Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger
Microscopic examinations done on fecal sample are shown in Figure 846.1.
A random specimen of stool (at least 4 ml or 4 cm³) is collected in a clean, dry, container with a tightly fitting lid (a tin box, plastic box, glass jar, or waxed cardboard box) and transported immediately to the laboratory (this is because trophozoites of Entameba histolytica rapidly degenerate and alter in morphology). About 20-40 grams of formed stool or 5-6 tablespoons of watery stool should be collected. Stool should not be contaminated with urine, water, soil, or menstrual blood. Urine and water destroy trophozoites; soil will introduce extraneous organisms and also hinder proper examination. Parasites are best detected in warm, freshly passed stools and therefore stools should be examined as early as possible after receipt in the laboratory (preferably within 1 hour of collection). If delay in examination is anticipated, sample may be refrigerated. A fixative containing 10% formalin (for preservation of eggs, larvae, and cysts) or polyvinyl alcohol (for preservation of trophozoites and cysts, and for permanent staining) may be used if specimen is to be transported to a distant laboratory.
Patient should not be receiving oily laxatives, antidiarrheal medications, bismuth, antibiotics like tetracycline, or antacids for 7 days before stool examination. Patient should not have undergone a barium swallow examination.
In the laboratory, macroscopic examination is done for consistency (watery, loose, soft or formed) (Figure 846.2), color, odor, and presence of blood, mucus, adult worms or segments of tapeworms.
Trophozoites are most likely to be found in loose or watery stools or in stools containing blood and mucus, while cysts are likely to be found in formed stools. Trophozoites die soon after being passed and therefore such stools should be examined within 1 hour of passing. Examination of formed stools can be delayed but should be completed on the same day.
After receipt in the laboratory, saline and iodine wet mounts of the sample are prepared (Figure 846.3).
A drop of normal saline is placed near one end of a glass slide and a drop of Lugol iodine solution is placed near the other end. A small amount of feces (about the size of a match-head) is mixed with a drop each of saline and iodine using a wire loop, and a cover slip is placed over each preparation separately. If the specimen contains blood or mucus, that portion should be included for examination (trophozoites are more readily found in mucus). If the stools are liquid, select the portion from the surface for examination.
Saline wet mount is used for demonstration of eggs and larvae of helminths, and trophozoites and cysts of protozoa. It can also detect red cells and white cells. Iodine stains glycogen and nuclei of the cysts. The iodine wet mount is useful for identification of protozoal cysts. Trophozoites become non-motile in iodine mounts. A liquid, diarrheal stool can be examined directly without adding saline.
Concentration of fecal specimen is useful if very small numbers of parasites are present. However, in concentrated specimens, amebic trophozoites can no longer be detected since they are destroyed. If wet mount examination is negative and there is clinical suspicion of parasitic infection, fecal concentration is indicated. It is used for detection of ova, cysts, and larvae of parasites.
Various concentration methods are available; the choice depends on the nature of parasites to be identified and the equipment/reagent available in a particular laboratory. Concentration techniques are of two main types:
The most commonly used sedimentation method is formol-ethyl acetate concentration method since: (i) it can detect eggs and larvae of almost all helminths, and cysts of protozoa, (ii) it preserves their morphology well, (iii) it is rapid, and (iv) risk of infection to the laboratory worker is minimal because pathogens are killed by formalin.
In this method, fecal suspension is prepared in 10% formalin (10 ml formalin + 1 gram feces). This suspension is then passed through a gauze filter till 7 ml of filtered material is obtained. To this, ethyl acetate (3 ml) is added and the mixture is centrifuged for 1 minute. Eggs, larvae, and cysts sediment at the bottom of the centrifuge tube (Figure 846.4). Above this deposit, there are layers of formalin, fecal debris, and ether. Fecal debris is loosened with an applicator stick and the supernatant is poured off. One drop of sediment is placed on one end of a glass slide and one drop is placed at the other end. One of the drops is stained with iodine, cover slips are placed, and the preparation is examined under the microscope.
Intestinal parasites of humans are classified into two main kingdoms: protozoa and metazoa (helminths) (Figure 846.5).
Chemical examination of feces is usually carried out for the following tests (Figure 845.1):
Presence of blood in feces which is not apparent on gross inspection and which can be detected only by chemical tests is called as occult blood. Causes of occult blood in stools are:
Occult blood test is recommended as a screening procedure for detection of asymptomatic colorectal cancer. Yearly examinations should be carried out after the age of 50 years. If the test is positive, endoscopy and barium enema are indicated.
Tests for detection of occult blood in feces: Many tests are available which differ in their specificity and sensitivity. These tests include tests based on peroxidase-like activity of hemoglobin (benzidine, orthotolidine, aminophenazone, guaiac), immunochemical tests, and radioisotope tests.
Principle: Hemoglobin has peroxidase-like activity and releases oxygen from hydrogen peroxide. Oxygen molecule then oxidizes the chemical reagent (benzidine, orthotolidine, aminophenazone, or guaiac) to produce a colored reaction product.
Benzidine and orthotolidine are carcinogenic and are no longer used. Benzidine test is also highly sensitive and false-positive reactions are common. Since bleeding from the lesion may be intermittent, repeated testing may be required.
These tests specifically detect human hemoglobin. Therefore there is no interference from animal hemoglobin or myoglobin (e.g. meat) or peroxidase-containing vegetables in the diet.
The test consists of mixing the sample with latex particles coated with anti-human haemoglobin antibody, and if agglutination occurs, test is positive. This test can detect 0.6 ml of blood per 100 grams of feces.
In this test, 10 ml of patient’s blood is withdrawn, labeled with 51Cr, and re-infused intravenously. Radioactivity is measured in fecal sample and in simultaneously collected blood specimen. Radioactivity in feces indicates gastrointestinal bleeding. Amount of blood loss can be calculated. Although the test is sensitive, it is not suitable for routine screening.
Apt test: This test is done to decide whether blood in the vomitus or in the feces of a neonate represents swallowed maternal blood or is the result of bleeding in the gastrointestinal tract. The test was devised by Dr. Apt and hence the name. The baby swallows blood during delivery or during breastfeeding if nipples are cracked. Apt test is based on the principle that if blood is of neonatal origin it will contain high proportion of hemoglobin F (Hb F) that is resistant to alkali denaturation. On the other hand, maternal blood mostly contains adult hemoglobin or Hb A that is less resistant.
Dietary fat is absorbed in the small intestine with the help of bile salts and pancreatic lipase. Fecal fat mainly consists of neutral fats (unsplit fats), fatty acids, and soaps (fatty acid salts). Normally very little fat is excreted in feces (<7 grams/day in adults). Excess excretion of fecal fat indicates malabsorption and is known as steatorrhea. It manifests as bulky, frothy, and foul-smelling stools, which float on the surface of water.
Tests for fecal fat are qualitative (i.e. direct microscopic examination after fat staining), and quantitative (i.e. estimation of fat by gravimetric or titrimetric analysis).
Fecal urobilinogen is determined by Ehrlich’s aldehyde test (see Article “Test for Detection of Urobilinogen in Urine”). Specimen should be fresh and kept protected from light. Normal amount of urobilinogen excreted in feces is 50-300 mg per day. Increased fecal excretion of urobilinogen is seen in hemolytic anemia. Urobilinogen is deceased in biliary tract obstruction, severe liver disease, oral antibiotic therapy (disturbance of intestinal bacterial flora), and aplastic anemia (low hemoglobin turnover). Stools become pale or clay-colored if urobilinogen is reduced or absent.
Deficiency of intestinal enzyme lactase is a common cause of malabsorption. Lactase converts lactose (in milk) to glucose and galactose. If lactase is deficient, lactose is converted to lactic acid with production of gas. In infants this leads to diarrhea, vomiting, and failure to thrive. Benedict’s test or Clinitest™ tablet test for reducing sugars is used to test freshly collected stool sample for lactose. In addition, oral lactose tolerance test is abnormal (after oral lactose, blood glucose fails to rise above 20 mg/dl of basal value) in lactase deficiency. Rise in blood glucose indicates that lactose has been hydrolysed and absorbed by the mucosa. Lactose tolerance test is now replaced by lactose breath hydrogen testing. In lactase deficiency, accumulated lactose in the colon is rapidly fermented to organic acids and gases like hydrogen. Hydrogen is absorbed and then excreted through the lungs into the breath. Amount of hydrogen is then measured in breath; breath hydrogen more than 20 ppm above baseline within 4 hours indicates positive test.
Fecal osmotic gap is calculated from concentration of electrolytes in stool water by formula 290-2([Na+] + [K+]). (290 is the assumed plasma osmolality). In osmotic diarrheas, osmotic gap is >150 mOsm/kg, while in secretory diarrhea, it is typically below 50 mOsm/kg. Evaluation of chronic diarrhea is shown in Figure 845.3.
Stool pH below 5.6 is characteristic of carbohydrate malabsorption.
In renal glycosuria, glucose is excreted in urine, while blood glucose level is normal. This is because of a specific tubular lesion which leads to impairment of glucose reabsorption. Renal glycosuria is a benign condition. Glycosuria can also occur in Fanconi syndrome.
In proximal renal tubular dysfunction, many amino acids are excreted in urine due to defective tubular reabsorption.
Normally, low molecular weight proteins (β2 –microglobulin, retinol-binding protein, lysozyme, and α1 –microglobulin) are freely filtered by glomeruli and are completely reabsorbed by proximal renal tubules. With tubular damage, these low molecular weight proteins are excreted in urine and can be detected by urine protein electrophoresis. Increased amounts of these proteins in urine are indicative of renal tubular damage.
If both BUN and serum creatinine are acutely increased, it is necessary to distinguish between prerenal azotemia (renal underperfusion) and acute tubular necrosis. In prerenal azotemia, renal tubules are functioning normally and reabsorb sodium, while in acute tubular necrosis, tubular function is impaired and sodium absorption is decreased. Therefore, in prerenal azotemia, urinay sodium concentration is < 20 mEq/L while in acute tubular necrosis, it is > 20 mEq/L.
Measurement of urinary sodium concentration is affected by urine volume and can produce misleading results. Therefore, to avoid this, fractional excretion of sodium is calculated. This refers to the percentage of filtered sodium that has been absorbed and percentage that has been excreted. Measurement of fractional sodium excretion is a better indicator of tubular absorption of sodium than quantitation of urine sodium alone.
This test is indicated in acute renal failure. In oliguric patients, this is the most reliable means of early distinction between pre-renal failure and renal failure due to acute tubular necrosis. It is calculated from the following formula:
In pre-renal failure this ratio is less than 1%, and in acute tubular necrosis it is more than 1%. In pre-renal failure (due to reduced renal perfusion), aldosterone secretion is stimulated which causes maximal sodium conservation by the tubules and the ratio is less than 1%. In acute tubular necrosis, maximum sodium reabsorption is not possible due to tubular cell injury and consequently the ratio will be more than 1%. Values above 3% are strongly suggestive of acute tubular necrosis.
Normal specific gravity is 1.003 to 1.030. It depends on state of hydration and fluid intake.
As a test of renal function, it gives information about the ability of renal tubules to concentrate the glomerular filtrate. This concentrating ability is lost in diseases of renal tubules.
Fixed specific gravity of 1.010, which cannot be lowered or increased by increasing or decreasing the fluid intake respectively, is an indication of chronic renal failure.
The most commonly employed test to evaluate tubular function is measurement of urine/plasma osmolality. This is the most sensitive method for determination of ability of concentration. Osmolality measures number of dissolved particles in a solution. Specific gravity, on the other hand, is the ratio of mass of a solution to the mass of water i.e. it measures total mass of solute. Specific gravity depends on both the number and the nature of dissolved particles while osmolality is exact number of solute particles in a solution. Specific gravity measurement can be affected by the presence of solutes of large molecular weight like proteins and glucose, while osmolality is not. Therefore measurement of osmolality is preferred.
When solutes are dissolved in a solvent, certain changes take place like lowering of freezing point, increase in boiling point, decrease in vapor pressure, or increase of osmotic pressure of the solvent. These properties are made use of in measuring osmolality by an instrument called as osmometer.
Osmolality is expressed as milliOsmol/kg of water.
Urine/plasma osmolality ratio is helpful in distinguishing pre-renal azotemia (in which ratio is higher) from acute renal failure due to acute tubular necrosis (in which ratio is lower). If urine and plasma osmolality are almost similar, then there is defective tubular reabsorption of water.
If the value of baseline osmolality of urine is inconclusive, then water deprivation test is performed. In this test, water intake is restricted for a specified period of time followed by measurement of specific gravity or osmolality. Normally, urine osmolality should rise in response to water deprivation. If it fails to rise, then desmopressin is administered to differentiate between central diabetes insipidus and nephrogenic diabetes insipidus. Urinary concentration ability is corrected after administration of desmopressin in central diabetes insipidus, but not in nephrogenic diabetes insipidus.
If urine osmolality is > 800 mOsm/kg of water or specific gravity is ≥1.025 following dehydration, concentrating ability of renal tubules is normal. However, normal result does not rule out presence of renal disease.
False result will be obtained if the patient is on low-salt, low-protein diet or is suffering from major electrolyte and water disturbance.
This test assesses the capacity of the kidney to make urine dilute after water loading.
After overnight fast, patient empties the bladder and drinks 20 ml/kg of water in 15-30 minutes. The urine is collected at hourly intervals for the next 4 hours for measurements of urine volume, specific gravity, and osmolality. Plasma levels of antidiuretic hormone and serum osmolality should be measured at hourly intervals.
Normally, more than 90% of water should be excreted in 4 hours. The specific gravity should fall to 1.003 and osmolality should fall to < 100 mOsm/kg. Plasma level of antidiuretic hormone should be appropriate for serum osmolality. In renal function impairment, urine volume is reduced (<80% of fluid intake is excreted) and specific gravity and osmolality fail to decrease. The test is also impaired in adrenocortical insufficiency, malabsorption, obesity, ascites, congestive heart failure, cirrhosis, and dehydration.
This test is not advisable in patients with cardiac failure or kidney disease. If there is failure to excrete water load, fatal hyponatremia can occur.
Diagnosis of renal tubular acidosis is usually considered after excluding other causes of metabolic acidosis. This test is considered as a ‘gold standard’ for the diagnosis of distal or type 1 renal tubular acidosis. Urine pH and plasma bicarbonate are measured after overnight fasting. If pH is less than 5.4, acidifying ability of renal tubules is normal. If pH is greater than 5.4 and plasma bicarbonate is low, diagnosis of renal tubular acidosis is confirmed. In both the above cases, further testing need not be performed. In all other cases in which neither of above results is obtained, further testing is carried out. Patient is given ammonium chloride orally (0.1 gm/kg) over 1 hour after overnight fast and urine samples are collected hourly for next 6-8 hours. Ammonium ion dissociates into H+ and NH3. Ammonium chloride makes blood acidic. If pH is less than 5.4 in any one of the samples, acidifying ability of the distal tubules is normal.