- 18 May 2018
Waste products discharged from the digestive tract are composed of up to 75% water, food which is digested but not absorbed, indigestible residue, undigested food, epithelial cells, bile, bacteria, secretion from the digestive tract and inorganic bacteria. Normally an adult human excretes 100-200 grams of feces in a day.
Examination of stool is very helpful in the diagnosis of disease of the gastrointestinal tract as listed below.
Detection of parasites
Stool examination is performed for the detection and identification of worms (adult worms, larvae, segments of worms, ova) and protozoa (cyst or trophozoites). See also: Microscopic Examination of Feces
Stool culture is performed for the evaluation of bacterial infection such as Clostridium difficile, Yersinia, Salmonella, Shigella or Vibrio. Bacterial toxins (such as those released by Clostridium difficile or Clostridium botulinum) can also be identified. See also: Microscopic Examination of Feces
Evaluation of chronic diarrhea
Chronic diarrhea defined as a passage of three or more liquid or loose stools in a day lasting for more than four weeks. Acute diarrhea refers to the passing of three or more liquid or loose stools in a day for less than four weeks. In diarrhea, stool examination is very important part of laboratory investigations. Depending on the nature of the investigation, either a random stool sample or 72- sample or 48-hour sample is collected. A random stool sample is used for the tests of occult blood, pH, fat, white blood cells, microscopy, or culture. A 72- or 48-hour sample is collected and examined for the weight, carbohydrate, fat content, osmolality, or chymotrypsin activity. Causes of chronic and acute diarrhea are listed in Table 988.1 and Figure 988.1 respectively.
1. Watery diarrhea
2. Inflammatory diarrhea
3. Fatty diarrhea
Evaluation of dysentery
Identification of Rotavirus
In infants and young children, Rotavirus is the most common cause of diarrhea. Rotavirus can be identified by the electron microscopic examination of stool. Other techniques, such as latex agglutination, immunofluorescence, or enzyme-linked immunosorbent assay (ELISA) are also used for the detection of Rotavirus in stool.
Chemical tests can be applied on feces to detect excess fat excretion (malabsorption syndrome), occult blood (in ulcerated lesions of the gastrointestinal tract, especially occult carcinoma of the colon) and presence or absence of urobilinogen (obstructive jaundice). See also: Chemical Examination of Feces
Differentiating infection by invasive bacteria (like Salmonella or Shigella) from that due to toxin-producing bacteria (like Vibrio cholerae or Escherichia coli)
Feces is examined for the presence of white blood cells. Increased numbers of polymorphonuclear neutrophils (identified by methylene blue stain from the presence of granules in their cytoplasm) are seen as shown in Figure 988.2. See also: Causes, symptoms, diagnosis, and treatment of Cholera
- 28 Sep 2017
- 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.
- 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.
- 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.
- 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.
- 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.
Box 864.1 Thyroid autoantibodies
- Hyperthyroidism due to Graves’ disease, toxic multinodular goiter, toxic adenoma, TSH-secreting tumor.
- Hyperthyroidism due to administration of thyroid hormone, factitious hyperthyroidism, subacute thyroiditis.
- 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.
|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 hyperthyroidism|
- 22 Sep 2017
1. Hypothalamic-pituitary dysfunction:
2. Ovarian dysfunction:
|3. Dysfunction in passages:|
|4. Dysfunction of sexual act: Dyspareunia|
- 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.
- 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.
- 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.
- 22 Sep 2017
2. Hypothalamic-pituitary dysfunction (hypogonadotropic hypogonadism)
3. Testicular dysfunction:
4. Dysfunction of passages and accessory sex glands:
5. Dysfunction of sexual act:
- 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.
- 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.
- 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.
- 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.
- Hormonal studies: This includes measurement of FSH, LH, and testosterone to detect hormonal abnormalities causing testicular failure (Table 861.2).
- 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).
|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|
- 07 Sep 2017
- 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.
- 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.
- 07 Sep 2017
- 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.
- 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.
- 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.
- 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).
- 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.
- 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%
- 07 Sep 2017
- 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.
- To determine the cause of raised fasting serum gastrin level: Hypergastrinemia can occur in achlorhydria, Zollinger-Ellison syndrome, and antral G cell hyperplasia.
- 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).
- 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.
- 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.
- 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.
- 05 Sep 2017
Box 855.1 Determination of basal acid output, maximum acid output, and peak acid output
- 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.
- 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.
- pH: Normal pH is 1.5 to 3.5. In pernicious anemia, pH is greater than 7.0 due to absence of HCl.
- 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.
- 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.
- 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.
|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|
- 30 Aug 2017
- 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.
- 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.
- 30 Aug 2017
- Occult blood
- Excess fat excretion (malabsorption)
- Reducing sugars
- Fecal osmotic gap
- Fecal pH
- Intestinal diseases: hookworms, amebiasis, typhoid fever, ulcerative colitis, intussusception, adenoma, cancer of colon or rectum.
- Gastric and esophageal diseases: peptic ulcer, gastritis, esophageal varices, hiatus hernia.
- Systemic disorders: bleeding diathesis, uremia.
- Long distance runners.
- 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.
- Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons.
- Foods containing large amounts of vitamin C.
- Conversion of all hemoglobin to acid hematin (which has no peroxidase-like activity) during passage through the gastrointestinal tract.
- Deficiency of pancreatic lipase (insufficient lipolysis): chronic pancreatitis, cystic fibrosis.
- Deficiency of bile salts (insufficient emulsification of fat): biliary obstruction, severe liver disease, bile salt deconjugation due to bacterial overgrowth in the small intestine.
- Diseases of small intestine: tropical sprue, celiac disease, Whipple’s disease.
- 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.
- 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.
- 28 Aug 2017
(Plasma sodium × Urine creatinine)
- Causes of increased specific gravity:
a. Reduced renal perfusion (with preservation of concentrating ability of tubules),
e. Urinary tract obstruction.
- Causes of reduced specific gravity:
a. Diabetes insipidus
b. Chronic renal failure
c. Impaired concentrating ability due to diseases of tubules.
- 27 Aug 2017
- 27 Aug 2017
- Pre-renal azotemia: shock, congestive heart failure, salt and water depletion
- Renal azotemia: impairment of renal function
- Post-renal azotemia: obstruction of urinary tract
- Increased rate of production of urea:
• High protein diet
• Increased protein catabolism (trauma, burns, fever)
• Absorption of amino acids and peptides from a large gastrointestinal hemorrhage or tissue hematoma
- Diacetyl monoxime urea method: This is a direct method. Urea reacts with diacetyl monoxime at high temperature in the presence of a strong acid and an oxidizing agent. Reaction of urea and diacetyl monoxime produces a yellow diazine derivative. The intensity of color is measured in a colorimeter or spectrophotometer.
- Urease- Berthelot reaction: This is an indirect method. Enzyme urease splits off ammonia from the urea molecule at 37°C. Ammonia generated is then reacted with alkaline hypochlorite and phenol with a catalyst to produce a stable color (indophenol). Intensity of color produced is then measured in a spectrophotometer at 570 nm.
- It is produced from muscles at a constant rate and its level in blood is not affected by diet, protein catabolism, or other exogenous factors;
- It is not reabsorbed, and very little is secreted by tubules.
Causes of Increased Serum Creatinine Level
- Pre-renal, renal, and post-renal azotemia
- Large amount of dietary meat
- Active acromegaly and gigantism
- Increasing age (reduction in muscle mass)
- Jaffe’s reaction (Alkaline picrate reaction): This is the most widely used method. Creatinine reacts with picrate in an alkaline solution to produce spectrophotometer at 485 nm. Certain substances in plasma (such as glucose, protein, fructose, ascorbic acid, acetoacetate, acetone, and cephalosporins) react with picrate in a similar manner; these are called as non-creatinine chromogens (and can cause false elevation of serum creatinine level). Thus ‘true’ creatinine is less by 0.2 to 0.4 mg/dl when estimated by Jaffe’s reaction.
- Enzymatic methods: These methods use enzymes that cleave creatinine; hydrogen peroxide produced then reacts with phenol and a dye to produce a colored product, which is measured in a spectrophotometer.
- Increased BUN with normal serum creatinine:
• Pre-renal azotemia (reduced renal perfusion)
• High protein diet
• Increased protein catabolism
• Gastrointestinal hemorrhage
- Increase of both BUN and serum creatinine with disproportionately greater increase of BUN:
• Post-renal azotemia (Obstruction to the outflow of urine)
Obstruction to the urine outflow causes diffusion of urinary urea back into the blood from tubules because of backpressure.
Causes of Decreased BUN/Creatinine Ratio (<10:1)
- Acute tubular necrosis
- Low protein diet, starvation
- Severe liver disease
(72 × Serum creatinine in mg/dl)
The agents used for measurement of GFR are:
- Exogenous: Inulin, Radiolabelled ethylenediamine tetraacetic acid (51Cr- EDTA), 125I-iothalamate
- Endogenous: Creatinine, Urea, Cystatin C
- A small amount of creatinine is secreted by renal tubules that increase even further in advanced renal failure.
- Collection of urine is often incomplete.
- Creatinine level is affected by intake of meat and muscle mass.
- Creatinine level is affected by certain drugs like cimetidine, probenecid, and trimethoprim (which block tubular secretion of creatinine).
- Establish the diagnosis
- Assess severity and activity of disease
- Assess prognosis by noting the amount of scarring
- To plan treatment and monitor response to therapy
- Nephrotic syndrome in adults (most common indication)
- Nephrotic syndrome not responding to corticosteroids in children.
- Acute nephritic syndrome for differential diagnosis
- Unexplained renal insufficiency with near-normal kidney dimensions on ultrasonography
- Asymptomatic hematuria, when other diagnostic tests fail to identify the source of bleeding
- Isolated non-nephrotic range proteinuria (1-3 gm/24 hours) with renal impairment
- Impaired function of renal graft
- Involvement of kidney in systemic disease like systemic lupus erythematosus or amyloidosis
- Uncontrolled severe hypertension
- Hemorrhagic diathesis
- Solitary kidney
- Renal neoplasm (to avoid spread of malignant cells along the needle track)
- Large and multiple renal cysts
- Small, shrunken kidneys
- Acute urinary tract infection like pyelonephritis
- Urinary tract obstruction
- Hemorrhage: As renal cortex is highly vascular, major risk is bleeding in the form of hematuria or perinephric hematoma. Severe bleeding may occasionally necessitate blood transfusion and rarely removal of kidney.
- Arteriovenous fistula
- Accidental biopsy of another organ or perforation of viscus (liver, spleen, pancreas, adrenals, intestine, or gallbladder)
- Death (rare).
- Patient’s informed consent is obtained.
- Ultrasound/CT scan is done to document the location and size of kidneys.
- Blood pressure should be less than 160/90 mm of Hg. Bleeding time, platelet count, prothrombin time, and activated partial thromboplastin time should be normal. Blood sample should be drawn for blood grouping and cross matching, as blood transfusion may be needed.
- Patient is sedated before the procedure.
- Patient lies in prone position and kidney is identified with ultrasound.
- The skin over the selected site is disinfected and a local anesthetic is infiltrated.
- A small skin incision is given with a scalpel (to insert the biopsy needle). Localization of kidney is done with a fine bore 21 G lumbar puncture needle. A local anesthetic is infiltrated down to the renal capsule.
- A tru-cut biopsy needle or spring loaded biopsy gun is inserted under ultrasound guidance and advanced down to the lower pole. Biopsy is usually obtained from lateral border of lower pole. Patient should hold his/her breath in full inspiration during biopsy. After obtaining the biopsy and removal of needle, patient is allowed to breath normally.
- The biopsy should be placed in a drop of saline and examined under a dissecting microscope for adequacy.
- Patient is turned to supine position. Vital signs and appearance of urine should be monitored at regular intervals. Patient is usually kept in the hospital for 24 hours.
- Hematoxylin and eosin (for general architecture of kidney and cellularity)
- Periodic acid Schiff: To highlight basement membrane and connective tissue matrix.
- Congo red: For amyloid.
- 17 Aug 2017
- Diagnosis of DM
- Screening of DM
- Assessment of glycemic control
- Assessment of associated long-term risks
- Management of acute metabolic complications.
- Chemical methods:
– Orthotoluidine method
– Blood glucose reduction methods using neocuproine, ferricyanide, or copper.
- Enzymatic methods: These are specific for glucose.
– Glucose oxidase-peroxidase
– Glucose dehydrogenase
- Fasting blood glucose: Sample for blood glucose is withdrawn after an overnight fast (no caloric intake for at least 8 hours).
- Post meal or postprandial blood glucose: Blood sample for glucose estimation is collected 2 hours after the subject has taken a normal meal.
- Random blood glucose: Blood sample is collected at any time of the day, without attention to the time of last food intake.
- Patient should be put on a carbohydrate-rich, unrestricted diet for 3 days. This is because carbohydrate-restricted diet reduces glucose tolerance.
- Patient should be ambulatory with normal physical activity. Absolute bed rest for a few days impairs glucose tolerance.
- Medications should be discontinued on the day of testing.
- Exercise, smoking, and tea or coffee are not allowed during the test period. Patient should remain seated.
- OGTT is carried out in the morning after patient has fasted overnight for 8-14 hours.
- A fasting venous blood sample is collected in the morning.
- Patient ingests 75 g of anhydrous glucose in 250-300 ml of water over 5 minutes. (For children, the dose is 1.75 g of glucose per kg of body weight up to maximum 75 g of glucose). Time of starting glucose drink is taken as 0 hour.
- A single venous blood sample is collected 2 hours after the glucose load. (Previously, blood samples were collected at ½, 1, 1½, and 2 hours, which is no longer recommended).
- Plasma glucose is estimated in fasting and 2-hour venous blood samples.
|Parameter||Normal||Impaired fasting glucose||Impaired glucose tolerance||Diabetes mellitus|
|(1) Fasting (8 hr)||< 100||100-125||—||≥ 126|
|(2) 2 hr OGTT||< 140||< 140||140-199||≥ 200|
- Low-risk pregnant women need not be tested if all of the following criteria are met, i.e. age below 25 years, normal body weight (before pregnancy), absence of diabetes in first-degree relatives, member of an ethnic group with low prevalence of DM, no history of poor obstetric outcome, and no history of abnormal glucose tolerance.
- Average-risk pregnant women (i.e. who are in between low and high risk) should be tested at 24-28 weeks of gestation.
- High-risk pregnant women i.e. those who meet any one of the following criteria should be tested immediately: marked obesity, strong family history of DM, glycosuria, or personal history of GDM.
- One step approach
- Two step approach
- Fasting: 95 mg/dl
- 1 hour: 180 mg/dl
- 2 hour: 155 mg/dl
- 3 hour: 140 mg/dl
- Periodic measurement of glycated hemoglobin (to assess long-term control).
- Daily self-assessment of blood glucose (to assess day-to- day or immediate control).
Numerous prospective studies have demonstrated that a good control of blood glucose reduces the development and progression of microvascular complications (retinopathy, nephropathy, and peripheral neuropathy) of diabetes mellitus. Mean glycated hemoglobin level correlates with the risk of these complications.
|Box 837.1 Glycated hemoglobin
Goal of tight glycemic control in type 1 DM patients on insulin can be achieved through self-monitoring of blood glucose by portable blood glucose meters.
- It is the earliest marker of diabetic nephropathy. Early diabetic nephropathy is reversible.
- It is a risk factor for cardiovascular disease in both type 1 and type 2 patients.
- It is associated with higher blood pressure and poor glycemic control.
- Albumin to creatinine ratio in a random urine sample
- Urinary albumin excretion in a 24-hour urine sample.
- Total cholesterol
- Low-density lipoprotein (LDL) cholesterol
- High-density lipoprotein (HDL) cholesterol
|Category||Low density lipoproteins||High density lipoproteins||Triglycerides|
|High-risk||≥130||< 35 (men)||≥ 400|
|< 45 (women)|
|Low-risk||< 100||> 45 (men)||< 200|
|> 55 (women)|
- Diabetic ketoacidosis (DKA)
- Hyperosmolar hyperglycemic state (HHS)
|Parameter||Diabetic ketoacidosis||Hyperosmolar hyperglycemic state|
|1. Type of DM in which more common||Type 1||Type 2|
|2. Age||Younger age||Older age|
|3. Prodromal clinical features||< 24 hrs||Several days|
|4. Abdominal pain, Kussmaul’s respiration||Yes||No|
|6. Plasma glucose||> 250 mg/dl||Very high (>600 mg/dl)|
|7. Serum bicarbonate||<15 mEq/L||>15 mEq/L|
|8. Blood/urine ketones||++++||±|
|9. β-hydroxybutyrate||High||Normal or raised|
|10. Arterial blood pH||Low (<7.30)||Normal (>7.30)|
|11. Effective serum osmolality*||Variable||Increased (>320)|
|12. Anion gap**||>12||Variable|
|Osmolality: Number of dissolved (solute) particles in solution; normal: 275-295 mOsmol/kg
** Anion gap: Difference between sodium and sum of chloride and bicarbonate in plasma; normal average value is 12
- Blood and urine glucose
- Blood and urine ketone
- Arterial pH, Blood gases
- Serum electrolytes (sodium, potassium, chloride, bicarbonate)
- Blood osmolality
- Serum creatinine and blood urea.
- At diagnosis of diabetes mellitus
- At regular intervals in all known cases of diabetes, during pregnancy with pre-existing diabetes, and in gestational diabetes
- In known diabetic patients: during acute illness, persistent hyperglycemia (> 300 mgs/dl), pregnancy, and clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain).
- Venous plasma glucose:
Fasting: 60-100 mg/dl
At 2 hours in OGTT (75 gm glucose): <140 mg/dl
- Glycated hemoglobin: 4-6% of total hemoglobin
- Lipid profile:
– Serum cholesterol: Desirable level: <200 mg/dl
– Serum triglycerides: Desirable level: <150 mg/dl
– HDL cholesterol: ≥60 mg/dl
– LDL cholesterol: <130 mg/dl
– LDL/HDL ratio: 0.5-3.0
- C-peptide: 0.78-1.89 ng/ml
- Arterial pH: 7.35-7.45
- Serum or plasma osmolality: 275-295 mOsm/kg of water.
Serum Osmolality can also be calculated by the following formula recommended by American Diabetes Association:
- Anion gap:
– Na+ – (Cl– + HCO3–): 8-16 mmol/L (Average 12)
– (Na+ + K+) – (Cl– + HCO3–): 10-20 mmol/L (Average 16)
- Serum sodium: 135-145 mEq/L
- Serum potassium: 3.5-5.0 mEq/L
- Serum chloride: 100-108 mEq/L
- Serum bicarbonate: 24-30 mEq/L
- 16 Aug 2017
Pregnancy tests detect human chorionic gonadotropin (hCG) in serum or urine. Although pregnancy is the most common reason for ordering the test for hCG, measurement of hCG is also indicated in other conditions as shown in Box 836.1.
Human chorionic gonadotropin is a glycoprotein hormone produced by placenta that circulates in maternal blood and excreted intact by the kidneys. It consists of two polypeptide subunits: α (92 amino acids) and β (145 amino acids) which are non-covalently bound to each other. Structurally, hCG is closely related to three other glycoprotein hormones, namely, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The α subunits of hCG, LH, FSH, and TSH are similar, while β subunits differ and confer specific biologic and immunologic properties. Immunological tests use antibodies directed against β-subunit of hCG to avoid cross-reactivity against LH, FSH, and TSH.
Box 836.1 Indications for measurement of β human chorionic gonadotropin
• Early diagnosis of pregnancy
• Diagnosis and management of gestational trophoblastic disease
• As a part of maternal triple test screen
Syncytiotrophoblastic cells of conceptus and later of placenta synthesize hCG. Human chorionic gonadotropin supports the corpus luteum of ovary during early pregnancy. Progesterone, produced by corpus luteum, prevents ovulation and thus maintains pregnancy. After 7-10 weeks of gestation, sufficient amounts of progesterone are synthesized by placenta, and hCG is no longer needed and its level declines.
CLINICAL APPLICATIONS OF TESTS FOR HUMAN CHORIONIC GONADOTROPIN
- Early diagnosis of pregnancy: Qualitative serum hCG test becomes positive 3 weeks after last menstrual period (LMP), while urine hCG test becomes positive 5 weeks after LMP.
- Exclusion of pregnancy before prescribing certain medications (like oral contraceptives, steroids, some antibiotics), and before ordering radiological studies, radiotherapy, or chemotherapy. This is necessary to prevent any teratogenic effect on the fetus.
- Early diagnosis of ectopic pregnancy: Trans-vaginal ultrasonography (USG) and quantitative estimation of hCG are helpful in early diagnosis of ectopic pregnancy (before rupture).
- Evaluation of threatened abortion: Serial quantitative estimation of hCG is helpful in following the course of threatened abortion.
- Diagnosis and follow-up of gestational trophoblastic disease (GTD).
- Maternal triple test screen: This consists of measurement of hCG, α-fetoprotein, and unconjugated estriol in maternal serum at 14-19 weeks of gestation. The maternal triple screen identifies pregnant women with increased risk of Down syndrome and major congenital anomalies like neural tube defects.
- Follow-up of ovarian or testicular germ cell tumors, which produce hCG.
In women with normal menstrual cycle, conception (fertilization of ovum to form a zygote) occurs on day 14 in the fallopian tube. Zygote travels down the fallopian tube into the uterus. Division of zygote produces a morula. At 50-60-cell stage, morula develops a primitive yolk sac and is then called as a blastocyst. About 5 days after fertilization, implantation of blastocyst occurs in the uterine wall. Trophoblastic cells (on the outer surface of the blastocyst) penetrate the endometrium and develop into chorionic villi. There are two main forms of trophoblasts—syncytiotrophoblast and cytotrophoblast. Placental development occurs from chorionic villi. After formation of placenta, the conceptus is called as an embryo. When embryo develops most major organs, it is called as fetus (after 10 weeks of gestation).
Box 836.2 Diagnosis of early pregnancy
• Positive serum hCG test: 8 days after conception or 3 weeks after last menstrual period (LMP)
• Positive urine hCG test: 21 days after conception or 5 weeks after LMP
• Ultrasonography for visualization of gestational sac:
– Transvaginal: 21 days after conception or 5 weeks after LMP
– Transabdominal: 28 days after conception or 6 weeks after LMP
Human chorionic gonadotropin is synthesized by syncytiotrophoblasts (of placenta) and detectable amounts (~5 mIU/ml) appear in maternal serum about 8 days after conception (3 weeks after LMP). In the first trimester (first 12 weeks, calculated from day 1 of LMP) of pregnancy, hCG levels rapidly rise with a doubling time of about 2 days. Highest or peak level is reached at 8-10 weeks (about 100,000 mIU/ml). This is followed by a gradual fall, and from 15-16 weeks onwards, a steady level of 10,000-20,000 mIU/ml is maintained for the rest of the pregnancy (Figure 836.1). After delivery, hCG becomes non-detectable by about 2 weeks.
Box 836.2 shows minimum time required for the earliest diagnosis of pregnancy by hCG test and ultrasonography (USG).
Two types of pregnancy tests are available:
- Qualitative tests: These are positive/negative result types that are done on urine sample.
- Quantitative tests: These give numerical result and are done on serum or urine. They are also used for evaluation of ectopic pregnancy, failing pregnancy, and for follow-up of gestational trophoblastic disease.
Ectopic pregnancy refers to the implantation of blastocyst at a site other than the cavity of uterus. The most common of such sites (>95% cases) is fallopian tube. Early diagnosis and treatment of tubal ectopic pregnancy is essential since it can lead to maternal mortality (from rupture and hemorrhage) and future infertility. Ectopic pregnancy is a leading cause of maternal death during first trimester. Diagnosis of ectopic pregnancy can be readily made in most cases by ultrasonography and estimation of β-subunit of human chorionic gonadotropin.
Early diagnosis of unruptured tubal pregnancy can be made by quantitative estimation of serum hCG and ultrasonography. In normal intrauterine pregnancy, hCG titer doubles every 2 days until first 40 days of gestation. If hCG rise is abnormally slow, then an unviable pregnancy (either ectopic or abnormal intrauterine pregnancy) should be suspected.
Transabdominal USG can detect gestational sac in intrauterine pregnancy 6 weeks after LMP. The level of hCG in serum at this stage is >6500 mIU/ml. If gestational sac is not visualized at this level of hCG, then there is a possibility of ectopic pregnancy. Transvaginal ultrasonography can detect ectopic pregnancy average 1 week earlier than abdominal ultrasonography; it can detect gestational sac if β-hCG level is 1000-1500 mIU/ml. Therefore, if gestational sac is not visualized in the presence of >1500 mIU/ml of β-hCG level, an ectopic pregnancy can be suspected.
Early diagnosis of ectopic pregnancy provides the option of administration of intramuscular methotrexate (rather than surgery), which causes dissolution of conceptus. This improves the chances of patient’s future fertility. Serial measurements of hCG after surgical removal of ectopic pregnancy can help in detecting persistence of trophoblastic tissue.
Termination of pregnancy before fetus becomes viable (i.e. before 20 weeks) is called as abortion.
In threatened abortion, vaginal bleeding is present but internal os is closed and process of abortion, though started, is still reversible. It is possible that pregnancy will continue.
Serial quantitative titers of hCG showing lack of expected doubling of hCG level and USG are helpful in diagnosis and management of abortion.
Gestational Trophoblastic Disease (GTD)
It is characterized by proliferation of pregnancyassociated trophoblastic tissue. The two main forms of GTD are hydatidiform (vesicular) mole (benign) and choriocarcinoma (malignant). Clinical features of GTD are as follows:
- Short history of amenorrhea followed by vaginal bleeding.
- Size of uterus larger than gestational age; uterus is soft and doughy on palpation with no fetal parts and no fetal heart sounds.
- Excessive nausea and vomiting due to high hCG.
- Characteristic snowstorm appearance on pelvic USG.
Quantitative estimation of hCG is helpful in diagnosis and management of GTD.
Trophoblastic cells of GTD produce more hCG as compared to the trophoblasts of normal pregnancy for the same gestational age. Concentration of hCG parallels tumor load. Also, hCG continues to rise beyond 10 weeks of gestation without reaching plateau (as expected at the end of first trimester).
After evacuation of uterus, weekly estimation of hCG is advised till subsequent three (weekly) results are negative; following evacuation of vesicular mole, hCG becomes undetectable (after 2-3 months) on follow-up in 80% of cases. Plateau or rising hCG indicates persistent GTD. In such cases, chemotherapy is indicated.
Negative results for hCG after therapy should be regularly followed up every 3 months for 1-2 years.
LABORATORY TESTS FOR HUMAN CHORIONIC GONADOTROPIN
These are classified into two main groups:
- Biological assays or bioassays
- Immunological assays
In bioassay, effect of hCG is tested on laboratory animals under standardized conditions. There are several limitations of bioassays like need for animal facilities, need for standardization of animals, long time required for the test results, low sensitivity, and high cost. Therefore, bioassays have been replaced by immunological assays.
In Ascheim-Zondek test, urine from pregnant woman is injected into immature female mice. Formation of hemorrhagic corpora lutea in ovaries (after 4 days) is a positive test. Friedman test is similar except that urine is injected into female rabbit. In rapid rat test, injection of urine containing hCG into female rats is followed by hyperaemia and hemorrhage in ovaries. Yet another test measures release of spermatozoa from male frog after injection of urine containing hCG.
These are rapid and sensitive tests for detection and quantitation of hCG. Variable results are obtained by different immunological tests with the same serum sample; this is due to differences in specificity of different immunoassays to complete hCG, β-subunit, and β-core fragment. A number of immunological tests are commercially available based on different principles like agglutination inhibition assay, enzyme immunoassay including enzyme linked immunosorbent assay or ELISA, radioimmunoassay (RIA), and immunoradiometric assay.
A commonly used qualitative urine test is agglutination inhibition assay. Early morning urine specimen is preferred because it contains the highest concentration of hCG. Causes of false-positive test include red cells, leukocytes, bacteria, some drugs, proteins, and excess luteinizing hormone (menopause, midcycle LH surge) in urine. Some patients have anti-mouse antibodies (that are used in the test), while others have hCG-like material in circulation, producing false-positive test. Anti-mouse antibodies also interfere with other antibody-based tests and are known as ‘heterophil’ antibodies. Fetal death, abortion, dilute urine, and low sensitivity of a particular test are causes of false-negative test. Renal failure leads to accumulation of interfering substances causing incorrect results.
In latex particle agglutination inhibition test (Figure 836.2), anti-hCG antibodies are incubated with patient’s urine. This is followed by addition of hCGcoated latex particles. If hCG is present in urine, anti-hCG serum is neutralized, and no agglutination of latex particles occurs (positive test). If there is no hCG in urine, there is agglutination of latex particles (negative test). This is commonly used as a slide test and requires only a few minutes.
Sensitivity of agglutination inhibition test is >200 units/liter of hCG.
Radioimmunoassay, enzyme immunoassay, and radioimmunometric assay are more sensitive and reliable than agglutination inhibition assay.
Quantitative tests are employed for detection of very early pregnancy, estimation of gestational age, diagnosis of ectopic pregnancy, evaluation of threatened abortion, and management of GTD.
- Serum human chorionic gonadotropin:
– Non-pregnant females: <5.0 mIU/ml
– Pregnancy: 4 weeks after LMP: 5-100 mIU/ml
– 5 weeks after LMP: 200-3000 mIU/ml
– 6 weeks after LMP: 10,000-80,000 mIU/ml
– 7-14 weeks: 90,000-500,000 mIU/ml
– 15-26 weeks: 5000-80000 mIU/ml
– 27-40 weks: 3000-15000 mIU/ml
Further Reading: SEMEN ANALYSIS FOR INVESTIGATION OF INFERTILITY
- 15 Aug 2017
Box 835.1 Contributions to semen volume
• Testes and epididymis: 10%
• Seminal vesicles: 50%
• Prostate: 40%
• Cowper’s glands: Small volume
- Testes: Male gametes or spermatozoa (sperms) are produced by testes; constitute 2-5% of semen volume.
- Epididymis: After emerging from the testes, sperms are stored in the epididymis where they mature; potassium, sodium, and glycerylphosphorylcholine (an energy source for sperms) are secreted by epididymis.
- Vas deferens: Sperms travel through the vas deferens to the ampulla which is another storage area. Ampulla secretes ergothioneine (a yellowish fluid that reduces chemicals) and fructose (source of nutrition for sperms).
- Seminal vesicles: During ejaculation, nutritive and lubricating fluids secreted by seminal vesicles and prostate are added. Fluid secreted by seminal vesicles consists of fructose (energy source for sperms), amino acids, citric acid, phosphorous, potassium, and prostaglandins. Seminal vesicles contribute 50% to semen volume.
- Prostate: Prostatic secretions comprise about 40% of semen volume and consist of citric acid, acid phosphatase, calcium, sodium, zinc, potassium, proteolytic enzymes, and fibrolysin.
- Bulbourethral glands of Cowper secrete mucus.
|1. Volume||≥2 ml|
|2. pH||7.2 to 8.0|
|3. Sperm concentration||≥20 million/ml|
|4. Total sperm count per ejaculate||≥40 million|
|5. Morphology||≥30% sperms with normal morphology|
|6. Vitality||≥75% live|
|7. White blood cells||<1 million/ml|
|8. Motility within 1 hour of ejaculation|
|• Class A||≥25% rapidly progressive|
|• Class A and B||≥50% progressive|
|9. Mixed antiglobuiln reaction (MAR) test||<50% motile sperms with adherent particles|
|10. Immunobead test||<50% motile sperms with adherent particles|
|1. Total fructose (seminal vesicle marker)||≥13 μmol/ejaculate|
|2. Total zinc (Prostate marker)||≥2.4 μmol/ejaculate|
|3. Total acid phosphatase (Prostate marker)||≥200U/ejaculate|
|4. Total citric acid (Prostate marker)||≥52 μmol/ejaculate|
|5. α-glucosidase (Epididymis marker)||≥20 mU/ejaculate|
|6. Carnitine (Epididymis marker)||0.8-2.9 μmol/ejaculate|
|Box 835.2 Tests done on seminal fluid
• Physical examination: Time to liquefaction, viscosity, volume, pH, color
• Microscopic examination: Sperm count, vitality, motility, morphology, and proportion of white cells
• Immunologic analysis: Antisperm antibodies (SpermMAR test, Immunobead test)
• Bacteriologic analysis: Detection of infection
• Biochemical analysis: Fructose, zinc, acid phosphatase, carnitine.
• Sperm function tests: Postcoital test, cervical mucus penetration test, Hamster egg penetration assay, hypoosmotic swelling of flagella, and computer-assisted semen analysis
- Investigation of infertility: Semen analysis is the first step in the investigation of infertility. About 30% cases of infertility are due to problem with males.
- To check the effectiveness of vasectomy by confirming absence of sperm.
- To support or disprove a denial of paternity on the grounds of sterility.
- To examine vaginal secretions or clothing stains for the presence of semen in medicolegal cases.
- For selection of donors for artificial insemination.
- For selection of assisted reproductive technology, e.g. in vitro fertilization, gamete intrafallopian transfer technique.
|Box 835.3 Semen analysis for initial investigation of infertility
• Microscopic examination for (i) percentage of motile spermatozoa, (ii) sperm count, and (iii) sperm morphology
| Box 835.4 Terminology in semen analysis
• Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration <20 million/ml (mild to moderate: 5-20 million/ml; severe: <5 million/ml)
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility; <50% of sperms showing class (a) and class (b) type of motility OR <25% sperms showing class (a) type of motility.
• Teratozoospermia: Spermatozoa with reduced proportion of normal morphology (or increased proportion of abnormal forms)
• Leukocytospermia: >1 million white blood cells/ml of semen
• Oligoasthenoteratozoospermia: All sperm variables are abnormal
• Necrozoospermia: All sperms are non-motile or non-viable
- PHYSICAL EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY
- BIOCHEMICAL ANALYSIS OF SEMEN FOR INVESTIGATION OF INFERTILITY
- MICROSCOPIC EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY
- IMMUNOLOGIC ANALYSIS OF SEMEN FOR INVESTIGATION OF INFERTILITY
- SPERM FUNCTION TESTS OR FUNCTIONAL ASSAYS
- EXAMINATION FOR THE PRESENCE OF SEMEN IN MEDICOLEGAL CASES
- 15 Aug 2017
Atleast 200 motile spermatozoa should be counted. If >50% of spermatozoa show attached latex particles, immunological problem is likely.
- Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
- A smear is made from a drop placed on a glass slide.
- The smear is air-dried and examined under oilimmersion objective. White sperms are classified as live or viable, and red sperms are classified as dead or non-viable. At least 200 spermatozoa are examined.
- The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.
- Semen is diluted 1:20 with sodium bicarbonateformalin diluting fluid (Take 1 ml liquefied semen in a graduated tube and fill with diluting fluid to 20 ml mark. Mix well).
- A coverslip is placed over the improved Neubauer counting chamber and the counting chamber is filled with the well-mixed diluted semen sample using a Pasteur pipette. The chamber is then placed in a humid box for 10-15 minutes for spermatozoa to settle.
- The chamber is placed on the microscope stage. Using the 20× or 40× objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large corner squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square.
- Sperm count per ml is calculated as follows:
Sperm count = Sperms counted × correction factor × 1000
Number of squares counted × Volume of 1 square
= Sperms counted × 20 1000
4 × 0.1
= Sperms counted × 50, 000
- Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 106/ml). Sperm count < 20 million/ml may be associated with infertility in males.
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
– Length: 3-5 μ
– Width: 2-3 μ
– Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
– Length: 3-5 μ
– Width: 1.0 μ
• Principal piece:
– Length: 40-50 μ
– Width: 0.5 μ
• End piece: 4-6 μ
- Normal sperm
- Defects in head:
• Large heads
• Small heads
• Tapered heads
• Pyriform heads
• Round heads
• Amorphous heads
• Vacuolated heads (> 20% of the head area occupied by vacuoles)
• Small acrosomes (occupying < 40% of head area)
• Double heads
- Defects in neck:
• Bent neck and tail forming an angle >90° to the long axis of head
- Defects in middle piece:
• Asymmetric insertion of midpiece into head
• Thick or irregular midpiece
• Abnormally thin midpiece
- Defects in tail:
• Bent tails
• Short tails
• Coiled tails
• Irregular tails
• Multiple tails
• Tails with irregular width
- Pin heads: Not to be counted
- Cytoplasmic droplets
• > 1/3rd the size of the sperm head
- Precursor cells: Considered abnormal
|1. Total fructose (seminal vesicle marker)||≥13 μmol/ejaculate|
|2. Total zinc (Prostate marker)||≥2.4 μmol/ejaculate|
|3. Total acid phosphatase (Prostate marker)||≥200U/ejaculate|
|4. Total citric acid (Prostate marker)||≥52 μmol/ejaculate|
|5. α-glucosidase (Epididymis marker)||≥20 mU/ejaculate|
|6. Carnitine (Epididymis marker)||0.8-2.9 μmol/ejaculate|
- Vagina: Direct aspiration or saline lavage
- Clothing: When scanned with ultraviolet light, semen produces green white fluorescence. A small piece (1 m2) of clothing from stained portion is soaked in 1-2 ml of physiologic saline for 1 hour. A similar piece of clothing distant from the stain is also soaked in saline as a control.
- 13 Aug 2017
- Contamination of urine by menstrual blood in females
- Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.
- Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
- Use of formalin as a preservative for urine
- 10 Aug 2017
The chemical examination is carried out for substances in urine are listed below:
- Bile salts
- Nitrite or leukocyte esterase
Normally, kidneys excrete scant amount of protein in urine (up to 150 mg/24 hours). These proteins include proteins from plasma (albumin) and proteins derived from urinary tract (Tamm-Horsfall protein, secretory IgA, and proteins from tubular epithelial cells, leucocytes, and other desquamated cells); this amount of proteinuria cannot be detected by routine tests.
(Tamm-Horsfall protein is a normal mucoprotein secreted by ascending limb of the loop of Henle).
Proteinuria refers to protein excretion in urine greater than 150 mg/24 hours in adults.
Causes of Proteinuria
Box 826.1: Causes of proteinuria
Causes of proteinuria can be grouped as shown in Box 826.1.
- Glomerular proteinuria: Proteinuria due to increased permeability of glomerular capillary wall is called as glomerular proteinuria.
There are two types of glomerular proteinuria: selective and nonselective. In early stages of glomerular disease, there is increased excretion of lower molecular weight proteins like albumin and transferrin. When glomeruli can retain larger molecular weight proteins but allow passage of comparatively lower molecular weight proteins, the proteinuria is called as selective. With further glomerular damage, this selectivity is lost and larger molecular weight proteins (γ globulins) are also excreted along with albumin; this is called as nonselective proteinuria.
Selective and nonselective proteinuria can be distinguished by urine protein electrophoresis. In selective proteinuria, albumin and transferrin bands are seen, while in nonselective type, the pattern resembles that of serum (Figure 826.1).
Causes of glomerular proteinuria are glomerular diseases that cause increased permeability of glomerular basement membrane. The degree of glomerular proteinuria correlates with severity of disease and prognosis. Serial estimations of urinary protein are also helpful in monitoring response to treatment. Most severe degree of proteinuria occurs in nephrotic syndrome (Box 826.2).Box 826.2: Nephrotic syndrome
- Tubular proteinuria: Normally, glomerular membrane, although impermeable to high molecular weight proteins, allows ready passage to low molecular weight proteins like β2-microglobulin, retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light chains. These low molecular weight proteins are actively reabsorbed by proximal renal tubules. In diseases involving mainly tubules, these proteins are excreted in urine while albumin excretion is minimal.
Urine electrophoresis shows prominent α- and β-bands (where low molecular weight proteins migrate) and a faint albumin band (Figure 826.1).
Tubular type of proteinuria is commonly seen in acute and chronic pyelonephritis, heavy metal poisoning, tuberculosis of kidney, interstitial nephritis, cystinosis, Fanconi syndrome and rejection of kidney transplant.
Purely tubular proteinuria cannot be detected by reagent strip test (which is sensitive to albumin), but heat and acetic acid test and sulphosalicylic acid test are positive.
- Overflow proteinuria: When concentration of a low molecular weight protein rises in plasma, it “overflows” from plasma into the urine. Such proteins are immunoglobulin light chains or Bence Jones proteins (plasma cell dyscrasias), hemoglobin (intravascular hemolysis), myoglobin (skeletal muscle trauma), and lysozyme (acute myeloid leukemia type M4 or M5).
- Hemodynamic proteinuria: Alteration of blood flow through the glomeruli causes increased filtration of proteins. Protein excretion, however, is transient. It is seen in high fever, hypertension, heavy exercise, congestive cardiac failure, seizures, and exposure to cold.
Postural (orthostatic) proteinuria occurs when the subject is standing or ambulatory, but is absent in recumbent position. It is common in adolescents (3-5%) and is probably due to lordotic posture that causes inferior venacaval compression between the liver and vertebral column. The condition disappears in adulthood. Amount of proteinuria is <1000 mg/day. First-morning urine after rising is negative for proteins, while another urine sample collected after patient performs normal activities is positive for proteins. In such patients, periodic testing for proteinuria should be done to rule out renal disease.
- Post-renal proteinuria: This is caused by inflammatory or neoplastic conditions in renal pelvis, ureter, bladder, prostate, or urethra.
Further reading: Tests for Detection of Proteinuria
The main indication for testing for glucose in urine is detection of unsuspected diabetes mellitus or follow-up of known diabetic patients.
Box 826.3: Urine glucose
Practically all of the glucose filtered by the glomeruli is reabsorbed by the proximal renal tubules and returned to circulation. Normally a very small amount of glucose is excreted in urine (< 500 mg/24 hours or <15 mg/dl) that cannot be detected by the routine tests. Presence of detectable amounts of glucose in urine is called as glucosuria or glycosuria (Box 826.3). Glycosuria results if the filtered glucose load exceeds the capacity of renal tubular reabsorption. Most common cause is hyperglycemia from diabetes mellitus.
Causes of Glycosuria
1. Glycosuria with hyperglycemia:
- Endocrine diseases: diabetes mellitus, acromegaly, Cushing’s syndrome, hyperthyroidism, pancreatic disease
- Non-endocrine diseases: central nervous system diseases, liver disorders
- Drugs: adrenocorticotrophic hormone, corticosteroids, thiazides
- Alimentary glycosuria (Lag-storage glycosuria): After a meal, there is rapid intestinal absorption of glucose leading to transient elevation of blood glucose above renal threshold. This can occur in persons with gastrectomy or gastrojejunostomy and in hyperthyroidism. Glucose tolerance test reveals a peak at 1 hour above renal threshold (which causes glycosuria); the fasting and 2-hour glucose values are normal.
2. Glycosuria without hyperglycemia
- Renal glycosuria: This accounts for 5% of cases of glycosuria in general population. Renal threshold is the highest glucose level in blood at which glucose appears in urine and which is detectable by routine laboratory tests. The normal renal threshold for glucose is 180 mg/dl. Threshold substances need a carrier to transport them from tubular lumen to blood. When the carrier is saturated, the threshold is reached and the substance is excreted. Up to this level glucose filtered by the glomeruli is efficiently reabsorbed by tubules. Renal glycosuria is a benign condition in which renal threshold is set below 180 mgs/dl but glucose tolerance is normal; the disorder is transmitted as autosomal dominant. Other conditions in which glycosuria can occur with blood glucose level remaining below 180 mgs/dl are renal tubular diseases in which there is decreased glucose reabsorption like Fanconi’s syndrome, and toxic renal tubular damage. During pregnancy, renal threshold for glucose is decreased. Therefore it is necessary to estimate blood glucose when glucose is first detected in urine.
Further reading: Tests for Detection of Glucose in Urine
Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric acid, and acetone) in urine is called as ketonuria. Ketones are breakdown products of fatty acids and their presence in urine is indicative of excessive fatty acid metabolism to provide energy.
Causes of Ketonuria
Box 826.4: Urine ketones in diabetes
Indications for testing
Normally ketone bodies are not detectable in the urine of healthy persons. If energy requirements cannot be met by metabolism of glucose (due to defective carbohydrate metabolism, low carbohydrate intake, or increased metabolic needs), then energy is derived from breakdown of fats. This leads to the formation of ketone bodies (Figure 826.2).
- Decreased utilization of carbohydrates:
a. Uncontrolled diabetes mellitus with ketoacidosis: In diabetes, because of poor glucose utilization, there is compensatory increased lipolysis. This causes increase in the level of free fatty acids in plasma. Degradation of free fatty acids in the liver leads to the formation of acetoacetyl CoA which then forms ketone bodies. Ketone bodies are strong acids and produce H+ ions, which are neutralized by bicarbonate ions; fall in bicarbonate (i.e. alkali) level produces ketoacidosis. Ketone bodies also increase the plasma osmolality and cause cellular dehydration. Children and young adults with type 1 diabetes are especially prone to ketoacidosis during acute illness and stress. If glycosuria is present, then test for ketone bodies must be done. If both glucose and ketone bodies are present in urine, then it indicates presence of diabetes mellitus with ketoacidosis (Box 826.4).
In some cases of diabetes, ketone bodies are increased in blood but do not appear in urine.
Presence of ketone bodies in urine may be a warning of impending ketoacidotic coma.
b. Glycogen storage disease (von Gierke’s disease)
- Decreased availability of carbohydrates in the diet:
b. Persistent vomiting in children
c. Weight reduction program (severe carbohydrate restriction with normal fat intake)
- Increased metabolic needs:
a. Fever in children
b. Severe thyrotoxicosis
d. Protein calorie malnutrition
Further reading: Tests for Detection of Ketones in Urine
BILE PIGMENT (BILIRUNIN)
Bilirubin (a breakdown product of hemoglobin) is undetectable in the urine of normal persons. Presence of bilirubin in urine is called as bilirubinuria.
There are two forms of bilirubin: conjugated and unconjugated. After its formation from hemoglobin in reticuloendothelial system, bilirubin circulates in blood bound to albumin. This is called as unconjugated bilirubin. Unconjugated bilirubin is not water-soluble, is bound to albumin, and cannot pass through the glomeruli; therefore it does not appear in urine. The liver takes up unconjugated bilirubin where it combines with glucuronic acid to form bilirubin diglucuronide (conjugated bilirubiun). Conjugated bilirubin is watersoluble, is filtered by the glomeruli, and therefore appears in urine.
Detection of bilirubin in urine (along with urobilinogen) is helpful in the differential diagnosis of jaundice (Table 826.1).
|Urine test||Hemolytic jaundice||Hepatocellular jaundice||Obstructive jaundice|
In acute viral hepatitis, bilirubin appears in urine even before jaundice is clinically apparent. In a fever of unknown origin bilirubinuria suggests hepatitis.
Presence of bilirubin in urine indicates conjugated hyperbilirubinemia (obstructive or hepatocellular jaundice). This is because only conjugated bilirubin is water-soluble. Bilirubin in urine is absent in hemolytic jaundice; this is because unconjugated bilirubin is water-insoluble.
Further reading: Tests for Detection of Bilirubin in Urine
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).
Further reading: Test for Detection of Bile Salts in Urine
Conjugated bilirubin excreted into the duodenum through bile is converted by bacterial action to urobilinogen in the intestine. Major part is eliminated in the feces. A portion of urobilinogen is absorbed in blood, which undergoes recycling (enterohepatic circulation); a small amount, which is not taken up by the liver, is excreted in urine. Urobilinogen is colorless; upon oxidation it is converted to urobilin, which is orange-yellow in color. Normally about 0.5-4 mg of urobilinogen is excreted in urine in 24 hours. Therefore, a small amount of urobilinogen is normally detectable in urine.
Urinary excretion of urobilinogen shows diurnal variation with highest levels in afternoon. Therefore, a 2-hour post-meal sample is preferred.
Causes of Increased Urobilinogen in Urine
- Hemolysis: Excessive destruction of red cells leads to hyperbilirubinemia and therefore increased formation of urobilinogen in the gut. Bilirubin, being of unconjugated type, does not appear in urine. Increased urobilinogen in urine without bilirubin is typical of hemolytic anemia. This also occurs in megaloblastic anemia due to premature destruction of erythroid precursors in bone marrow (ineffective erythropoiesis).
- Hemorrhage in tissues: There is increased formation of bilirubin from destruction of red cells.
Causes of Reduced Urobilinogen in Urine
- Obstructive jaundice: In biliary tract obstruction, delivery of bilirubin to the intestine is restricted and very little or no urobilinogen is formed. This causes stools to become clay-colored.
- Reduction of intestinal bacterial flora: This prevents conversion of bilirubin to urobilinogen in the intestine. It is observed in neonates and following antibiotic treatment.
Testing of urine for both bilirubin and urobilinogen can provide helpful information in a case of jaundice (Table 826.1).
Further reading: Tests for Detection of Urobilinogen in Urine
The presence of abnormal number of intact red blood cells in urine is called as hematuria. It implies presence of a bleeding lesion in the urinary tract. Bleeding in urine may be noted macroscopically or with naked eye (gross hematuria). If bleeding is noted only by microscopic examination or by chemical tests, then it is called as occult, microscopic or hidden hematuria.
Causes of Hematuria
1. Diseases of urinary tract:
- Glomerular diseases: Glomerulonephritis, Berger’s disease, lupus nephritis, Henoch-Schonlein purpura
- Nonglomerular diseases: Calculus, tumor, infection, tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney disease, trauma, after strenuous physical exercise, diseases of prostate (benign hyperplasia of prostate, carcinoma of prostate).
2. Hematological conditions:
Coagulation disorders, sickle cell disease Presence of red cell casts and proteinuria along with hematuria suggests glomerular cause of hematuria.
Further reading: Tests for Detection of Blood in Urine
Presence of free hemoglobin in urine is called as hemoglobinuria.
Causes of Hemoglobinuria
- Hematuria with subsequent lysis of red blood cells in urine of low specific gravity.
- Intravascular hemolysis: Hemoglobin will appear in urine when haptoglobin (to which hemoglobin binds in plasma) is completely saturated with hemoglobin. Intravascular hemolysis occurs in infections (severe falciparum malaria, clostridial infection, E. coli septicemia), trauma to red cells (march hemoglobinuria, extensive burns, prosthetic heart valves), glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant drugs, immune hemolysis (mismatched blood transfusion, paroxysmal cold hemoglobinuria), paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and disseminated intravascular coagulation.
Tests for Detection of Hemoglobinuria
Tests for detection of hemoglobinuria are benzidine test, orthotoluidine test, and reagent strip test.
Hemosiderin in urine (hemosiderinuria) indicates presence of free hemoglobin in plasma. Hemosiderin appears as blue granules when urine sediment is stained with Prussian blue stain (Figure 826.3). Granules are located inside tubular epithelial cells or may be free if cells have disintegrated. Hemosiderinuria is seen in intravascular hemolysis.
Myoglobin is a protein present in striated muscle (skeletal and cardiac) which binds oxygen. Causes of myoglobinuria include injury to skeletal or cardiac muscle, e.g. crush injury, myocardial infarction, dermatomyositis, severe electric shock, and thermal burns.
Chemical tests used for detection of blood or hemoglobin also give positive reaction with myoglobin (as both hemoglobin and myoglobin have peroxidase activity). Ammonium sulfate solubility test is used as a screening test for myoglobinuria (Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while hemoglobin is insoluble and is precipitated. A positive chemical test for blood done on supernatant indicates myoglobinuria).
Distinction between hematuria, hemoglobinuria, and myoglobinuria is shown in Table 826.2
|1. Urine color||Normal, smoky, red, or brown||Pink, red, or brown||Red or brown|
|2. Plasma color||Normal||Pink||Normal|
|3. Urine test based on peroxidase activity||Positive||Positive||Positive|
|4. Urine microscopy||Many red cells||Occasional red cell||Occasional red cell|
|5. Serum haptoglobin||Normal||Low||Normal|
|6. Serum creatine kinase||Normal||Normal||Markedly increased|
Chemical Tests for Significant Bacteriuria (Indirect Tests for Urinary Tract Infection)
In addition to direct microscopic examination of urine sample, chemical tests are commercially available in a reagent strip format that can detect significant bacteriuria: nitrite test and leucocyte esterase test. These tests are helpful at places where urine microscopy is not available. If these tests are positive, urine culture is indicated.
1. Nitrite test: Nitrites are not present in normal urine; ingested nitrites are converted to nitrate and excreted in urine. If gram-negative bacteria (e.g. E.coli, Salmonella, Proteus, Klebsiella, etc.) are present in urine, they will reduce the nitrates to nitrites through the action of bacterial enzyme nitrate reductase. Nitrites are then detected in urine by reagent strip tests. As E. coli is the commonest organism causing urinary tract infection, this test is helpful as a screening test for urinary tract infection.
Some organisms like Staphylococci or Pseudomonas do not reduce nitrate to nitrite and therefore in such infections nitrite test is negative. Also, urine must be retained in the bladder for minimum of 4 hours for conversion of nitrate to nitrite to occur; therefore, fresh early morning specimen is preferred. Sufficient dietary intake of nitrate is necessary. Therefore a negative nitrite test does not necessarily indicate absence of urinary tract infection. The test detects about 70% cases of urinary tract infections.
2. Leucocyte esterase test: It detects esterase enzyme released in urine from granules of leucocytes. Thus the test is positive in pyuria. If this test is positive, urine culture should be done. The test is not sensitive to leucocytes < 5/HPF.
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Microscopic examination of urine is also called as the “liquid biopsy of the urinary tract”.
Urine consists of various microscopic, insoluble, solid elements in suspension. These elements are classified as organized or unorganized. Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. The unorganized substances are crystalline and amorphous material. These elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; therefore they are known as urinary deposits or urinary sediments. Examination of urinary deposit is helpful in diagnosis of urinary tract diseases as shown in Table 825.1.
|1. Normal||0-trace||0-2||0-2||Occasional (Hyaline)||–|
|2. Acute glomerulonephritis||1-2+||Numerous;dysmorphic||0-few||Red cell, granular||Smoky urine or hematuria|
|3. Nephrotic syndrome||> 4+||0-few||0-few||Fatty, hyaline, Waxy, epithelial||Oval fat bodies, lipiduria|
|4. Acute pyelonephritis||0-1+||0-few||Numerous||WBC, granular||WBC clumps, bacteria, nitrite test|
Different types of urinary sediments are shown in Figure 825.1. The major aim of microscopic examination of urine is to identify different types of cellular elements and casts. Most crystals have little clinical significance.
The cellular elements are best preserved in acid, hypertonic urine; they deteriorate rapidly in alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is the most concentrated. The specimen should be examined within 2 hours of voiding because cells and casts degenerate upon standing at room temperature. If preservative is required, then 1 crystal of thymol or 1 drop of formalin (40%) is added to about 10 ml of urine.
A well-mixed sample of urine (12 ml) is centrifuged in a centrifuge tube for 5 minutes at 1500 rpm and supernatant is poured off. The tube is tapped at the bottom to resuspend the sediment (in 0.5 ml of urine). A drop of this is placed on a glass slide and covered with a cover slip (Figure 825.2). The slide is examined immediately under the microscope using first the low power and then the high power objective. The condenser should be lowered to better visualize the elements by reducing the illumination.
Cellular elements in urine are shown in Figure 825.3.
Red Blood Cells
Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9-10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of > 80% of dysmorphic red cells is strongly suggestive of glomerular pathology.
The quantity of red cells can be reported as number of red cells per high power field.
Causes of hematuria have been listed earlier.
White Blood Cells (Pus Cells)
White blood cells are spherical, 10-15 μ in size, granular in appearance in which nuclei may be visible. Degenerated white cells are distorted, smaller, and have fewer granules. Clumps of numerous white cells are seen in infections. Presence of many white cells in urine is called as pyuria. In hypotonic urine white cells are swollen and the granules are highly refractile and show Brownian movement; such cells are called as glitter cells; large numbers are indicative of injury to urinary tract.
Normally 0-2 white cells may be seen per high power field. Pus cells greater than 10/HPF or presence of clumps is suggestive of urinary tract infection.
Increased numbers of white cells occur in fever, pyelonephritis, lower urinary tract infection, tubulointerstitial nephritis, and renal transplant rejection.
In urinary tract infection, following are usually seen in combination:
- Clumps of pus cells or pus cells >10/HPF
- Positive nitrite test
Simultaneous presence of white cells and white cell casts indicates presence of renal infection (pyelonephritis).
Eosinophils (>1% of urinary leucocytes) are a characteristic feature of acute interstitial nephritis due to drug reaction (better appreciated with a Wright’s stain).
Renal Tubular Epithelial Cells
Presence of renal tubular epithelial cells is a significant finding. Increased numbers are found in conditions causing tubular damage like acute tubular necrosis, pyelonephritis, viral infection of kidney, allograft rejection, and salicylate or heavy metal poisoning.
These cells are small (about the same size or slightly larger than white blood cell), polyhedral, columnar, or oval, and have granular cytoplasm. A single, large, refractile, eccentric nucleus is often seen.
Renal tubular epithelial cells are difficult to distinguish from pus cells in unstained preparations.
Squamous Epithelial Cells
Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus.
Transitional Epithelial Cells
Transitional cells line renal pelvis, ureters, urinary bladder, and upper urethra. These cells are large, and diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization and in transitional cell carcinoma.
Oval Fat Bodies
These are degenerated renal tubular epithelial cells filled with highly refractile lipid (cholesterol) droplets. Under polarized light, they show a characteristic “Maltese cross” pattern. They can be stained with a fat stain such as Sudan III or Oil Red O. They are seen in nephrotic syndrome in which there is lipiduria.
They may sometimes be seen in urine of men.
Telescoped urinary sediment: This refers to urinary sediment consisting of red blood cells, white blood cells, oval fat bodies, and all types of casts in roughly equal proportion. It occurs in lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis.
Organisms detectable in urine are shown in Figure 825.4.
Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a cleancatch midstream sample, >104 colony forming units/ml of urine in catheterized sample, and >103 colonyforming units/ml of urine in a suprapubic aspiration sample.
- Microscopic examination: In a wet preparation, presence of bacteria should be reported only when urine is fresh. Bacteria occur in combination with pus cells. Gram’s-stained smear of uncentrifuged urine showing 1 or more bacteria per oil-immersion field suggests presence of > 105 bacterial colony forming units/ml of urine. If many squamous cells are present, then urine is probably contaminated with vaginal flora. Also, presence of only bacteria without pus cells indicates contamination with vaginal or skin flora.
- Chemical or reagent strip tests for significant bacteriuria: These are given earlier.
- Culture: On culture, a colony count of >105/ml is strongly suggestive of urinary tract infection, even in asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to Gram-negative enteric bacteria, particularly Escherichia coli.
If three or more species of bacteria are identified on culture, it almost always indicates contamination by vaginal flora.
Negative culture in the presence of pyuria (‘sterile’ pyuria) occurs with prior antibiotic therapy, renal tuberculosis, prostatitis, renal calculi, catheterization, fever in children (irrespective of cause), female genital tract infection, and non-specific urethritis in males.
Yeast Cells (Candida)
These are round or oval structures of approximately the same size as red blood cells. In contrast to red cells, they show budding, are oval and more refractile, and are not soluble in 2% acetic acid.
Presence of Candida in urine may suggest immunocompromised state, vaginal candidiasis, or diabetes mellitus. Usually pyuria is present if there is infection by Candida. Candida may also be a contaminant in the sample and therefore urine sample must be examined in a fresh state.
These are motile organisms with pear shape, undulating membrane on one side, and four flagellae. They cause vaginitis in females and are thus contaminants in urine. They are easily detected in fresh urine due to their motility.
Eggs of Schistosoma haematobium
Infection by this organism is prevalent in Egypt.
They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel.
Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts. They take the shape and diameter of the lumina (molds or ‘casts’) of the renal tubules. They have parallel sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein). Since casts form only in renal tubules their presence is indicative of disease of the renal parenchyma. Although there are several types of casts, all urine casts are basically hyaline; various types of casts are formed when different elements get deposited on the hyaline material (Figure 825.5). Casts are best seen under low power objective (×10) with condenser lowered down to reduce the illumination.
Casts are the only elements in the urinary sediment that are specifically of renal origin.
Casts (Figure 825.6) are of two main types:
- Noncellular: Hyaline, granular, waxy, fatty
- Cellular: Red blood cell, white blood cell, renal tubular epithelial cell.
Hyaline and granular casts may appear in normal or diseased states. All other casts are found in kidney diseases.
Hyaline casts: These are the most common type of casts in urine and are homogenous, colorless, transparent, and refractile. They are cylindrical with parallel sides and blunt, rounded ends and low refractive index. Presence of occasional hyaline cast is considered as normal. Their presence in increased numbers (“cylinduria”) is abnormal. They are composed primarily of Tamm-Horsfall protein. They occur transiently after strenuous muscle exercise in healthy persons and during fever. Increased numbers are found in conditions causing glomerular proteinuria.
Granular casts: Presence of degenerated cellular debris in a cast makes it granular in appearance. These are cylindrical structures with coarse or fine granules (which represent degenerated renal tubular epithelial cells) embedded in Tamm-Horsfall protein matrix. They are seen after strenuous muscle exercise and in fever, acute glomerulonephritis, and pyelonephritis.
Waxy cast: These are the most easily recognized of all casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have homogenous, smooth glassy appearance, cracked or serrated margins and irregular broken-off ends. The ends are straight and sharp and not rounded as in other casts. They are light yellow in color. They are most commonly seen in end-stage renal failure.
Fatty casts: These are cylindrical structures filled with highly refractile fat globules (triglycerides and cholesterol esters) in Tamm-Horsfall protein matrix. They are seen in nephrotic syndrome.
To be called as cellular, casts should contain at least three cells in the matrix. Cellular casts are named according to the type of cells entrapped in the matrix.
Red cell casts: These are cylindrical structures with red cells in Tamm-Horsfall protein matrix. They may appear brown in color due to hemoglobin pigmentation. These have greater diagnostic importance than any other cast. If present, they help to differentiate hematuria due to glomerular disease from hematuria due to other causes. RBC casts usually denote glomerular pathology e.g. acute glomerulonephritis.
White cell casts: These are cylindrical structures with white blood cells embedded in Tamm-Horsfall protein matrix. Leucocytes usually enter into tubules from the interstitium and therefore presence of leucocyte casts indicates tubulointerstitial disease like pyelonephritis.
Renal tubular epithelial cell casts: These are composed of renal tubular epithelial cells that have been sloughed off. They are seen in acute tubular necrosis, viral renal disease, heavy metal poisoning, and acute allograft rejection. Even an occasional renal tubular cast is a significant finding.
Crystals are refractile structures with a definite geometric shape due to orderly 3-dimensional arrangement of its atoms and molecules. Amorphous material (or deposit) has no definite shape and is commonly seen in the form of granular aggregates or clumps.
Crystals in urine (Figure 825.7) can be divided into two main types: (1) Normal (seen in normal urinary sediment), and (2) Abnormal (seen in diseased states).
However, crystals found in normal urine can also be seen in some diseases in increased numbers.
Most crystals have no clinical importance (particularly phosphates, urates, and oxalates). Crystals can be identified in urine by their morphology. However, before reporting presence of any abnormal crystals, it is necessary to confirm them by chemical tests.
Crystals present in acid urine:
- Uric acid crystals: These are variable in shape (diamond, rosette, plates), and yellow or red-brown in color (due to urinary pigment). They are soluble in alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid crystals may be mistaken for cysteine crystals that also form in acid urine.
- Calcium oxalate crystals: These are colorless, refractile, and envelope-shaped. Sometimes dumbbell-shaped or peanut-like forms are seen. They are soluble in dilute hydrochloric acid. Ingestion of certain foods like tomatoes, spinach, cabbage, asparagus, and rhubarb causes increase in their numbers. Their increased number in fresh urine (oxaluria) may also suggest oxalate stones. A large number are seen in ethylene glycol poisoning.
- Amorphous urates: These are urate salts of potassium, magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are soluble in alkali or saline at 60°C.
Crystals present in alkaline urine:
- Calcium carbonate crystals: These are small, colorless, and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve.
- Phosphates: Phosphates may occur as crystals (triple phosphates, calcium hydrogen phosphate), or as amorphous deposits.
• Phosphate crystals
Triple phosphates (ammonium magnesium phosphate): They are colorless, shiny, 3-6 sided prisms with oblique surfaces at the ends (“coffinlids”), or may have a feathery fern-like appearance.
Calcium hydrogen phosphate (stellar phosphate): These are colorless, and of variable shape (starshaped, plates or prisms).
• Amorphous phosphates: These occur as colorless small granules, often dispersed.
All phosphates are soluble in dilute acetic acid.
- Ammonium urate crystals: These occur as cactus-like (covered with spines) and called as ‘thornapple’ crystals. They are yellow-brown and soluble in acetic acid at 60°C.
They are rare, but result from a pathological process.
These occur in acid pH, often in large amounts. Abnormal crystals should not be reported on microscopy alone; additional chemical tests are done for confirmation.
- Cysteine crystals: These are colorless, clear, hexagonal (having 6 sides), very refractile plates in acid urine. They often occur in layers. They are soluble in 30% hydrochloric acid. They are seen in cysteinuria, an inborn error of metabolism. Cysteine crystals are often associated with formation of cysteine stones.
- Cholesterol crystals: These are colorless, refractile, flat rectangular plates with notched (missing) corners, and appear stacked in a stair-step arrangement. They are soluble in ether, chloroform, or alcohol. They are seen in lipiduria e.g. nephrotic syndrome and hypercholesterolemia. They can be positively identified by polarizing microscope.
- Bilirubin crystals: These are small (5 μ), brown crystals of variable shape (square, bead-like, or fine needles). Their presence can be confirmed by doing reagent strip or chemical test for bilirubin. These crystals are soluble in strong acid or alkali. They are seen in severe obstructive liver disease.
- Leucine crystals: These are refractile, yellow or brown, spheres with radial or concentric striations. They are soluble in alkali. They are usually found in urine along with tyrosine in severe liver disease (cirrhosis).
- Tyrosine crystals: They appear as clusters of fine, delicate, colorless or yellow needles and are seen in liver disease and tyrosinemia (an inborn error of metabolism). They dissolve in alkali.
- Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.
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Methods for detection of bilirubin in urine are foam test, Gmelin’s test, Lugol iodine test, Fouchet’s test, Ictotest tablet test, and reagent strip test.
- Foam test: About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also obtained with proteins and highly concentrated urine. In normal urine, foam is white.
- Gmelin’s test: Take 3 ml of concentrated nitric acid in a test tube and slowly place equal quantity of urine over it. The tube is shaken gently; play of colors (yellow, red, violet, blue, and green) indicates positive test (Figure 823.1).
- Lugol iodine test: Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test tube and add 4 drops of urine. Mix by shaking. Development of green color indicates positive test.
- Fouchet’s test: This is a simple and sensitive test.
i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride, and mix well. A precipitate of sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex).
ii. Filter to obtain the precipitate on a filter paper.
iii. To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent. (Fouchet’s reagent consists of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml).
iv. Immediate development of blue-green color around the drop indicates presence of bilirubin (Figure 823.2).
- Reagent strips or tablets impregnated with diazo reagent: These tests are based on reaction of bilirubin with diazo reagent; color change is proportional to the concentration of bilirubin. Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests are less sensitive (0.5 mg/dl).