MICROSCOPIC EXAMINATION OF FECES

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

Additional Info

  • Reference(s):
    • American Gastroenterological Association. AGA technical review on the evaluation and management of chronic diarrhea. Gastroenterology 1999;116: 1464-86.
    • American Gastroenterological Association Medical Position Statement: Guidelines for the evaluation and management of chronic diarrhoea. Gastroenterology 1999;116:1461-3.
    • Haque R, Huston CD, Hughes M, Houpt E, Petri, WA Jr. Amebiasis. New Engl J Med 2003;348:1565:73.
    • Kucik CJ, Martin GL, Sortor BV. Common intestinal parasites. Am Fam Physician 2004;69:1161-8.
Last modified on Thursday, 31 August 2017 00:41
Dayyal Dg.

Medical Laboratory Technician at National Institute of Cardiovascular Diseases, Karachi. | Author/Writer/Blogger

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    Figure 859.1 Parts of stomach and their lining cells
    Figure 859.1 Parts of stomach and their lining cells 
     
    In the stomach, ingested food is mechanically and chemically broken down to form semi-digested liquid called chyme. Following relaxation of pyloric sphincter, chyme passes into the duodenum.
     
    There are three phases of gastric acid secretion: cephalic, gastric, and intestinal.
     
    • Cephalic or neurogenic phase: This phase is initiated by the sight, smell, taste, or thought of food that causes stimulation of vagal nuclei in the brain. Vagus nerve directly stimulates parietal cells to secrete acid; in addition, it also stimulates antral G cells to secrete gastrin in blood (which is also a potent stimulus for gastric acid secretion) (Figure 859.2). Cephalic phase is abolished by vagotomy.
    • Gastric phase: Entry of swallowed food into the stomach causes gastric distension and induces gastric phase. Distension of antrum and increase in pH due to neutralization of acid by food stimulate antral G cells to secrete gastrin into the circulation. Gastrin, in turn, causes release of hydrochloric acid from parietal cells.
    • Intestinal phase: Entry of digested proteins into the duodenum causes an increase in acid output from the stomach. It is thought that certain hormones and absorbed amino acids stimulate parietal cells to secrete acid.
     
    The secretion from the stomach is called as gastric juice. The chief constituents of the gastric juice are:
     
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    • Pepsin: Pepsin is secreted by chief cells in stomach. Pepsin causes partial digestion of proteins leading to the formation of large polypeptide molecules (optimal function at pH 1.0 to 3.0). Its secretion is enhanced by vagal stimulation.
    • Mucus
    • Intrinsic factor (IF): IF is necessary for absorption of vitamin B12 in the terminal ileum. It is secreted by parietal cells of stomach.
     
    Figure 859.2 Stimulation of gastric acid secretion
    Figure 859.2 Stimulation of gastric acid secretion. Three receptors on parietal cells stimulate acid secretion: histamine (H2) receptor, acetylcholine or cholinergic receptor, and gastrin/CCK-B receptor. Histamine is released by enterochromaffin-like cells in lamina propria. Acetylcholine is released from nerve endings. Gastrin is released from G cells in antrum (in response to amino acids in food, antral distention, and gastrin-releasing peptide). After binding to receptors, H+ is secreted in exchange for K+ by proton pump
  • CONTRAINDICATIONS TO GASTRIC ANALYSIS
    • Gastric intubation for gastric analysis is contraindicated in esophageal stricture or varices, active nasopharyngeal disease, diverticula, malignancy, recent history of severe gastric hemorrhage, hypertension, aortic aneurysm, cardiac arrhythmias, congestive cardiac failure, or non-cooperative patient.
    • Pyloric stenosis: Obstruction of gastric outlet can elevate gastric acid output due to raised gastrin (following antral distension).
    • Pentagastrin stimulation is contraindicated in cases with allergy to pentagastrin, and recent severe gastric hemorrhge due to peptic ulcer disease.
     
    Gastric analysis is not a commonly performed procedure because of following reasons:
     
    • It is an invasive and cumbersome technique that is traumatic and unpleasant for the patient.
    • Information obtained is not diagnostic in itself.
    • Availability of better tests for diagnosis such as endoscopy and radiology (for suspected peptic ulcer or malignancy); serum gastrin estimation (for ZE syndrome); vitamin assays, Schilling test, and antiparietal cell antibodies (for pernicious anemia); and tests for Helicobacter pylori infection (in duodenal or gastric ulcer).
    • Availability of better medical line of treatment that obviates need for surgery in many patients.
  • LABORATORY TESTS FOR GASTRIC ANALYSIS
    1. Hollander’s test (Insulin hypoglycemia test): In the past, this test was used for confirmation of completeness of vagotomy (done for duodenal ulcer).

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

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

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

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

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

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

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

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

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