TEST FOR DETECTION OF BILE SALTS IN URINEWritten by Dayyal Dg.
Published in Clinical PathologyThursday, 10 August 2017 00:34
Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).
Bile salts along with bilirubin can be detected in urine in cases of obstructive jaundice. In obstructive jaundice, bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their detection is Hay’s surface tension test. The property of bile salts to lower the surface tension is utilized in this test.
Take some fresh urine in a conical glass tube. Urine should be at the room temperature. Sprinkle on the surface particles of sulphur. If bile salts are present, sulphur particles sink to the bottom because of lowering of surface tension by bile salts. If sulphur particles remain on the surface of urine, bile salts are absent.
Thymol (used as a preservative) gives false positive test.
Last modified on Sunday, 20 August 2017 15:20
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TOTAL THYROXINE (T4)Total serum thyroxine includes both free and protein-bound thyroxine and is usually measured by competitive immunoassay. Normal level in adults is 5.0-12.0 μg/dl.Test for total thyroxine or free thyroxine is usually combined with TSH measurement and together they give the best assessment of thyroid function.Causes of Increased Total T4
Causes of Decreased Total T4
- Hyperthyroidism: Elevation of both T4 and T3 values along with decrease of TSH are indicative of primary hyperthyroidism.
- Increased thyroxine-binding globulin: If concentration of TBG increases, free hormone level falls, release of TSH from pituitary is stimulated, and free hormone concentration is restored to normal. Reverse occurs if concentration of binding proteins falls. In either case, level of free hormones remains normal, while concentration of total hormone is altered. Therefore, estimation of only total T4 concentration can cause misinterpretation of results in situations that alter concentration of TBG.
- Factitious hyperthyroidism
- Pituitary TSH-secreting tumor.
Free Thyroxine (FT4)FT4 comprises of only a small fraction of total T4, is unbound to proteins, and is the metabolically active form of the hormone. It constitutes about 0.05% of total T4. Normal range is 0.7 to 1.9 ng/dl. Free hormone concentrations (FT4 and FT3) correlate better with metabolic state than total hormone levels (since they are not affected by changes in TBG concentrations).Measurement of FT4 is helpful in those situations in which total T4 level is likely to be altered due to alteration in TBG level (e.g. pregnancy, oral contraceptives, nephrotic syndrome).Total and Free Triiodothyronine (T3)Uses
- Primary hypothyroidism: The combination of decreased T4 and elevated TSH are indicative of primary hypothyroidism.
- Secondary or pituitary hypothyroidism
- Tertiary or hypothalamic hypothyroidism
- Hypoproteinaemia, e.g. nephrotic syndrome
- Drugs: oestrogen, danazol
- Severe non-thyroidal illness.
A low T3 level is not useful for diagnosis of hypothyroidism since it is observed in about 25% of normal individuals.For routine assessment of thyroid function, TSH and T4 are measured. T3 is not routinely estimated because normal plasma levels are very low.Normal T3 level is 80-180 ng/dl.Free T3: Measurement of free T3 gives true values in patients with altered serum protein levels (like pregnancy, intake of estrogens or oral contraceptives, and nephrotic syndrome). It represents 0.5% of total T3.Thyrotropin Releasing Hormone (TRH) Stimulation TestUses
- Diagnosis of T3 thyrotoxicosis: Hyperthyroidism with low TSH and elevated T3, and normal T4/FT4 is termed T3 thyrotoxicosis.
- Early diagnosis of hyperthyroidism: In early stage of hyperthyroidism, total T4 and free T4 levels are normal, but T3 is elevated.
This test is not much used nowadays due to the availability of sensitive TSH assays.Procedure
- Confirmation of diagnosis of secondary hypothyroidism
- Evaluation of suspected hypothalamic disease
- Suspected hyperthyroidism
- A baseline blood sample is collected for estimation of basal serum TSH level.
- TRH is injected intravenously (200 or 500 μg) followed by measurement of serum TSH at 20 and 60 minutes.
Antithyroid AntibodiesBox 864.1 Thyroid autoantibodies
- Normal response: A rise of TSH > 2 mU/L at 20 minutes, and a small decline at 60 minutes.
- Exaggerated response: A further significant rise in already elevated TSH level at 20 minutes followed by a slight decrease at 60 minutes; occurs in primary hypothyroidism.
- Flat response: There is no response; occurs in secondary (pituitary) hypothyroidism.
- Delayed response: TSH is higher at 60 minutes as compared to its level at 20 minutes; seen in tertiary (hypothalamic) hypothyroidism.
Various autoantibodies (TSH receptor, antimicrosomal, and antithyroglobulin) are detected in thyroid disorders like Hashimoto’s thyroiditis and Graves’ disease. Antimicrosomal (also called as thyroid peroxidase) and anti-thyroglobulin antibodies are observed in almost all patients with Hashimoto’s disease. TSH receptor antibodies (TRAb) are mainly tested in Graves’ disease to predict the outcome after treatment (Box 864.1).Radioactive Iodine Uptake (RAIU) TestThis is a direct test that assesses the trapping of iodide by thyroid gland (through the iodine symporters or pumps in follicular cells) for thyroid hormone synthesis. Patient is administered a tracer dose of radioactive iodine (131I or 123I) orally. This is followed by measurement of amount of radioactivity over the thyroid gland at 2 to 6 hours and again at 24 hours. RAIU correlates directly with the functional activity of the thyroid gland. Normal RAIU is about 10-30% of administered dose at 24 hours, but varies according to the geographic location due to differences in dietary intake.Causes of Increased Uptake
- Useful for diagnosis and monitoring of autoimmune thyroid diseases.
- Antimicrosomal or antithyroid peroxidase antibodies: Hashimoto’s thyroiditis
- Anti-TSH receptor antibodies: Graves’ disease
Causes of Decreased Uptake
- Hyperthyroidism due to Graves’ disease, toxic multinodular goiter, toxic adenoma, TSH-secreting tumor.
UsesRAIU is most helpful in differential diagnosis of hyperthyroidism by separating causes into those due to increased uptake and due to decreased uptake.Thyroid ScintiscanningAn isotope (99mTc-pertechnetate) is administered and a gamma counter assesses its distribution within the thyroid gland.Interpretation
- Hyperthyroidism due to administration of thyroid hormone, factitious hyperthyroidism, subacute thyroiditis.
Interpretation of thyroid function tests is shown in Table 164.1.Table 864.1 Interpretation of thyroid function tests
- Differential diagnosis of high RAIU thyrotoxicosis:
– Graves’ disease: Uniform or diffuse increase in uptake
– Toxic multinodular goiter: Multiple discrete areas of increased uptake
– Adenoma: Single area of increased uptake
- Evaluation of a solitary thyroid nodule:
– ‘Hot’ nodule: Hyperfunctioning
– ‘Cold’ nodule: Non-functioning; about 20% cases are malignant.
Test results Interpretations 1. TSH Normal, FT4 Normal Euthyroid 2. Low TSH, Low FT4 Secondary hypothyroidism 3. High TSH, Normal FT4 Subclinical hypothyroidism 4. High TSH, Low FT4 Primary hypothyroidism 5. Low TSH, Normal FT4, Normal FT3 Subclinical hyperthyroidism 6. Low TSH, Normal FT4, High FT3 T3 toxicosis 7. Low TSH, High FT4 Primary hyperthyroidismNeonatal Screening for HypothyroidismThyroid hormone deficiency during neonatal period can cause severe mental retardation (cretinism) that can be prevented by early detection and treatment. Estimation of TSH is done on dry blood spots on filter paper or cord serum between 3rd to 5th days of life. Elevated TSH is diagnostic of hypothyroidism. In infants with confirmed hypothyroidism, RAIU (123I) scan should be done to distinguish between thyroid agenesis and dyshormonogenesis.
DISORDERS OF THYROID
Box 863.1 Terminology in thyroid disorders
Among the endocrine disorders, disorders of thyroid are common and are only next in frequency to diabetes mellitus. They are more common in women than in men. Functional thyroid disorders can be divided into two types depending on activity of the thyroid gland: hypothyroidism (low thyroid hormones), and hyperthyroidism (excess thyroid hormones). Any enlargement of thyroid gland is called as a goiter. Terminology related to thyroid disorders is shown in Box 863.1.HyperthyroidismHyperthyroidism is a condition caused by excessive secretion of thyroid hormone. Causes of hyperthyroidism are listed in Table 863.1.Table 863.1 Causes of hyperthyroidismClinical CharacteristicsClinical characteristics of hyperthyroidism are nervousness, anxiety, irritability, insomnia, fine tremors; weight loss despite normal or increased appetite; heat intolerance; increased sweating; dyspnea on exertion; amenorrhea and infertility; palpitations, tachycardia, cardiac arrhythmias, heart failure (especially in elderly); and muscle weakness, proximal myopathy, and osteoporosis (especially in elderly).The triad of Graves’ disease consists of hyperthyroidism, ophthalmopathy (exophthalmos, lid retraction, lid lag, corneal ulceration, impaired eye muscle function), and dermopathy (pretibial myxoedema).
Primary hyper-/hypothyroidism: Increased or decreased function of thyroid gland due to disease of thyroid itself and not due to increased or decreased levels of TRH or TSH.
Secondary hyper-/hypothyroidism: Increased or decreased function of thyroid gland due to increased or decreased levels of TSH.
Tertiary hypothyroidism: Decreased function of thyroid gland due to decreased function of hypothalamus.
Subclinical thyroid disease: A condition with abnormality of thyroid hormone levels in blood but without specific clinical manifestations of thyroid disease and without any history of thyroid dysfunction or therapy.
Subclinical hyperthyroidism: A condition with normal thyroid hormone levels but with low or undetectable TSH level.
Subclinical hypothyroidism: A condition with normal thyroxine and triiodothyronine level along with mildly elevated TSH level.
Box 863.2 Thyroid function tests in hyperthyroidism
Laboratory FeaturesIn most patients, free serum T3 and T4 are elevated. In T3 thyrotoxicosis (5% cases of thyrotoxicosis), serum T4 levels are normal while T3 is elevated. Serum TSH is low or undetectable (< 0.1 mU/L) (Box 863.2).Undetectable or low serum TSH along with normal levels of T3 and T4 is called as subclinical hyperthyroidism; subtle signs and symptoms of thyrotoxicosis may or may not be present. Subclinical hyperthyroidism is associated with risk of atrial fibrillation, osteoporosis, and progression to overt thyroid disease.Features of primary and secondary hyperthyroidism are compared in Table 863.2.Table 863.2 Differences between primary and secondary hyperthyroidism
– Serum TSH low or undetectable
– Raised total T4 and free T4.
- T3 toxicosis:
– Serum TSH undetectable
– Normal total T4 and free T4
– Raised T3
Parameter Primary hyperthyroidism Secondary hyperthyroidism 1. Serum TSH Low Normal or high 2. Serum free thyroxine High High 3. TSH receptor antibodies May be positive Negative 4. Causes Graves’ disease, toxic multinodular goiter, toxic adenoma Pituitary adenomaEvaluation of hyperthyroidism is presented in Figure 863.1.Figure 863.1 Evaluation of hyperthyroidism. TSH: thyroid stimulating hormone; FT4: free T4; FT3: free T3; TRAb: TSH receptor antibody; TRH: Thyrotropin releasing hormoneHypothyroidismHypothyroidism is a condition caused by deficiency of thyroid hormones. Causes of hypothyroidism are listed in Table 863.3. Primary hypothyroidism results from deficient thyroid hormone biosynthesis that is not due to disorders of hypothalamus or pituitary. Secondary hypothyroidism results from deficient secretion of TSH from pituitary. Deficient or loss of secretion of thyro-tropin releasing hormone from hypothalamus results in tertiary hypothyroidism. Secondary and tertiary hypothyroidism are much less common than primary. Plasma TSH is high in primary and low in secondary and tertiary hypothyroidism. Differences between primary and secondary hypothyroidism are shown in Table 863.4.Table 863.3 Causes of hypothyroidism
Table 863.4 Differences between primary and secondary hypothyroidism
- Primary hypothyroidism (Increased TSH)
• Iodine deficiency
• Hashimoto’s thyroiditis
• Exogenous goitrogens
• Iatrogenic: surgery, drugs, radiation
- Secondary hypothyroidism (Low TSH): Diseases of pituitary
- Tertiary hypothyroidism (Low TSH, Low TRH): Diseases of hypothalamus
Parameter Primary hypothyroidism Secondary hypothyroidism 1. Cause Hashimoto’s thyroiditis Pituitary disease 2. Serum TSH High Low 3. Thyrotropin releasing hormone stimulation test Exaggerated response No response 4. Antimicrosomal antibodies Present AbsentBox 863.3 Thyroid function tests in hypothyroidism
Clinical features of primary hypothyroidism are: lethargy, mild depression, disturbances in menstruation, weight gain, cold intolerance, dry skin, myopathy, constipation, and firm and lobulated thyroid gland (in Hashimoto’s thyroiditis).In severe cases, myxoedema coma (an advanced stage with stupor, hypoventilation, and hypothermia) can occur.Laboratory FeaturesLaboratory features in hypothyroidism are shown in Box 863.3.Normal serum thyroxine (T4 and FT4) coupled with a moderately raised TSH (>10 mU/L) is referred to as subclinical hypothyroidism. It is associated with bad obstetrical outcome, poor cognitive development in children, and high risk of hypercholesterolemia and progression to overt hypothyroidism.Evaluation of hypothyroidism is presented in Figure 863.2Figure 863.2 Evaluation of hypothyroidism. TSH: thyroid stimulating hormone; FT4: free T4; TRH: Thyrotropin releasing hormone
- Primary hypothyroidism
– Serum TSH: Increased (proportional to degree of hypofunction)
– Free T4: Decreased
– TRH stimulation test: Exaggerated response
- Secondary hypothyroidism
– Serum TSH: Decreased
– Free T4: Decreased
– TRH stimulation test: Absent response
- Tertiary hypothyroidism
– Serum TSH: Decreased
– FT4: Decreased
– TRH stimulation test: Delayed response
FEMALE INFERTILITY: CAUSES AND INVESTIGATIONSThe ovaries are the sites of production of female gametes or ova by the process of oogenesis. The ova are released by the process of ovulation in a cyclical manner at regular intervals. Ovary contains numerous follicles that contain ova in various stages of development. During each menstrual cycle, up to 20 primordial follicles are activated for maturation; however, only one follicle becomes fully mature; this dominant follicle ruptures to release the secondary oocyte from the ovary. Maturation of the follicle is stimulated by follicle stimulating hormone (FSH) secreted by anterior pituitary (Figure 862.1). Maturing follicle secretes estrogen that causes proliferation of endometrium of the uterus (proliferative phase). Follicular cells also secrete inhibin which regulates release of FSH by the anterior pituitary. Fall in FSH level is followed by secretion of luteinizing hormone (LH) by the anterior pituitary (LH surge). This causes follicle to rupture and the ovum is expelled into the peritoneal cavity near the fimbrial end of the fallopian tube. The fallopian tubes conduct ova from the ovaries to the uterus. Fertilization of ovum by the sperm occurs in the fallopian tube.Figure 862.1 The hypothalamus-pituitary-ovarian axisThe ovum consists of the secondary oocyte, zona pellucida and corona radiata. The ruptured follicle in the ovary collapses and fills with blood clot (corpus luteum). LH converts granulose cells in the follicle to lutein cells which begin to secrete progesterone. Progesterone stimulates secretion from the endometrial glands (secretory phase) that were earlier under the influence of estrogen. Rising progesterone levels inhibit LH production from the anterior pituitary. Without LH, the corpus luteum regresses and becomes functionless corpus albicans. After regression of corpus luteum, production of estrogen and progesterone stops and endometrium collapses, causing onset of menstruation. If the ovum is fertilized and implanted in the uterine wall, human chorionic gonadotropin (hCG) is secreted by the developing placenta into the maternal circulation. Human chorionic gonadotropin maintains the corpus luteum for secetion of estrogen and progesterone till 12th week of pregnancy. After 12th week, corpus luteum regresses to corpus albicans and the function of synthesis of estrogen and progesterone is taken over by placenta till parturition.The average duration of the normal menstrual cycle is 28 days. Ovulation occurs around 14th day of the cycle. The time interval between ovulation and menstruation is called as luteal phase and is fairly constant (14 days) (Figure 862.2).Figure 862.2 Normal menstrual cycleCauses of Female InfertilityCauses of female infertility are shown in Table 862.1.Table 862.1 Causes of female infertility1. Hypothalamic-pituitary dysfunction:2. Ovarian dysfunction:
- Polycystic ovarian disease (Stein-Leventhal syndrome)
- Luteinized unruptured follicle
- Turner’s syndrome
- Radiation or chemotherapy
- Surgical removal of ovaries
3. Dysfunction in passages: 4. Dysfunction of sexual act: DyspareuniaInvestigationsEvaluation of female infertility is shown in Figure 862.3.Figure 862.3 Evaluation of female infertility. FSH: Follicle stimulating hormone; LH: Luteinizing hormone; DHEA-S: Dihydroepiandrosterone; TSH: Thyroid stimulating hormone; ↑ : Increased; ↓ : DecreasedTests for OvulationMost common cause of female infertility is anovulation.
Figure 862.4 Ferning of cervical mucosaFigure 862.5 Serum progesterone during normal menstrual cycleTests to Determine the Cause of Anovulation
- Regular cycles, mastalgia, and laparoscopic direct visualization of corpus luteum indicate ovulatory cycles. Anovulatory cycles are clinically characterized by amenorrhea, oligomenorrhea, or irregular menstruation. However, apparently regular cycles may be associated with anovulation.
- Endometrial biopsy: Endometrial biopsy is done during premenstrual period (21st-23rd day of the cycle). The secretory endometrium during the later half of the cycle is an evidence of ovulation.
- Ultrasonography (USG): Serial ultrasonography is done from 10th day of the cycle and the size of the dominant follicle is measured. Size >18 mm is indicative of imminent ovulation. Collapse of the follicle with presence of few ml of fluid in the pouch of Douglas is suggestive of ovulation. USG also is helpful for treatment (i.e. timing of coitus or of intrauterine insemination) and diagnosis of luteinized unruptured follicle (absence of collapse of dominant follicle). Transvaginal USG is more sensitive than abdominal USG.
- Basal body temperature (BBT): Patient takes her oral temperature at the same time every morning before arising. BBT falls by about 0.5°F at the time of ovulation. During the second (progestational) half of the cycle, temperature is slightly raised above the preovulatory level (rise of 0.5° to 1°F). This is due to the slight pyrogenic action of progesterone and is therefore presumptive evidence of functional corpus luteum.
- Cervical mucus study:
• Fern test: During estrogenic phase, a characteristic pattern of fern formation is seen when cervical mucus is spread on a glass slide (Figure 862.4). This ferning disappears after the 21st day of the cycle. If previously observed, its disappearance is presumptive evidence of corpus luteum activity.
• Spinnbarkeit test: Cervical mucus is elastic and withstands stretching upto a distance of over 10 cm. This phenomenon is called Spinnbarkeit or the thread test for the estrogen activity. During the secretory phase, viscosity of the cervical mucus increases and it gets fractured when stretched. This change in cervical mucus is evidence of ovulation.
- Vaginal cytology: Karyopyknotic index (KI) is high during estrogenic phase, while it becomes low in secretory phase. This refers to percentage of super-ficial squamous cells with pyknotic nuclei to all mature squamous cells in a lateral vaginal wall smear. Usually minimum of 300 cells are evaluated. The peak KI usually corresponds with time of ovulation and may reach upto 50 to 85.
- Estimation of progesterone in mid-luteal phase (day 21 or 7 days before expected menstruation): Progesterone level > 10 nmol/L is a reliable evidence of ovulation if cycles are regular (Figure 862.5). A mistimed sample is a common cause of abnormal result.
Investigations to Assess Tubal and Uterine Status
- Measurement of LH, FSH, and estradiol during days 2 to 6: All values are low in hypogonadotropic hypogonadism (hypothalamic or pituitary failure).
- Measurement of TSH, prolactin, and testosterone if cycles are irregular or absent:
Increased TSH: Hypothyroidism
Increased prolactin: Pituitary adenoma
Increased testosterone: Polycystic ovarian disease (PCOD), congenital adrenal hyperplasia (To differentiate PCOD from congenital adrenal hyperplasia, ultrasound and estimation of dihydroepiandrosterone or DHEA are done).
- Transvaginal ultrasonography: This is done for detection of PCOD.
Possible pregnancy and active pelvic or vaginal infection are contraindications to tubal patency tests.Figure 862.6 Hysterosalpingography
- Infectious disease: These tests include endometrial biopsy for tuberculosis and test for chlamydial IgG antibodies for tubal factor in infertility.
- Hysterosalpingography (HSG): HSG is a radiological contrast study for investigation of the shape of the uterine cavity and for blockage of fallopian tubes (Figure 862.6). A catheter is introduced into the cervical canal and a radiocontrast dye is injected into the uterine cavity. A real time X-ray imaging is carried out to observe the flow of the dye into the uterine cavity, tubes, and spillage into the uterine cavity.
- Hysterosalpingo-contrast sonography: A catheter is introduced into the cervical canal and an echocontrast fluid is introduced into the uterine cavity. Shape of the uterine cavity, filling of fallopian tubes, and spillage of contrast fluid are noted. In addition, ultrasound scan of the pelvis provides information about any fibroids or polycystic ovarian disease.
- Laparoscopy and dye hydrotubation test with hysteroscopy: In this test, a cannula is inserted into the cervix and methylene blue dye is introduced into the uterine cavity. If tubes are patent, spillage of the dye is observed from the ends of both tubes. This technique also allows visualization of pelvic organs, endometriosis, and pelvic adhesions. If required, endometriosis and tubal blockage can be treated during the procedure.
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