FEMALE INFERTILITY: CAUSES AND INVESTIGATIONS

Published in Clinical Pathology
Friday, 22 September 2017 13:37
The 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 axis
Figure 862.1 The hypothalamus-pituitary-ovarian axis 
 
The 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 cycle
Figure 862.2 Normal menstrual cycle
 
Causes of Female Infertility
 
Causes of female infertility are shown in Table 862.1.
 
Table 862.1 Causes of female infertility
1. Hypothalamic-pituitary dysfunction:
  • Hypothalamic causes
    – Excessive exercise
    – Excess stress
    – Low weight
    – Kallman’s syndrome
    Idiopathic
  • Pituitary causes
    – Hyperprolactinemia
    Hypopituitarism (Sheehan’s syndrome, Simmond’s disease)
    – Craniopharyngioma
    – Cerebral irradiation
 2. Ovarian dysfunction:
  • Polycystic ovarian disease (Stein-Leventhal syndrome)
  • Luteinized unruptured follicle
  • Turner’s syndrome
  • Radiation or chemotherapy
  • Surgical removal of ovaries
  • Idiopathic
 3. Dysfunction in passages:
  • Fallopian tubes
    Infections: Tuberculosis, gonorrhea, Chlamydia
    – Previous surgery (e.g. laparotomy)
    – Tubectomy
    Congenital hypoplasia, non-canalization
    Endometriosis
  • Uterus
    – Uterine malformations
    – Asherman’s syndrome
    – Tuberculous endometritis
    Fibroid
  • Cervix: Sperm antibodies
  • Vagina: Septum
 4. Dysfunction of sexual act: Dyspareunia
 
Investigations
 
Evaluation of female infertility is shown in Figure 862.3.
 
Figure 862.3 Evaluation of female infertility
Figure 862.3 Evaluation of female infertility. FSH: Follicle stimulating hormone; LH: Luteinizing hormone; DHEA-S: Dihydroepiandrosterone; TSH: Thyroid stimulating hormone; ↑ : Increased; ↓ : Decreased
 
Tests for Ovulation
 
Most common cause of female infertility is anovulation.
 
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
 
Figure 862.4 Ferning of cervical mucosa
Figure 862.4 Ferning of cervical mucosa
 
Figure 862.5 Serum progesterone during normal menstrual cycle
Figure 862.5 Serum progesterone during normal menstrual cycle
 
Tests to Determine the Cause of Anovulation
 
  1. Measurement of LH, FSH, and estradiol during days 2 to 6: All values are low in hypogonadotropic hypogonadism (hypothalamic or pituitary failure).
  2. 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).
  3. Transvaginal ultrasonography: This is done for detection of PCOD.
 
Investigations to Assess Tubal and Uterine Status
 
  1. Infectious disease: These tests include endometrial biopsy for tuberculosis and test for chlamydial IgG antibodies for tubal factor in infertility.
  2. 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.
  3. 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.
  4. 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.
 
Possible pregnancy and active pelvic or vaginal infection are contraindications to tubal patency tests.
 
Figure 862.6 Hysterosalpingography
Figure 862.6 Hysterosalpingography

MALE INFERTILITY: CAUSES AND INVESTIGATIONS

Published in Clinical Pathology
Friday, 22 September 2017 00:03
The male reproductive system consists of testes (paired organs located in the scrotal sac that produce spermatozoa and secrete testosterone), a paired system of ducts comprising of epididymis, vasa deferentia, and ejaculatory ducts (collect, store, and conduct spermatozoa), paired seminal vesicles and a single prostate gland (produce nutritive and lubricating seminal fluid), bulbourethral glands of Cowper (secrete lubricating mucus), and penis (organ of copulation).
 
The hypothalamus secretes gonadotropin releasing hormone (GnRH) that regulates the secretion of the two gonadotropins from the anterior pituitary: luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Figure 861.1). Luteinizing hormone primarily stimulates the production and secretion of testosterone from Leydig cells located in the interstitial tissue of the testes. Testosterone stimulates spermatogenesis, and plays a role in the development of secondary sexual characters. Testosterone needs to be converted to an important steroidal metabolite, dihydrotestosterone within cells to perform most of its androgenic functions. Testosterone inhibits LH secretion by negative feedback. Follicle stimulating hormone acts on Sertoli cells of seminiferous tubules to regulate the normal maturation of the sperms. Sertoli cells produce inhibin that controls FSH secretion by negative feedback.
 
Figure 861.1 Hypothalamus-pituitary-testis axis. + indicates stimulation; – indicates negative feedback
Figure 861.1 Hypothalamus-pituitary-testis axis. + indicates stimulation; – indicates negative feedback
 
During sexual intercourse, semen is deposited into the vagina. Liquefaction of semen occurs within 20-30 minutes due to proteolytic enzymes of prostatic fluid. For fertilization to occur in vivo, the sperm must undergo capacitation and acrosome reaction. Capacitation refers to physiologic changes in sperms that occur during their passage through the cervix of the female genital tract. With capacitation, the sperm acquires (i) ability to undergo acrosome reaction, (ii) ability to bind to zona pellucida, and (iii) hypermotility. Sperm then travels through the cervix and uterus up to the fallopian tube. Binding of sperm to zona pellucida induces acrosomal reaction (breakdown of outer plasma membrane by enzymes of acrosome and its fusion with outer acrosomal membrane, i.e. loss of acrosome). This is necessary for fusion of sperm and oocyte membranes. Acrosomal reaction and binding of sperm and ovum surface proteins is followed by penetration of zona pellucida of ovum by the sperm. Following penetration by sperm, hardening of zona pellucida occurs that inhibits penetration by additional sperms. A sperm penetrates and fertilizes the egg in the ampullary portion of the fallopian tube (Figure 861.2).
 
Figure 861.2 Steps before and after fertilization of ovum
Figure 861.2 Steps before and after fertilization of ovum
 
Causes of Male Infertility
 
Causes of male infertility are listed in Table 861.1.
 
Table 861.1 Causes of male infertility 
2. Hypothalamic-pituitary dysfunction (hypogonadotropic hypogonadism)
3. Testicular dysfunction:
  • Radiation, cytotoxic drugs, antihypertensives, antidepressants
  • General factors like stress, emotional factors, drugs like marijuana, anabolic steroids, and cocaine, alcoholism, heavy smoking, undernutrition
  • Mumps orchitis after puberty
  • Varicocele (dilatation of pampiniform plexus of scrotal veins)
  • Undescended testes (cryptorchidism)
  • Endocrine disorders like diabetes mellitus, thyroid dysfunction
  • Genetic disorders: Klinefelter’s syndrome, microdeletions in Y chromosome, autosomal Robertsonian translocation, immotile cilia syndrome (Kartagener’s syndrome), cystic fibrosis, androgen receptor gene defect
4. Dysfunction of passages and accessory sex glands:
 5. Dysfunction of sexual act:
  • Defects in ejaculation: retrograde (semen is pumped backwards in to the bladder), premature, or absent
  • Hypospadias
 
Investigations of Male Infertility
 
  1. History: This includes type of lifestyle (heavy smoking, alcoholism), sexual practice, erectile dysfunction, ejaculation, sexually transmitted diseases, surgery in genital area, drugs, and any systemic illness.
  2. Physical examination: Examination of reproductive system should includes testicular size, undescended testes, hypospadias, scrotal abnormalities (like varicocele), body hair, and facial hair. Varicocele can occur bilaterally and is the most common surgically removable abnormality causing male infertility.
  3. Semen analysis: See article Semen Analysis. Evaluation of azoospermia is shown in Figure 861.3. Evaluation of low semen volume is shown in Figure 861.4.
  4. Chromosomal analysis: This can reveal Klinefelter’s syndrome (e.g. XXY karyotype) (Figure 861.5), deletion in Y chromosome, and autosomal Robertsonian translocation. It is necessary to screen for cystic fibrosis carrier state if bilateral congenital absence of vas deferens is present.
  5. Hormonal studies: This includes measurement of FSH, LH, and testosterone to detect hormonal abnormalities causing testicular failure (Table 861.2).
  6. Testicular biopsy: Testicular biopsy is indicated when differentiation between obstructive and non-obstructive azoospermia is not evident (i.e. normal FSH and normal testicular volume).
 
Table 861.2 Interpretation of hormonal studies in male infertility 
Follicle stimulating hormone Luteinizing hormone Testosterone Interpretation
Low Low Low Hypogonadotropic hypogonadism (Hypothalamic or pituitary disorder)
High High Low Hypergonadotropic hypogonadism (Testicular disorder)
Normal Normal Normal Obstruction of passages, dysfunction of accessory glands
 
Figure 861.3 Evaluation of azoospermia
Figure 861.3 Evaluation of azoospermia. FSH: Follicle stimulating hormone; LH: Luteinizing hormone
 
Figure 861.4 Evaluation of low semen volume
Figure 861.4 Evaluation of low semen volume
 
Figure 861.5 Karyotype in Klinefelter's Syndrome
 Figure 861.5 Karyotype in Klinefelter’s syndrome (47, XXY)
 
Common initial investigations for diagnosis of cause of infertility are listed below.
 

CONTRAINDICATIONS TO GASTRIC ANALYSIS

Published in Clinical Pathology
Thursday, 07 September 2017 23:53
  • Gastric intubation for gastric analysis is contraindicated in esophageal stricture or varices, active nasopharyngeal disease, diverticula, malignancy, recent history of severe gastric hemorrhage, hypertension, aortic aneurysm, cardiac arrhythmias, congestive cardiac failure, or non-cooperative patient.
  • Pyloric stenosis: Obstruction of gastric outlet can elevate gastric acid output due to raised gastrin (following antral distension).
  • Pentagastrin stimulation is contraindicated in cases with allergy to pentagastrin, and recent severe gastric hemorrhge due to peptic ulcer disease.
 
Gastric analysis is not a commonly performed procedure because of following reasons:
 
  • It is an invasive and cumbersome technique that is traumatic and unpleasant for the patient.
  • Information obtained is not diagnostic in itself.
  • Availability of better tests for diagnosis such as endoscopy and radiology (for suspected peptic ulcer or malignancy); serum gastrin estimation (for ZE syndrome); vitamin assays, Schilling test, and antiparietal cell antibodies (for pernicious anemia); and tests for Helicobacter pylori infection (in duodenal or gastric ulcer).
  • Availability of better medical line of treatment that obviates need for surgery in many patients.

LABORATORY TESTS FOR GASTRIC ANALYSIS

Published in Clinical Pathology
Thursday, 07 September 2017 23:53
  1. Hollander’s test (Insulin hypoglycemia test): In the past, this test was used for confirmation of completeness of vagotomy (done for duodenal ulcer).

    Hypoglycemia is a potent stimulus for gastric acid secretion and is mediated by vagus nerve. This response is abolished by vagotomy.

    In this test, after determining BAO, insulin is administered intravenously (0.15-0.2 units/kg) and acid output is estimated every 15 minutes for 2 hours (8 post-stimulation samples). Vagotomy is considered as complete if, after insulin-induced hypoglycemia (blood glucose < 45 mg/dl), no acid output is observed within 45 minutres.

    The test gives reliable results only if blood glucose level falls below 50 mg/dl at some time following insulin injection. It is best carried out after 3-6 months of vagotomy.

    The test is no longer recommended because of the risk associated with hypoglycemia. Myocardial infarction, shock, and death have also been reported.

  2. Fractional test meal: In the past, test meals (e.g. oat meal gruel, alcohol) were administered orally to stimulate gastric secretion and determine MAO or PAO. Currently, parenteral pentagastrin is the gastric stimulant of choice.

  3. Tubeless gastric analysis: This is an indirect and rapid method for determining output of free hydrochloric acid in gastric juice. In this test, a cationexchange resin tagged to a dye (azure A) is orally administered. In the stomach, the dye is displaced from the resin by the free hydrogen ions of the hydrochloric acid. The displaced azure A is absorbed in the small intestine, enters the bloodstream, and is excreted in urine. Urinary concentration of the dye is measured photometrically or by visual comparison with known color standards. The quantity of the dye excreted is proportional to the gastric acid output. However, if kidney or liver function is impaired, false results may be obtained. The test is no longer in use.

  4. Spot check of gastric pH: According to some investigators, spot determination of pH of fasting gastric juice (obtained by nasogastric intubation) can detect the presence of hypochlorhydria (if pH>5.0 in men or >7.0 in women).

  5. Congo red test during esophagogastroduodenoscopy: This test is done to determine the completeness of vagotomy. Congo red dye is sprayed into the stomach during esophagogastroduodenoscopy; if it turns red, it indicates presence of functional parietal cells in stomach with capacity of producing acid.
 
REFERENCE RANGES
 
  • Volume of gastric juice: 20-100 ml
  • Appearance: Clear
  • pH: 1.5 to 3.5
  • Basal acid output: Up to 5 mEq/hour
  • Peak acid output: 1 to 20 mEq/hour
  • Ratio of basal acid output to peak acid output: <0.20 or < 20%

INDICATIONS FOR GASTRIC ANALYSIS

Published in Clinical Pathology
Thursday, 07 September 2017 23:18
In gastric analysis, amount of acid secreted by the stomach is determined on aspirated gastric juice sample. Gastric acid output is estimated before and after stimulation of parietal cells (i.e. basal and peak acid output). This test was introduced in the past mainly for the evaluation of peptic ulcer disease (to assess the need for operative intervention). However, diminishing frequency of peptic ulcer disease and availability of safe and effective medical treatment have markedly reduced the role of surgery.
 
  1. To determine the cause of recurrent peptic ulcer disease:
    To detect Zollinger-Ellison (ZE) syndrome: ZE syndrome is a rare disorder in which multiple mucosal ulcers develop in the stomach, duodenum, and upper jejunum due to gross hypersecretion of acid in the stomach. The cause of excess secretion of acid is a gastrin-producing tumor of pancreas. Gastric analysis is done to detect markedly increased basal and pentagastrinstimulated gastric acid output for diagnosis of ZE syndrome (and also to determine response to acidsuppressant therapy). However, a more sensitive and specific test for diagnosis of ZE syndrome is measurement of serum gastrin (fasting and secretin-stimulated).
    To decide about completeness of vagotomy following surgery for peptic ulcer disease: See Hollander’s test.
  2. To determine the cause of raised fasting serum gastrin level: Hypergastrinemia can occur in achlorhydria, Zollinger-Ellison syndrome, and antral G cell hyperplasia.
  3. To support the diagnosis of pernicious anemia (PA): Pernicious anemia is caused by defective absorption of vitamin B12 due to failure of synthesis of intrinsic factor secondary to gastric mucosal atrophy. There is also absence of hydrochloric acid in the gastric juice (achlorhydria). Gastric analysis is done for demonstration of achlorhydria if facilities for vitamin assays and Schilling’s test are not available (Achlorhydria by itself is insufficient for diagnosis of PA).
  4. To distinguish between benign and malignant ulcer: Hypersecretion of acid is a feature of duodenal peptic ulcer, while failure of acid secretion (achlorhydria) occurs in gastric carcinoma. However, anacidity occurs only in a small proportion of cases with advanced gastric cancer. Also, not all patients with duodenal ulcer show increased acid output.
  5. To measure the amount of acid secreted in a patient with symptoms of peptic ulcer dyspepsia but normal X-ray findings: Excess acid secretion in such cases is indicative of duodenal ulcer. However, hypersecretion of acid does not always occur in duodenal ulcer.
  6. To decide the type of surgery to be performed in a patient with peptic ulcer: Raised basal as well as peak acid outputs indicate increased parietal cell mass and need for gastrectomy. Raised basal acid output with normal peak output is an indication for vagotomy.

METHOD OF GASTRIC ANALYSIS

Published in Clinical Pathology
Tuesday, 05 September 2017 23:51
To assess gastric acid secretion, acid output from the stomach is measured in a fasting state and after injection of a drug which stimulates gastric acid secretion.
 
Basal acid output (BAO) is the amount of hydrochloric acid (HCl) secreted in the absence of any external stimuli (visual, olfactory, or auditory).
 
Maximum acid output (MAO) is the amount of hydrochloric acid secreted by the stomach following stimulation by pentagastrin. MAO is calculated from the first four 15-minute samples after stimulation.
 
Peak acid output (PAO) is calculated from the two highest consecutive 15-minute samples. It indicates greatest possible acid secretory capacity and is preferred over MAO as it is more reproducible.
 
Acidity is estimated by titration.
 
Collection of Sample
 
All drugs that affect gastric acid secretion (e.g. antacids, anticholinergics, cholinergics, H2-receptor antagonists, antihistamines, tranquilizers, antidepressants, and carbonic anhydrase inhibitors) should be withheld for 24 hours before the test. Proton pump inhibitors should be discontinued 5 days prior to the test. Patient should be relaxed and free from all sources of sensory stimulation.
 
Patient should drink or eat nothing after midnight.
 
Gastric juice can be aspirated through an oral or nasogastric tube (polyvinyl chloride, silicone, or polyurethane) or during endoscopy.
 
Oral or nasogastric tube (Figure 855.1) is commonly used. It is a flexible tube having a small diameter and a bulbous end that is made heavy by a small weight of lead. The end is perforated with small holes to allow entry of gastric juice into the tube. As the end is radiopaque, the tube can be positioned in the most dependent part of the stomach under fluoroscopic or X-ray guidance. The tube is lubricated and can be introduced either through the mouth or the nose. The patient is either sitting or reclining on left side. The tube has three or four markings on its outer surface that correspond with distance of the tip of the tube from the teeth, i.e. 40 cm (tip to cardioesophageal junction), 50 cm (body of stomach), 57 cm (pyloric antrum), and 65 cm (duodenum). The position of the tube can be verified either by fluoroscope or by ‘water recovery test’. In the latter test, 50 ml of water is introduced through the tube and aspirated again; recovery of > 90% of water is indicative of proper placement. The tube is usually positioned in the antrum. A syringe is attached to the outer end of the tube for the aspiration of gastric juice.
 
Figure 855.1 Oral or nasogastric Ryles tube
Figure 855.1 Oral or nasogastric Ryle’s tube. The tube is marked at 40, 50, 57, and 65 cm with radiopaque lines for accurate placement. The tip is bulbous and contains a small weight of lead to assist the passage during intubation and to know the position under fluoroscopy or X-ray guidance. There are four perforations or eyes to aspirate contents from the stomach through a syringe attached to the base
 
For estimation of BAO, sample is collected in the morning after 12-hour overnight fast. Gastric secretion that has accumulated overnight is aspirated and discarded. This is followed by aspiration of gastric secretions at 15-minute intervals for 1 hour (i.e. total 4 consecutive samples are collected). All the samples are centrifuged to remove any particulate matter. Each 15-minute sample is analyzed for volume, pH, and acidity. The acid output in the four samples is totaled and the result is expressed as concentration of acid in milliequivalents per hour or in mmol per hour.
 
After the collection of gastric juice for determination of BAO, patient is given a subcutaneous or intramuscular injection of pentagastrin (6 μg/kg of body weight), and immediately afterwards, gastric secretions are aspirated at 15-minute intervals for 1 hour (for estimation of MAO or PAO). MAO is calculated from the first four 15-minute samples after stimulation. PAO is calculated from two consecutive 15-minute samples showing highest acidity.
 
Titration
 
Box 855.1 Determination of basal acid output, maximum acid output, and peak acid output
 
  • Basal acid output (BAO)= Total acid content in all four 15-minute basal samples in mEq/L
  • Maximum acid output (MAO) = Total acid content in all four 15-minute post-pentagastrin samples in mEq/L
  • Peak acid output (PAO) = Sum of two consecutive 15-minute post-pentagastrin samples showing highest acidity ×2 (mEq/L)
Gastric acidity is estimated by titration, with the end point being determined either by noting the change in color of the indicator solution or till the desired pH is reached.
 
In titration, a solution of alkali (0.1 N sodium hydroxide) is added from a graduated vessel (burette) to a known volume of acid (i.e. gastric juice) till the end point or equivalence point of reaction is reached. The concentration of acid is then determined from the concentration and volume of alkali required to neutralize the particular volume of gastric juice. Concentration of acid is expressed in terms of milliequivalents per liter or mmol per liter.
 
Free acidity refers to the concentration of HCl present in a free, uncombined form in a solution. The volume of alkali added to the gastric juice till the Topfer’s reagent (an indicator added earlier to the gastric juice) changes color or when the pH (as measured by the pH meter) reaches 3.5 is a measure of free acidity. A screening test can be carried out for the presence of free HCl in the gastric juice. If red color develops after addition of a drop of Topfer’s reagent to an aliquot of gastric juice, free HCl is present and the diagnosis of pernicious anaemia (achlorhydria) can be excluded.
 
Combined acidity refers to the amount of HCl combined with proteins and mucin and also includes small amount of weak acids present in gastric juice.
 
Total acidity is the sum of free and combined acidity. The amount of alkali added to the gastric juice till phenolphthalein indicator (added earlier to the gastric juice) changes color is a measure of total acidity (Box 855.1).
 
Interpretation of Results
 
  1. Volume: Normal total volume is 20-100 ml (usually < 50 ml). Causes of increased volume of gastric juice are—
    • Delayed emptying of stomach: pyloric stenosis
    • Increased gastric secretion: duodenal ulcer, Zollinger-Ellison syndrome.
  2. Color: Normal gastric secretion is colorless, with a faintly pungent odor. Fresh blood (due to trauma, or recent bleeding from ulcer or cancer) is red in color. Old hemorrhage produces a brown, coffee-ground like appearance (due to formation of acid hematin). Bile regurgitation produces a yellow or green color.
  3. pH: Normal pH is 1.5 to 3.5. In pernicious anemia, pH is greater than 7.0 due to absence of HCl.
  4. Basal acid output:
    • Normal: Up to 5 mEq/hour.
    • Duodenal ulcer: 5-15 mEq/hour.
    • Zollinger-Ellison syndrome: >20 mEq/hour.
    Normal BAO is seen in gastric ulcer and in some patients with duodenal ulcer.
  5. Peak acid output:
    • Normal: 1-20 mEq/hour.
    • Duodenal ulcer: 20-60 mEq/hour.
    • Zollinger-Ellison syndrome: > 60 mEq/hour.
    • Achlorhydria: 0 mEq/hour.
    Normal PAO is seen in gastric ulcer and gastric carcinoma. Values up to 60 mEq/hour can occur in some normal individuals and in some patients with Zollinger-Ellison syndrome.
    In pernicious anemia, there is no acid output due to gastric mucosal atrophy. Achlorhydria should be diagnosed only if there is no free HCl even after maximum stimulation.
  6. Ratio of basal acid output to peak acid output (BAO/PAO):
    • Normal: < 0.20 (or < 20%).
    • Gastric or duodenal ulcer: 0.20-0.40 (20-40%).
    • Duodenal ulcer: 0.40-0.60 (40-60%).
    • Zollinger-Ellison syndrome: > 0.60 (> 60%).
    Normal values occur in gastric ulcer or gastric carcinoma.
 
Conditions associated with change in gastric acid output are listed in Table 855.1.
 
It is to be noted that values of acid output are not diagnostic by themselves and should be correlated with clinical, radiological, and endoscopic features.
 
Table 855.1 Causes of alterations in gastric acid output
Increased gastric acid output Decreased gastric acid output
• Duodenal ulcer Chronic atrophic gastritis
• Zollinger-Ellison syndrome     1. Pernicious anemia
Hyperplasia of antral G cells     2. Rheumatoid arthritis
Systemic mastocytosis     3. Thyrotoxicosis
• Basophilic leukemia • Gastric ulcer
  • Gastric carcinoma
  • Chronic renal failure
  • Post-vagotomy
  • Post-antrectomy

MICROSCOPIC EXAMINATION OF FECES

Published in Clinical Pathology
Wednesday, 30 August 2017 23:26
Microscopic examinations done on fecal sample are shown in Figure 846.1.
 
Figure 846.1 Microscopic examinations carried out on fecal sample
Figure 846.1 Microscopic examinations carried out on fecal sample
 
Collection of Specimen for Parasites
 
A random specimen of stool (at least 4 ml or 4 cm³) is collected in a clean, dry, container with a tightly fitting lid (a tin box, plastic box, glass jar, or waxed cardboard box) and transported immediately to the laboratory (this is because trophozoites of Entameba histolytica rapidly degenerate and alter in morphology). About 20-40 grams of formed stool or 5-6 tablespoons of watery stool should be collected. Stool should not be contaminated with urine, water, soil, or menstrual blood. Urine and water destroy trophozoites; soil will introduce extraneous organisms and also hinder proper examination. Parasites are best detected in warm, freshly passed stools and therefore stools should be examined as early as possible after receipt in the laboratory (preferably within 1 hour of collection). If delay in examination is anticipated, sample may be refrigerated. A fixative containing 10% formalin (for preservation of eggs, larvae, and cysts) or polyvinyl alcohol (for preservation of trophozoites and cysts, and for permanent staining) may be used if specimen is to be transported to a distant laboratory.
 
One negative report for ova and parasites does not exclude the possibility of infection. Three separate samples, collected at 3-day intervals, have been recommended to detect all parasite infections.
 
Patient should not be receiving oily laxatives, antidiarrheal medications, bismuth, antibiotics like tetracycline, or antacids for 7 days before stool examination. Patient should not have undergone a barium swallow examination.
 
In the laboratory, macroscopic examination is done for consistency (watery, loose, soft or formed) (Figure 846.2), color, odor, and presence of blood, mucus, adult worms or segments of tapeworms.
 
Figure 846.2 Consistency of feces
Figure 846.2 Consistency of feces
 
Trophozoites are most likely to be found in loose or watery stools or in stools containing blood and mucus, while cysts are likely to be found in formed stools. Trophozoites die soon after being passed and therefore such stools should be examined within 1 hour of passing. Examination of formed stools can be delayed but should be completed on the same day.
 
Color/Appearance of Fecal Specimens
 
  • Brown: Normal
  • Black: Bleeding in upper gastrointestinal tract (proximal to cecum), Drugs (iron salts, bismuth salts, charcoal)
  • Red: Bleeeding in large intestine, undigested tomatoes or beets
  • Clay-colored (gray-white): Biliary obstruction
  • Silvery: Carcinoma of ampulla of Vater
  • Watery: Certain strains of Escherichia coli, Rotavirus enteritis, cryptosporidiosis
  • Rice water: Cholera
  • Unformed with blood and mucus: Amebiasis, inflammatory bowel disease
  • Unformed with blood, mucus, and pus: Bacillary dysentery
  • Unformed, frothy, foul smelling, which float on water: Steatorrhea.
 
Preparation of Slides
 
After receipt in the laboratory, saline and iodine wet mounts of the sample are prepared (Figure 846.3).
 
Figure 846.3 Saline and iodine wet mounts of fecal sample
Figure 846.3 Saline and iodine wet mounts of fecal sample 
 
A drop of normal saline is placed near one end of a glass slide and a drop of Lugol iodine solution is placed near the other end. A small amount of feces (about the size of a match-head) is mixed with a drop each of saline and iodine using a wire loop, and a cover slip is placed over each preparation separately. If the specimen contains blood or mucus, that portion should be included for examination (trophozoites are more readily found in mucus). If the stools are liquid, select the portion from the surface for examination.
 
Saline wet mount is used for demonstration of eggs and larvae of helminths, and trophozoites and cysts of protozoa. It can also detect red cells and white cells. Iodine stains glycogen and nuclei of the cysts. The iodine wet mount is useful for identification of protozoal cysts. Trophozoites become non-motile in iodine mounts. A liquid, diarrheal stool can be examined directly without adding saline.
 
Concentration Procedure
 
Concentration of fecal specimen is useful if very small numbers of parasites are present. However, in concentrated specimens, amebic trophozoites can no longer be detected since they are destroyed. If wet mount examination is negative and there is clinical suspicion of parasitic infection, fecal concentration is indicated. It is used for detection of ova, cysts, and larvae of parasites.
 
Various concentration methods are available; the choice depends on the nature of parasites to be identified and the equipment/reagent available in a particular laboratory. Concentration techniques are of two main types:
 
  • Sedimentation techniques: Ova and cysts settle at the bottom. However, excessive fecal debris may make the detection of parasites difficult. Example: Formolethyl acetate sedimentation procedure.
  • Floatation techniques: Ova and cysts float on surface. However, some ova and cysts do not float at the top in this procedure. Examples: Saturated salt floatation technique and zinc sulphate concentration technique.
 
The most commonly used sedimentation method is formol-ethyl acetate concentration method since: (i) it can detect eggs and larvae of almost all helminths, and cysts of protozoa, (ii) it preserves their morphology well, (iii) it is rapid, and (iv) risk of infection to the laboratory worker is minimal because pathogens are killed by formalin.
 
In this method, fecal suspension is prepared in 10% formalin (10 ml formalin + 1 gram feces). This suspension is then passed through a gauze filter till 7 ml of filtered material is obtained. To this, ethyl acetate (3 ml) is added and the mixture is centrifuged for 1 minute. Eggs, larvae, and cysts sediment at the bottom of the centrifuge tube (Figure 846.4). Above this deposit, there are layers of formalin, fecal debris, and ether. Fecal debris is loosened with an applicator stick and the supernatant is poured off. One drop of sediment is placed on one end of a glass slide and one drop is placed at the other end. One of the drops is stained with iodine, cover slips are placed, and the preparation is examined under the microscope.
 
Figure 846.4 Formol ethyl acetate concentration technique
Figure 846.4 Formol-ethyl acetate concentration technique
 
Classification of Intestinal Parasites of Humans
 
Intestinal parasites of humans are classified into two main kingdoms: protozoa and metazoa (helminths) (Figure 846.5).
 
Figure 846.5 Classification of intestinal parasites of humans
Figure 846.5 Classification of intestinal parasites of humans

CHEMICAL EXAMINATION OF FECES

Published in Clinical Pathology
Wednesday, 30 August 2017 01:21
Chemical examination of feces is usually carried out for the following tests (Figure 845.1):
 
  • Occult blood
  • Excess fat excretion (malabsorption)
  • Urobilinogen
  • Reducing sugars
  • Fecal osmotic gap
  • Fecal pH
 
Figure 845.17 Chemical examinations done on fecal sample
Figure 845.1 Chemical examinations done on fecal sample
 
Test for Occult Blood in Stools
 
Presence of blood in feces which is not apparent on gross inspection and which can be detected only by chemical tests is called as occult blood. Causes of occult blood in stools are:
 
  1. Intestinal diseases: hookworms, amebiasis, typhoid fever, ulcerative colitis, intussusception, adenoma, cancer of colon or rectum.
  2. Gastric and esophageal diseases: peptic ulcer, gastritis, esophageal varices, hiatus hernia.
  3. Systemic disorders: bleeding diathesis, uremia.
  4. Long distance runners.
 
Occult blood test is recommended as a screening procedure for detection of asymptomatic colorectal cancer. Yearly examinations should be carried out after the age of 50 years. If the test is positive, endoscopy and barium enema are indicated.
 
Tests for detection of occult blood in feces: Many tests are available which differ in their specificity and sensitivity. These tests include tests based on peroxidase-like activity of hemoglobin (benzidine, orthotolidine, aminophenazone, guaiac), immunochemical tests, and radioisotope tests.
 
Tests Based on Peroxidase-like Activity of Hemoglobin
 
Principle: Hemoglobin has peroxidase-like activity and releases oxygen from hydrogen peroxide. Oxygen molecule then oxidizes the chemical reagent (benzidine, orthotolidine, aminophenazone, or guaiac) to produce a colored reaction product.
 
Benzidine and orthotolidine are carcinogenic and are no longer used. Benzidine test is also highly sensitive and false-positive reactions are common. Since bleeding from the lesion may be intermittent, repeated testing may be required.
 
Causes of False-positive Tests
 
  1. Ingestion of peroxidase-containing foods like red meat, fish, poultry, turnips, horseradish, cauliflower, spinach, or cucumber. Diet should be free from peroxidase-containing foods for at least 3 days prior to testing.
  2. Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons.
 
Causes of False-negative Tests
 
  1. Foods containing large amounts of vitamin C.
  2. Conversion of all hemoglobin to acid hematin (which has no peroxidase-like activity) during passage through the gastrointestinal tract.
 
Immunochemical Tests
 
These tests specifically detect human hemoglobin. Therefore there is no interference from animal hemoglobin or myoglobin (e.g. meat) or peroxidase-containing vegetables in the diet.
 
The test consists of mixing the sample with latex particles coated with anti-human haemoglobin antibody, and if agglutination occurs, test is positive. This test can detect 0.6 ml of blood per 100 grams of feces.
 
Radioisotope Test Using 51Cr
 
In this test, 10 ml of patient’s blood is withdrawn, labeled with 51Cr, and re-infused intravenously. Radioactivity is measured in fecal sample and in simultaneously collected blood specimen. Radioactivity in feces indicates gastrointestinal bleeding. Amount of blood loss can be calculated. Although the test is sensitive, it is not suitable for routine screening.
 
Apt test: This test is done to decide whether blood in the vomitus or in the feces of a neonate represents swallowed maternal blood or is the result of bleeding in the gastrointestinal tract. The test was devised by Dr. Apt and hence the name. The baby swallows blood during delivery or during breastfeeding if nipples are cracked. Apt test is based on the principle that if blood is of neonatal origin it will contain high proportion of hemoglobin F (Hb F) that is resistant to alkali denaturation. On the other hand, maternal blood mostly contains adult hemoglobin or Hb A that is less resistant.
 
Test for Malabsorption of Fat
 
Dietary fat is absorbed in the small intestine with the help of bile salts and pancreatic lipase. Fecal fat mainly consists of neutral fats (unsplit fats), fatty acids, and soaps (fatty acid salts). Normally very little fat is excreted in feces (<7 grams/day in adults). Excess excretion of fecal fat indicates malabsorption and is known as steatorrhea. It manifests as bulky, frothy, and foul-smelling stools, which float on the surface of water.
 
Causes of Malabsorption of Fat
 
  1. Deficiency of pancreatic lipase (insufficient lipolysis): chronic pancreatitis, cystic fibrosis.
  2. Deficiency of bile salts (insufficient emulsification of fat): biliary obstruction, severe liver disease, bile salt deconjugation due to bacterial overgrowth in the small intestine.
  3. Diseases of small intestine: tropical sprue, celiac disease, Whipple’s disease.
 
Tests for fecal fat are qualitative (i.e. direct microscopic examination after fat staining), and quantitative (i.e. estimation of fat by gravimetric or titrimetric analysis).
 
  1. Microscopic stool examination after staining for fat: A random specimen of stool is collected after putting the patient on a diet of >80 gm fat per day. Stool sample is stained with a fat stain (oil red O, Sudan III, or Sudan IV) and observed under the microscope for fat globules (Figure 845.2). Presence of ≥60 fat droplets/HPF indicates steatorrhea. Ingestion of mineral or castor oil and use of rectal suppositories can cause problems in interpretation.
  2. Quantitative estimation of fecal fat: The definitive test for diagnosis of fat malabsorption is quantitation of fecal fat. Patient should be on a diet of 70-100 gm of fat per day for 6 days before the test. Feces are collected over 72 hours and stored in a refrigerator during the collection period. Specimen should not be contaminated with urine. Fat quantitation can be done by gravimetric or titrimetric method. In gravimetric method, an accurately weighed sample of feces is emulsified, acidified, and fat is extracted in a solvent; after evaporation of solvent, fat is weighed as a pure compound. Titrimetric analysis is the most widely used method. An accurately weighed stool sample is treated with alcoholic potassium hydroxide to convert fat into soaps. Soaps are then converted to fatty acids by the addition of hydrochloric acid. Fatty acids are extracted in a solvent and the solvent is evaporated. The solution of fat made in neutral alcohol is then titrated against sodium hydroxide. Fatty acids comprise about 80% of fecal fat. Values >7 grams/day are usually abnormal. Values >14 grams/day are specific for diseases causing fat malabsorption.
 
Figure 845.2 Sudan stain on fecal sample
Figure 845.2 Sudan stain on fecal sample: (A) Negative; (B) Positive
 
Test for Urobilinogen in Feces
 
Fecal urobilinogen is determined by Ehrlich’s aldehyde test (see  Article “Test for Detection of Urobilinogen in Urine). Specimen should be fresh and kept protected from light. Normal amount of urobilinogen excreted in feces is 50-300 mg per day. Increased fecal excretion of urobilinogen is seen in hemolytic anemia. Urobilinogen is deceased in biliary tract obstruction, severe liver disease, oral antibiotic therapy (disturbance of intestinal bacterial flora), and aplastic anemia (low hemoglobin turnover). Stools become pale or clay-colored if urobilinogen is reduced or absent.
 
Test for Reducing Sugars
 
Deficiency of intestinal enzyme lactase is a common cause of malabsorption. Lactase converts lactose (in milk) to glucose and galactose. If lactase is deficient, lactose is converted to lactic acid with production of gas. In infants this leads to diarrhea, vomiting, and failure to thrive. Benedict’s test or Clinitest™ tablet test for reducing sugars is used to test freshly collected stool sample for lactose. In addition, oral lactose tolerance test is abnormal (after oral lactose, blood glucose fails to rise above 20 mg/dl of basal value) in lactase deficiency. Rise in blood glucose indicates that lactose has been hydrolysed and absorbed by the mucosa. Lactose tolerance test is now replaced by lactose breath hydrogen testing. In lactase deficiency, accumulated lactose in the colon is rapidly fermented to organic acids and gases like hydrogen. Hydrogen is absorbed and then excreted through the lungs into the breath. Amount of hydrogen is then measured in breath; breath hydrogen more than 20 ppm above baseline within 4 hours indicates positive test.
 
Fecal Osmotic Gap
 
Fecal osmotic gap is calculated from concentration of electrolytes in stool water by formula 290-2([Na+] + [K+]). (290 is the assumed plasma osmolality). In osmotic diarrheas, osmotic gap is >150 mOsm/kg, while in secretory diarrhea, it is typically below 50 mOsm/kg. Evaluation of chronic diarrhea is shown in Figure 845.3.
 
Figure 845.3 Evaluation of chronic diarrhea
Figure 845.3 Evaluation of chronic diarrhea
 
Fecal pH
 
Stool pH below 5.6 is characteristic of carbohydrate malabsorption.

LABORATORY TESTS TO EVALUATE TUBULAR FUNCTION

Published in Clinical Pathology
Monday, 28 August 2017 01:46
Tests to Assess Proximal Tubular Function
 
Renal tubules efficiently reabsorb 99% of the glomerular filtrate to conserve the essential substances like glucose, amino acids, and water.
 
1. Glycosuria: In renal glycosuria, glucose is excreted in urine, while blood glucose level is normal. This is because of a specific tubular lesion which leads to impairment of glucose reabsorption. Renal glycosuria is a benign condition. Glycosuria can also occur in Fanconi syndrome.
 
2. Generalized aminoaciduria: In proximal renal tubular dysfunction, many amino acids are excreted in urine due to defective tubular reabsorption.
 
3. Tubular proteinuria (Low molecular weight proteinuria): Normally, low molecular weight proteins2 –microglobulin, retinol-binding protein, lysozyme, and α1 –microglobulin) are freely filtered by glomeruli and are completely reabsorbed by proximal renal tubules. With tubular damage, these low molecular weight proteins are excreted in urine and can be detected by urine protein electrophoresis. Increased amounts of these proteins in urine are indicative of renal tubular damage.
 
4. Urinary concentration of sodium: If both BUN and serum creatinine are acutely increased, it is necessary to distinguish between prerenal azotemia (renal underperfusion) and acute tubular necrosis. In prerenal azotemia, renal tubules are functioning normally and reabsorb sodium, while in acute tubular necrosis, tubular function is impaired and sodium absorption is decreased. Therefore, in prerenal azotemia, urinay sodium concentration is < 20 mEq/L while in acute tubular necrosis, it is > 20 mEq/L.
 
5. Fractional excretion of sodium (FENa): Measurement of urinary sodium concentration is affected by urine volume and can produce misleading results. Therefore, to avoid this, fractional excretion of sodium is calculated. This refers to the percentage of filtered sodium that has been absorbed and percentage that has been excreted. Measurement of fractional sodium excretion is a better indicator of tubular absorption of sodium than quantitation of urine sodium alone.
 
This test is indicated in acute renal failure. In oliguric patients, this is the most reliable means of early distinction between pre-renal failure and renal failure due to acute tubular necrosis. It is calculated from the following formula:
 
 
(Urine sodium × Plasma creatinine) × 100%
(Plasma sodium × Urine creatinine)
 
 
In pre-renal failure this ratio is less than 1%, and in acute tubular necrosis it is more than 1%. In pre-renal failure (due to reduced renal perfusion), aldosterone secretion is stimulated which causes maximal sodium conservation by the tubules and the ratio is less than 1%. In acute tubular necrosis, maximum sodium reabsorption is not possible due to tubular cell injury and consequently the ratio will be more than 1%. Values above 3% are strongly suggestive of acute tubular necrosis.
 
Tests to Assess Distal Tubular Function
 
1. Urine specific gravity: Normal specific gravity is 1.003 to 1.030. It depends on state of hydration and fluid intake.
 
  1. Causes of increased specific gravity:
    a. Reduced renal perfusion (with preservation of concentrating ability of tubules),
    b. Proteinuria,
    c. Glycosuria,
    d. Glomerulonephritis.
    e. Urinary tract obstruction.
  2. Causes of reduced specific gravity:
    a. Diabetes insipidus
    b. Chronic renal failure
    c. Impaired concentrating ability due to diseases of tubules.
 
As a test of renal function, it gives information about the ability of renal tubules to concentrate the glomerular filtrate. This concentrating ability is lost in diseases of renal tubules.
 
Fixed specific gravity of 1.010, which cannot be lowered or increased by increasing or decreasing the fluid intake respectively, is an indication of chronic renal failure.
 
2. Urine osmolality: The most commonly employed test to evaluate tubular function is measurement of urine/plasma osmolality. This is the most sensitive method for determination of ability of concentration. Osmolality measures number of dissolved particles in a solution. Specific gravity, on the other hand, is the ratio of mass of a solution to the mass of water i.e. it measures total mass of solute. Specific gravity depends on both the number and the nature of dissolved particles while osmolality is exact number of solute particles in a solution. Specific gravity measurement can be affected by the presence of solutes of large molecular weight like proteins and glucose, while osmolality is not. Therefore measurement of osmolality is preferred.
 
When solutes are dissolved in a solvent, certain changes take place like lowering of freezing point, increase in boiling point, decrease in vapor pressure, or increase of osmotic pressure of the solvent. These properties are made use of in measuring osmolality by an instrument called as osmometer.
 
Osmolality is expressed as milliOsmol/kg of water.
 
Urine/plasma osmolality ratio is helpful in distinguishing pre-renal azotemia (in which ratio is higher) from acute renal failure due to acute tubular necrosis (in which ratio is lower). If urine and plasma osmolality are almost similar, then there is defective tubular reabsorption of water.
 
3. Water deprivation test: If the value of baseline osmolality of urine is inconclusive, then water deprivation test is performed. In this test, water intake is restricted for a specified period of time followed by measurement of specific gravity or osmolality. Normally, urine osmolality should rise in response to water deprivation. If it fails to rise, then desmopressin is administered to differentiate between central diabetes insipidus and nephrogenic diabetes insipidus. Urinary concentration ability is corrected after administration of desmopressin in central diabetes insipidus, but not in nephrogenic diabetes insipidus.
 
If urine osmolality is > 800 mOsm/kg of water or specific gravity is ≥1.025 following dehydration, concentrating ability of renal tubules is normal. However, normal result does not rule out presence of renal disease.
False result will be obtained if the patient is on low-salt, low-protein diet or is suffering from major electrolyte and water disturbance.
 
4. Water loading antidiuretic hormone suppression test: This test assesses the capacity of the kidney to make urine dilute after water loading.
 
After overnight fast, patient empties the bladder and drinks 20 ml/kg of water in 15-30 minutes. The urine is collected at hourly intervals for the next 4 hours for measurements of urine volume, specific gravity, and osmolality. Plasma levels of antidiuretic hormone and serum osmolality should be measured at hourly intervals.
 
Normally, more than 90% of water should be excreted in 4 hours. The specific gravity should fall to 1.003 and osmolality should fall to < 100 mOsm/kg. Plasma level of antidiuretic hormone should be appropriate for serum osmolality. In renal function impairment, urine volume is reduced (<80% of fluid intake is excreted) and specific gravity and osmolality fail to decrease. The test is also impaired in adrenocortical insufficiency, malabsorption, obesity, ascites, congestive heart failure, cirrhosis, and dehydration.
 
This test is not advisable in patients with cardiac failure or kidney disease. If there is failure to excrete water load, fatal hyponatremia can occur.
 
5. Ammonium chloride loading test (Acid load test): Diagnosis of renal tubular acidosis is usually considered after excluding other causes of metabolic acidosis. This test is considered as a ‘gold standard’ for the diagnosis of distal or type 1 renal tubular acidosis. Urine pH and plasma bicarbonate are measured after overnight fasting. If pH is less than 5.4, acidifying ability of renal tubules is normal. If pH is greater than 5.4 and plasma bicarbonate is low, diagnosis of renal tubular acidosis is confirmed. In both the above cases, further testing need not be performed. In all other cases in which neither of above results is obtained, further testing is carried out. Patient is given ammonium chloride orally (0.1 gm/kg) over 1 hour after overnight fast and urine samples are collected hourly for next 6-8 hours. Ammonium ion dissociates into H+ and NH3. Ammonium chloride makes blood acidic. If pH is less than 5.4 in any one of the samples, acidifying ability of the distal tubules is normal.

MICROALBUMINURIA AND ALBUMINURIA

Published in Clinical Pathology
Sunday, 27 August 2017 13:15
Normally, a very small amount of albumin is excreted in urine. The earliest evidence of glomerular damage in diabetes mellitus is occurrence of microalbuminuria (albuminuria in the range of 30 to 300 mg/24 hours). An albuminuria > 300-mg/24 hour is termed clinical or overt and indicates significant glomerular damage. (See “Proteinuria” under Article “Chemical Examination of Urine”).
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