Week 2 - Laboratory Handout-1 PDF

DEPARTMENT OF MEDICAL TECHNOLOGY INSTITUTE OF HEALTH SCIENCES AND NURSING FAR EASTERN UNIVERSITY, MANILA MTY1206 CLINICAL PARASITOLOGY...

DEPARTMENT OF MEDICAL TECHNOLOGY INSTITUTE OF HEALTH SCIENCES AND NURSING FAR EASTERN UNIVERSITY, MANILA MTY1206 CLINICAL PARASITOLOGY 2nd SEMESTER, A.Y. 2024-2025 SPECIMEN COLLECTION, TRANSPORT, PRESERVATION AND PROCESSING: ® Diagnosis of parasitic infections is done by: 1. Demonstration of parasite or parasite components. → Definitive diagnosis; adults, egg, larvae, cysts, oocysts, trophozoites, and antigen 2. Detection of host immune response to the parasites. → Presumptive evidence for infection; antibodies ® Stool is the most utilized specimen for examination. ® Other specimens: urine, blood, CSF, sputum, tissue aspirates and biopsies, orifice swabs SPECIMEN COLLECTION: ® Clean, wide-mouthed containers, plastic with tight-fitting lid ® Typical stool collection protocol: 1. Three (3) specimens: One every other day for 10 days 2. Patients with Amoebiasis: Six specimens in 14 days ® Requires a thumb-sized specimen (formed stool) ® 5-6 tablespoons of watery stool SPECIMEN SHOULD BE SUBMITTED WITH THE FOLLOWING: 1. Patient’s name 6. Requested physician 2. Age 7. Requested procedure 3. Gender/ sex 8. Presumptive diagnosis 4. Date of collection 9. Prior infections 5. Time of collection 10. Travel history FACTORS TO CONSIDER IN STOOL EXAM: 1. Intake of drugs/ medicinal substance: antacids, anti-diarrheals 2. Antibiotics and anti-malarials intake 3. Amount of stool collected 4. Contamination with toilet water, urine, and soil 5. Age of stool samples 6. Delay in examination may require preservation 7. Temporary storage of fecal samples in a (3-5℃) refrigerator 8. Never freeze and incubate stool samples STOOL PRESERVATION: ® Prevent possible destruction of helminth eggs, and larvae ® Appropriate fixation will preserve protozoan morphological features ® Stool to fixative ratio: One part stool to 3 parts preservative 1. FORMALIN: ® An all-purpose fixative, with 5% and 10% concentrations ® 5% is for protozoan cysts, 10% is for helminth eggs and larvae ® FECT/ FEACT 2. SCHAUDINN’S SOLUTION: 3. POLYVINYL ALCOHOL: ® Used to preserve fresh stool for staining the smears ® Plastic resin which serves to adhere stool onto a slide ® Contains Mercuric Chloride (toxic) ® Incorporated to Schaudinn’s solution (fixation) ® FECT/ FEACT ® Preserves protozoan cysts and trophozoite for staining 4. MERTHIOLATE-IODINE FORMALIN: 5. SODIUM ACETATE-ACETIC ACID FORMALIN: ® Contains Merthiolate/ Iodine which act as staining ® Do not contain Mercuric chloride ® Formalin acts as the preservative ® A liquid fixative ® Intestinal protozoans, helminth eggs, and larvae ® Long shelf-life Unauthorized possession, duplication, and reproduction of this document in any form or media is strictly prohibited. Anyone caught shall be 1 sanctioned according to the University’s guidelines and policies. This document is NOT FOR SALE. FOR CLASS USE ONLY. – KMRP, RMT  DEPARTMENT OF MEDICAL TECHNOLOGY INSTITUTE OF HEALTH SCIENCES AND NURSING FAR EASTERN UNIVERSITY, MANILA MTY1206 CLINICAL PARASITOLOGY 2nd SEMESTER, A.Y. 2024-2025 MACROSCOPIC EXAMINATION: ® Includes gross examination, stool color, and consistency STOOL CONSISTENCY: 1. Liquid or soft ® Trophozoites ® Process within 30 mins. 2. Formed ® Cysts ® Process within 3-4 hrs. 3. Liquid or formed ® Helminth eggs STOOL COLOR/ GROSS EXAMINATION: 1. Dark red ® Upper GI bleeding 2. Bright red ® Distal location bleeding 3. Blood-tinged mucus (formed) ® Presence of trophic amoebae 4. Bloody mucus (loose/ liquid) ® Amoebic ulcerations 5. Tapeworm proglottids, scolex 6. Adult nematodes (Ascaris, Enterobius) MICROSCOPIC EXAMINATION: ® Includes gross examination, stool color, and consistency 1. WBCs (PMNs, Eosinophils) 6. Vegetable spirals 2. RBCs 7. Fungal spores 3. Macrophages 8. Plant cells/ fibers, pollen grains 4. Charcot-Leyden Crystal 9. Starch granules 5. Eggs of arthropods 10. Plant and animal hair PARASITOLOGIC TECHNIQUES: 1. DIRECT FECAL SMEAR: ® Small amount of unfixed stool + Saline/ Iodine ® Detection of trophic forms of amoeba and flagellates A. Unstained Saline Mount: 2mg of stool + 1 drop of 0.85% NSS B. Iodine-stained Preparation: Lugol’s Iodine/ D’Antoni Iodine Iodine kills trophozoite, enhances protozoan cyst 2. KATO THICK SMEAR: ® Glycerine is a clearing solution. ® Malachite green minimizes the brightness ® Good in detecting eggs with thick shell ® Not able to detect protozoans ® Useful in mass stool examinations 3. CONCENTRATION TECHNIQUES: ® Used in cases of light infections ® Used to recover, cysts, oocysts, helminth eggs and larvae ® Larger amount to stool (usually 1g) ® Based on differences in specific gravity A. SEDIMENTATION: AECT FECT/ FEACT B. FLOTATION: Zinc Sulfate Brine Sheather’s Sugar Unauthorized possession, duplication, and reproduction of this document in any form or media is strictly prohibited. Anyone caught shall be 2 sanctioned according to the University’s guidelines and policies. This document is NOT FOR SALE. FOR CLASS USE ONLY. – KMRP, RMT  DEPARTMENT OF MEDICAL TECHNOLOGY INSTITUTE OF HEALTH SCIENCES AND NURSING FAR EASTERN UNIVERSITY, MANILA MTY1206 CLINICAL PARASITOLOGY 2nd SEMESTER, A.Y. 2024-2025 A. 1. ACID ETHER CONCENTRATION TECHNIQUE (AECT): ® Reagents: 40% HCl (dissolves albuminous material), Ether (neutral fat) ® Recovery of Trichuris, Capillaria, Schistosoma A. 2. FORMALIN ETHER CONCENTRATION TECHNIQUE (FECT): ® Reagents: 10% Formalin, Ether ® Recovery of helminth eggs and protozoan cysts ® Ethyl acetate is more efficient in recovery of cestode eggs ® Other fixative: Polyvinyl Alcohol (PVA) B.1. ZINC FLOTATION TECHNIQUE: ® Reagents: 33% Zinc Sulfate solution ® Ideal specific gravity: 1.18 to 1.20 B.2. BRINE FLOTATION ® Reagents: Saturated table salt solution ® Low cost, but simple ® Hookworm and Schistosome eggs are badly shrunken ® Not useful in operculated eggs (Clonorishis, Opistorchis, Heterophyids) B.3. SHEATHER’S SUGAR FLOTATION TECHNIQUE: ® Reagents: Boiled sugar solution with Phenol ® Best for recovery of Coccidian oocysts (Cryptosporidium, Cyclospora, Cystoisospora) 4. CULTURE METHODS: ® Used for hookworms and Strongyloides, Trichostrongylus spp. ® Copro culture, Harada Mori (test tube culture method) ® Culture media is also used for cultivation of the intestinal protozoans. 5. EGG COUNTING PROCEDURES: ® Helps correlate severity of disease, intensity of infection/ worm burden ® Done to assess the efficacy of anthelminthics ® Kato-Katz Method, Stoll Egg Count 6. PERMANENT STAINS: ® Useful in examination of the nuclear characteristics of amoeba ® For identification of intestinal protozoans: B. coli, G. intestinalis ® STAINS: A. Iron-Hematoxylin B. Trichrome stain C. Periodic Acid Schiff (PAS) D. Chlorazol Black E E. Acid fast stain 7. PERIANAL SWAB: ® Used to recover eggs of Enterobius vermicularis, Taenia spp. ® Collected early in the morning, before patient washes/ defecates ® Cellophane tape/ Cellulose tape/ Scotch tape method OTHER SPECIMENS: OTHER TECHNIQUES: 1. Blood 6. Urine 1. Thick and thin smears 6. Schistosome egg harvest 2. CSF 1. Eye specimens 2. Knott’s conc. technique 7. Serological testing 3. Tissue aspirates, biopsies 2. Mouth scrapings 3. Xenodiagnosis 8. Molecular testing 4. Sputum 3. Nasal discharge 4. India ink preparation 9. Enterotest 5. Genital secretions 4. Skin snips 5. Buffy coat films 10. Acid-fast stain Unauthorized possession, duplication, and reproduction of this document in any form or media is strictly prohibited. Anyone caught shall be 3 sanctioned according to the University’s guidelines and policies. This document is NOT FOR SALE. FOR CLASS USE ONLY. – KMRP, RMT  DEPARTMENT OF MEDICAL TECHNOLOGY INSTITUTE OF HEALTH SCIENCES AND NURSING FAR EASTERN UNIVERSITY, MANILA MTY1206 CLINICAL PARASITOLOGY 2nd SEMESTER, A.Y. 2024-2025 QUALITY ASSURANCE IN THE PARASITOLOGIC LABORATORY: ® A system of continuous improvement in reliability, efficiency, and utilization of laboratory services. ® Encompasses all factors that affect laboratory performance such as procedure manuals, quality control for test reagents, and equipment, workload, workplace conditions, training, and staff support. ® Internal and external quality control must be observed regularly to monitor and verify the reliability of test results. ® Internal Quality Control and External Quality Assessment A. INTERNAL QUALITY CONTROL (IQC): ® Allows the laboratory to look into its own process. ® Ensures the staff performed the test to the best of their ability ® It is more economical than EQA. B. EXTERNAL QUALITY ASSESSMENT (EQA): ® Assessment of laboratory performance by an outside party or agency. ® Provides an early warning for laboratory processes ® Provides early warning for systematic problems in the laboratory processes, indicates areas that need improvement, identifies training needs, allows comparison of performance and results among different test sites. ® Types: Proficiency testing, Rechecking/ Retesting, On-site evaluation COMPONENTS OF QUALITY ASSURANCE: 1. Proficiency of laboratory personnel. 2. Use of standardized techniques. 3. Choice of procedure and reagents, collection of samples. 4. Proper processing of samples. 5. Accurate reading 6. Correct reporting of results. QUALITY ASSESSMENT PROGRAM (QAP): ® A clinical laboratory requirement for procurement of license. ® Includes Internal and External Quality Assessment Program ® In EQAP, the clinical laboratory is required to participate in NEQAS ® NEQAS: National External Quality Assessment Scheme ® NEQAS is administered by the NRL (National Reference Laboratory) ® NRL for Parasitology: Research Institute for Tropical Medicine THREE STAGES OF QUALITY ASSURANCE: 1. PRE-ANALYTICAL: 2. ANALYTICAL: 3. POST-ANALYTICAL: ® Specimen collection ® Type of test and reagent ® Accurate reporting of results ® Quality ® Equipment ® Results assessments ® Volume ® SOP ® LIS ® Handling ® Microscopic examination ® Transmission of results ® Patient preparation ® Specimen concentration ® Data transcription ® Specimen identification ® Molecular diagnostics ® Final reports *SOP = Standard Operating Procedure; LIS = Laboratory Information System PERSONNEL: ® Procedure ® Records are properly kept ® Availability of controls ® Equipment and instruments ® Clerical, analytical errors ® Unusual laboratory results ® Standardized procedures Unauthorized possession, duplication, and reproduction of this document in any form or media is strictly prohibited. Anyone caught shall be 4 sanctioned according to the University’s guidelines and policies. This document is NOT FOR SALE. FOR CLASS USE ONLY. – KMRP, RMT  DEPARTMENT OF MEDICAL TECHNOLOGY INSTITUTE OF HEALTH SCIENCES AND NURSING FAR EASTERN UNIVERSITY, MANILA MTY1206 CLINICAL PARASITOLOGY 2nd SEMESTER, A.Y. 2024-2025 PROCEDURE MANUAL: ® Instructions for proper collection ® Information on when to reject samples ® Reagent and solution preparation ® Detailed description of techniques ® Criteria for parasite identification ® Quality control procedures ® Reporting and interpretation of results ® General safety precautions INSTRUMENTS AND EQUIPMENT: ® Microscope is the most important instrument used in a diagnostic parasitology laboratory. ® In identification of protozoan cysts, microscopes should be calibrated using an ocular and a stage micrometer. ® Stereoscopic microscope is used for easier examination of adult worms, worm segments, and arthropods. ® Centrifuge is mostly used in concentration techniques. ® Temperature of incubators, refrigerators, freezers REAGENTS: ® Reagents for concentration techniques requires checking of specific gravity ® Check also pH of buffer reagents ® Reagents must be kept in sealed, dark bottles to avoid degradation ® Special precaution in handling flammable solvents ® Batch number of purchased reagents must be noted ® Quality of stain can be checked using a positive slide as control ® Control reagents are strongly recommended in seroimmunodiagnosis APPROPRIATE PARASITOLOGIC TECHNIQUES: 1. Routine stool examination: ® DFS, modified Kato thick method 2. Diarrheic stool examination: ® DFS 3. Screening of food handlers: ® FECT/ FEACT 4. STH/ Schistosomiasis surveillance: ® Kato-Katz technique 5. Epidemiologic investigations: ® DFS, Kato-thick, Kato-Katz, FECT 6. Intestinal Coccidian infections: ® Modified Kinyoun (Acid fast staining) 7. Quantitative diagnosis, egg counts: ® Kato-Katz technique *DFS = Direct Fecal Smear; FECT = Formalin Ether Concentration Technique; FEACT = Formalin Ethyl-Acetate Concentration Technique REPORTING: EPITHELIAL CELL: ® Complete name (include genus and species) and specific stage/s ® Presence of Charcot-Leyden crystals, and budding yeast must be noted ® Presence of macrophages, eosinophils, PMNs must be reported ® Quantitative reporting of fecal leukocytes (WBC/ hpf) *PMNs = Polymorphonuclears PITTFALLS IN THE DIAGNOSIS OF PARASITIC INFECTION: ® Diagnosis of parasitic infections is dependent on O and P examination. ® Artifacts such as mite egg, pollen grain might be mistaken as parasite ova. ® Plant hair can be confused as helminth larvae. ® Howell-Jolly bodies and nRBCs are misidentified as Malaria parasites. ® Fungal spores of Helicosporium is mistaken as microfilariae. QUALITY ASSURANCE IN MICROSCOPY: ® Microscopic examination is still considered as the “gold standard” procedure in helminth infections and Malaria. ® Quantification of malarial parasites is reported as parasites/ 𝝁L. ® Quality Control (QC) for the following: Malaria microscopy → “Blinded” cross-checking of 10 slides STH/ Schistosomiasis → All negative slides and 10% of positives may be re-read by a reference microscopist. Unauthorized possession, duplication, and reproduction of this document in any form or media is strictly prohibited. Anyone caught shall be 5 sanctioned according to the University’s guidelines and policies. This document is NOT FOR SALE. FOR CLASS USE ONLY. – KMRP, RMT  DEPARTMENT OF MEDICAL TECHNOLOGY INSTITUTE OF HEALTH SCIENCES AND NURSING FAR EASTERN UNIVERSITY, MANILA MTY1206 CLINICAL PARASITOLOGY 2nd SEMESTER, A.Y. 2024-2025 MICROSCOPY: ® An instrument that magnifies objects that are too small to be seen by the naked eye, producing an image in which the objects appear larger. MICROSCOPE: ® Produces enlarged images of minute objects/ structures for examination, and analysis. THE PARTS AND FUNCTIONS OF THE MICROSCOPE: HEAD: ® Carries the optical parts in the upper part of the microscope BASE: ® Acts as support; also carries the microscopic illuminators ARM: ® Connects the base and the head of the microscope EYEPIECE: ® Ocular: part used to look through the microscope EYEPIECE TUBE: ® Holds the eyepiece OBJECTIVE LENS: ® Major lenses used for visualization (Scanner, LPO, HPO, OIO) NOSE PIECE: ® Revolving turret; holds the objective lenses STAGE: ® Holds the specimen for viewing STAGE CLIPS: ® Holds the specimen slides in place APERTURE: ® Hole in the stage in which the light is transmitted ADJUSTMENT KNOBS: ® Coarse and fine; used to focus MICROSCOPIC ILLUMINATOR: ® Light source; used instead of a mirror CONDENSER: ® Used to collect and focus light DIAPHRAGM: ® Controls the amount of light reaching the specimen PROPER USE OF MICROSCOPES: 1. Be conversant with the parts of the microscope and their functioning for efficient use. 2. Turn on the light source and adjust the optimum light setting to ensure the correct level of brightness by turning or sliding the brightness adjustment knob at the base. 3. Rotate the LPO into position. Remove the eyepiece, look down the body tube and adjust the mirror and diaphragm setting so that light is reflected up the tube and a circle of evenly illuminated light is visible in the field of view. 4. View the specimen with the 10x objective, then with the 40x and then with the 100x oil immersion objective. DAILY MAINTENANCE: 1. Inspect the microscope for damage or malfunction. 2. Record any damage or malfunction in the daily microscopy maintenance sheet. Clean the parts of 3. the microscope with a clean cloth. 4. Clean the objectives after each day’s work. 5. Ensure that immersion oil residues are removed. Do not clean any part of the microscope with xylene, which will damage the microscope and is toxic. 6. Clean the objective lens with lens cleaning tissue only. Never clean lenses with alcohol, ordinary 7. tissues, cleaning paper, toilet paper, cotton wool or hand towels, which will scratch the lens surface. 8. Cover the microscope with a dust cover when not in use. 9. Turn off the power at the end of the day and unplug the microscope to protect it from a power surge. 10. Do not leave lens ports uncovered; use the port cover or sealing tape. REFERENCES: 1. Use, Care and Maintenance of Microscopes: https://www.who.int/publications/i/item/HTM-GMP-MM-SOP-12 2. Analysis of Urine and Other Body Fluids, Strasinger 7th ed., Microscopy 3. Markell and Voge’s Medical Parasitology 10th SEA ed. 2020 4. Belizario, Vincent Jr. Medical Parasitology in the Philippines. University of the Philippines Press 2013 5. Centers for Disease Control and Prevention, DPDX – Laboratory Identification of Parasitic Diseases of Public Health Concern, http://www.cdc.gov/dpdx/ Unauthorized possession, duplication, and reproduction of this document in any form or media is strictly prohibited. Anyone caught shall be 6 sanctioned according to the University’s guidelines and policies. This document is NOT FOR SALE. FOR CLASS USE ONLY. – KMRP, RMT