias. A rise in temperature eventually occurs in nearly all patients (some cases have been reported without temperature elevation), first slowly and then rapidly. The characteristic temperature elevation may not be present in the early stages, or the initial elevation may be gradual. A defect in muscle cell membrane calcium release mechanism (“calcium channel”) has been postulated, leading to increased intracellular calcium. The majority of cases have been familial. There is frequent association with various hereditary muscle diseases, especially the muscular dystrophies.

    Laboratory tests. Biochemical abnormalities include metabolic acidosis (markedly elevated lactic acid value) and respiratory acidosis (increased partial pressure of carbon dioxide [P CO2] due to muscle CO2 production). The anion gap is increased due to the lactic acid. In the early phases, venous P CO2 is markedly increased, whereas arterial P CO2 may be normal or only mildly increased (widening of the normal arteriovenous [AV] CO2 dissociation). The same accentuation of normal AV differences also occurs with the PO2 values. Later, arterial blood gas values also show increased PCO2, decreased pH, and decreased PO2. In addition to blood gas changes there typically is greatly increased CK levels (from muscle contraction), and myoglobin appears in the urine. In the later stages there may be hyperkalemia, hypernatremia, muscle edema, pulmonary edema, renal failure, disseminated intravascular coagulation, and shock. The serum calcium level may be normal or increased, but the ionized calcium level is increased.

    Diagnosis. CK elevation without known cause has been proposed as a screening test; there is marked difference of opinion among investigators as to the usefulness of the procedure, either in screening for surgery or in family studies (literative reports vary from 0%-70% regarding the number of persons susceptible to develop MH that have elevated baseline total CK. About 30% may be a reasonable estimate. Also, elevated CK can be caused by a variety of conditions affecting muscle). Likewise, disagreement exists as to the CK isoenzyme associated with abnormality; BB has been reported by some, although the majority found MM to be responsible. However, CK assay is still the most widely used screening test. Muscle biopsy with special in vitro testing of muscle fiber sensitivity to such agents as caffeine and halothane (caffeine-halothane contractive test) is considered a definitive diagnostic procedure but is available only in a few research centers. Since the test should be performed less than 5 hours after the muscle biopsy, it is preferable (if possible) to have this biopsy performed at the institution that will do the test. Microscopic examination of muscle biopsy specimens shows only nonspecific abnormalities, most often described as compatible with myopathy.

  • Chromosomal Abnormalities

    Chromosome analysis. There are several conditions, some relatively common and some rare, that result from either abnormal numbers of chromosomes, defects in size or configuration of certain single chromosomes, or abnormal composition of the chromosome group that determines sexual characteristics. Laboratory diagnosis, at present, takes three forms. First, chromosome charts may be prepared on any individual by culturing certain body cells, such as WBCs from peripheral blood or bone marrow, and by introducing a chemical such as colchicine, which kills the cells at a specific stage in mitosis when the chromosomes become organized and separated, and then photographing and separating the individual chromosomes into specific groups according to similarity in size and configuration. The most widely used system is the Denver classification. The 46 human chromosomes are composed of 22 chromosome pairs and, in addition, 2 unpaired chromosomes, the sex chromosomes (XX in the female and XY in the male). In a Denver chromosome chart (karyotype) the 22 paired chromosomes are separated into 7 groups, each containing 2 or more individually identified and numbered chromosomes. For example, the first group contains chromosomes 1 to 3, the seventh group contains chromosomes 21 to 22. In addition, there is an eighth group for the two unpaired sex chromosomes. Chromosome culture requires substantial experience and care in preparation and interpretation. Material for chromosome analysis can be obtained in the first trimester of pregnancy by means of chorionic villus biopsy.

    Barr body test. The other, more widely used technique provides certain useful information about the composition of the sex chromosome group. Barr found that the nuclei of various body cells contain a certain stainable sex chromatin mass (Barr body) that appears for each X chromosome more than one that the cell possesses. Therefore, a normal male (XY) cell has no Barr body because there is only one X chromosome, a normal female (XX) cell has one Barr body, and a person with the abnormal configuration XXX has two Barr bodies. The most convenient method for Barr body detection at present is the buccal smear. This is obtained by scraping the oral mucosa, smearing the epithelial cells thus collected onto a glass slide in a monolayer, and, after immediate chemical fixation, staining with special stains. Comparison of the results, together with the secondary sex characteristics and genitalia of the patient, allows presumptive diagnosis of certain sex chromosome abnormalities. The results may be confirmed, if necessary, by chromosome karyotyping.

    Specimens for buccal smear should not be obtained during the first week of life or during adrenocorticosteroid or estrogen therapy, because these situations falsely lower the incidence of sex chromatin Barr bodies. Certain artifacts may be confused with the nuclear Barr bodies. Poor slide preparations may obscure the sex chromatin mass and lead to false negative appearance. Only about 40%-60% of normal female cells contain an identifiable Barr body. The buccal smear by itself does not reveal the true genetic sex; it is only an indication of the number of female (X) chromosomes present. Many labs no longer do this test.

    The third method is nucleic acid probe, more sensitive than either Barr body or standard chromosome analysis. However, the chromosome abnormality must be known and a probe must be available for that specific gene or chromosome area.

    Klinefelter’s syndrome. In this condition the patient looks outwardly like a male, but the sex chromosome makeup is XXY instead of XY. The external genitalia are usually normal except for small testes. There is a tendency toward androgen deficiency and thus toward gynecomastia and decreased body hair, but these findings may be slight or not evident. There also is a tendency toward mental deficiency, but most affected persons have perfectly normal intelligence. Patients with Klinefelter’s syndrome are almost always sterile. Testicular biopsy used to be the main diagnostic method, with histologic specimens showing marked atrophy of the seminiferous tubules. A buccal smear can be done; it shows a “normal female” configuration with one Barr body (due to the two XX chromosomes). In the presence of unmistakably male genitalia, this usually is sufficient for clinical diagnosis. Since 10% of cases have a mosaic cell pattern, chromosome karyotyping is now the procedure of choice.

    Turner’s syndrome (ovarian agenesis). Turner’s syndrome is the most frequent chromosomal sexual abnormality in females, just as Klinefelter’s syndrome is in males. In Turner’s syndrome there is a deletion of 1 female (X) chromosome so that the patient has only 45 chromosomes instead of 46 and only 1 female sex chromosome instead of 2. Typically the affected female has relatively short stature but normal body proportions. There is deficient development of secondary sex characteristics and small genitalia, although body hair usually is female in distribution. Some affected persons have associated anomalies such as webbing of the neck, coarctation of the aorta, and short fingers. They do not menstruate and actually lack ovaries. A buccal smear should be “sex-chromatin negative,” since Barr bodies appear only when the female sex chromosomes number more than one. If the buccal smear is “chromatin positive,” a chromosome karyotype should be ordered, because some patients with Turner’s syndrome have mixtures of normal cells and defective cells (mosaicism). Some investigators believe that in patients with short stature only, chromosome karyotyping should be done without a buccal smear, since most of the “nonphenotypic” Turner’s syndrome patients have mosaicism rather than XO genotype. Most geneticists karyotype without buccal smear due to smear interpretation problems.

    Down’s syndrome. Down’s syndrome is a relatively frequent disorder associated with two different chromosome abnormalities. Most patients (about 92%) have an extra number 21 chromosome in the number 21-22 chromosome group (therefore having 3 chromosomes in this group instead of 2, a condition known as trisomy 21). These patients have a total of 47 chromosomes instead of 46. The chromosome abnormality has nothing to do with the sex chromosomes, which are normal. This type of Down’s syndrome apparently is spontaneous, not inherited (i.e., there is no family history of Down’s syndrome and there is very little risk the parents will produce another affected child). This nonfamilial (sporadic) type of Down’s syndrome occurs with increased frequency when the mother is over age 35. About 5% of patients have familial Down’s syndrome; the patient has an extra 21-type chromosome, but it is attached to one of the other chromosomes, most often in the 13-15 group (called the “D group” in some nomenclatures). This type of arrangement is called a “translocation.” The translocation attachment is most frequent on the number 14 chromosome, but it may attach elsewhere. The translocation abnormality can be inherited; it means that one parent has a normal total number of chromosomes, but one of the pair of number 21 chromosomes was attached to one of the number 14 chromosomes. The other number 21 and the other number 14 chromosome are normal. The two-chromosome (14 + 21) cluster behaves in meiosis as though it were a single number 14 chromosome. If the abnormal chromosome cluster is passed to a child, two situations could result: a child with clinical Down’s syndrome who received the translocated 14 + 21 chromosome plus the normal number 21 chromosome from one parent (and another number 21 chromosome from the other parent, making a total of three number 21 chromosomes), or a carrier who received the translocated 14 + 21 chromosome but did not receive the other (normal) number 21 chromosome from the same parent (the translocated 14 + 21 chromosome plus a number 21 chromosome from the other parent make a total of two number 21 chromosomes). The translocation Down’s syndrome patient has a total of 46 chromosomes (the two-chromosome unit counts as a single chromosome).

    Clinically, an infant or child with Down’s syndrome usually has some combination of the following: prominent epicanthal folds at the medial aspect of the eyes, flattened facies, flat bridge of the nose, slanted lateral aspect of the eyes, mental retardation or deficiency, broad hands and feet, and a single long transverse crease on the palm instead of several shorter transverse creases. Other frequent but still less common associated abnormalities are umbilical hernia, webbing of the toes, and certain types of congenital heart disease. There also is an increased incidence of acute leukemia.

    Diagnosis usually can be made clinically, but chromosome karyotyping is a valuable means of confirmation and of diagnosis in equivocal cases. It probably is advisable to do chromosome karyotyping in most children with Down’s syndrome, because the type of chromosome pattern gives an indication of the prognosis for future children.

    Prenatal diagnosis can be made in the first trimester by chorionic villus biopsy with chromosome analysis. Screening for Down’s syndrome can be done using maternal serum during the 16th to 18th gestation week. If the maternal alpha-fetoprotein serum level is lower than normal, the unconjugated estriol (E3) lower than normal, and the beta human chorionic gonadotropin (beta-hCG) higher than normal, this suggests possible Down’s syndrome. This would have to be confirmed with fetal cells obtained by amniocentesis (chorionic villus biopsy is not done after the 12th week of pregnancy).

    Fragile X chromosome. The fragile X chromosome refers to a narrowing in the X chromosome, at which point the chromosome breaks more easily than usual when cultured in a medium that is deficient in thymidine and folic acid. The syndrome is said to be second only to Down’s syndrome as a cause of hereditary mental retardation. The fragile X abnormality is reported to be associated with 30%-50% of cases of X-linked mental retardation as part of a syndrome which also includes certain mild facial changes. About 30%-35% of female carriers may have mild mental retardation, which is unusual for heterozygotic status in most genetic illnesses and very unusual for an X-linked inherited disorder (in which the carrier female seldom has clinical symptoms). In addition, about 20% of males with the chromosome defect are asymptomatic and not detectable by standard chromosome analysis. Male offspring of a (heterozygous) carrier female would have a 50% chance of developing the syndrome. Unfortunately, only about 30%-56% of heterozygotic females demonstrate the fragile X defect using current laboratory methods. Sensitivity of these methods is age dependent, and best detection rates occur testing women less than 30 year old. There have also been reports of some affected men with normal range IQ who would qualify as carriers. It has been estimated that as many as 20% of male offspring with normal IQS born to female carriers actually are themselves carriers. DNA probe methods are now available that can often detect fragile X presence when standard chromosome analysis is equivocal or negative.

    Adult polycystic kidney disease (PKD-1). This autosomal dominant condition is reported to be present in 1 of 1,000 live births. Multiple cysts form in the kidney and eventually enlarge, destroying nearby renal parenchyma and in many cases eventually resulting in renal failure. The genetic abnormality is located on chromosome 16. DNA probes are used that bracket the gene area (gene linkage analysis using restriction fragment length polymorphism).

    Other chromosomal disorders. A wide variety of syndromes, usually consisting of multiple congenital deformities and anomalies, are now found to be due to specific chromosomal abnormalities. The most common of these involve trisomy in the 13-15 (D) group and in the 16-18 (E) group. Some patients with repeated spontaneous abortions have abnormal karyotypes. Various tumors have yielded abnormal chromosome patterns, but no one type of tumor is associated with any consistent pattern (except for chronic myelogenous leukemia).

    Commonly accepted indications for buccal smear. These include the following:

    1. Ambiguous or abnormal genitalia
    2. Male or female infertility without other known cause
    3. Symptoms suggestive of Turner’s syndrome or Klinefelter’s syndrome, such as primary amenorrhea

    Indications for chromosome karyotyping

    These include the patients in the buccal smear groups just described for confirmation or initial diagnosis and the following:

    1. Down’s syndrome infants or possible carriers
    2. Mentally defective persons
    3. Persons with multiple congenital anomalies

  • Defects in Amino Acid metabolism (Aminoacidopathies)

    Primary (metabolic) aminoacidopathies

    Phenylketonuria (PKU). Classic PKU is inherited as an autosomal recessive trait. It is uncommon in Asians and African Americans and is due to deficiency of a liver enzyme known as “phenylalanine hydroxylase,” which is needed to convert the amino acid phenylalanine to tyrosine. With its major utilization pathway blocked, phenylalanine accumulates in the blood and leads to early onset of progressive mental deficiency. This disease is one of the more common causes of hereditary mental deficiency and one of the few whose bad effects can be prevented by early treatment of the infant. At birth the infant usually has normal serum levels of phenylalanine (<2 mg/100 ml) due to maternal enzyme activity, although some instances of mental damage in utero occur. In the neonatal period, after beginning a diet containing phenylalanine (e.g., milk), serum phenylalanine levels begin to rise. When the phenylalanine to tyrosine pathway is blocked, some phenylalanine metabolism is shunted to ordinarily little-used systems such as transamination to phenylpyruvic acid.

    Urine screening tests. When the serum phenylalanine level reaches 12-15 mg/100 ml, sufficient phenylpyruvic acid is produced that it begins to appear in the urine. This becomes detectable by urine screening tests (Phenistix or the older ferric chloride test) at some time between ages 3 and 6 weeks. Unfortunately, by then some degree of irreversible mental damage may have occurred. Therefore, it is highly desirable to make an earlier diagnosis to begin treatment as soon as possible after birth.

    Blood screening tests. The most widely used screening test for elevated serum phenylalanine levels is the Guthrie test. This is a bacterial inhibition procedure. A certain substance that competes with phenylalanine in Bacillus subtilis metabolism is incorporated into culture medium; this essentially provides a phenylalanine-deficient culture medium. Bacillus subtilis spores are seeded into this medium; but to produce significant bacterial growth, a quantity of phenylalanine equivalent to more than normal blood levels must be furnished. Next, a sample of the patient’s blood is added, and the presence of abnormal quantities of serum phenylalanine is reflected by bacterial growth in the area where the specimen was applied. The Guthrie test, if properly done, is adequately sensitive and accurate and reliably detects definitely abnormal levels of serum phenylalanine (і4 mg/100 ml). It also fulfills the requirements for an acceptable screening method. However, there are two controversial aspects to this test. First, the standard practice is to obtain a blood specimen from the infant (usually as a filter paper blood spot using a heel puncture) before discharge from the hospital. In some cases this may result in the specimen being obtained 48 hours or less after birth, with an even shorter duration of phenylalanine intake if milk feeding is not begun soon after birth. There is some controversy in the literature as to whether a significant percentage (about 5%-10%) of infants with PKU will be missed if the specimen is obtained in the first 48 hours of life. A few studies indicate that this is unlikely, but the number of patients was not large enough to conclusively establish this point. Some screening programs recommend second testing of infants discharged before 48 hours of life, and most authorities recommend retesting if the initial test specimen was obtained before 24 hours of life.

    Second, PKU is not the only cause of elevated serum phenylalanine levels in the neonate. In fact, more positive (abnormally elevated) Guthrie test results are reportedly caused by non-PKU etiologies than by PKU. A major etiology is temporary (“late enzyme development”) hypertyrosinemia associated with low birth weight, high-protein formulas, and vitamin C deficiency. Some infants with severe liver disease and some with galactosemia have been reported to have elevated serum phenylalanine levels. Since hyperphenylalaninemia is not diagnostic of PKU, and since a long-term low-phenylalanine diet could be harmful to some persons who do not have PKU, an abnormal Guthrie test result should be followed up by more detailed investigation, including, as a minimum, both the serum phenylalanine and tyrosine levels. The typical PKU patient has a serum phenylalanine level greater than 15 mg/100 ml, with a serum tyrosine level less than 5 mg/100 ml. The tests may have to be repeated in 1-2 weeks if values have not reached these levels. DNA probe diagnosis is also available for equivocal or problem cases or prebirth diagnosis.

    Phenylketonuria variants. About 10% of infants with apparent PKU have been found to have a PKU variant. The enzyme system for alteration of phenylalanine to tyrosine is actually a group of at least four enzymes and coenzymes. Deficiency in the hydroxylase enzyme produces classic PKU. Deficiency in one of the other components of the system produces variant PKU. In particular, the variant caused by deficiency of the cofactor tetrahydrobiopterin (estimated to account for 0.5%-3.0% of persistent hyperphenylalaninemias) requires therapy in addition to a phenylalanine-deficient diet. Some patients with other variants of PKU do not require a phenylalanine-free diet.

    Diagnosis of PKU variants was originally made with a tolerance test using oral phenylalanine (in milk or other materials). Persistence of elevated blood phenylalanine levels greater than 20 mg/100 ml for more than 72 hours was considered indicative of classic PKU, whereas a decrease below the 20-ml level before 72 hours was considered indicative of variant PKU. This association has been challenged by others, and sophisticated tests have been devised to determine the exact etiology of the different forms of persistent hyperphenylalaninemia. Some of these procedures are available only in pediatric research centers. It would seem reasonable to recommend that an abnormal Guthrie test result be immediately followed up with a blood specimen for assay of phenylalanine and tyrosine levels. Afterward, pending results, the infant should be placed on a low-phenylalanine diet. If both phenylalanine and tyrosine levels are high, the patient probably does not have PKU. If only the phenylalanine level is high, and especially if it is greater than 15 mg/100 ml, the infant should be referred to a specialized PKU center for additional studies while the low-phenylalanine diet is continued.

    Alkaptonuria (ochronosis). The typical manifestations of this uncommon disease are the triad of arthritis, black pigmentation of cartilage (ochronosis), and excretion of homogentisic acid in the urine. Arthritis usually begins in middle age and typically involves the spine and the large joints. Black pigmentation of cartilage is most apparent in the ears but may be noticed in cartilage elsewhere or may even appear in tendons. The intervertebral disks often become heavily calcified and thus provide a characteristic x-ray picture. The disease is caused by abnormal accumulation of homogentisic acid, an intermediate metabolic product of tyrosine; this, in turn, is caused by a deficiency of the liver enzyme homogentisic acid oxidase, which mediates the further breakdown of the acid. Most of the hemogentisic acid is excreted in the urine, but enough slowly accumulates in cartilage and surrounding tissues to cause the characteristic changes previously described. Diagnosis is established by demonstration of homogentisic acid in the urine. Addition of 10% sodium hydroxide turns the urine black or gray-black. A false positive urine glucose test result is produced by copper reduction methods such as Benedict’s test or Clinitest, whereas glucose oxidase dipstick methods are negative.

    Other primary aminoacidopathies. These are numerous, varied, and rare. They are mostly diagnosed by paper chromatography of urine (or serum), looking for abnormal quantities of the particular amino acid involved whose metabolic pathway has been blocked. The most widely known diseases (apart from PKU and alkaptonuria) are maple syrup disease and histidinemia. The most common is homocystinuria. Many of the primary aminoacidopathies can be diagnosed in the first trimester of pregnancy by means of chorionic villus biopsy.

    Secondary aminoacidopathies. Secondary aminoacidopathies are associated with a renal defect, usually of reabsorption, rather than a primary defect in the metabolic pathway of the amino acid in question. The serum levels are normal. The most common cause is a systemic disease such as Wilson’s disease, lead poisoning, or Fanconi’s syndrome. In such cases, several amino acids usually are found in the urine. Aminoaciduria may occur normally in the first week of life, especially in premature infants. A much smaller number of patients have a more specific amino acid renal defect with one or more specific amino acids excreted; the most common of these diseases is cystinuria. Patients with cystinuria develop cystine renal calculi. Cystine crystals may be identified in acidified urine, proving the diagnosis. Otherwise, combined urine and serum paper chromatography is the diagnostic method of choice.

  • Mucopolysaccharidoses (Disorders of Connective Tissue and Bone)

    The best known of this group are Hunter’s and Hurler’s syndromes. In these conditions there is inability to metabolize certain mucopolysaccharides, resulting in accumulation and storage of these substances in various body organs and tissues and excretion of some stored material in the urine.

    Hurler’s syndrome. Hurler’s syndrome is caused by deficiency of the enzyme alpha-L-iduronase. Affected infants appear normal for the first 6-8 months of life but then develop skeletal abnormalities (short stature, kyphosis, broad hands, saddle-shaped deformity of the bridge of the nose), clouding of the cornea leading to severe loss of vision, a tendency toward umbilical and inguinal hernias, hepatosplenomegaly, thick tongue, and mental retardation. Diagnosis of Hurler’s syndrome (or any of the mucopolysaccharide disorders) can be made by chromatographic identification of the mucopolysaccharide excreted in the urine. A more conclusive diagnosis can be established by tissue culture of skin biopsy fibroblasts with assay for the specific enzyme involved.

    Other connective tissue disorders. There is an important group of hereditary connective tissue disorders, including Marfan’s syndrome, Ehlers-Danlos syndrome, and osteogenesis imperfecta, for which no laboratory screening test or specific diagnostic biochemical test is available. Diagnosis is made by clinical criteria, in some cases supported by x-ray findings.

  • Lysosomal Storage Diseases

    Lysosomal storage diseases are the result of genetic deficiency in certain enzymes found in tissue cell cytoplasmic lysosomes. These enzymes help metabolize certain glycoproteins, glycolipids, and mucopolysaccharides. The substance normally altered by the deficient enzyme accumulates in the cell lysosome area, and a storage disease results. The nonmetabolized substance either is stored in the tissue cell cytoplasm or is taken up by phagocytes. Most of these conditions can be diagnosed by rather sophisticated assays for the enzyme that is deficient. In some instances the assay can be carried out on plasma or serum; in other cases, on peripheral blood white blood cells (WBCs); and for some enzymes, it is necessary to use patient fibroblasts obtained by skin biopsy and grown in tissue culture. In some cases the diagnosis can be made from fetal cells obtained by amniocentesis and grown in tissue culture. In most cases the assays are available only at university medical centers or specialized clinics. A few are available at certain large reference laboratories. It is recommended that a university medical center specializing in such problems or the Neurological Diseases branch of the National Institutes of Health be contacted for details on how to proceed with any patient suspected of having a lipid storage disease. It is highly preferable that the patient be sent directly to the center for biopsy or required specimen collection to avoid unnecessary and costly delays and to prevent damage to the specimen in transport.

    The lysosomal storage diseases can be divided into two major categories: the sphingolipidoses and the mucopolysaccharidoses. A short summary of these conditions is presented. Several are described below in more detail. Many of these disorders can be diagnosed in the first trimester of pregnancy by chorionic villus biopsy.

    Sphingolipidoses (disordered lipid metabolism) The best known of this group are glycolipid storage diseases, including ganglioside storage (Tay-Sachs disease and metachromatic leukodystrophy) and ceramide storage diseases (Gaucher’s disease and Niemann-Pick disease).

    Tay-Sachs disease. This condition is due to accumulation of the ganglioside GM2 in various tissues but most notably in the brain. The defective enzyme responsible is known as “hexaminidase.” There are two forms (similar to isoenzymes) of hexaminidase, commonly abbreviated as hex-A and hex-B. The ethnic groups most often affected by classic Tay-Sachs disease are Ashkenazic (Central or Eastern European) Jews and, to a lesser extent, other Jews and inhabitants of the Middle East country of Yemen. There are several other very similar disorders involving hexaminidase deficiency that are generally considered variants of Tay-Sachs disease and are not commoner in Jews. In the classic and commonest form of Tay-Sachs disease, the infant appears normal at birth and for the first 5-6 months but then fails to develop any further mentally, loses some motor ability, and develops a “cherry-red spot” on the macula of the eye. The disease proceeds to dementia, flaccid muscles followed by spastic muscles, blindness, and death by age 3 years. There are less common variants that proceed more swiftly or in a somewhat more prolonged fashion.

    Diagnosis. The classic form of Tay-Sachs disease is due to deficiency in hex-A enzyme. The hex-A enzyme can be assayed in serum; peripheral blood WBCs and patient fibroblasts can also be used. The diagnosis can be established in the fetus by amniocentesis. The hex-A assay can be used to detect carriers of the Tay-Sachs gene. The disease is transmitted as an autosomal recessive trait, so that if one parent is tested and has normal hex-A levels, the infant will not be homozygous. Therefore, the infant will not be clinically affected, even if the other parent has the gene. DNA probe diagnosis is available in addition to hex-A enzyme measurement. Screening for Tay-Sachs disease can be performed on the fetus in utero by chorionic villus biopsy in the first trimester.

    Gaucher’s disease. Gaucher’s disease is a disorder in which the glycolipid cerebroside compound kerasin is phagocytized by the reticuloendothelial system. There seem to be two subgroups of this disorder: a fatal disease of relatively short duration in infancy accompanied by mental retardation, and a more slowly progressive disease found in older children and young adults and not accompanied by mental retardation. Splenomegaly is the most characteristic finding, but the liver and occasionally the lymph nodes also may become enlarged. The most characteristic x-ray findings are aseptic necrosis of the femoral heads and widening of the femoral marrow cavities; although typical, these findings may be absent. Anemia is frequent, and there may be leukopenia and thrombocytopenia due to hypersplenism. The serum acid phosphatase level usually is elevated if the chemical method used is not reasonably specific for prostatic acid phosphatase. (There are several widely used chemical methods, and although none is completely specific for prostatic acid phosphatase, some are considerably more so than others.)

    Before the late 1970s, diagnosis was made by bone marrow aspiration. Wright-stained bone marrow smears frequently would contain characteristic Gaucher’s cells, which are large mononuclear phagocytes whose cytoplasm is filled with a peculiar linear or fibrillar material. Splenic aspiration or liver biopsy was also done in problematic cases. The diagnosis is now made by assay of peripheral blood leukocytes for beta-glucosidase, the enzyme whose deficiency is the cause of the disease. Skin biopsy with tissue culture of skin fibroblasts followed by beta-glucosidase assay is also possible.

    Niemann-Pick disease. Niemann-Pick disease is similar clinically and pathologically to the fatal early childhood form of Gaucher’s disease, except that the abnormal lipid involved is the phospholipid sphingomyelin. As in Gaucher’s disease, diagnosis used to be made by bone marrow aspiration, although the phagocytic cells are not as characteristic as those of Gaucher’s disease. Splenic biopsy with tissue lipid analysis was also done. The diagnosis is now made by skin biopsy with tissue culture of the fibroblasts and assay of the fibroblasts for sphingomyelinase, the enzyme whose deficiency is the cause of the disease.

  • Diseases of Carbohydrate Metabolism

    Galactosemia. Galactosemia results from congenital inability to metabolize galactose to glucose. The most common source of galactose is milk, which contains lactose. Lactose is converted to glucose and galactose in the gastrointestinal (GI) tract by the enzyme lactase. There are three forms of galactosemia, each with autosomal recessive inheritance, and each caused by an enzyme defect in the galactose-glucose metabolic pathway. This enzyme system is located primarily in RBCs. The classic and most common type of abnormality is found in 1 in 62,000 infants and is due to deficiency of the enzyme galactose-1-phosphate uridyltransferase (Gal-1-PUT); the defect is called transferase deficiency galactosemia (TD-galactosemia). Normally, galactose is metabolized to galactose-1-phosphate, and Gal-1-PUT mediates conversion to the next step in the sequence toward glucose-1-phosphate.

    TD-galactosemia is not clinically evident at birth, but symptoms commence within a few days after the infant starts a milk diet. Failure to thrive occurs in 50%-95% of patients. Vomiting (about 50% of cases), diarrhea (about 30%), or both occur in nearly 90% of patients. Evidence of liver dysfunction develops in about 90% of patients, consisting of hepatomegaly (about 70%), jaundice (about 60%), or both. Physiologic jaundice may seem to persist, or jaundice may develop later. Splenomegaly may appear in 10%-30% of patients. Individual signs and symptoms are sufficiently variable that a complete full-blown classic picture does not appear in a substantial number of affected infants. Cataracts develop in several weeks in about 50%, and mental retardation is a sequel in about one third of the patients. The disease is treatable with a lactose-free diet, if the diet is begun early enough.

    Laboratory abnormalities. Transferase deficiency galactosemia has multiple laboratory test abnormalities. Urinalysis typically reveals protein and galactose, although in one series only two thirds of patients demonstrated urine galactose. Urine galactose excretion depends on lactose ingestion and may be absent if the infant refuses milk or vomits persistently. Galactose in urine can be detected by a positive copper sulfate-reducing substance test (e.g., Clinitest) plus a negative test specific for glucose (such as glucose oxidase test strips). A nonglucose reducing substance must be identified by more specific tests, because lactose and various other sugars can produce the same reaction as galactose on the screening procedure. There is also abnormal amino acid urine excretion that can be detected by chromatography, although such information adds little to the diagnosis. Positive urine galactose test results do not mean that the infant has galactosemia, since occasionally normal newborns have transient galactosuria. However, urine screening is important, because detection of galactose enables a tentative diagnosis to be made and treatment started, pending results of confirmatory tests.

    Other nonspecific abnormalities in classic transferase-deficient galactosemia include hepatomegaly and jaundice, although jaundice may not be present. Liver function test results are variable, although the aspartate aminotransferase (AST, formerly serum glutamate oxaloacetate) and possibly alkaline phosphatase levels are frequently elevated. Liver function test results must be interpreted with knowledge that the reference ranges are different in the neonate than in adults. Liver biopsy has been used in certain problematic patients (mostly before Gal-1-PUT assays were available). The histologic changes are suggestive but not conclusive and consist of early fatty metamorphosis, with a type of cirrhosis pattern often developing after about 3 months of age.

    Diagnosis. Diagnosis of classic TD-galactosemia depends on assay of Gal-1-PUT in the RBCs of the infant. Several methods have been described, all of which are sufficiently difficult that the test is available only in university centers and a few large reference laboratories. The specimen presents certain problems. RBCs from anticoagulated whole blood are tested, so the specimen cannot be frozen. On the other hand, one report indicates that 25% of the enzyme activity is lost after 24 hours at either room temperature or refrigerator temperature. For this and other reasons, many physicians rely on screening tests for transferase enzyme deficiency and, after starting therapy, refer the patient to a specialized center for definitive diagnosis.

    The galactose tolerance test was once the most widely used method for confirmation of galactosemia. However, there is considerable danger of hypoglycemia and hypokalemia during the test, and it has been replaced by chromatography and RBC enzyme assay.

    Screening tests. Several screening tests are available. The most popular are a bacterial inhibition method (Paigen assay, roughly similar to the PKU Guthrie test) and the Beutler fluorometric method. The Paigen assay measures elevated blood galactose (or galactose-6-phosphate) levels, and the Beutler assay measures Gal-1-PUT activity. Both can be performed on filter paper blood spots. The Paigen test depends on elevated blood galactose levels, and therefore milk feeding is necessary. Specimens from patients not receiving milk or specimens drawn several hours after a milk feeding may yield false negative (normal) results. If the Paigen test is adapted to detect galactose-1-phosphate rather than galactose, length of time after feeding is not a problem, and even most cases in patients on a galactose-free diet are reportedly detected. The Paigen test (either type) detects galactokinase deficiency as well as transferase deficiency. The Beutler test does not depend on milk feeding. However, the test does not detect galactokinase deficiency and is more subject to effects of heat and humidity on the filter paper specimen. The Beutler test also can detect certain nonpathologic variants of transferase enzyme such as the Duarte variant.

    Variants. There are at least four variants of the transferase enzyme. The Duarte variant is the most frequent. Patients with this disorder exhibit about 50% of normal Gal-1-PUT activity on RBC assay and are clinically asymptomatic. In classic (homozygous) transferase deficiency there is almost no Gal-1-PUT activity on assay.

    Other forms of galactosemia. There are two other forms of galactosemia. One consists of deficiency in the enzyme galactokinase. Development of cataracts is the only symptom. The incidence of galactokinase deficiency has been variously reported as equal to that of transferase deficiency or less. The third type is deficiency of the enzyme erythrocyte epimerase, of which very few cases have been reported. These patients seem to be completely asymptomatic.

    Disaccharide malabsorption. Certain enzymes are present in the lumen border of small intestinal mucosal cells that aid in absorption of various complex sugars by preliminary hydrolyzation. Deficiency of one or more of these enzymes may impair absorption of the affected sugar, depending on the degree of enzyme deficiency. The most common deficiency affects the disaccharide sugar lactose. Lactose is present in milk or milk products such as ice cream, yogurt, and many types of cheese. Northern Europeans as a group usually have normal intestinal lactase throughout life and only about 10%-15% develop lactose intolerance. Many other ethnic populations have a high incidence of lactase deficiency. The highest incidences are reported in Asians (such as Chinese and Japanese) and Native Americans (over 90% are said to develop lactose intolerance). Eastern European (Ashkenazic) Jews, African Americans, and persons of Mediterranean or South American ancestry have a lesser but still high rate of deficiency (60%-70% incidence). Besides primary (genetic) lactase deficiency, secondary deficiency may be induced, more or less temporarily, by certain diseases affecting the small intestine. These include primary small intestine mucosal disease (sprue), short bowel syndrome, severe acute gastroenteritis, prolonged protein-calorie malnutrition, and certain antibiotics (e.g., neomycin and kanamycin). Lactase deficiency may also occur due to prematurity. Between 26 and 34 weeks of gestation there is only about one third of full-term lactase activity present. This increases to about 70% between 35 and 38 weeks. Full activity levels are attained by birth at 40 weeks.

    Persons who inherit the trait for lactase deficiency usually have normal lactase activity levels at (full-term) birth. However, at about age 3-5 years there is a fall in lactase activity. After this time there is some individual variation in degree of clinical tolerance to milk, even when intestinal lactase activity measurement is low.

    Symptoms of lactose intolerance include some combination of abdominal cramps, diarrhea, bloating, and flatulence, usually occurring at or becoming worse after meals. One study involving children from age 4 years to the teen-age years who had intermittent abdominal pain found their symptoms could be explained by lactose malabsorption in a high proportion of those from ethnic groups with a high incidence of lactase deficiency. Lactase deficiency in full-term newborns or young children is thought to be rare. Milk allergy may produce similar symptoms but is uncommon, usually includes allergy symptoms such as asthma with any GI symptoms, and the parents usually have a history of allergy.

    Screening tests for lactase deficiency include testing of stool for pH and sugar at a time when the patient is symptomatic. Normal stool pH is 7.0-8.0. A stool pH below 6.0 raises the question of lactase deficiency. The stool can be tested for sugar by either a reducing substance method or a glucose oxidase paper strip method. The presence of glucose in the stool suggests lactase deficiency. However, in one study, parenteral antibiotics administered to neonates caused an increase in fecal reducing substances beginning within 48 hours after starting the antibiotics. Negative test results for pH and sugar do not exclude the diagnosis, and positive test results do not conclusively establish the diagnosis. Acidic stool pH can be found in certain other conditions associated with diarrhea, especially steatorrhea.

    Diagnostic tests for lactase deficiency include the lactose tolerance test, hydrogen breath test, and small intestine biopsy with tissue assay for lactase. The lactose tolerance test is performed in a similar manner to the oral glucose tolerance test. After overnight fasting, 50 gm of oral lactose (in children, 1-2 gm of lactose/kg of body weight) is given in some type of flavored liquid. Serum glucose levels are assayed before lactose administration and 15, 30, 60, and 90 minutes afterward (some investigators use different time intervals; some beginning at 30 minutes instead of 15 and some ending at 120 minutes instead of 90 minutes). Normal lactase activity results in a postdose glucose elevation more than 20 mg/100 ml (1.1 mmol/L) over baseline. This assumes that malabsorption from some other cause is excluded. Some investigators measure galactose instead of glucose; 150 mg of ethanol/kg of body weight is administered with the lactose dose to inhibit liver conversion of galactose to glucose. A single blood or urine sample is obtained 40 minutes after the test dose and is assayed for galactose. The advantage of this procedure is need for only one test specimen. However, this method has not been evaluated as thoroughly as the standard lactose tolerance procedure. The hydrogen breath test is currently considered the most accurate of the tolerance tests. Briefly, it consists of analysis of expiratory breath for hydrogen, followed by administration of oral lactose and retesting of breath samples at either 30- or 60-minute intervals for 2-4 hours. Lactase deficiency results in deposition of excess lactose into the colon, where bacterial fermentation produces excess hydrogen, which is excreted through the lungs. The hydrogen breath test can be performed only by specialized medical centers. Small intestine biopsy with quantitation of tissue levels of intestinal lactase is performed during an endoscopy procedure. Tissue biopsy has the added advantage that some of the secondary causes of lactase deficiency (e.g., sprue) can be detected. However, intestinal lactase measurement is available only at specialized medical centers.

    Lactosuria. Some patients with lactase deficiency absorb lactose from the GI tract after oral intake of lactose and excrete the lactose in the urine. However, other lactase-deficient persons do not exhibit lactosuria. Premature infants are said to be predisposed to temporary lactosuria (presumably due to their relatively lactase-deficient state). Lactosuria is apparently not uncommon in the last trimester of pregnancy and for several days following delivery.

    Sucrase enzyme deficiency. The disaccharide sugar sucrose is commonly used as a carbohydrate supplement. Sucrose is hydrolyzed by the enzyme sucrase in small intestine mucosa cells. Congenital sucrase deficiency has been reported but is not common. Most cases became clinically manifest within the first year of life, with symptoms predominantly of failure to thrive, diarrhea, or both. Stool pH is usually acidic. Reducing substance sugar test methods are not accurate for stool testing, since sucrose is not a biochemical reducing substance. Fructose may also be malabsorbed by some persons. Diagnosis is most often made by the hydrogen breath test. Definitive diagnosis usually requires small intestine biopsy with tissue assay for sucrase (or fructose) activity.

    Glycogen storage disease. Glycogen storage disease includes a spectrum of syndromes resulting from defective synthesis or use of glycogen. Clinical manifestations depend on the organ or tissue primarily affected and the specific enzyme involved. The disease in one or another of its clinical syndromes may affect the liver, heart, or skeletal muscle. The most common is von Gierke’s disease, whose clinical manifestations primarily involve the liver. Classic cases usually have elevated levels of triglyceride, cholesterol, and uric acid. Some have hypoglycemia. Patients typically have a diabetic type of oral glucose tolerance curve.

    Diagnosis. Diagnosis of the various forms of glycogen storage disease requires biopsy of liver or muscle (depending on the particular disease variant) for glycogen and enzyme analysis. In some cases, enzyme assay can be performed on other tissues. These are specialized tests, and it is best to contact a pediatric research center rather than expose the patient to inappropriate or incomplete diagnostic procedures.

  • Diagnostic Methods

    Various diagnostic modalities are available. Some genetic disorders involve chromosomes. Many chromosomal abnormalities are autosomal (not involving the sex chromosome); others are sex-linked (inherited through the sex chromosomes). In some cases there may be total or partial deletion of a chromosome, presence of an extra chromosome attached to a chromosome pair (trisomy), or translocation, when a portion of a chromosome breaks off and attaches to another chromosome. These disorders require chromosome analysis. Other genetic disorders are due to a defective or missing enzyme and are diagnosed by measurement of the enzyme responsible. Sometimes it is necessary to demonstrate that a normal quantity of enzyme does not produce its expected degree of activity. Finally, genetic abnormalities may be due to a gene defect not shown by standard chromosome analysis. In some of these cases, diagnosis can be made by methods using nucleic acid probes. This technique is described in Chapter 14. Briefly, a specific amino acid sequence is obtained from ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) using an enzyme known as “restriction endonuclease.” This probe fragment is spliced into a DNA strand (recombinant DNA) using an enzyme called “reverse transcriptase.” The probe is then used to identify patient DNA that contains the same amino acid sequence as the probe. It is also possible to construct a probe for certain gene deletions (chromosome regions where part of a gene area has been deleted) and for certain mutations (if the mutation has eliminated a restriction endonuclease cleavage site). In some cases the gene can be detected directly. This is generally the most sensitive and accurate technique. When this cannot be done, a technique known as gene linkage analysis using restriction fragment length polymorphism can be used. This refers to abnormal length of a DNA fragment produced by an enzyme (restriction enzyme) that cuts the DNA chain at a specific location near to the gene in question (an area closely linked to the gene location). This technique is said to be 94%-99% accurate.

    Genetic diagnosis has been extended to the fetus by means of amniotic cells obtained through amniocentesis. This is usually carried out at 16-18 weeks’ gestation. It is also possible to obtain a small sample of chorionic villi from the fetal side of placenta at 9-12 weeks’ gestation (before the amniotic sac completely fills the uterine cavity) using suction from a catheter introduced through the cervix into the uterine cavity and then into the placenta with the guidance of ultrasound. There appears to be about a 2%-4% (range, 1%-6.4%) incidence of fetal death after the procedure. An impressive and continually increasing number of conditions can be diagnosed prenatally.

    Neonatal genetic diagnosis is being increasingly mandated by state governments. Currently, for example, the state of Illinois requires neonatal screening for phenylketonuria (PKU), galactosemia, biotinidase deficiency, sickle cell hemoglobinopathy, congenital adrenal hyperplasia, and hypothyroidism. All of these disorders can be diagnosed using heelstick blood spotted on filter paper.

  • Miscellaneous Cancer Tests

    Serum lactic dehydrogenase. Serum LDH levels are sometimes elevated in extensive carcinomatosis, often without any obvious reason. This is especially true in lymphoma, where it may be abnormal in up to 50% cases. However, LDH levels can be elevated in many conditions, which considerably lessens its usefulness in cancer diagnosis.

    Carcinoma antigen 19-9. CA 19-9 is a carbohydrate antigen segment of a glycoprotein that appears to be a sialylated derivative of the Lewis A blood group. It is detected by monoclonal antibody immunoassay and is reasonably (but not highly) specific for GI tract origin (Table 33-14).

    CA 19-9 in various conditions

    Table 33-14 CA 19-9 in various conditions

    Currently, although there is considerable research activity, CA 19-9 assay is not being widely used. Although it has reasonably good sensitivity in pancreatic carcinoma, it does not detect these tumors early enough to improve their current dismal prognosis, and it is not specific for pancreatic neoplasms. It might be useful in the workup of patients when pancreatic or gastric carcinoma is a possibility and more informative diagnostic procedures (e.g., ultrasound or CT) are not available or do not provide a definite answer. A definitely elevated CA 19-9 level result would favor carcinoma of GI or GI accessory organs. The specimen has to be sent to a reference laboratory in most cases, and the results would not be available for several days. The CA 19-9 assay could be used to follow pancreatic carcinomas after surgical resection to detect recurrence, but the question then arises as to what could be done if it does recur. Finally, CA 19-9 has been used in addition to CEA in order to detect recurrence of colon cancer. In some cases, CA 19-9 values become elevated before CEA, and the combination of the two tests is said to be more reliable than either test alone. However, at present CA 19-9 is not widely used for this purpose. As noted in Table 33-14, CA 19-9 may be elevated in some persons who do not have cancer. Also, 5%–10% of the population is Lewis A (blood group) negative and will not react with CA 19-9, even if they have cancer.

    Centocor CA 15-3 and Hybritech CA 549. CA 15-3 detects membrane antigens against human milk globules. It is said to be elevated in 57% of preoperative breast cancer patients, 75% (68%–80%) of patients with breast cancer with metastases, 3%–9% of patients with benign breast tumors, 4.5%–10.5% of patients with various nonmalignant conditions, and 1% of clinically normal persons. It is said to correlate with the clinical course of about 75% of patients with metastatic breast cancer, more frequently correlating with tumor progression than tumor regression. CA 549 detects any antigen present both in milk fat globule membranes and also in tumor cytoplasm. CA 549 is reported to be abnormal in 53%–90% of patients with metastatic breast cancer and 1%–13% of patients with benign breast disease. Certain other metastatic tumors are also detected.

    Flow cytometry test for bladder cancer. One study reports that standard urine cytology detected 58% of high-grade bladder carcinomas and 33% of low-grade bladder carcinomas. Flow cytometry methods detected 76% of low-grade tumors and 100% of high-grade tumors. However, one study suggests that the technique is not reliable when intravesical chemotherapy is given (which is also the problem with standard urine cytology).

  • Multiple Endocrine Neoplasia (MEN) Syndromes

    These syndromes have been mentioned in the discussion of certain tumors that may be associated with MEN. These syndromes are familial, with types I and II being inherited as an autosomal dominant disorder. About one half of type III cases are sporadic. Some cases of incomplete or overlapping organ tumor patterns have been reported.

    Although carcinoid tumors are not considered part of the MEN syndromes, carcinoid of foregut origin (bronchial and gastric) may be associated with MEN I. Gastrinomas of the Z-E syndrome may be associated with MEN I, which includes pancreatic islet cell tumors (these tumors may produce insulin, gastrin, or vasoactive intestinal polypeptide). About 5%–10% of pheochromocytomas are associated with MEN types II and III.

    Many patients with the MEN syndromes do not have all the tumor types considered part of the syndromes.

  • Effusions and Tests for Cancer

    In general, when an effusion occurs, the problem is differentiation among neoplastic, infectious, and fluid leakage etiologies. Effusions due to neoplasms or infection are frequently termed exudates and those due to hydrostatic leakage from vessels are called transudates. Several criteria have been proposed to separate transudates and exudates and to differentiate among the three major diagnostic categories. Most work has been done on pleural fluids. The significance of tests performed on pleural fluid may not be the same if the tests are performed on ascitic fluid.

    Etiology. The two most common causes of pleural effusions are congestive heart failure and neoplasm. Infection (tuberculosis or pneumonia) is the third most frequent etiology. In some cases it is necessary to establish the diagnosis of chylous effusion. Chylous effusions usually have a triglyceride content of 110 mg/100 ml (1.2 mmol/L) or greater and are usually more than twice the serum triglyceride value. Centrifugation does not clear a supernate area, and it may be possible to demonstrate fat droplets with a fat stain such as Sudan III. Another problem that occasionally arises is to differentiate urine from effusion fluid. Urine almost always has a creatinine concentration twice that of serum or more, whereas effusion fluid usually has the same creatinine concentration as the patient’s serum or at least is not elevated as much as twice the serum level. Rarely, recurrent fluid in or draining from the nose or ear has to be differentiated between cerebrospinal fluid (CSF) leakage from the central nervous system (CNS) subarachnoid space versus a serum transudate or local mucosa secretion. The usual diagnostic test is injection of a radioisotope into the CSF and subsequent analysis of a specimen of the draining fluid. Some other tests may be the ratio between serum and CSF total protein (which is usually more than 100), serum albumin/CSF albumin ratio (which is usually over 200), and serum prealbumin/ CSF prealbumin ratio (which is usually over 14).

    Specific gravity. Exudates typically have a specific gravity of 1.016 or more and transudates less than 1.015. One study found about 25% error in misclassification of either transudates or exudates.

    Protein content. Pleural fluid total protein levels higher than 3 gm/100 ml (30 gm/L) are characteristic of exudates. Transudates have total protein content of less than 3 and usually less than 2 gm. Two studies found that 8% of exudates and 11%–15% of transudates would be misdiagnosed if 3 gm/100 ml were used as the dividing line. Most exudates that were misdiagnosed as transudates were neoplastic. A pleural fluid/serum protein ratio of 0.5 may be a slightly better dividing line; exudates usually have a ratio greater than 0.5. With this criterion, accuracy in identifying transudates improved, but 10% of the exudates, mostly of malignant origin, were incorrectly classified as transudates. Pulmonary infarct, rheumatoid-collagen diseases, acute pancreatitis, cirrhosis with high-protein ascites (12%–19% of cases), and other conditions may produce effusions with protein content compatible with exudates.

    Several investigators report that the albumin gradient between serum and ascitic fluid differentiates between transudate or exudate nature of ascites better than total protein content. In one study, total protein ascitic values produced 64% overlap between etiologies of the exudate and transudate groups, whereas the serum albumin-ascitic fluid albumin gradient (SA-AFAG) produced 38% overlap. Another study produced only 7% overlap. The SA-AFAG consists of subtracting the ascitic albumin value from the serum albumin value. A SA-AFAG value of 1.1 gm/100 ml (11 gm/L) or more suggests a transudate, usually caused by portal hypertension due to cirrhosis. A SA-AFAG value less than 1.1 gm suggests an exudate but will not differentiate malignancy from infection or inflammation and occasionally may occur in nonmalignant, nonalcoholic cirrhosis. Another problem may arise when two conditions coexist such as liver metastases in a patient with cirrhotic ascites.

    Patients with ascites due to cirrhosis develop bacterial infection of the ascitic fluid without known cause (“spontaneous bacterial peritonitis”) in about 15% of cases (range, 4%–20%). Spontaneous ascitic infection typically has an ascitic total protein less than 1.0 gm/100 ml (10 gm/L). Other types of ascitic fluid infection (“secondary peritonitis”) usually have an ascitic fluid total protein level greater than 1.0 gm/100 ml, ascitic fluid glucose less than 50mg/100 ml (2.78 mmol/L), and more than one organism obtained by culture. Gram stains of ascitic fluid are said to be positive in only 10% of spontaneous peritonitis, but more frequently in peritoneal fluid due to intestinal perforation.

    Effusion lactic dehydrogenase. A pleural fluid to serum lactic dehydrogenase (LDH) ratio greater than 0.6 is reported to be typical of exudates. One study found that most transudates were correctly identified but that nearly 30% of exudates were misclassified.

    Combinations of criteria. The more criteria that favor one category as opposed to the other, the more accurate the results become. One study found that the combination of pleural fluid/serum protein ratio and pleural fluid/serum LDH ratio correctly identified most transudates and exudates.

    pH. An effusion fluid pH higher than 7.40 usually is associated with a transudate, whereas a pH of less than 7.40 is more likely to be an exudate caused by infection, inflammation, or tumor.

    Glucose. A pleural fluid glucose level more than 10 mg/100 ml below lower limits of normal for serum, especially when the actual pleural fluid value is less than 20 mg/100 ml, is reported to be suggestive of neoplasm or infection. Possibly 15%–20% of malignant effusions have decreased glucose levels. Patient hypoglycemia, rheumatoid arthritis, and infection are other etiologies.

    Cell count and differential. In ascites, a total WBC count of 250 mm 3 or more strongly suggests infection, especially when neutrophils exceed 50% of total WBCs (some use 500 WBCs as the cutoff point). In any body fluid, presence of many segmented granulocytes suggests infection (empyema); many mononuclear cells raise the question of lymphoid malignancy, carcinoma, or tuberculosis. However, several investigators state that sufficient exceptions occur to severely limit the usefulness of differential counts in the diagnosis of individual patients. One study reported that peripheral blood WBCs did not affect ascitic fluid WBC counts.

    Culture. Culture is frequently performed for tuberculosis, fungus, and ordinary bacteria. Pleural fluid culture for tuberculosis is said to be positive only in approximately 25% of known cases of tuberculosis effusion. Some believe that tuberculosis culture should be limited to high-risk patients or patients who have a positive skin test result. Whereas tuberculosis is an important cause of idiopathic pleural effusion, although less common in the United States than in the past, fungus is an uncommon cause of pulmonary infection except in patient groups with compromised immunologic defenses. Studies have shown about 85% sensitivity of culture in ascitic infection using blood culture bottles inoculated at the time of paracentesis versus only 50% sensitivity when ascitic fluid is streaked on agar plates or inoculated onto broth media in the laboratory.

    Cytology. About 30%–40% (literature range, 25%–52%) of all pleural effusions are associated with neoplasms. About 35%–40% are caused by lung carcinoma (most often adenocarcinoma), and about 20%–25% are due to breast carcinoma, with lymphoma or leukemia, ovary, or unknown primary in third place. Cytologic study is reported to detect tumor cells in about 50%–65% (literature range, 30%–98%) of patients with malignant pleural effusions. One problem that sometimes occurs is poor cytologic preparations due to blood in the pleural fluid. We have obtained better results by using cytologic spray fixative when the cytologic slides are prepared rather than fixing the slides by the usual technique of dipping them in alcohol.

    Pleural effusion carcinoembryonic antigen (CEA) CEA is discussed in detail elsewhere. Pleural fluid CEA levels may be elevated in various malignant and some benign conditions. When a cutoff level approximately 4 times the upper reference limit (corresponding to 10 ng/ml with the Hansen technique, whose upper normal limit is 2.5 ng/ml) is used, most elevations due to nonmalignant cause are eliminated (< 5% false positive results; literature range, 1%–12%). About 35%–50% of malignancies are detected (25%–89%). Therefore, CEA by itself is less sensitive than cytology. Addition of CEA to cytology (using a CEA cutoff value sufficient to exclude benign disease) improves detection of malignancy about 10%–15% over cytology alone. Carcinoembryonic antigen assay can also be used for ascitic fluid, with similar results.

    Tests for cancer-related ascites Among many tests proposed to detect malignancy causing ascites or accumulation of peritoneal fluid are the serum albumin-ascitic albumin gradient (SAAAG), ascitic fluid cholesterol, ascitic fluid fibronectin, cytology, CEA, flow cytometry (FCM), CA 125, and the monoclonal antibody immunohistochemical stains. In general, SAAAG less than 1.1 appears to be the best single overall relatively simple test, with sensitivity in detecting malignancy about 93% (range, 85%–100%) and accuracy of about 95% (range, 93%–97%). The main drawback is inability to detect those cases of ascites due to liver metastases or hepatocellular carcinoma without peritoneal implants (since the intrahepatic malignant cells are infrequently in direct contact with ascitic fluid) or differentiate these cases from ascities due to cirrhosis. Ascitic fluid cholesterol greater than 45 in two reports had 90%–100% sensitivity, but not enough studies are available, and patients with cardiac or pancreatic-origin ascities may in some cases have elevated ascitic cholesterol. Fibronectin had sensitivity of about 90% (range, 86%–93%) in three studies, but specimens usually would have to be sent to a reference laboratory. Serum CEA has been discussed earlier. Ascitic fluid CEA has a reported sensitivity of about 50% (range, 36%–80%). Cytology of ascitic fluid has sensitivity of about 60% (range, 40%–70%). Adding CEA assay to cytology increases cytologic sensitivity about 10%–20%. FCM estimates the amount of nucleic acid in cell nuclei; in general, an abnormal quantity of nucleic acid (aneuploidy) suggests malignancy. In one study, FCM aneuploidy increased the number of patients found to have malignancy by 39% over results of cytology alone. However, not all aneuploid cells are malignant, and not all malignant cells are aneuploid . Therefore, flow cytometry has been reported to produce about 30% (range, 0%–43%) false negative results and some false positive results. CA 125 assay in serum is discussed earlier in this chapter. It has much less often been applied to ascitic fluid. In a few reports, CA 125 has been reported to increase detection of ovarian carcinoma (and occasionally, uterine or fallopian tube carcinoma) over detection rates from cytology with or without CEA. Disadvantages of ascitic fluid CA 125 assay is frequent elevation of the antigen in ascities due to cirrhosis and to some extent with endometriosis. Monoclonal antibody stains against various tumor antigens have been applied to cell blocks or smears or by FCM in body cavity fluid specimens. The most useful antibodies in peritoneal fluid appear to be CA 125 and B72.3 for ovarian carcinoma, and EMA and CEA for adenocarcinoma in general. In one representative study, peritoneal washings from patients with stage I and II ovarian carcinoma were positive by cytology in 41% of patients and by immunohistology in 56%. In stage III and IV ovarian carcinoma, immunohistology also added an additional 14% positive patients to results from cytology.

    Peritoneal lavage for traumatic injury. Although this subject does not involve cancer, it does fit with discussions on tests for effusions, and thus it is included here. The standard criteria leading to high expectation of intraabdominal bleeding are one or more of the following: aspiration of gross blood (the quantity required is not uniform, but at least 10 ml or 20 ml are most often mentioned), fluid with an RBC count greater than 100,000/mm 3, or a WBC count greater than 500/mm 3. Other criteria that have been proposed but that are not widely accepted are abdominal fluid bilirubin or creatinine values higher than serum values or elevated effusion amylase. In most series the standard criteria detect significant intraabdominal bleeding in about 90% of cases and falsely suggest significant bleeding in about 10%–15% of cases (some of these patients may have bleeding that retrospectively is not considered sufficient to warrant laparotomy). CT scanning has proved extremely useful in trauma patients, with a sensitivity equal to that of lavage and a false positive rate significantly less than that of lavage. In addition, CT can often demonstrate what organs are affected.

    General considerations. Three anticoagulated tubes of effusion fluid should be sent to the laboratory, one tube containing ethylenediamine tetraacetic acid (EDTA) anticoagulant, one tube containing 0.05% sodium polyanetholesulfonate (SPS; Liquoid), and the third containing heparin. The EDTA tube is used for cell count and differential, the SPS tube for culture, and the heparinized tube for cytology. Without anticoagulant there may be sufficient protein in the specimen to induce spontaneous clotting, which can trap WBCs and bacteria and produce erroneous cell counts and falsely negative cultures. Some use of the heparinized tube both for culture and for cytology, but too much heparin may inhibit bacterial growth. Nonanticoagulated effusion fluid should also be sent to perform biochemical tests. As noted previously, when the effusion is ascites it is better to inoculate blood culture bottles when the ascitic fluid is obtained rather than to perform routine culture methods.