VARIOUS LABORATORY TECHNIQUE USED IN MEDICAL PARASITOLOGY

THIS TOPIC IS FOR MEDICAL PARASITOLOGY

1. 4.3 Various laboratory technique: Feacel (stool) examination: physical, chemical-reducing substances and occult blood, and microscopic. Blood examination by wet and stained smears preparation for blood parasites. Urine , sputum examination for urinary and respiratory tract parasites. Various concentration methods (floatation and sedimentation) to detect the blood and intestinal parasites .

2.  Feacel (stool) examination: physical, chemical-reducing substances and occult blood, and microscopic . Stool Examination is carried out in laboratories for various diagnostic purposes. Examination of stool is very helpful in the diagnosis of disease of the gastrointestinal tract. Mostly a clean container which does not contain any detergent or disinfectant is sufficient for all types of stool examinations including stool culture. It consist the following tests:-

3. A.PHYSICAL EXAMINATION OF STOOL: Sample should be examined immediately after collection. Samples left standing prolonged will deteriorate helminthes, Ovum, other parasites and increase the numbers of monilia and bacteria which gives wrong results, however the following aspects of stool should be examined: (1) Quantity: the adult person excretions about 150-250 gm. /day of feces, about (1/3- 1/2) of feces dry weight is bacteria.

4. (2) Consistency and form: Normal stool is well formed. But in constipation (Dehydration) the stool is solid (Hard) and the semi-solid (soft or loose) seen when taking certain medications and laxatives. In abnormal cases such as diarrhea and dysentery the stool appear liquid, or watery in nature. In cholera the stools have a rice water appearance. In cases of malabsorption of fats the stools are pale bulky and semi-solid.

5. (3) Colour : 1- Normal colours of stools are light to dark brown due to the Presence of bile pigments. 2- Dark black: In cases with bleeding into the intestinal tract the stools become dark tarry in nature due to the formation of acid hematin , if the bleeding is in the small intestines. In case of bleeding in large intestines or rectum stool color may be bright red due to fresh blood. 3- Red color: Resulted from eating certain colorful foods such as red beets. 4- Clay colour : The stool may be clay coloured due to absence of stercobilinogen in biliary tract obstruction.

6. (4) Odor: The normal fecal odor of stool resulted from Indole and Skatol . Odor of stools may become offensive in conditions like, Intestinal amoebiasis . In cases of bacillary dysentery and cholera the stools are not foul smelling due to the absence of fecal matter. (5) Blood: 1- The blood is present on the outer surface of the feces and this caused either by contamination from menstrual cycle blood in women or bleeding hemorrhoids from the blood vessels. 2- Blood should be noted in stools if present as it is indicative of Ulceration or presence of any other pathology like malignancy

7. (6) Mucus: Is present in certain conditions like amoebic or bacillary dysentery (7) Parasite : Stools may contain adult helminthes. Nematodes like Ascaris are easily visible as their size is large. Hook worms and Proglottids of cetodes may also present . These may be visible to the naked eye.

8. B. MICROSCOPIC EXAMINATION OF STOOL: The laboratory diagnosis of most parasitic infections is by the demonstration of ova of the parasite in the stools of the infected person . The stool is collected in a clean container. The stool can be examined by the following techniques (a)Wet mounts examination. (b) Iodine examination.

9. (a) Saline wet mount examination: The stool is emulsified in normal saline and a large drop is placed on a glass slide and is then covered with a cover slide. Then examined under a light microscope, it is important to examine specimen under 10X objective lens at first to observe large molecules, cells, ova and helminthes, then to the 40X objective to complete the test . It is preferable to keep the condenser down and the intensity of the light low for proper visualization of the ova and cysts. The thickness of the film should be such that one is able to see the printed letters of the newspaper through it.

10. (b)Iodine examination: Iodine preparation leads to better visualization of morphological details of ova and cysts as it stains the glycogen in them. However it has the disadvantage that the live trophozoites of Entamoeba histolytica and other live parasites cannot be seen as the iodine kills them. The examination instructions in normal saline must be followed the same in iodine test.

11. Microscopic examination include the following: (1) Pus cells: Observed in stool the same procedure as in urine. (2) RBCs: Observed in stool the same procedure as in urine. (3) Monilia (Vaginal yeast) : Observed in stool the same procedure as in urine. (4) Protozoa: (a) Entamoeba histolytica : To investigate the vegetative phase ( trophozoite ) and cyst, causing amoebic dysentery disease. (b) Entamoeba coli: trophozoite + cyst Note: - most of children diarrhea less than 2 years cause by Entamoeba coli. (c) Giardia lamblia , trophozoite + cyst, Cause watery diarrhea disease in children, especially. (d) Balantidium coli, trophozoite + cyst, causing Balantidiasis in colon.

12. (5) Worms : (a) Enterobius vermicularis (pinworm): investigating the eggs that are of convex and flat surface and a pointed end. (b) Ascaris lumbricoides : investigating for eggs which characterized by the content of granular yellow to Brown irregular albumin membrane. (c) Hookworm ( Ancylostoma duodenale ): investigating the eggs where the egg yolk is divided and surrounded by a thin membrane. (d) Tapeworms, ( Taenia solium ): investigating the worm pieces called (gravid segments or Proglottids ) that comes out with the feces. (e) Schistosoma mansoni : Investigating the eggs distinct by lateral spin.

13. C. CHEMICAL EXAMINATION OF STOOL: (a) pH: The pH of stools is acidic in amoebic dysentery and is alkaline in bacillary dysentery. (b) Reducing factors: mono sugar and di sugar ,there level in stool (6mg/g) any increase in that level indicate disturbance in enzymes that digest sugar ( e.g.Lactase,Sucrase ). Benedict’s test: Benedict’s test is a chemical test that can be used to check for the presence of reducing sugars in a given sample. Therefore, simple carbohydrates containing a free ketone or aldehyde functional group can be identified with this test. The test is based on Benedict’s reagent (also known as Benedict’s solution), which is a complex mixture of sodium citrate, sodium carbonate, and the pentahydrate of copper(II) sulfate. When exposed to reducing sugars, the reactions undergone by Benedict’s reagent result in the formation of a brick-red precipitate, which indicates a positive Benedict’s test.

14. Principle: When a reducing sugar is subjected to heat in the presence of an alkali, it gets converted into an enediol (which is a relatively powerful reducing agent). Therefore , when reducing sugars are present in the analyte , the cupric ions (Cu2+) in Benedict’s reagent are reduced to cuprous ions (Cu+ ). These cuprous ions form copper(I) oxide with the reaction mixture and precipitate out as a brick-red coloured compound.

15. Testing for Reducing Sugars: One ml of the analyte sample must be mixed with 2 ml of Benedict’s reagent and heated in a bath of boiling water for 3 to 5 minutes. The development of a brick-red coloured precipitate of cuprous oxide confirms the presence of reducing sugars in the analyte . Interpreting the Results: Colour of the Precipitate g% of Reducing Sugar Green 0.5% Yellow 1% Orange 1.5% Red 2%

16. (c) Occult blood: 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 present in a number of diseases including malignancy of the gastrointestinal tract. The reagents used are: 1- Benzidine reagent: - Development of blue colour is indicative of presence of occult blood in the stool specimen. 2- Orthotolidine : Development of green colour 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.

17. PRINCIPLE: The test is composed of guaiac impregnated paper enclosed in a cardboard frame which permits sample application to one side with development and interpretation on the reverse side. The process involves placing two specimens, collected from three successive evacuations, onto the guaiac paper. Like all guaiac paper tests for occult blood, it is based on the oxidation of phenolic compounds present in the guaiac (i.e. guaiaconic acids) to quinones resulting in production of the blue color. Because of its similarity to the prosthetic group of peroxidase , the hematin portion of the hemoglobin molecule can function in a pseudoenzymatic manner, catalyzing the oxidation of guaiac . When a fecal specimen containing occult blood is applied to the test paper, contact is made between hemoglobin and the guaiac . A pseudoperoxidase reaction will occur upon the addition of the developer solution, with a blue chromagen formed proportional to the concentration of hemoglobins . The color reaction will occur after thirty seconds

18. Hemoglobin + Developer Hb + 2H2O2 2H2O + 02 Oxidation of Guaiac O2 + Guaiac Oxidized Guaiac (Colorless) (Blue) The kits include Positive/Negative Monitors which provide a quality control system for each test. The Monitors are incorporated into each slide.

19. PROCEDURE: Materials Provided: The following materials are provided for the performance of fecal occult blood tests: Occult Blood Slides with Monitors Occult Blood Developer Specimen Applicators Mailing Envelopes METHOD A. Sample Collection and Application 1. Supply all information listed on the front flap of the Occult Blood Slide. Open the front flap. 2. Using the applicator provided, collect a small amount of specimen from different areas (e.g. surface or interior) of the stool on one end of applicator. Apply a very thin smear in Box A.

20. 3. Reuse applicator to obtain a second sample from a different part of the stool specimen. Apply a very thin smear inside Box B. (On subsequent bowel movements, repeat above steps on additional slides.) 4. Flush stool, and discard stick in waste container. 5. Allow the specimen to air dry, then close the cover B. Development 1. Open perforated Open perforated window on the back of the slide. 2. Apply two (2) drops of Occult Blood Developer to the back side of boxes A and B. 3. Read results after 30 seconds and within 2 minutes. 4. Record the results; any trace of blue color, within or on the outer rim of the specimen, is positive for occult blood.

21. INTERPRETATION OF RESULTS: Any trace of blue color within the specimen application area, within the specified time, is positive for occult blood An absence of blue color indicates no detectable occult blood in the specimen. Causes of False-positive Tests : 1. Ingestion of peroxidase -containing foods like red meat, fish, poultry, turnips, horseradish, cauliflower, spinach, or cucumber. Diet should be free from peroxidase -containing foods for at least 3 days prior to testing. 2. Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons Causes of False-negative Tests : 1. Foods containing large amounts of vitamin C. 2. Conversion of all hemoglobin to acid hematin (which has no peroxidase -like activity) during passage through the gastrointestinal tract.

22.  Blood examination by wet and stained smears preparation for blood parasites. Blood Smear Preparation: Cleaning new slides: Slides should be absolutely grease free and clean. Procedure: To make it absolutely greasefree the slides are soaked overnight in 2% glacial acetic acid in ethyl alcohol, washed in distilled water, dried and cleaned with dry muslin cloth before use Blood collection vials : EDTA containing blood collecting vial Types of blood smear: Wet smear ( Wet blood film) Thin smear Thick Blood Smear

23. Wet smear ( Wet blood film) A wet blood film is used for the detection of living trypanosomes, microfilariae of filarial worms etc. Preparation Method : Place a small drop of blood on to a clean grease free slide and put a cover slip on it and examine under low power (10x or 40x) of the microscope. This is an easy and rapid method of detection of live Trypanosoma spp. and microfilaria or first larval stage of blood filarial worms in acute infection.

24. Thin smear Preparation Method : The site of the vein is cleared with non-fluffy cotton and ethyl alcohol to remove the contaminants and the slide is dried. The vein is punctured using a clean needle A small drop of blood, less than a pin's head is placed in the middle, near one end of the slide. Slide is held firmly between the middle finger and thumb of the left hand and another clean slide with straight and smooth edges (spreader slide), is placed on the center of the examination slide. Lower edge of the spreader slide is held at an angle of 30 to 45 degrees and is drawn up to make contact with the drop of blood and wait until drop of blood flows both end of the spreader slide. Draw the spreader slide away from the blood drop with a smooth rapid movement. This action results in thin and even blood smear. Blood film is dried by waiving it in the air. Stain and examine under microscope

25. Thick Blood Smear The diagnosis of milder infection or chronic infections when only a few Trypanosoma spp. are present in the entire blood smear. Procedure: In such cases prepare a thick blood smear in which large quantity of blood can be examined. A large drop of fresh blood is placed on a glass slide. Thick smear must be dehaemoglobinised (removal of hemoglobin) before it is stained. This is done by placing the slides in water until the colour disappear. Thick blood smears cannot be used with fowl or camel blood because of the nucleated R.B.C.

26. Points of a good blood film or smear : The film should occupy about 1/3 of the length of the slide. The greater part of it should consist of a uniform single layer of blood cells. The edges of the film should be as unbroken as possible. The film should not be so thin to break the continuity of the film.

27. Smears preparation for blood parasites: Stained blood smears are used to visualize and identify blood parasites under a microscope.  The process involves preparing a thin smear of blood on a slide, fixing it, and then staining it with a specific dye, commonly Giemsa or Leishman's stain.  This allows for the differentiation of various blood cells and the detection of parasitic organisms. 

28. Steps in Stained Smear Preparation: 1. Smear Preparation: A small drop of blood is placed on a clean microscope slide.  A second slide is used to spread the blood thinly across the first slide, creating a thin smear.  Alternatively, a thick smear can be prepared by spreading the blood drop into a circular pattern.  Thick and thin smears can be prepared on separate slides or together on one slide.  2. Smear Drying: Allow the smear to dry completely at room temperature.  Do not heat the smear, as this can damage the parasites.  3. Smear Fixation: Thin smears are typically fixed with absolute methanol for a short time (e.g., 1 minute) to preserve cell morphology.  Thick smears are not fixed with methanol to allow for proper lysis of red blood cells, aiding in parasite detection.  Staining: Giemsa stain:  This stain is commonly used for blood parasites, especially malaria.  Leishman's stain:  Another option for staining blood smears, often used for detecting malarial parasites in thick films. 

29. Giemsa stain:   Giemsa stain was a name adopted from a Germany Chemist scientist, for his application of a combination of reagents in demonstrating the presence of parasites in malaria. It belongs to a group of stains known as  Romanowsky stains . These are neutral stains made up of a mixture of oxidized methylene blue, azure, and Eosin Y and they performed on an air-dried slide that is post-fixed with methanol. Romanowsky stains are applied in the differentiation of cells, pathological examinations of samples like blood and bone marrow films and demonstration of parasites e.g malaria. There are four types of Romanoswsky stains: Giemsa stain Jenner Stain Wright stain May- Grunwald Stain

30. Objectives of Giemsa stain: To accurately prepare the Giemsa stain stock solution To stain and identify blood cells To differentiate blood cells nuclei from the cytoplasm Principle: Giemsa stain is a gold standard staining technique that is used for both thin and thick smears to examine blood for malaria parasites, a routine check-up for other blood parasites and to morphologically differentiate the nuclear and cytoplasm of Erythrocytes, leucocytes and Platelets and parasites. Like any type of Romanowsky stains, it composed of both the Acidic and Basic dyes, in relation to affinities of acidity and basicity for  blood  cells. Azure and methylene blue, a basic dye binds to the acid nucleus producing blue-purple color. Eosin is an acidic dye that is attracted to the cytoplasm and cytoplasmic granules which are alkaline-producing red coloration. The stain must be buffered with water to pH 6.8 or 7.2, to precipitate the dyes to bind simple materials.

31. Reagents Used: Methanol Giemsa powder Glycerin Water (Buffer) Procedure: Preparation of the Giemsa Stain Stock solution (500ml) Into 250ml of methanol, add 3.8g of Giemsa powder and dissolve. Heat the solution up to ~60 o C Then, add 250ml of glycerin to the solution, slowly. Filter the solution and leave it to stand for about 1-2 months before use.

32. Preparation of Working solution Add 10ml of stock solution to 80ml of distilled water and 10ml of methanol Staining procedure 1: Thin Film staining On a clean dry microscopic glass slide, make a thin film of the specimen (blood) and leave to air dry. dip the smear (2-3 dips) into pure methanol for fixation of the smear, leave to air dry for 30seconds Flood the slide with 5% Giemsa stain solution for 20-30 minutes. Flush with tap water and leave to dry

33. Staining Procedure 2: Thick Film Staining Add a thick smear of blood and air dry for 1 hour on a staining rack. Dip the thick blood smear into diluted Giemsa stain (prepared by taking 1ml of the stock solution and adding to 49ml of phosphate buffer or distilled water, but the results may vary differently). Wash the smear by dipping in in buffered water of distilled water for 3-5 minutes Leave it to dry Results: The Cytoplasm and cytoplasmic granules of blood cells appear red in color while the nucleus appears blue-purple in color. The erythrocytes will appear pink in clour Eosinophils will have a blue-purple nucleus, a pale pink cytoplasm, and orange-red granules. Neutrophils will appear purple-red nucleus and a pink cytoplasm. Basophils will have a purple nucleus and bluish granules. Lymphocytes have a dark blue nucleus and a light blue cytoplasm. Monocytes will have a purple nucleus and a pink cytoplasm. Platelets will have purple granules.

34. Leishman's stain:   Use: Leishman stain and Wrights stain are most commonly used stains in haematology laboratory, especially for visualizing malarial parasites. They can be also used for routine blood smear staining procedures. Preparation: 1. Methanol Solution: Dissolve 0.15g of Leishman's powder in 100ml of methanol. Some sources suggest using 1.5g of powder in 500ml of methanol and adding glass beads for better mixing.  2. Dilution: Add an equal volume of distilled water or a phosphate buffer (pH 6.8) to the methanol solution. Alternatively, add double the volume of distilled water to the stain after it's applied to the slide.  3. Incubation: Allow the stain to incubate at 37°C overnight. 

35. Principle: The polychromic staining solutions (Wrights, Leishmans ) contain methylene blue and eosin. These basic and acidic dyes induce multiple colours when applied to cells. Methanol acts as fixative and also as a solvent. The fixative does not allow any further change in the cells and makes them adhere to the glass slide. The basic component of white cells (i.e. cytoplasm) is stained by acidic dye and they are described as eosinophilic or acidophilc The acidic components (e.g. nucleus with nucleic acid) take blue to purple shades by the basic dye and they are called basophilic. The neutral components of the cell are stained by both the dyes Storage And Stability: 1. Store the bottle in dry, cool and dark place. 2. The shelf life of reagents is as per the expiry date mentioned on the reagent bottle labels.

36. Procedure: Fixing and staining procedure for blood smears 1. Stain provided contains methanol so it does not require separate fixing. 2. If staining is to be done later, smear can be fixed using methanol for 2-3 min 3. Cover the slide with Leishman Stain for 5 min. This also allows fixation of the smear. 4. Add on the slide Buffered Water(pH 7.0) of about double the volume of the stain, allow staining to continue for 5-7 min., a metallic sheen should be formed on top of this mixture. Staining time may have to be adjusted according to the reaction of the stain. Reduce the time if over stained increase the time if poorly stained. 5. Wash the stain off in the stream of buffered water until it has acquired a pinkish tinge. Do not tip off the stain, this will leave a deposit of stain on the blood film and will hamper microscopic examination

37. Drying of blood film: 1. Shake off the buffered water adhering to the slide and set the slide in an upright position in a drying rack. Keep the smeared surface of the slide facing down. This will avoid picking up dust. 2. After complete drying, observe the stained slide under oil immersion lens. Result Morphology and staining properties : Granulocytes: These are cells with granulated cytoplasm which stain faint pink. These include Neutrophils , and Basophils . Neutrophils : Pale pink cytoplasm with fine mauve coloured granules, include band and segmented forms (normally 3 lobed) of nucleus. Eosinophils : Cytoplasm stains faint pink, contains large red orange granules and bilobed nucleu Basophils : Cytoplasmic granules appear large, dark blue black which fill the cell and obscular nucleus Lymphocytes: Large size lymphocytes have clear blue cytoplasm on the margins of the nucleus. In smaller lymphocytes, dark violet coloured nucleus fills the entire cell and has a rim of clear cytoplasm Malaria parasites appear as red (or pinkish-red) with a blue (or bluish-purple) cytoplasm. The nucleus of the parasite will stain red, and the cytoplasm will stain blue. 

38.  Urine, sputum examination for urinary and respiratory tract parasites. Urinalysis: A urinalysis (UA), also known as routine and microscopy (R&M), is an array of tests performed on urine. A part of a urinalysis can be performed by using urine test strips, in which the test results can be read as color changes. Another method is light microscopy of urine samples. Macroscopic Urinalysis: - The first part of a urinalysis is direct visual observation. - Normal, fresh urine is pale to dark yellow or amber in color and clear. - Normal urine volume is750 to 2000 ml/24hr. - Turbidity or cloudiness may be caused by excessive cellular material or protein in the urine. - A red or red-brown (abnormal) color could be from a food dye, a drug, or the presence of hemoglobin. - If the sample contained many red blood cells, it would be cloudy as well as red.

39. Urine Dipstick Chemical Analysis: pH: Urinary pH may range from as low as 4.5 to as high as 8.0. Urine pH generally reflects the blood pH but in renal tubular acidosis (RTA) this is not the case Specific Gravity (sp gr ): • Specific gravity (which is directly proportional to urine osmolality which measures solute concentration) measures urine density, or the ability of the kidney to concentrate or dilute the urine over that of plasma. • Specific gravity between 1.002 and 1.035 on a random sample should be considered normal if kidney function is normal. • Any measurement below this range indicates hydration and any measurement above it indicates relative dehydration

40. Protein: Dipstick screening for protein is done on whole urine. Asmall amount of filtered plasma proteins and protein secreted by the nephron ( mucoprotein ) (Tamm- Horsfall protein) can be found in normal urine. Normal total protein excretion does not usually exceed 150 mg/24 hours (or 10 mg/100 ml in any single specimen). More than 150 mg/day is defined as proteinuria . Proteinuria > 3.5 gm/24 hours is severe and known as nephrotic syndrome.

41. Glucose: Nearly all glucose filtered by the glomeruli is reabsorbed in the proximal tubules and only undetectable amounts appear in urine in healthy patients. Above renal threshold (10 mmol /L) glucose will appear in urine. Glycosuria (excess sugar in urine) generally means diabetes mellitus (DM). Ketones : Ketones (acetone, acetoacetic acid, beta- hydroxybutyric acid) resulting from either diabetic ketoacidosis or some other form of caloric deprivation (starvation), are easily detected using either dipsticks or test tablets containing sodium nitroprusside .

42. Nitrite: This test relies on the breakdown of urinary nitrates to nitrites, which are not found in normal urine. Many Gram-negative and some Gram-positive bacteria are capable of producing this reaction and a positive test suggests their presence in significant numbers ( ie more than 10,000 per ml). A negative result does not rule out a UTI. Microscopic Urinalysis: Methodology - A sample of well-mixed urine (usually 10-15 ml) is centrifuged in a test tube at relatively low speed (about 2-3,000 rpm) for 5-10 minutes until a moderately cohesive button is produced at the bottom of the tube. - The supernatant is decanted and a volume of 0.2 to 0.5 ml is left inside the tube. The sediment is re_ suspended in the remaining supernatant by flicking the bottom of the tube several times. A drop of resuspended sediment is poured onto a glass slide and cover slipped

43. Examination The sediment is first examined under low power to identify most crystals, casts, squamous cells, and other large objects. The numbers of casts seen are usually reported as number of each type found per low power field (LPF). Example: 5-10 hyaline casts/LPF. Next, examination is carried out at high power to identify crystals, cells, and bacteria. The various types of cells are usually described as the number of each type found per average high power field (HPF). Example: 1-5 WBC/HPF.

44. Red Blood Cells: Hematuria is the presence of abnormal numbers of red cells in urine due to: • glomerular damage. • kidney trauma. • urinary tract stones. • upper and lower urinary tract infections. • nephrotoxins . • physical stress. • Red cells may also contaminate the urine from the vagina in menstruating women. Theoretically, no red cells should be found, but some find their way into the urine even in very healthy individuals. RBC's may appear normally shaped, swollen by dilute urine (in fact, only cell ghosts and free hemoglobin may remain). Both swollen, partly hemolyzed RBC's and are sometimes difficult to distinguish from WBC's in the urine. The presence of dysmorphic RBC's in urine suggests a glomerular disease such as a glomerulonephritis . Dysmorphic RBC's have odd shapes as a consequence of being distorted via passage through the abnormal glomerular structure

45. White Blood Cells • Pyuria refers to the presence of abnormal numbers of leukocytes that may appear with infection in either the upper or lower urinary tract or with acute glomerulonephritis . • Usually, the WBC's are granulocytes. White cells from the vagina, especially in the presence of vaginal and cervical infections. • If two or more leukocytes per each high power field appear in non-contaminated urine, the specimen is probably abnormal. • Leukocytes have lobed nuclei and granular cytoplasm.

46. Epithelial Cells Renal tubular epithelial cells, usually larger than granulocytes, contain a large round or oval nucleus and normally slough into the urine in small numbers. However, with nephrotic syndrome and in conditions leading to tubular degeneration, the number sloughed is increased. Casts: • They are solid and cylindrical structures formed by precipitation of debris in the renal tubules. • Urinary casts are formed only in the distal convoluted tubule (DCT) or the collecting duct (distal nephron ). The proximal convoluted tubule (PCT) and loop of Henle are not locations for cast formation. • Hyaline casts are composed primarily of a mucoprotein secreted by tubule cells, hyalin cast are seen in healthy individuals. • RBCs casts are formed when RBCs stick together and in glomerular disease. • WBCs casts are seen in acute pylonephritis and glomerulonephritis . • Granular and waxy casts are seen in nephrotic syndrome.

47. Bacteria: Bacteria are common in urine specimens because of the abundant normal microbial flora of the vagina or external urethral and because of their ability to rapidly multiply in urine standing at room temperature. • Therefore, microbial organisms found in all but the most carefully collected urines should be interpreted in view of clinical symptoms. Yeast: Yeast cells may be contaminants or represent a true yeast infection. They are often difficult to distinguish from red cells and amorphous crystals but are distinguished by their tendency to bud. Most often they are Candida, which may colonize bladder, urethra, or vagina . Crystals: Common crystals seen even in healthy patients include calcium oxalate, triple phosphate crystals and amorphous phosphates.

48. Five parasites commonly found in urine include  Trichomonas vaginalis , Schistosoma haematobium , microfilaria of Wuchereria bancrofti , Enterobius vermicularis , and Dioctophyma renale .  Trichomonas vaginalis : This parasitic protozoan is a common cause of urinary tract infections.  Schistosoma haematobium : This parasitic worm is the cause of urinary schistosomiasis , a disease characterized by the presence of worm eggs in urine. 

49. Microfilaria of Wuchereria bancrofti : These are the larvae of a parasitic worm, and their presence in urine can indicate lymphatic filariasis .  Enterobius vermicularis : While primarily an intestinal parasite (pinworm), its eggs can sometimes be found in urine, often due to fecal contamination.  Dioctophyma renale : This giant kidney worm is a nematode parasite, primarily found in dogs and other animals, but can also infect humans. It releases eggs into the urine. 

50. To prepare a urine slide for parasite identification: 1. Sample Collection and Centrifugation: Collect a midstream urine sample in a clean container.  Transfer 5-10 mL of urine to a conical centrifuge tube.  Centrifuge the sample for 5-10 minutes at 450g (approximately 1500-2000 RPM).  2. Removing Supernatant: Carefully decant or aspirate the supernatant (the liquid above the sediment). Leave approximately 0.5 mL of supernatant to resuspend the sediment pellet. 

51. 3. Resuspending the Sediment: Gently resuspend the sediment pellet in the remaining supernatant by gently swirling or tapping the tube.  4. Preparing the Slide: Using a pipette, place one drop of the resuspended sediment on a clean microscope slide. Cover the drop with a coverslip .  6. Microscopic Examination: Examine the prepared slide under a microscope using a low-power objective (e.g., 10x) to scan the slide for any parasites or their eggs. If needed, increase magnification to 40x for closer examination of suspicious structures. 

52. Sputum examination for respiratory tract parasites: Sputum examination, particularly microscopic analysis, can be used to detect some respiratory tract parasites. While not the primary method for diagnosing parasitic infections, it can be helpful in identifying certain parasites like Paragonimus westermani eggs, Strongyloides stercoralis larvae, Ascaris lumbricoides larvae, and hookworm larvae.  Sputum slide preparation for respiratory tract parasite detection involves collecting a sputum sample, preparing a smear, and then examining it under a microscope. The specimen is typically collected in the morning after gargling and rinsing the mouth, with the individual coughing deeply into a sterile container. The sputum is then processed, and a smear is made by spreading a thin layer of the sample on a glass slide. 

53. 1. Specimen Collection: Collect the sputum specimen first thing in the morning.  Gargle and rinse the mouth with water to remove debris.  Cough deeply and expel sputum into a sterile container.  Collect at least 5 ml of sputum.  Ensure the specimen reaches the 5 ml line on the container.  2. Specimen Processing (Concentration if needed): If concentration is needed (e.g., for protozoan cysts), use methods like formalin-ethyl acetate concentration.  If protozoa are suspected, preserve the specimen in PVA (polyvinyl alcohol) and stain with trichrome stain. 

54. 3. Slide Preparation (Smear): Place a small amount of the processed sputum (or concentrated sediment) on a clean glass slide.  Spread the sample thinly and evenly across the slide, avoiding smearing.  4. Fixation and Staining: Allow the smear to air dry completely.  Fix the smear using heat or alcohol fixation.  Stain the smear with appropriate stains (e.g., Giemsa , trichrome stain).  5. Microscopic Examination: Examine the stained slide under a microscope using appropriate objective lenses.  Look for parasites, including eggs, larvae, and trophozoites .  Note the morphology, size, and movement of any identified parasites. 

55. Various concentration methods (floatation and sedimentation) to detect the blood and intestinal parasites. Sample is Needed for Stool Examination: Can take a random stool sample. More than 2 grams of stool is needed, ideally 2 to 5 grams, which is sometimes called a pigeon’s egg. To rule out worm infestation, three consecutive stools are tested. Collect three stools in a span of 10 days. Two samples on alternate days. The hospitalized patient can take a stool sample every day. Multiple samples are needed to rule out the parasitic infestation. One sample after purgation (a substance, often a medication, that induces or speeds up the process of defecation, essentially emptying the bowels.) Collect the sample in a clean, water-tight, dry, urine-free container with a tight lid. In the case of Infants, collect from the diaper.

56. Concentration methods: It is used for: Protozoan cysts and helminth eggs. It consists of the following: Sedimentation method. Floatation technique. The methods to prepare the stool smears Saline wet preparation: Take one drop of 0.85% saline. Take a small amount of stool and mix well. The smear should be thin to see the newsprint under the slide. Put cover glass and see the 100x and 400x objective under the microscope. This is best to see helminth eggs, larvae, and trophozoites .

57. Iodine wet preparation: This is also called wet preparation. The stool is mixed with an iodine solution. Prepare Lugol’s iodine solution Contents of the Iodine solution Potassium iodide (KI) = 10 grams Iodine powder crystals = 5 grams Distilled water = 100 ml Prepare Lugol’s iodine solution Dissolve KI (Potassium iodide) in D.water (Distilled water) . Slowly add iodine crystals. Shake the solution gently until it dissolves. Filter the solution before use, and this is the stock solution. Dilute the stock solution 1:5 with D. water. Make this working solution before use.

58. Make a stool smear with Lugol’s Iodine Take a drop of Lugol’s iodine solution. Take a small amount of stool and mix it well.  Make a thin smear. Put the cover glass on it and gently press it to get an evenly thin smear. See under 100 x and 400 x objective lenses. Too weak iodine solution; in that case, organisms will not stain properly. Too strong an iodine solution will clump the stool.

59. Concentration methods for stool Purpose of the concentration method: The main aim of the concentration method is to remove debris. Also, when the parasite is low in number. The possibilities of the concentration of stool method Formalin-ethyl-acetate concentration method. Zinc-floatation method. Sheather sugar floatation method. The formalin-ethyl-acetate concentration method for stool The formalin-ethyl-acetate concentration method is most commonly used. This method recovers the helminth eggs and larvae and, to a lesser extent, trophozoites .

60. Principle: This is based on specific gravity. After centrifugation, the stool’s parasites are heavier and settle at the bottom as sediments. Debris is lighter and rises to the upper layers. Advantages are: It is easy to prepare the solution. This is inexpensive. The procedure is easy to perform. There is a rare distortion of parasite forms (eggs).

61. The procedure of formalin-ethyl-acetate concentration The stool should be fixed in formalin for at least 30 minutes. Mix 2 to 5 grams of the stool thoroughly in the 10% formalin. Filter the above stool in the formalin. This can be done by two layers of gauze or a wire screen and collecting around 3 mL. Add 10 to 12  mL of 0.85% saline and mix it well. Centrifuge for 2 minutes at 2000 RPM (or 2500 RPM). Discard the supernatant and leave 1 to 1.5 mL of the sediment. If the supernatant is cloudy, then repeat the above steps of saline. Add 9 mL of 10% formalin to the sediment. Now add 3 mL of ethyl acetate. Cap the test tube and shake well for 30 seconds. Centrifuge the tubes for 1 minute at 2000 RPM. Four layers will form.  The bottom is the sediment that is needed to prepare the smear.

62. Remove the debris with a wooden applicator stick. Decant the upper three layers carefully and leave the sediments in the test tube. Clean the sides of the test tube with a swab. Giardia cyst may stick to the side of the test tube. Add a few drops of the formalin and mix the sediment thoroughly. This will preserve the sediment. Now, we can make the smears in saline and iodine wet preparation. Examine under the microscope

63. The Zinc sulfate floatation method Some believe it is superior for concentrating and identifying eggs and protozoan cysts. The parasites are lighter and float on the surface, while the debris settles at the bottom. Principle : The zinc sulfate flotation technique relies on the difference in specific gravity between parasite eggs, cysts, and oocysts compared to the surrounding fecal matter. The zinc sulfate solution, having a higher specific gravity than these parasite components, causes them to float to the surface, while the heavier debris settles at the bottom. This allows for easier collection and identification of the parasites under a microscope. 

64. The procedure for Zinc sulfate floatation Fix the stool in the formalin. Make a dilution of the above specimen (1 mL) with tap water from 1:10 to 15. Pour the above suspension through a funnel with two layers of gauze into a small test tube. Add 2 mL of ether to the test tube with a stopper and gently shake. Add water to the above test tube to the top, just 1 cm above. Centrifuge at 2500 rpm for 45 seconds. Decant the supernatant. Add 2.5 ml of water to the sediment, shaking well to resuspend it. Repeat steps 5 and 6. Add 2.5 ml of zinc sulfate (ZnSO4) to the sediment to resuspend it. Add zinc sulfate solution to the top of the test tube, leaving only 1 to 0.5 cm open on top. Centrifuge the test tube for 2 minutes at 2000 RPM. Take the surface material with a wire loop, where you can see the parasites. Make a wet preparation with saline and iodine. Examine under the microscope.

65. Collection, Precautions, and Handling of the Stool samples The precautions before collecting the stool: Advise patients about the following things for at least 48 hours before the collection of the stool: Avoid mineral oils. Do not take bismuth. Don’t take antibiotics like tetracyclines . Don’t take anti-diarrheal drugs that are non-absorbent. Avoid anti-malarial drugs. The patient should not have a barium swallow examination before the stool examination. Stop iron-containing drugs, meat, and fish 48 hours before the collection of occult blood. Take a sample before or after one week for antibiotics or contrast media. These substances produce unknown objects or mask the parasites. Warm stools are better for the ova and parasites. Don’t refrigerate the stool for ova and parasites. Stools for ova and parasites can be collected in formalin and polyvinyl alcohol, which are used as fixatives.

66. If there is blood or mucus, it should be included in the stool because most pathogens are found in this substance. Examine the stool before giving antibiotics or other drugs. The semi-formed stool should be examined within 60 minutes of collection. The liquid stool should be examined within the first 30 minutes. The solid stool should be examined within the first hour of collection. Trophozoites degenerate in liquid stool rapidly, so examine the stool within 30 minutes. In constipated cases, use non-residual purgatives the night before collecting the stool. Avoid urine contamination because the urine destroys some of the parasites. Water destroys some of the parasites like Schistosome eggs and amoebic trophozoites . Toilet paper in the stool makes it difficult to examine the stool and may mask the parasites.

67. The stool preservative: In routine, stool preservatives used are: Preserve the stool by Formalin 5% is ideal for protozoan cysts. 10% preserves eggs and cysts. Advantages are: It is easy to prepare. It can preserve the stool for several years. It has a long shelf life. Disadvantages are: It is not good for a permanent smear. Details of eggs and cysts fade away. Trophozoites can not be recovered.

68. Preserve the stool with Polyvinyl alcohol This is an effective parasitic fixative. This can be used along with Schaudinn’s solution. Advantages are: This is easy to do with stool. This helps in gluing the sample on the slide. It has a long shelf life when stored at room temperature. Smears can stain with trichrome , iron, and hemotoxylin . Disadvantages are: A large amount of mercury is present in the solution, which is hazardous to health. It is not easy to prepare in the lab.

69. Formula of Polyvinyl alcohol

70. Perform the PVA Procedure  Add glycerol to PVA in a large beaker. Blend it with a glass rod by stirring. Gradually add water and keep on mixing. Leave it overnight before use.

71. Preserve the stool using a sodium-acetate formalin mixture (SAF) There is increased interest in the use of this fixative. Advantages are: This is good for intestinal protozoa and coccidia -like bodies. This fixative eliminates the use of mercury compounds. This is an inexpensive fixative. This is easy to prepare in the lab. It has a buffering effect to decrease the distortion of the protozoa. This can be used to concentrate the stool smear. It has good results when used with iron and hematoxylin permanent stain. Disadvantages are: This does not have adhesive properties, so you may need albumin. It is diluted with water.

72. The formula of Sodium acetate formalin: If the sample needs delay, use stool preservatives; otherwise, reject the sample. Send sample in two vials: One contains 5% or 10% formalin. The second vial contains either polyvinyl or sodium acetate formalin.

73. The preservatives for the wet preparation of stool 10% formol -saline  for the wet preparation. This is the best preservative as it kills bacteria and preserves protozoa and helminths . Another preservative is  Sodium acetate formalin (SAF). Methionate iodine formalin.  This is a good preservative for the field collection of the stool. For staining, use Polyvinyl alcohol. Avoid preservatives for the culture of stool. Usually, three parts of the preservatives and one part of the stool are used.

74. The permanent stains for the stool smears The sample of choice for stains is a thinly prepared slide from a PVA preservative (polyvinyl alcohol). There are three methods for permanent stain: Wheatley trichrome . Iron hematoxylin . Modified acid-fast stain. The most commonly used is the Wheatley trichrome .

75. Various concentration methods to detect the blood parasite: Buffy Coat- Definition, Preparation, Uses Buffy Coat Definition: A buffy coat suspension is a concentrated suspension of leukocytes and platelets that make up a part of the anticoagulated blood sample obtained by the process of density gradient centrifugation. The term buffy coat arose from the fact that the suspension has a color (yellowish beige) that is similar to buff. Buffy coats primarily contain white  blood cells  and platelets when separated from the whole blood sample via  centrifugation . The generation of buffy coat from the whole blood sample helps to concentrate large volumes of blood samples so that it decreases the downstream during cell separation and handling. In a test tube with whole blood, a thin grey-white layer of buffy coat is formed between the plasma and the hematocrit , consisting of leukocytes and platelets, both less dense than erythrocytes.

76. Buffy coat accounts for about less than 1% of the total blood volume taken in a tube. Whole blood samples are generally fractioned as a pretreatment in order to separate the buffy coat from the plasma and erythrocytes. After centrifugation, the buffy coat remains between the plasma and the erythrocytes depending on its density. The color of the buffy coat usually remains between yellow to light brown, but there might be some variations in the color as the color depends on the concentration of neutrophils . The color is more greenish when the concentration of neutrophils is high, whereas it remains yellow if the concentration is low.

77. Buffy Coat Preparation: Buffy coat preparation in laboratories is usually performed for the concentration and observation of parasites in blood samples. The following is the protocol for the preparation of buffy coat to concentrate parasites: A small narrow test tube or a bore plastic is taken and filed with an EDTA-treated anticoagulated blood sample with a Pasteur pipette. The blood sample is then centrifuged for a total of 15 minutes at the RCF of 1000g. The time might differ with laboratories as the time might range between 10 to 25 minutes. Following  centrifugation , the supernatant containing the plasma above the buffy coat layer is removed onto a new tube.

78. The buffy coat layer, along with some red cells present immediately below the buffy coat is transferred to one end of a slide. Buffy coat and the red cells are mixed with the tip of the pipette. Note:  In the case of some parasites like trypanosomes and microfilariae , a small amount of plasma is withdrawn together with the buffy coat as these parasites might be concentrated in the plasma. A thin preparation is made on the slide with a smooth-edged spreader, and the slide is allowed to air dry. When dry, the preparation is fixed with absolute methanol or ethanol for 2 minutes. The slide is then stained using either the Field’s thin-film staining technique or with the  Giemsa staining  method. The stained preparation is examined first with a 40X objective and then with a 100X objective.

79. Buffy Coat Uses: Buffy coats are important for DNA isolation from blood samples. Especially in the case of the mammalian blood sample with non-nucleated RBCs, DNA extraction is performed from white blood cells as leukocytes are about ten times more concentrated source of nucleated cells. The technique is also useful for the purification of large amounts of gDNA from relatively small sample sizes. Buffy coat preparation also allows the differentiation of white blood cells as they are more concentrated in the buddy coat than in the whole blood sample. The generation of buffy coat from the whole blood sample helps to concentrate large volumes of blood samples so that it decreases the downstream during cell separation and handling.

80. The use of a buffy coat reduces donor variability as donor-specific soluble serum factors are eliminated with the rest of the discard. The platelet-rich buffy coats have become an alternative source for the platelet concentration method as the buffy coat preparation causes less platelet activation and damage. A quantitative buffy coat is a standard laboratory test to detect infection with malaria or other blood parasites like trypanosomes, Leishmania , and Histoplasma . Buffy coat preparation is a cheaper method of blood cell separation, and it is also the ideal method to meet the emergency requirement for platelets.

81. Knott's concentration technique: Principle: The Knott's Technique detects circulating  microfilariae  and is used for the identification of  Dirofilaria   immitis microfilariae . It is the only microfilarial test that allows differentiation between Dirofilaria immitis and other filarial parasites such as the non- pathogenic Acanthocheilonema reconditum . The method is used for the detection of microfilariae in the blood. The method is more sensitive than a direct smear with fresh blood as it concentrates the microfilariae .

82. Reagents 2% formalin 1% methylene blue Procedure Mix 1 ml blood with 9 ml of 2% formalin in a conical centrifuge tube Invert the tube gently 4 times to mix the solution Centrifuge at 500  g  for 5 min Discard supernatant Stain sediment for 1-2 min with 1-2 drops of 0.1% methylene blue Add a drop of the sample on a glass slide and cover with a coverslip Examine under a light microscope at low power (10x) for microfilariae

83. Filtration methods: Filtration methods for detecting blood parasites involve concentrating parasites from a blood sample using a membrane filter, followed by microscopic examination or staining. This method is particularly useful for parasites like Wuchereria bancrofti and Brugia malayi where traditional microscopy can be less sensitive.  1. Blood Sample Preparation: A blood sample, typically 1 mL, is mixed with a lysing solution (like 10% Teepol or 2% formalin) to break down red blood cells and release parasites. The mixture is then transferred to a syringe. 

84. 2. Filtration: A syringe is connected to a filter holder containing a membrane filter with a specific pore size (e.g., 5 µm). The blood sample is forced through the filter, retaining the parasites while allowing other blood components and debris to pass through. The filter is then washed with water to remove any remaining debris, followed by air to clear excess fluid.  3. Examination: The filter is removed from the holder and examined under a microscope. For some parasites, staining with dyes like Giemsa or methylene blue is used to enhance visibility. Alternatively, the filter can be mounted on a slide and stained before examination,

85. Advantages of Filtration: Enhanced Sensitivity: Filtration can be more sensitive than traditional microscopic examination of blood smears, especially for parasites like microfilariae . Concentration of Parasites: The filtration process concentrates parasites, making them easier to identify. Reduced Interference: Filtration removes red blood cells and other debris, reducing interference with parasite identification. 

86. Thick and thin blood smears: Introduction A well-prepared blood smear is important to produce good results on analysis after doing a Giemsa stain, in identifying blood cells or/and demonstrating the presence of parasites in a sample. Below, we discuss the procedures for preparing both thin and thick smear for  Giemsa staining  technique, Importance, and applications of blood smears, in detail. Blood smears are mostly done for Differential Leukocyte count (DLC) i.e it quantifies the white blood cells and specifies the morphologies of each leukocyte. Normally, peripheral blood is used to prepare smears and depending on the function of the smear, two types of smear can be prepared. a. Thin blood smear –  for demonstration and differentiation of leukocytes. b. Thick blood smear –  for diagnosis of blood protozoan parasites and blood abnormalities eg anemiae .

87. Requirements  Sterile syringe & needle EDTA vials Tourniquet 70% isopropyl alcohol Sterile Lancet Microscopic glass slides Thick Blood Smear Preparation Specimen : Venous blood sample Principle Thick blood smears require larger volumes of blood than the thin blood smears. this allows them to be used for the detection of blood parasites in the blood samples. A thick blood smear is made by spreading a large blood drop in a small area of about 1 cm which provides a better opportunity to detect various parasitic forms against a more transparent background.

88. Procedure Collect blood sample by venipuncture and put in a clean test tube Using a capillary tube collects blood from the tube and put two large drops at the center of a sterile microscopic slide. Holding the slide between your thumb and index finger, gently shake the slide to spread the blood about 10mm in diameter. Air-dry the smear for 20-30 minutes till its completely dry then apply the appropriate Romanowski stain.

89. Thin Blood Smear Preparation Specimen : Peripheral Blood sample Principle The Thin Blood smear is prepared by making a drop of well-mixed venous blood, 2mm in diameter at the center of a sterilized microscopic glass slide. Some borders are left around the smear for easy counting and differentiating of the cells. A second glass slide is used as a spreader, streaking the blood into a thin film across the glass slide. This preparation is allowed to dry and then fixed with an appropriate Romanowski stain, depending on your objective.

90. Procedure Using a sterile pricking needle, make a prick on the index finger apply some pressure on the finger and put two drops of blood at the edge, leaving a margin on a sterile Microscopic slide. Place the edge of the sterile microscopic slide over the drops of blood, at an angle of 30-45 , and make two streaks rapidly but smoothly forward from the blood sample and spread it. This will leave a thin film of blood resembling a tongue-shape. Allow the slide to air dry and stain with an appropriate staining technique.

91. Applications of blood smears: For classification of blood disorders including types of anemia, bleeding disorders To characterize blood-related disorders such as leukemias To detect immune-mediated inflammatory disorders and infections To detect protozoal parasites:  Plasmodium falciparum ,   Mycoplasma spp ( Mycoplasma haemofelis  and  Mycoplasms haemominutus  and Bacteria such as  Bartonella  spp.

92. Advantages: It is a rapid simple technique which requires basic equipment It can be performed with very small volumes of blood. Disadvantages: Use clean slides to avoid the formation of grease spots (holes in the smear). Rapid air drying of smear to preserve cell morphologies Regular use of the technique to produce useful blood smears