ROUTINE STOOLE EXAMINATIN

Dr. ajit kumar singh

1. Routine stool examination Dr. Ajit Kumar Singh (MD Laboratory Medicine) PGT 1st year Guided by Dr. Subhranshu Mandal M.B.B.S., M.D (Microbiology) Associate Professor Department Of Laboratory Medicine CHITTRANJAN NATIONAL CANCER INSTITUTE , KOLKATA

2. Introduction Normal amount of stools in an adult is 100-200 g/day (soft and formed). Stools are composed of : – water (up to 75%) – indigestible residue – undigested food – food which is digested but not absorbed – bile, epithelial cells, secretions from digestive tract – inorganic material – Bacteria

3. Indications for stool examination To evaluate the function and integrity of the GI tract. Rule out the presence of WBCs and RBCs. Find ova or parasites. To see the presence of fat for malabsorption syndrome. For screening for colon cancer. For asymptomatic ulceration of GI tract. Bacteriologic examination. Evaluation of chronic diarrhea. Identification of Rotavirus.

4. Collection of stool samples A random specimen of stool (at least 4 ml or 4 cm 3 ) is collected in a clean, dry, container with a tightly fitting lid & transported immediately to the laboratory About 20-40 grams of formed stool or 5-6 tablespoons of watery stool should be collected Parasites are best detected in warm, freshly passed stools which should be examined as early as possible (preferably within 1 hour of collection) Stool should not be contaminated with urine, water, or menstrual blood 3 separate samples collected at 3-day intervals are recommended to detect all parasite infections One negative report for ova and parasites does not exclude the possibility of infection

5. Stool preservative Preservative for the wet preparation are : 10% formol-saline . Sodium acetate formalin. Methionate iodine formalin. Sample may be refrigerated if delay in examination is anticipated. A fixative may be used if specimen is to be transported to a distant laboratory. 10% formalin for preservation of eggs, larvae & cysts. For staining , use Polyvinyl alcohol , iron hematoxylin , modified acid-fast stain and trichrome (most commonly used) Avoid preservatives for the culture of stool (usually , three parts of the preservative and one part of the stool)

6. Macroscopic examination (Gross stool examination) Findings :- Color Consistency Quantity Odor Presence of blood Presence of mucus Presence of adult worms or segments of tapeworms

7. Consistency of Stool samples 1. Hard 2. Formed 3. Semi-formed 4. Soft 5. Loose 6. Diarrhea like 7. Watery or liquid •Cysts are likely to be found in formed stools. • Trophozoites are most likely to be found in loose or watery stools or in stools containing blood and mucus. • 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.

8. Color/Appearance of Stool samples

9. Macroscopic examination Odor Stool odor – Indole and skatole which are formation by bacterial fermentation and putrefaction. Foul odor – Undigested protein & by excessive intake of carbohydrate. Sickly odor – Undigested lactose & fatty acids. Putrid odor – severe diarrhea of malignancy or gangrenous dysentery.

10. Macroscopic examination Presence of mucus in stools: Translucent gelatinous material clinging to surface of stool. Produced by colonic mucosa in response to parasympathetic stimulation. Seen in – Severe constipation – Mucous colitis

11. Macroscopic examination Presence of mucus and blood in stools Seen in Bacillary dysentery Ulcerative Colitis Intestinal tuberculosis Amoebiasis Enteritis Mucus with blood clinging to stool is seen in Lower GIT malignancy. Inflammatory lesions of anal canal

12. Preparation of slides 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 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 that portion from the surface for examination.

13. Preparation of slides Saline wet mount is used for demonstration of eggs and larvae of helminths, and trophozoites and cysts of protozoa. Saline wet mount can also detect red cells and white cells. The iodine wet mount is useful for identification of protozoal cysts as iodine stains glycogen and nuclei of the cysts. Trophozoites become non-motile in iodine mounts. A liquid, diarrheal stool can be examined directly without adding saline.

14. Concentration of stool samples Useful if very small numbers of parasites are present. Indicated in cases of negative wet mount examination and there is clinical suspicion of parasitic infection. Used for detection of ova, cysts, and larvae of parasites. Demerit - Amoebic trophozoites are destroyed in concentrated specimens and therefore cannot be detected. Concentration techniques are of two main types: Sedimentation techniques : – Ova and cysts settle at the bottom. – Excessive stool debris may make the detection of parasites difficult. e.g. Formol ethyl acetate sedimentation procedure.

15. Concentration of stool samples Floatation techniques : – Ova and cysts float on surface. – Some ova and cysts do not float at the top in this procedure. e.g. Saturated salt floatation technique, zinc sulphate concentration technique The most commonly used sedimentation method is formol-ethyl acetate sedimentation method because: 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. iv. risk of infection to the laboratory worker is minimal because pathogens are killed by formalin.

16. Concentration of stool samples Formol-ethyl acetate sedimentation method • Stool sample suspension is prepared in 10% formalin (10 ml formalin + 1 gram stool sample) & passed through a gauze filter till 7 ml of filtered material is obtained. • Ethyl acetate (3 ml) is added to this filtered material & the mixture is centrifuged for 1 minute. • Eggs, larvae & cysts sediment at the bottom of the centrifuge tube with layers of formalin, stool debris, and ether above. • Stool debris is loosened with an applicator stick & the supernatant is poured off. • One drop of sediment is placed on one end of a glass slide & one drop is placed at the other end. • One of the drops is stained with iodine, cover slips are placed, & the preparation is examined under the microscope.

17. Microscopic examination Findings :- Leukocytes (WBCs) Red Blood Cells (RBCs) Macrophages Epithelial cells Bacteria Ova/ Cysts/ Trophozoites of parasites Meat/muscle fibres Fat

18. Microscopic examination 1 . Leukocytes (WBCs) Normal stool may contain occasional (0-1) WBCs. To look for WBCs, the smears should be prepared from areas of mucous or watery stools. Increased no of WBCs in stools is associated with : Bacillary dysentery Chronic ulcerative colitis Shigellosis Salmonella infections Invasive E-Coli infections Anal/Rectal Fistula Localised abscess Amoebiasis & typhoid

19. Microscopic examination 2.Red Blood Cells (RBCs) • Bright red stool is seen in cases of lower GIT bleeding. • Black and tarry blood are seen in cases of – Upper GIT bleeding. – Occult bleeding. •RBCs Present in – Dysentery – Hemorrhoids – GIT Malignancies

20. Microscopic examination 3.Macrophages • seen in – Bacillary dysentery – Ulcerative colitis 4.Epithelial cells • Seen in inflammatory conditions of the bowel 5.Bacteria At least 3 stool cultures collected on separate days are recommended if the patient’s clinical picture suggests bacterial involvement, despite previous negative cultures. Stool cultures are commonly done to identify bacteria associated with enteric infection. Epithelial cell

21. Microscopic examination 6.Fat Present in : • Malabsorption • Deficiency of pancreatic digestive enzyme • Deficiency of bile 7.Meat/muscle fibres in stools : Their presence show impaired intraluminal digestion. Increased amount of meat fibres are found in • Malabsorption syndrome • Pancreatic functional defect like cystic fibrosis. 8.Ova/ Cysts/ Trophozoites of parasites • Normally there are no parasites/eggs in the stool sample. • Multiple stool samples should be examined to rule out parasitic infestations • At least 3 consecutive days’ stool samples are examined to rule out parasitic infestations

22. Microscopic examination

23. It is also important to identify artefacts during microscopic examination of stool samples which could be confused with ova & cysts of various protozoa & helminths Microscopic examination - artefacts

24. Microscopic examination - protozoa Entamoeba histolytica • Demonstration of trophozoites of E. histolytica in stool samples is required for diagnosis of amoebic dysentery. • For diagnosis, at least three fresh stool samples should be examined to increase sensitivity. • Trophozoites vary from 15 to 40 μ in diameter. • In saline wet mounts, trophozoites show motility in one direction via pseudopodia, which form rapidly.

25. Microscopic examination - protozoa Entamoeba histolytica : Diagnostic feature of E. histolytica trophozoites is the presence of ingested red cells. E.histolytica trophozoites in dysentery faecal specimen

26. Relative sizes of trophozoites and cysts of intestinal protozoa, common nematode eggs and larvae of Strongyloides seen in microscope Field using the 40X objective(with 10X eyepieces) 1. I. belli oocyst, 2. A. lumbricoides egg, 3. Leucocytes, 4. E.histolytica/E. dispar cyst, 5. E.histolytica trophozoites(motile), 6. Red cells, 7. S. stercoralis larva (motile), 8. E.coli cyst (mature), 9. G. lamblia cyst, 10. C. mesnili cyst, 11. Hookworm egg, 12. G. lamblia trophozoites (motile) Relative sizes of helminth eggs as seen in microscope Field using the 10X objective(with 10X eyepieces) eggs are as seen in saline preparation 1. E. vermicularis, 2. A. lumbricoides 3. S. stercoralis larva (motile), 4. Hookworm, 5. T.trichiura, , 6 . D.latum , 7. O.sinensis, 8. Fasciola sp , 9. S.mansoni , 10. Paragonimus sp , 11. S.japonicum , 12. S.intercalatum , 13. Taenia sp , 14. V.nana , 15. H.dimineta

27. Fig 1 . E.histolytica / E.dispar cysts showing chromatoid bodies; (a) immature single nucleus cyst in saline, (b) Iodine stained mature nuclei, (c) Burrow stained chromatoid body in cyst, left Acrydine orange stained chromatoid bars. Fig 2. Left: G.lamlia trophozoites in saline preparation, Right: Stained G.lamblia trophozoites seen with 100X Fig 4. Cryptosporidium oocysts in faecal smear as seen with 40X objective Fig 3. Cysts of G. lamblia (a) In eosin preparation, (b) In saline preparation, (c) In iodine preparation

28. Fig 7. Egg of T. trichiura Egg of C. philippineensis Fig 5. Cyclospora oocyst in Ziehl - Neelsen stained faecal smear Fig 6. Left : Infertile egg of A. lumbricoides . Right : Fertile egg of A. lumbricoides Fig 8. Hookworm eggs; Left segmented; Right ;Embryonated Egg of Trichostrongylus

29. Fig 9. Larva of S.stercoralis seen with 100X objective Fig 10. Egg of S.mansoni with lateral spine Fig 11. Egg of S.intercalatum with terminal spines Fig 12. Egg of S. japonicum Fig 13. Egg of O.sinensis Fig 14. Eggs of F. hepatica

30. Fig 15. Taenia species showing small hooklets Fig 16. Egg of D. latum containing yolk cells Fig 17. Egg capsule of D. caninum

31. Chemical examination Chemical examination of feces is usually carried out for the following tests : • Occult blood ( IBD & neoplasm) • Excess fat excretion (malabsorption) • Urobilinogen (liver disease) • Reducing sugars( carbohydrate malabsorption) • stool osmotic gap(distinction b/w secretory and osmotic disease) • stool pH (carbohydrate malabsorption)

32. Test for Occult Blood in Stools Presence of blood in feces which is not apparent on gross inspection and which can be detected only by chemical tests is called as occult blood. Principle: Hemoglobin has peroxidase-like activity & 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.

33. Test for Occult Blood in Stools

34. Test for Occult Blood in Stools Immunochemical Tests • These tests specifically detect human hemoglobin. • 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 & if agglutination occurs, test is positive. • This test can detect 0.6 ml of blood per 100 grams of feces.

35. Test for Occult Blood in Stools Apt test This test devised by Dr. Apt 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 GIT. • 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

36. Microscopic stool examination for fat Test for Malabsorption of Fat qualitative (i.e. direct microscopic examination after fat staining) quantitative (i.e. estimation of fat by gravimetric or titrimetric analysis) 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. Presence of ≥ 60 fat droplets/HPF indicates steatorrhea. Ingestion of mineral or castor oil and use of rectal suppositories can cause problems in interpretation .

37. Quantitative estimation of fecal fat 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.

38. Test for Urobilinogen in Feces Fecal urobilinogen is determined by Ehrlich’s aldehyde test Sample 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 decreased in biliary tract obstruction, severe liver disease, oral antibiotic therapy (disturbance of intestinal bacterial flora) & aplastic anemia (low hemoglobin turnover). Stools become pale or clay-colored if urobilinogen is reduced or absent.

39. Test for Reducing Sugars Deficiency of intestinal enzyme lactase which converts lactose (in milk) to glucose and galactose, is a common cause of malabsorption. If lactase is deficient, lactose is converted to lactic acid with production of gas which leads to diarrhea, vomiting, and failure to thrive in infants. Benedict’s test for reducing sugars is used to test freshly collected stool sample for lactose. Fecal pH ( 7.0 – 7.5 ) Stool pH below 5.6 is characteristic of carbohydrate malabsorption.

40. Trypsin & Chymotrypsin in Stools Trypsin is a proteolytic enzyme formed in the small intestine . Inadequate trypsin secretion can lead to malabsorption & abdominal discomfort. No trypsin activity is detectable in constipated stools owing to prolonged exposure to intestinal bacteria, which inactivates trypsin. Chymotrypsin, an intestinal proteolytic enzyme secreted by the pancreas, can be used to assess pancreatic function. Fecal chymotrypsin is a more reliable measurement of pancreatic function than trypsin.

41. Stool Electrolytes: Sodium, Chloride, Potassium & Osmolality Stool electrolyte tests are used to assess electrolyte imbalance in patients with diarrhea. Stool electrolytes must be evaluated along with the serum and urine electrolytes as well as clinical findings in the patient. Stool osmolality is used in conjunction with blood serum osmolality to calculate the osmotic gap and to diagnose intestinal disaccharide deficiency Fecal Osmotic Gap : Fecal osmotic gap is calculated from concentration of electrolytes in stool water by the formula 290-2([Na+] + [K+]) (290 is the assumed plasma osmolality). • In osmotic diarrhea, osmotic gap >150 mOsm /kg. • In secretory diarrhea, osmotic gap < 50 mOsm /kg

42. Reference ranges in stool examination Bulk: 100-200 grams/day Color: Brown Water: Up to 75% pH: 7.0-7.5 Red blood cells: Absent White blood cells: Few Epithelial cells: Present Crystals: Calcium oxalate, triple phosphate

43. Reference ranges in stool examination Fat (Adults): <7 grams/day (gravimetric method) , <6grams/day (titrimetric method) Fat droplets: Average 2.5 per high power field in random sample Urobilinogen: 50-300 mg/24 hours Parasites: Nil Ova, cysts, trophozoites: Nil

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