Human chorionic gonadotropin (HCG) is a two-chain glycoprotein hormone (MW -37,000) normally found in blood and urine only during pregnancy. It is secreted by placental tissue, beginning with the primitive trophoblast, almost before and / or from the time of implantation, and serves to support the corpus luteum during the early weeks of pregnancy. Circulating – HCG typically reaches levels of approximately 2,000 mIU/mL one month after conception. A peak level on the order of 100,000 mIU/mL is attained in the third month, after which a gradual decline sets in. Following delivery, the HCG level normally undergoes rapid descent, reaching nonpregnant concentrations (usually less than 5 mIU/mL) some two weeks later. Ectopic pregnancies and pregnancies terminating in spontaneous abortion tend to have lower than normal circulating HCG levels, while somewhat higher levels are often seen in multiple pregnancies. Many publications describe the value of detecting multiple HCG-related molecules in abnormal pregnancies, aneuploidy (trisomy-21 pregnancies), spontaneous and threatened abortions, preeclampsia, cancers and trophoblastic diseases.

Choriocarcinomas, one of the gestational trophoblastic diseases, arise mostly in the uterus from hydatidiform mole, following abortion, or during normal pregnancy. They can also occur in the testis. Although choriocarcinomas represent less than 1 percent of gynecological malignancies, they are important to recognize: early treatment of this life- threatening cancer results in a high cure rate. Metastases are commonly found at diagnosis and are taken into account in disease staging. HCG and especially the free-β-HCG subunit can aid in the diagnosis and monitoring of choriocarcinomas.

In testicular tumors, measurement of HCG in conjunction with α-fetoprotein  is used as an aid in determining tumor type (seminoma, nonseminoma), prognosis and therapy. These tumor markers are essential monitoring tools; failure of either marker to return to normal after therapy indicates residual tumor. In certain testicular tumors, the ratio of free-β-HCG subunit to HCG can be quite high; occasional tumors secrete only the free-β subunit and virtually no detectable intact HCG.

Hepatitis-B virus (HBV) is the sole human pathogen in the family of hepatitis-associated DNA viruses, and is found world-wide. Distribution of HBV infection will vary among geographical areas and population groups. Transmission of the virus is due to parenteral contact, through the exchange of blood or blood products, sexual contact, and perinatal spread from mother to newborn. Clinical manifestations range from mild asymptomatic infections to severe fulminant hepatitis. Over 90% of infected adults will have an acute self- limiting infection, with jaundice and abnormal liver function. Recovery occurs without any chronic sequelae. Chronic liver disease, a condition in which infection persists for more than six months, a known sequel of a hepatitis-B infection, is usually progressive. The risk of developing the chronic carrier state is more likely to follow infection acquired in childhood than as an adult. In chronic HBV carriers, there is no evidence of continued hepatic damage, however, the infection persists and the carrier maintains the ability to transmit the virus.

Availability of recombinant HBV vaccines, and the recommendation of universal immunization for infants and other high-risk persons has aided in the prevention of HBV infections. In addition, treatment with alpha-interferon to relieve symptoms is available. Results have shown positive response to treatment in 40-50% of selected individuals with chronic active hepatitis-B.

Classification of a hepatitis B infection requires the identification of several serological markers expressed during three phases (incubation, acute and convalescent) of the infection. The first marker to appear in serum is hepatitis B surface antigen (HBsAg). Presence of this antigen indicates an ongoing infection with HBV and is detectable in the acutely ill and in chronic carriers.

The C gene of the HBV viral genome can express two distinct proteins 1) core protein (HBc Ag) which forms the nucleocapsid 2 e non-particulate protein (HBe Ag). HBe Ag can be detected in the serum of patients  with hepatitis B wild-type virus during active viral replication . The function of this antigen in the viral replication cycle is not clearly defined. It is not indispensable to the virus, however this antigen is a major immunological target in the clearance of the virus. Generally, HBe Ag can be detected early in an acute HBV infection.It coincides with or follows the appearance of HBs Ag. In acute cases which evolve to recovery, HBe Ag disappears after several weeks and seroconversion to anti-HBe usually follows. For chronic HBV cases, HBe Ag can persist from several months to as long as several years, indicating on-going viral replication. The presence of HBe Ag in serum indicates active replication of HBV and people with HBe Ag- positive results are considered highly infectious for hepatitis B.

HBe Ag is used to monitor chronic hepatitis and anti- viral therapy. The objective of anti-viral therapy is to prevent the progression to liver cirrhosis. HBe Ag seroconversion to anti-Hbe  is generally considered as an indicator of transition to a state of viral latency accompanied by normalization of aminotransferase levels. HBe Ag seroconversion to Anti-HBe reduces the risk of developing cirrhosis and decompensated liver disease .

A positive result for anti-HBe in patients recovering from acute hepatitis indicates normal recovery, particularly if HBsAg and HBe Ag are no longer detectable. In an HBV carrier, a positive anti-HBeV result usually indicates inactivity of the virus and low infectivity of the patient. However a positive anti-HBe result in the presence of a positive HBV-DNA test result, as found in HBV mutants, can indicate active viral replication and progression of liver disease in a carrier. HBV mutants unable to secrete HBe Ag, can prevail over wild-type HBVs in patients with severe acute and chronic hepatitis B and in chronic HBs Ag carriers at the time of HBe Ag/anti-HBe seroconversion. These mutants are positively selected by the anti-viral immunoreaction.

Because this mutation affects only a portion of the viral population, serum HBe Ag and anti-HBe antibodies can be simultaneously detected during a period which varies depending on the individual.

Perinatal transmission of hepatitis B virus is frequent in areas with a high endemicity of the infection or in countries where screening of pregnant women is not undertaken regularly. About 90% of HBe Ag positive mothers transmit HBV to their infants and 90% become chronic HBs Ag carriers.

The hepatitis B virus causes acute, and occasionally fulminant viral hepatitis, but also chronic hepatitis which may result in cirrhosis and cancer of the liver, proving fatal for over 2 million carriers every year. In a study completed in 1990 , more than 2000 million people worldwide have been infected, including approximately 280 million chronic carriers. Acute hepatits B virus carriers are usualiy asymptomatic. Infection will lead to jaundice in approximate 10 % of infected subjects. One percent of icteric patients will develop fulminant hepatitis, which has an 80 % mortality rate . Chronic infection may lead to cirrhosis in 15 to 30 % of patients, which can be further complicated by hepatocellular carcinoma in 30 to 40 % of patients.

The normal course of chronic infection comprises three phases :

– an active replication phase, which can last for several years, during which viral replication is high, but the destruction of hepatocytes is moderate,

– an inactivation phase, during which viral replication is slow, but the destruction of hepatocytes increases, due to the HBc antigen present on the cell surface, and the strong immune response,

– an inactive phase, during which HBc Ag is no longer synthesized and viral replication no longer occurs. One or several phases of reactivation are possible.

The hepatitis B virus has a very limited cytopathogenic effect. Hepatocellular necrosis is caused by the immunocellular response to HBc Ag on the surface of the hepatocytes.

Diagnosis of hepatitis B relies on the detection of HBs surface antigen and IgM anti-HBc. The level of infectivity, and infection evolution will be assessed by other markers; they will also enable a discrimination between patients that need to be screened. The HBs antigen will either disappear in case of a positive evolution after 2 or 6 months or remain in chronic carriers.

In the case of fulminant hepatitis, HBs antigen can disappear at an early stage. Determination of anti-HBcIgM is then the only indication of HBV infection. The ultra sensitive quantitative tests enable patients to be monitored from the time HBs Ag appear up to the appearance of anti-HBs Ab, by titering the anti-HBc IgM, and also a distinction to be made between healthy carriers and those with active chronic hepatitis. In cases of chronic hepatitis, anti-HBc IgM is a very sensitive indirect marker of viral replication, but also of the host response to HBc Ag in the liver. As with serum HBe Ag and HBV DNA, a positive result for anti-HBc IgM is of considerable assistance in the diagnosis of chronic hepatitis, but a negative result does not necessarily exclude this possibility. The diagnosis should therefore be made using both histological and serological criteria. In patients with a significant response, titration of anti-HBc IgM enables anti-viral treatments to be monitored by a specific marker of HBV.

85 % of patients infected with the hepatitis B virus evolve towards recovery (appearance of anti-HBs Ab, considered to be immunity markers) and the remaining 15 % become chronic carriers. In areas where hepatitis B is endemic, all individuals are at risk. Elsewhere, indviduals with multiple sexual partners, drug-abusers, medical workers, immunosuppressed patients, and children with HBs antigen positive mothers are concerned. Vaccination strategies against the hepatitis B virus depend on the epidemiologic: situation Vaccination is highly recommended for patients at risk.

In cases of acquired immunity following infection by the hepatitis B virus, presence of anti-HBs antibodies is associated with anti-HBc antibodies.

Patients vaccinated against HBV will only be anti-HBs positwe. Post-vaccine protection is limited to a period of time, which is directly related to the concentration of anti- HBs currently admitted of at least 10 mIU/ml. Regular controls are essential to determine the need for booster injections.

Total (tHcy) has emerged as an important risk factor in the assessment of cardiovascular disease.3-7,12 Hcy, a thiol-containing amino acid, is produced by the intracellular demethylation of methionine. Hcy thereby serves as a pool that can be later scavenged for use in the remanufacture of either methionine through the action of the folate-dependent enzyme methionine synthase or cysteine using the B6 dependent transsulphuration pathway. 1,2Hcy in plasma is found primarily in a protein bound form but free, oxidized and disulfide forms are also present. Highly elevated levels of tHcy are found in patients with homocystinuria, a rare genetic disorder of the enzymes involved in Hcy metabolism 1,2,3 Homocystinuria patients exhibit arterial thromboembolism, mental retardation and early arteriosclerosis.1 Less severe genetic defects are also associated with moderate levels of Hcy.

Homocysteine has been identified as a indicator of cardiovascular disease. A meta-analysis of 27 epidemiological studies has suggested that a 5 µmol/L increase in tHcy could be associated with an odds ratio for coronary artery disease (CAD) of 1.6 for men and 1.8 for women, the same increase in risk as a 0.5 mmol/L increase in cholesterol.7 In addition, patients with chronic renal disease complicated by arteriosclerotic cardiovascular disease show elevated tHcy due to the inability of the kidney to remove Hcy from the blood.

Human immunodeficiency viruses (HIV) are RNA retroviruses transmitted via sexual contact, parenteral and perinatal pathways or the placenta. HIV1 and HIV2 were respectively isolated in 1983 and 1985 in patients infected by AIDS (Acquired Immunodeficiency Syndrome). Since then numerous genetic variants have  been characterized. These mutations seemed to be  without consequence for serological diagnosis until HIV1 variants of group O (Outlier) were isolated, since they have only 50 % homology at the env gene level with  those of group M (Major)

During 1995, 2.7 million adults were newly infected by HIV and 500,000 children were born infected with HIV. From the beginning of the pandemic until the middle of 1996, an estimated 27.9 million people were infected by the HIV virus. This figure includes 7.6 million people who contracted AIDS.

Current diagnosis of HIV infection relies on the detection of anti-HIV serum antibodies using an ELISA method. : However, there is a mean period of 3 weeks between contamination and the appearance of the first antibodies. Recent studies have shown that during this period p24 antigen was present in most people infected by , HIV1, regardless of their origin. Thus simultaneous detection of p24 antigenemia, anti-HIV1 and anti-HIV2  antibodies enables the time lapse between contamination and diagnosis of the infection to be decreased.

Hepatitis E Virus (HEV) is a recently discovered agent of enterically transmitted non-A, non-B hepatitis. The HEV infection has a worldwide distribution and a predominat waterborn route of transmission. The first reported outbreak of this infection occured in New Delhi; since this time, epidemics on hepatitis E have been documented in many countries. HEV shows morphological and biophysical properties similar to viruses found in the family Caliviridae. The HEV genom consists of a single stranded RNA molecule that contains 3 open reading frames (ORF1, ORF2, ORF3) That encode for non-structural and structural proteins.

Epitope mapping of the viral genome have lead to identify the presence of highly immunoreactive type common epitopes. Synthetic peptides or recombinant expressed proteins, derived from these regions, have been used to develop enzyme immunoassay for the detection of IgG and IgM antibody. Antibody to HEV of IgM class appears associated to acute stage of infection, while antibody to IgG class characterizes previous HEV exposure.

Herpes simplex virus (HSV) is an ancient and ubiquitous virus, known to cause acute and recurrent infections in humans. The virus enters the mucous membranes (ocular, genital or oral) and replicates locally. Infection of neonates during passage through the birth canal may result in neurological damage and death. In a small portion of infected individuals, the virus may enter the sensory root ganglion, resulting in latent, recurrent infections.

In the 1960s it was recognized that HSV consisted of two distinct types, HSV-I and HSV-II. HSV-I is considered to be primarily associated with ocular and oral infection, while HSV-II is considered to be a genital infection. Clinical cases primarily are 1) Eczema herpeticum, with eczematous skin changes with numerous lesions, 2) Gingivo-stomatitis and 3) Herpes sepsis, almost only found in newly born of premature infants. However, HSV types I and II share several common antigens, and the use of specific monoclonal antibodies or restriction endonuclease mapping may be required to type individual strains.

Infections with HSV type I or type II can differ in their clinical manifestations and severity. The immune response of the host can play an important role in controlling the severity of primary or reactivated infections. Those at highest risk are neonates, who contact the infection during passage through the birth canal, and immunocompromised patients.

While isolation of the virus in tissue culture is recommended for the diagnosis of active infections, serological testing can provide valuable information in the management of at-risk populations, such as pregnant women,

Human insulin is a polypeptide hormone originating in the beta cells of the pancreas and serving as a principal regulator for the storage and production of carbohydrates. Its secretion is normally stimulated by increases in the amount of glucose in circulation. This leads to higher insulin levels and more rapid tissue-assimilation of glucose – followed by a decline in the insulin level as the glucose level subsides.

In a number of conditions, notably insulinoma and diabetes, this relationship is impaired. Insulin tends to circulate at inappropriately high levels in patients with insulin-secreting pancreatic tumors; such tumors can thus be a cause of hypoglycemia. Accordingly, insulin immunoassays  used sometimes in connection with provocative doses of tolbutamide or calcium, play an essential role in the identification (and localization) of insulinomas. The finding of fasting hypoglycemia in association with an inappropriately high serum insulin coentration is considered diagnostic.

Insulin levels do not figure in the subclassification of diabetes worked out by the National Diabetes Data Group. Nevertheless, when obtained in the course of a glucose tolerance test, they appear to be of some prognostic value in predicting the benefits of insulin therapy and the likelihood of progression to insulin-dependence and the complications (such as retinopathy) characteristic of  diabetes.

The application of insulin immunoassay to patients already undergoing insulin therapy is complicated by the fact that such therapy typically leads to the formation of anti-insulin antibodies capable of interfering with the assay. Some investigators have sought therefore to measure insulin in urine, or in serum samples subjected to column chromatography or PEG precipitation. But the measurement of “free” insulin remains of limited interest as a technique for – monitoring insulin therapy in the absence of statistics establishing therapeutic or toxic ranges. So far it appears that glucose control in diabetics cannot in general be achieved by normalizing the.insulin profile. Nor is it known at what point abnormally high insulin levels become dangerous.

Insulin assay also finds a significant diagnostic role in anovulatory hyperandrogenic women. Insulin resistance (an impaired glucose response to a specific amount of insulin) is frequently observed in association with polycystic ovarian disease in women. In many of these women normal glucose levels are maintained at the expense of increased circulating insulin to overcome the underlying defect. Elevated insulin perhaps acting through the IGF-1 receptors on the theca interstitial cells of the ovary produces excess androgens and induces dysovulation. Assessment of insulin levels in fasting and subsequently after glucose load may help identify these hyperinsulinemic anovulatory women

The autoimmune disorders are a group of acquired diseases in which genetic factors appear to play a role. In autoimmune disease, the patient’s immune system reacts to normal components of the body as if they were foreign substances by producing antibodies (auto antibodies) against them. These diseases share certain clinical features and differentiation among them is often difficult. The most common findings associated with them include wide-spread immunologic and inflammatory alterations of connective tissue causing synovitis, pleuritis, myocarditis, endocarditis, pericarditis, peritonitis, vasculitis, myositis, skin rash and nephritis.

Generally, the autoimmune diseases are regarded as acquired diseases; however, their causes cannot be determined in most instances. Consequently, quantitation of specific protein assays, such as immunoglobulin G (IgG), immunoglobulin A (IgA),immunoglobulin M (IgM), C-reactive protein (CRP), and rheumatoid factor (RF), while not diagnostic of the underlying cause, can be used to assess disease activity and response to therapy.

Modern laboratory methods for allergy testing are based on the comparatively recent discovery that many allergies are mediated by immunoglobulins of the IgE class acting as points of contact between the allergen and specialized cells. The IgE molecules, which have a molecular mass of approximately 200 kd bind to the surface of mast cells and basophilic granulocytes. Subsequent binding of allergens to cell-bound IgE causes these cells to release histamines and other vasoactive substances, thereby initiating the events which we recognize as an allergic reaction.

In deciding on a Course of therapy, it is important to distinguish between IgE-mediated and non-lgE-mediated reactions. Measurement of the total circulating IgE level, in conjunction with other supporting diagnostic information, can aid in making this diagnosis. The supporting information should include appropriate tests for allergen-specific IgE. Measurement of the total circulating IgE level may also be of value in the early detection of allergy in infants, and as a means for predicting future atopic manifestations.

IgE levels normally show a slow increase during childhood, reaching adult levels in the second decade of life. In general, the total IgE level increases with the number of allergies which a person has and with the amount of exposure to relevant allergens.

Significant elevations may be encountered not only in sensitized individuals, but also in cases of IgE myeloma, pulmonary aspergillosis, and during the active stage of parasitic infestations.

Luteinizing hormone (Iutropin, LH), a glycoprotein of 28,000 daltons, is secreted by the anterior pituitary under the control of the hypothalamic gonadotropin releasing hormone (GnRH). LH consists of two polypeptide chains, alpha and beta. The alpha chains of LH, FSH, TSH and HCG are biochemically identical, whereas the beta chains are :biochemically unique, conferring bioactivity and biological and immunological specificity. In females, LH causes ovulation and steroid (estrogen and progesterone) production by the corpus luteum. Small quantities of LH are also necessary to promote estrogen production by the maturing follicle. In males, it stimulates interstitial (Leydig) cells to produce androgens and estrogens. Circulating levels of LH are controlled by a negative feedback effect on the hypothalamus by the steroid hormones. LH secretion, different for the two sexes and required for normal sexual function, occurs in pulses with rapid fluctuations over the entire reference range. Values for samples obtained in a single day from the same patient may therefore vary widely.

LH  measurements are used to define the hypothalamic-pituitary-gonadal axis. Serum gonadotropin determinations permit distinguishing between primary gonadal failure and deficient gonadal stimulation. If LH and FSH levels are elevated, primary gonadal failure is present, whereas if gonadotropin levels are low, deficient gonadal stimulation resulted in the hypogonadal state. LH measurement is also clinically important because LH and growth hormone are frequently the first hormones to be affected by pituitary disease.

Serum LH determinations have been very useful in the diagnosis and treatment of infertility in women. A midcycle rise is a good indication that ovulation will occur approximately 24 hours later. Subfertile couples, and women being treated with gonadotropins for infertility, can be informed that ovulation is imminent.

LH determination is also sometime in certain forms of dysovulatory infertility primarily polycystic overian (PCO) disease. In many PCO women LH:FSH ratio is increased, and under the influence of excess LH follicular theca cells produce excess androgenus and follicules become atretic and results is anovulation. 

Parathyroid hormone (parathyrin, PTH), a single-chain polypeptide (molecular mass of ~ 9500 daltons) containing 84 amino acids, exerts significant influence in the maintenance of optimal calcium ion concentrations. PTH raises serum ionized calcium levels through direct action on bone and the kidneys: it increases the rate of calcium ion flow from bone to the extra cellular fluid, and increases both the renal tubular reabsorption of ionized calcium and the renal excretion of phosphate. Long-term regulation of total body calcium by PTH occurs through its stimulation of vitamin D metabolism, which results in enhanced intestinal absorption of ionized calcium.

In healthy individuals, PTH is secreted in response to circulating calcium ion levels. Any dip below an individual’s normal level triggers a pronounced increase in PTH secretion. Calcium levels returning to normal exert a negative feedback effect, thus inhibiting PTH secretion by the parathyroid glands.

PTH undergoes proteolysis to a lesser extent in the parathyroid glands but mostly peripherally – especially in the liver but also in the kidneys and bone – to yield N-terminal fragments and longer lived C-terminal and midregion fragments. The N-terminal fragment contains the region that confers bioactivity. For the intact hormone, the in vivo half-life is 2 to 5 minutes.

In hypercalcemia due to primary hyperparathyroidism or to ectopic PTH production (pseudohyperparathyroidism),the majority of patients have elevated PTH levels. By contrast, in hypercalcemia due to malignancy or other causes, the concentration of PTH in circulation is typically low or within normal reference range limits. PTH levels are also characteristically high in secondary hyperparathyroidism – usually associated  with renal failure – as a result of constant stimulation of the parathyroid gland by low calcium levels. Hypocalcemia accompanied by a low PTH level, on the other hand, is to be expected in hypoparathyroidism, either postsurgical or idiopathic.

Immunoassays specific for various PTH fragments have been developed. However, recent assays for intact PTH have the necessary sensitivity for detecting circulating intact PTH in normals and for discriminating between normals, those with primary hyperparathyroidism and nonparathyroidal hypercalcemic patients.

Human prolactin is a polypeptide hormone of the anterior pituitary with a molecular mass of about 22 kd. It plays an essential role in the secretion of milk and has the ability to suppress gonadal function. Determination of prolactin is an important tool in the investigation of amenorrhea, galactorrhea and hypothalamic-pituitary disorders.

As a reference range for circulating prolactin, the literature suggests concentrations up to approximately 20 ng/mL. Values are distinctly elevated at birth but decline to adult levels in less than three months. Women are reported to have slightly higher mean levels than men, with a slight rise at puberty – apparently estrogen related – and a corresponding fall at menopause. During pregnancy, the prolactin level climbs steadily to ten or twenty times its former value, then drops back down to normal after delivery – within three weeks in nonnursing mothers. In those who breast-feed, the decline to normal is more gradual because of the prompt and dramatic surges in prolactin release induced by suckling. Women taking oral contraceptives or under estrogen treatment may have prolactin levels higher than normal.In assessing the significance of moderate elevations, it is important to keep in mind that prolactin is a stress hormone. Not only surgery, but events no more distressing than venipuncture or a clinical interview have been reported to occasion a transient rise. Moreover, the release of prolactin is inherently episodic, and day- to-day fluctuations with CVs as high as 30% have been encountered. Finally, there is a sleep-related diurnal variation: prolactin levels increase during sleep and reach their lowest a few hours after waking. The advice sometimes given to draw samples “between nine and noon” is based on the assumption that subjects observe reasonably normal waking hours.

Under normal circumstances the 22 kd  prolactin predominates, however, prolactin may also be produced in higher molecular mass forms (50-150 kd). These prolactins have markedly reduced bioactivity. In certain forms of macroprolactinemia the high mw prolactin variant may represent 85% or more of the total prolactin. Because of difference in the binding affinity and avidity between the macroprolactins and the employed antibody in an assay (it is desirable that macroprolactins should have negligible cross-reactivity with the antibody), results of commercial prolactin assays in the presence of macroprolactis may widely differ between different assay systems and, therefore, between the laboratories.

17α-Hydroxyprogesterone is a twenty-one carbon steroid ” produced in the adrenals – and also in the ovaries, testes and placenta – which serves as a biosynthetic precursor to cortisol. Its measurement is of value in the diagnosis and management of congenital adrenal hyperplasia, hirsutism and infertility. Circulating 17α-hydroxyprogesterone normally exhibits a diurnal pattern similar to that of cortisol, with higher values in the early morning than in the late afternoon. Hence, the time of collection should be standardized. Like progesterone, l7a-hydroxyprogesterone increases during the luteal phase of menstruating women and rises during pregnancy.

In the most common form of congenital adrenal hyperplasia, deficiency of the enzyme 21-hydroxylase blocks normal synthesis of cortisol, leading to a compensatory increase of ACTH secretion. This results in adrenal hyperplasia with increased levels of 17α-hydroxyprogesterone, DHEA and other adrenal steroids. Measurement of I7α-hydroxyprogesterone in blood samples collected on filter paper has been used to screen for congenital adrenal hyperplasia in newborns. In addition, serum measurements have been used in the differential diagnosis of hirsutism and infertility where 21-hydroxylase deficiency is suspected, since late-onset congenital adrenal hyperplasia can sometimes mimic the polycystic ovary syndrome. Untreated congenital adrenal hyperplasia in newborns is usually associated with markedly elevated 17α-hydroxyprogesterone levels ranging from 10 to 400 times the upper limit of normal, but lesser elevations may also occur. Some women with late-onset congenital adrenal hyperplasia may present modestly elevated basal 17α-hydroxyprogesterone levels (greater than 3 or4 ng/mL), but in others measurement of 17α-hydroxyprogesterone after ACTH stimulation may be required to demonstrate the disorder.

Prostate specific antigen (PSA) is a glycoprotein monomer with protease activity. PSA has an isoelectric point of approximately 6.9 and a molecular weight of approximately 33-34 kilodaltons; it contains approximately 10% carbohydrate by weight. The amino acid sequence of PSA was reported, and the gene has been cloned. PSA is biochemically and immunologically distinct from Prostatic Acid Phosphatase (PAP) and does not exhibit enzymatic phosphatase activity.

PSA is localized in the cytoplasm of prostatic ductal epithelium and in secretions of the ductallumina. Because PSA is a secretory protein of the prostate, it can be recovered and purified both from prostatic tissue and from seminal plasma. PSA has been found to be exclusively associated with prostate tissue; an elevated serum PSA has been found In patients with prostate cancer, benign prostatic hypertrophy, and inflammatory conditions of other adjacent genitourinary tissues, but not in healthy men, men with nonprostatic carcinoma, healthy women or  women with cancer.  Measurement of serum PSA is not suitable as a screen for prostate cancer because elevated PSA concentrations are also observed in patients with benign prostatic hypertrophy; nor is it recommended as a guide in disease staging. The combination of PSA measurement and rectal examination with ultrasonography, in the event of abnormal findings, may provide a better method of detecting prostate cancer than rectal examination alone. Determinations of total immunoreactive PSA can be useful in detecting metastatic or persistent disease in patients following surgical or medical treatment of prostate cancer. Persistent elevation of PSA following treatment or an increase in the pretreatment PSA concentrations is indicative of recurrent or residual disease. Hence, PSA is widely accepted as an aid in the management of prostate cancer patients.

Progesterone is a steroid hormone which plays an important role in the preparation for and maintenance of pregnancy. It is synthesized from cholesterol via pregnenolone – then rapidly metabolized to pregnanediol, for the most part, in the liver. The ovary and placenta are the major production sites; but a small amount is also synthesized by the adrenal cortex in both men and women.

Circulating progesterone levels, which are characteristically low during the follicular phase, increase sharply during the luteal phase of menstrual cycles, reaching a maximum some 5 to 10 days after the midcycle LH peak. Unless pregnancy occurs, a steep decline to follicular levels sets in about 4 days before the next menstrual period. This pattern constitutes the rationale behind the well-established use of serum progesterone measurements as a simple and reliable method for ovulation detection.

There is growing literature on luteal phase defects. Daily progesterone levels are considered the most accurate means for documenting a defective luteal phase. However, some investigators have found that three samples or even a single sample (if well timed) can provide valuable information on the adequacy of the luteal phase.

Measurements of serum progesterone have also been used to check the effectiveness of ovulation induction, to monitor progesterone replacement therapy and to detect and evaluate patients at risk for abortion during the early weeks of pregnancy. On the other hand, although progesterone levels increase throughout pregnancy, they are not considered a suitable means for monitoring fetal well-being during the third trimester.

Phenytoin (5,5-diphenylhydantoin MW: 252.3 Da) is an antiepileptic drug widely used for treating several types of seizure. It is typically administered orally as phenytoin itself, or else by intravenous infusion (IV) or intramuscular injection (IM) of the prodrug fosphenytoin, a relatively water-soluble, phosphate ester of phenytoin. Depending on the route of administration, conversion of fosphenytoin to phenytoin is expected: to be essentially complete within 2 to 4 hours. Most of the phenytoin in circulation is tightly bound to plasma proteins, chiefly albumin. Normally, only about 10% circulates in the pharmacologically active free form, though in some conditions, such as renal failure, this fraction may increase to 30% or more. The amount circulating in free form is expected to be increased in hypoalbuminemia and in conditions where other compounds compete with phenytoin for protein binding sites. Examples include valproic acid and other anticonvulsants which may be administered concurrently with phenytoin; salicylates and nonsteroidal anti-inflammatory drugs such as oxaprozin as well as many other drugs which bind strongly to albumin. Likewise. high free fatty acid  levels may displace phenytoin from plasma proteins.

Phenytoin is cleared through the liver, where it is converted primarily to 5-(p- hydroxyphenyl)-5-phenylhydantoin, which is subsequently glucuronidated and excreted in the urine.  Though inactive, these metabolites-HPPH and its glucuronide HPPG – are structurally similar to phenytoin and may compete with phenytoin for protein binding sites. The clearance rate declines somewhat with age, and is reduced in renal insufficiency. Drugs enhancing or inhibiting the liver’s ability to metabolize phenytoin can be expected to affect circulating levels. Moreover, metabolic capacity is limited; hence the relationship between dosage and circulating levels is not uniform: at higher levels, even within the therapeutic range, small increases in administered dose may result in large increases in circulating levels.

Phenytoin is commonly monitored by immunoassay because (total) circulating levels of the drug correlate more strongly with therapeutic effect and toxicity than does the administered dose. The generally accepted therapeutic range for total circulating phenytoin is 10 to 20 μg/mL(40 to 80 μmol/L), but these limits should be considered as guidelines only.  (There is no established range for free circulating phenytoin.) Dosage optimization must take into account the patient’s clinical response to the drug. Low levels may be associated with inadequate control, as manifested in the frequency and/or severity of the seizures. High levels may be associated with symptoms of Ineurotoxicity – including involuntary rhythmic eye movements (nystagmus),  nausea, vomiting, impaired coordination (ataxia), speech interference (dysarthia), severe lethargy, and so on – or even with  a worsening of the seizures.

Rubella, also known as German measles,occurs throughout the world. The prodromal stage includes malaise and a low-grade fever, followed by characteristic lymphadenitis and finally a macular or maculopapular rash. While rubella is primarily an infection of children, rubella infections during the first trimester of pregnancy can result in congenital infections with disastrous consequences. Birth defects, such as deafness and/or congenital heart defects are the most common. Multiple defects such as mental retardation, cataracts and hepatosplenomegaly may also occur and contribute to death of the infant in the first year of life. Infants with congenital infections may secrete virus for up to two years. Contact with these children poses a risk to susceptible pregnant women.

Clinical symptoms of rubella infection are frequently mild or nonspecific, making the infection difficult to diagnose clinically.For these reasons, pregnant women with an undiagnosed illness with rash should be evaluated for the possibility of an acute, primary rubella infection. While the virus can be cultured in vitro, serology remains the principal means for , establishing a clinical diagnosis of acquired rubella and congenital infections. As two-thirds of defects are not apparent at birth, it is recommended that children exposed in utero be monitored for clinical and serological status until school age. A rubella vaccine, introduced in 1969. has significantly lowered the incidence of both acute disease and congenital rubella syndrome: However, as the vaccination program may vary with geographical location and as the level of immunity provided by vaccination is not always adequate, pregnant women and women of child-bearing age are routinely screened for immune status.

The presence of rubella IgG antibodies indicates a previous vaccination or infection and is indicative of presumptive immunity. Patients suspected of having primary, acute rubella infection should be tested for the presence of IgM antibodies.

In the male, testosterone is mainly synthesized in the interstitial Leydig cells of the testis, and is luteinizing hormone of the anterior pituitary. Testosterone is responsible for the development of secondary sex characteristics, such as the accessory sex organs, the prostate, seminal vessicles and the growth of facial, pubic and axillary hair. Testosterone measurements have been very helpful in evaluating hypogonadal states. Increased testosterone levels in males can be found in complete androgen resistance (testicular feminization). Common causes of decreased testosterone levels in males include: hypogonadism, orchidectomy, estrogen therapy, Klinefelter’s syndrome, hypopituitarism, and hepatic cirrhosis. In the female, testosterone comes from three sources. It is secreted in small quantities by both the adrenal glands and the ovaries, and in healthy women 50-60% of the daily testosterone production arises from peripheral metabolism of prehormones, chiefly androstenedione. Assessment of testosterone measurement finds a major role in women with dysovulatory infertility, hirsutism, polycystic ovarian disease and related hyperandrogenic states.

Testosterone circulates almost entirely bound to transport proteins: normally less than one percent is free. The principal transport protein for testosterone is known as sex hormone binding globulin (SHBG) or testosterone-estradiol binding globulin(TeBG),since it binds not only to testosterone but also to estradiol (and dihydrotestosterone). Albumin and cortisol binding globulin (CBG) are the other testosterone transport proteins. For three reasons, SHBG is the most important of these transport proteins. It carries, at least in females, a higher percentage of the testosterone in circulation than either albumin or CBG. Furthermore, it binds to testosterone with a much higher affinity than do the other two transport proteins. (The testosterone bound to albumin and CBG is collectively known as “weakly bound” testosterone.) Finally, SHBG has a responsiveness, not associated with albumin, insofar as the SHBG level is sensitive to changes in the ratio of circulating estrogens to circulating androgens. SHBG thus plays a greater role in determining the level of free testosterone in circulation.

In pregnancy, SHBG levels show a progressive increase that mirrors the rise in estradiol levels. Although the total testosterone level also shows some increase during pregnancy, the rise in estradiol, and hence also in SHBG, is more dramatic. The net result is a decrease in free testosterone as a percent of total testosterone. Women taking oral contraceptives likewise have increased SHBG levels, at least if they are taking high dose ethinyl estradiol preparations. Conversely, the administration of androgens to prepubertal boys, or the elevation of endogenous testosterone by HCG stimulation, induces a significant decrease in the SHBG level. Obesity is also associated with a decrease in the SHBG level.

Other things being equal, we expect the concentration of free testosterone in circulation to increase if the testosterone level (T) increases or if the SHBG level decreases. The T/SHBG ratio, ie. the ratio of total testosterone to (the concentration or binding capacity of) SHBG, thus serves as a rough guide to the concentration of free testosterone in circulation. The T/SHBG ratio is sometimes referred to as the Testosterone Free Index (TFI).

The Testosterone Free Index is often increased in severe acne, male androgenic alopecia (balding), hirsutism and other conditions. A low SHBG level, often in combination with a normal total testosterone level, is a common finding in these conditions. An increase in the testosterone production rate typically induces a decrease in the SHBG level. This stimulates cellular uptake and metabolism of testosterone, by making more available in the free form. The result is that the total testosterone level is often normal, because the increased production rate is offset by an increased rate of clearance.

Total testosterone measurements have traditionally been used to help screen for hirsutism. In view of the mechanism just described, it is natural to expect free testosterone levels, measured directly or indexed by the T/SHBG ratio, to correlate better with hirsutism. It is therefore logical to measure free testosterone, rather than total testosterone, for the assessment of true androgenic status.

Thyroglobulin (TG) is a heterogeneous iodoglycoprotein which has a molecular mass of approximately 660,000 daltons. Thyroglobulin is normally synthesized in the follicular cells of the the thyroid gland, under the influence of thyrotropin and represents the precursor to thyroxine and the other iodothyronines. The expected upper limit of normal for circulating thyroglobulin is approximately 40 to 60 ng/mL, with a median of 5 to 10 ng/mL. Somewhat higher values are encountered in new borns and during the third trimester of pregnancy. Thyroglobunin levels also tend to be elevated in regions of endemic goiter. The major clinical applications for measurement of this prohormone is derived from the fact that functioning thyroid tissue appears to be the only source of circulating thyroglobulin. Accordingly, thyroglobulin determinations have been widely used to complement radioiodine scanniog and other technique (such as ultrasound or immunohistocbemical staining) as an aid in identifying the presence or absence of functioning thyroid tissue, or an increase in such tissue relative to an individually established baseline. The differential diagnosis of congenital hypothyroidism is a well-established context of use for this application of serum thyroglobulin measurement Congoniltal hypothyroidism.

Thyroglobulin determinations have been used, sometimes in conjunction with ultrasound and radioiodine scanning, to help clarify the type of thyroid defect in previously diagnosed congenital hypothyroidism. Very low or undetectable thyroglobulin levels are expected in infants born without thyroid tissue (thyroid agenesis), whereas higher, but widely varying levels are generally encountered in infants with hypoplastic thyroid gland, ectopic thyroid tissue, dyshormonogenic goiter, congenital TBG deficiency or trasient hypothyrodism.

Thyroglobulin measurements may also be value in helping to distinguish subacute thyroiditis from thyrotoxicosis caused by cover administration of thyroid hormones. In te lattr event low levels of thyroglobulin are expected due to thyroid hormone suppression of thyrotropin.

Thyroglobulin is produced only by the thyroid gland and is a major component of the thyroid follicular colloid. The thyroid hormones 3,5,3′,5′-tetraiodothyronine (thyroxine, T4) and 3,5,3′-triiodothyronine (T3) are synthesized from thyroglobulin.  Autoantibodies to thyroglobulin (TG autoantibodies) are often present in patients with autoimmune thyroid disease. Approximately 10 percent of healthy individuals have TG autoantibodies at low levels; higher concentrations are found in 30 and 85 percent of patients with Graves’ disease and Hashimoto’s thyroiditis, respectively. Elevated levels of antibodies to thyroid peroxidase (TPO autoantibodies) occur more frequently than high anti-TG levels in these diseases. However, anti-TG determinations therefore do not seem to add to the diagnostic information provided by anti- TPO results.

TG autoantibody measurements are most useful for evaluating samples submitted for thyroglobulin measurements because TG autoantibodies can interfere with both competitive immunoassays and immunometric assays for thyroglobulin.

Antithyroid peroxidase (TPO) antibodies are autoantibodies directed against the thyroid peroxidase enzyme. This enzyme catalyzes the iodination of tyrosine in thyroglobulin during the biosynthesis of T3 and T4.

Historically, these antibodies were referred to as antimicrosomal antibodies (AMA) because the antibodies bind to the microsomal part of the thyroid cells. Recent research has identified thyroid peroxidase as the primary antigenic component of microsomes. Thyroid autoimmune disease is the major factor underlying hypothyroidism and hyperthyroidism and tends to occur in a genetically predisposed population. 

The major thyroid autoimmune diseases are Hashimoto’s thyroiditis and Graves’ disease. In virtually all cases of Hashimoto’s disease and in the majority of Graves’ disease, TPO autoantibodies are elevated. High levels of TPO autoantibodies, in the context of the clinical presentation of hypothyroidism, confirms the diagnosis of Hashimoto’s disease.

The principal thyroid hormone, thyroxine (T4), circulates almost entirely bound to carrier proteins, chief of which is thyroxine-binding globulin (TBG). Altered carrier protein concentrations induce changes in total T4levels, and free T4 concentrations tend to stay within a tight range. For this reason, total T4 measurements do not always reflect thyroid status. TBG levels may vary under different physiological conditions, such as during pregnancy, oral contraceptive use, and estrogen therapy. Total T4 1evels may increase above the normal range while free T4 remains normal. Alternatively, patients with a dysfunctional thyroid gland and altered TBG levels can have normal total T4levels, masking the illness. Since abnormal T4 1evels may signify either abnormal thyroid function or carrier protein variation (physiological or pathological), free T4 measurements more highly correlate with thyroid status than total T₄ measurements.

Thyroid stimulating hormone (thyrotropin, TSH) is a pituitary hormone which, through its action on the thyroid gland, plays a major role in maintaining normal circulating levels of the iodothyronines, T4 and T3. TSH is controlled by negative feedback from circulating T4 and T3, and by the hypothalamic hormone TRH (thyrotropin releasing hormone).

In primary hypothyroidism, where there is impaired production of thyroid hormones, the TSH level is typically highly elevated. In secondary or tertiary hypothyroidism, on the other hand, where thyroid hormone production is low as a consequence of pituitary or hypothalamic lesions, the TSH level is usually low. In hyperthyroidism, the TSH level is typically suppressed to subnormal levels. Less often, this condition may result from hyperstimulation of the thyroid, due to hypothalamic or pituitary lesions, in which case the TSH level is usually increased.

Measurement of circulating TSH has been used as a primary test for differential diagnosis of hypothyroidism and as an aid in monitoring the adequacy of thyroid hormone replacement therapy. It should be remembered that hyperthyroidism and hypothyroidism are graded conditions. This implies that not all patients in these disease categories can be expected to have TSH levels far outside the euthyroid range. On the other hand, TSH levels exit the euthyroid reference range in the very early phases  developing thyroid disease, while the patient’s disease is still subclinical and thyroid hormone levels remain within their  euthyroid reference ranges.

Toxoplasma gondii is an obligate intracellular parasite capable of infecting most mammals, including humans. The organism is transmitted through ingestion of insufficiently cooked meat. Throughout the world, 1 to 90 percent of populations may be infected, with 25 to 30 percent of the adult population infected in the United States. While toxoplasmosis may be manifested in several forms, infections are usually clinically not apparent, and latent infections usually persist for life. Overt clinical symptoms are similar to infectious mononucleosis, with lymphadenopathy, fever, headache, malaise and sometimes pneumonia and myocarditis.

As with other latent infections, acute toxoplasma infection can pose a serious threat to immunocompromised individuals and newborns who acquire the infection in utero. Immuno-suppressed patients may develop encephalitis, myocarditis or pneumonitis. Congenital infections usually result as a consequence of asymptomatic acute maternal infection. This infection can cause premature delivery, spontaneous abortion or stillbirth. Neonates may manifest chorioretinitis, hydrocephaly, microcephaly, cerebral calcification and psychomotor retardation. The majority of congenitally infected children will not exhibit any symptoms until later in life. Management of toxoplasmosis requires serological monitoring of infected individuals as the organism is not readily available for culture. Quantitative testing for the presence of toxoplasma IgG can be useful to determine prior infection and indicate reactivation of the infection. Qualitative testing for the presence of toxoplasma IgM is useful to determine acute infection. Accurate diagnostic information is important, particularly during pregnancy, as treatment with spiramycin can reduce the  risk to the fetus.

Accurate diagnostic information is important, particularly during pregnancy, as treatment with spiramycin can reduce the risk to the fetus.

Under normal physiological conditions triiodothyronine (T3) represents approximately 5 percent of the thyroid hormone in plasma. Although present in lower concentration than thyroxine (T4), T3 has greater metabolic activity, faster turnover and a larger volume of distribution. It is produced largely through extrathyroidal conversion of T4. Like T4, it circulates almost entirely bound to the carrier proteins TBG, pre-albumin and albumin. Free T3 constitutes only about 0.25% of the total T3 in circulation.

Measurement of total T3 by immunoassay has a number of well-established uses. In the presence of elevated free or total T4, T3 measurements help confirm a hyperthyroidism diagnosis. Abnormal elevation of total T3 may also occur when the total T4 concentration is normal- a condition known as “T3 toxicosis”.

For the most part, free T3 levels correlate closely with total T3 levels. Total T3, however, depends not only on thyroid status and the peripheral conversion of T4 to T3, but also on the concentration of thyroid hormone-binding proteins. Free T3, on the other hand, is largely unaffected by variations in these carrier proteins. Thus, the TBG elevations typical of pregnancy, oral contraceptive use, and estrogen therapy effects an increase in the total T3 level while leaving the free T3 concentration basically unchanged. The free T3 concentration therefore typically reflects a patient’s actual status more reliable than the total T3 concentration.

Under normal physiological conditions, T3 represents approximately 5 percent of the thyroid hormones in serum. Although present in lower concentration, T3 has a greater intrinsic metabolic activity, faster turnover and larger volume of distribution than circulating T4. Reports that thyrotoxicosis may be caused by abnormally high concentrations of T3, rather than T4, have reinforced the importance of T3 measurements. In addition, T3 determination is an important tool for monitoring hypothyroid patients receiving sodium liothyronine therapy. Unlike “T3 Uptake” tests, which estimate the saturation of thyroid hormone binding proteins, total T3 analysis actually measures circulating levels of triiodothyronine. Most reports indicate that T3 levels distinguish clearly between euthyroid and hyperthyroid subjects, but , provide a less clear-cut separation between hypothyroid and euthyroid subjects.

Numerous conditions unrelated to thyroid disease may cause abnormal T3 values. Consequently, total T3 values should not be used on their own in establishing the thyroid status of an individual. The levels of serum T4, TBG (thyroid binding globulin), TSH (thyroid stimulating hormone) and other clinical findings must be considered as well.

According to the American College of Obstetricians and Gynecologists, it has become standard in prenatal care to offer screening tests for neural tube defects and genetic abnormalities. There have been some changes in the recommended method of prenatal screening over the past few years, and research to improve detection rates with better combinations of maternal serum analytes is ongoing. The issues facing physicians are the sensitivity and specificity of multiple serum analyte combinations. The current maternal serum analytes in use in most areas are alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG) and unconjugated estriol. Measurement of AFP alone can detect the vast majority of neural tube defects (NTDs) and a small portion of trisomy 21–affected pregnancies in patients of all ages. Adding hCG and unconjugated estriol to this screen increases the rate of detection of tri-somies 21 and 18. Counseling patients about the risks and benefits of such screening is important to provide a balanced discussion of screening issues.

Interpreting Triple Analyte Screening Results

Vitamin B12 (cobalamin) and folate are nutrients essential to hematopoiesis. Megaloblastic anemia is almost always due to lack of one of these two vitamins. Vitamin B12 deficiency can also result in severe neurological impairment. Circulating levels of vitamin B12 are usually a good index to tissue stores. That is, vitamin B12 levels as measured in serum or plasma by an optimized assay system are typically low in vitamin B12 deficiency, and normal or elevated otherwise. Exceptions to this rule can occur in those relatively uncommon situations where levels of vitamin B12 transport proteins are abnormal. Thus, low circulating vitamin B12 levels can occur in the absence of vitamin B12 deficiency where the level of Transcobalamin-I (a physiologically inactive transport protein) is low. Conversely, vitamin B12 deficiency can occur in the presence of normal or even elevated plasma vitamin B12 levels, where transcobalamin-II levels are low or where levels of inactive vitamin B12 transport proteins are high, as in chronic myelogenous leukemia.Circulating folate levels are usually normal or elevated in vitamin B12 deficiency, but red cell folate levels are frequently low in this condition.

Vitamin B12 deficiency occurs only rarely as a result of dietary lack of this vitamin. More commonly, it results from impaired absorption, as in partial or total gastrectomy, or in pernicious anemia, a condition characterized by absence or near absence of intrinsic factor. Since roughly two thirds of all patients with pernicious anemia have blocking antibodies to intrinsic factor (IFbAb), while IFbAb are only very rarely encountered in other situations, IFbAb determinations represent a useful follow-up test for the differential diagnosis of vitamin B 12 deficiency. (Circulating intrinsic factor antibodies are present in more than half of all pernicious anemia patients. Increased transport protein levels can occur, for example, in chronic myelogenous leukemia.)

Common causes of high vitamin B12 levels include liver disease, myeloproliferative disease (with chronic myelogenous leukemia as a special case) and the use of multivitamin supplements.

Valproic acid is used primarily in the treatment of simple and complex absence seizures. It can be administered alone or in combination with other drugs such as phenobarbital or phenytoin. Valproic acid increases the concentration of γ-amino- butyric acid (GABA) by inhibiting GABA transaminase. GABA is a potent inhibitor of pre- and postsynaptic discharges in the central nervous system. Valproic acid circulates mainly bound to protein. It competes with phenytoin for protein-binding sites. Valproic acid also inhibits the renal clearance of phenobarbital. When these two drugs are used in combination, the concentration of phenobarbital can be increased by up to  40 percent. Valproic acid is rapidly and almost completely absorbed after oral administration, reaching its peak concentration after 1 to 2 hours. In adults, its half-life is 16 hours. The therapeutic range for valproic acid is 50 to 100 μg/mL. Levels above 100 μg/mL are considered toxic. Plasma concentrations should be monitored to help maintain an effective dosage and prevent side effects caused by an excessive concentration.