Specimen Collection, Transport, and Processing PDF

REAN STAR PAULINE C. CORTINA, RMT INSTRUCTOR, CSU-CAHS  GENERAL GUIDELINES FOR SPECIMEN COLLECTION 1. Collected during the acute or early phase of an illness 2. Select the correct anatomic site 3. Collect using the proper technique and supplies with minimal contamination from normal bi...

REAN STAR PAULINE C. CORTINA, RMT INSTRUCTOR, CSU-CAHS  GENERAL GUIDELINES FOR SPECIMEN COLLECTION 1. Collected during the acute or early phase of an illness 2. Select the correct anatomic site 3. Collect using the proper technique and supplies with minimal contamination from normal biota 4. Collect the appropriate quantity of specimen 5. Collected before the administration of any antimicrobial agent 6. Package in a container or transport medium designed to maintain the viability and avoid hazards 7. Label the specimen accurately 8. Transport promptly or make provisions to store the specimen that will not degrade the suspected organism 9. Swabs are generally poor specimens compared with tissue or needle aspirates 10. Lesions, wounds and abcesses are collected from advancing margin of the lesion preferably by ASPIRATION  REJECTION OF UNACCEPTABLE SPECIMENS 1. Unidentified or improperly labelled specimen 2. Information on the label does not match the information on the requisition 3. Specimen transported at the improper temperature 4. Specimen has not been transported in the proper medium/container 5. Quantity of specimen is insufficient for testing 6. Specimen is leaking 7. Grossly contaminated specimen 8. Transport time exceeds 2 hours postcollection 9. Received in a fixative (formalin) 10. Received for anaerobic culture from a site known to have anaerobes as part of the normal flora 11. Specimen is dried 12. Not the proper specimen for the test 13. Processing the specimen would produce information of questionable medical value 14. If duplicate specimen is received 15. More than one specimen from the same source was submitted from the same patient on the same day 16. One swab was submitted with multiple requests for various organisms 17. Gram stain of expectorated sputum reveals 10 epithelial cells per LPF and mixed bacterial flora COLLECTION PROCEDURES üSterile containers Stool specimens üSwabs  upper respiratory tract, external ear, eye, and genital tract  COTTON, DACRON, OR CALCIUM ALGINATE LABELING AND REQUISITIONS Patient Identification: üName, Identification number, Room number, Physician, Culture site, Date of collection and Time of collection Requisition Form: üPatient’s name, age, date of birth, and gender, room number or location, Physician’s name and address, specific anatomic site, date and time of specimen collection, clinical diagnosis or relevant patient history, antimicrobial agents, and name of individual transcribing orders SPECIMEN TRANSPORT ü ideally within 30 minutes of collection, preferably within 2 hours üAnaerobic Bacteria  not more than 10 minutes üCSF  within 15 minutes üSpecimen containes should be leak-proof üSpecimen containers should be placed in a sealable, leak-proof plastic bags  TRANSPORT CONDITION SPECIMEN Immediately at Room Body fluids, Bone, Inner ear, Corneal scrapings, Foreign bodies, Gastric Temperature aspirate, Suprapubic aspirarte of urine, Prostatic sample Within 15 minutes at Room CSF Temperature Within 2 hours at Room Blood and Bone Marrow specimens Temperature Within 24 hours at Room Abcess, Outer ear, Conjunctiva specimens, Genital tract specimens (for Temperature urethral specimens, within 2 hours using JEMBEC), Hair, Nails or Skin Scrapings, Respiratory Tract specimens, Tissue specimens Immediately at 4°C Gastric Biopsy Within 2 hours at 4°C Indwelling catheter (urine sample), Straight catheter (urine sample) Within 24 hours at 4°C Rectal swab, Stool culture, clean voided midstream urine Kinds of Swabs According to Tip Used a. Cotton-tipped Swab b. Calcium Alginate-tipped Swab c. Polyester-tipped Swab (DACRON or RAYON) Kinds of Swabs According to Applicator Used a. Wooden Applicator Stick b. Nichrome or Thin Aluminum Wire HANDLING OF SPECIMENS refrigeration at 4°C-6°C a. Swabs from wounds, urogenital tract, throat and rectum and samples of feces or sputum - 2-3 hours b. Urine specimen- at least 24 hours c. C S F f r o m p a t i e n t s w i t h s u s p e c t e d m e n i n g i t i s - immediately examined d. Specimens for viral isolation- refrigerated immediately but never frozen SPECIMEN PRESERVATION Urine  used of boric acid Stool  refrigerated  delay is longer than 2 hours- added to CARY- BLAIR TRANSPORT MEDIA  Clostridium difficile toxin assay TRANSPORT or HOLDING MEDIUM üSTUART’S MEDIUM üAMIES MEDIUM üCARY and BLAIR MEDIUM üTRANSGROW MEDIUM üJOHN E. MARTIN BIOLOGICAL ENVIRONMENTAL CHAMBER (JEMBEC) ANTICOAGULANTS 1. 0.025% SODIUM POLYANETHOL SULFONATE (SPS) Adult: 10-30 ml Children: 5 to 10 ml a. Inhibit phagocytosis and complement activation b. Neutralizes aminoglycosides antibiotics c. Neutralize the bactericidal effect of plasma d. Precipitates fibrinogen, β-lipoproteins, β-1-C globulin SPS may inhibit the following bacteria: üNeisseria gonorrhoea üNeisseria meningitidis üGardnerella vaginalis üStreptobacillus moniliformis üPeptostreptococcus anaerobius 2. Heparin  viral cultures and for isolation of Mycobacterium spp. from blood 3. Citrate and Ethylenediaminetetraacetic Acid (EDTA)  should not be used for microbiology SPECIMEN STORAGE a. Refrigerator Temperature (4°C) Urine, stool, viral specimens, sputa, swabs, and foreign devices (catheters) and viral specimens b. Incubator Temperature (35°C) CSF samples intended for Gram staining, culture and sensitivity tests c. Ambient (Room) Temperature (22°C-25°C) specimens for anaerobic culture, sterile body fluids, genital specimens and swabs d. Freezer Temperature (-20°C or -70°C) serum for serologic studies- (-20°C) Tissues or specimens that stored for a long time - (-70°C) Fecal specimens for studying C. difficile toxin- (-70°C) SPECIMEN PRIORITY SPECIMEN PROCESSING ØGross Examination of Specimen ØPreparation of Smears üprepared by rolling the swab back and forth over contiguous areas of the glass slide to deposit a thin layer of sample material üSmears from Thick Liquids or Semisolids üSmears from Thick, Granular, or Mucoid Materials üSmears from Thin Fluids DIRECT MICROSCOPIC EXAMINATION Purpose: 1. Quality of the specimen can be assessed 2. Give the microbiology technologist and the physician an indication of the infectious process involved 3. Routine culture workup can be guided by the results of the smear 4. Can dictate the need for nonroutine or additional testing Methods of Direct Examination 1. Gram Staining 2. Acid Fast Staining-Ziehl-Neelsen, Kinyoun and Fluorescent Method PROCESSING OF SPECIMENS a. Homogenization b. Concentration (centrifugation or filtration) c. Decontamination SPECIMENS USED FOR BACTERIOLOGICAL STUDIES 1. BLOOD üVenipuncture site should be cleansed with 70% alcohol then with 2% iodine or iodophore allowed to dry üMost advantageous time to draw blood cultures will be just before the anticipated rise of temperature üCollection of 2 up to 4 sets ü2 sets should be drawn from the same arm üFever of unknown origin 4 separate blood culture, 2 drawn on each arm for 2 days üAerobic and anaerobic culture bottles are used ü0.025%-0.05% SPS üTSB, BHIA, Supplemental Peptone, THIO, Columbia Broth, Brucella Broth, and Castaneda Medium üPenicillinase üIncubated at 35°C (7 days) üEndocarditis, brucellosis and fungemia (2-4 weeks) Growth is usually indicated by the following: üHemolysis of red cell üGas bubbles in the medium üTurbidity üColony formation Newer Methods of Blood Culture üLysis-Centrifugation (Isolator) üSelf-contained Subculture(Septi-Check and Vacutainer Agar Slant) üDetection of microorganisms through changes in electrical impedence (BACTOMETER) üDetection of CO2 end product metabolism(BACTEC System) 2. CEREBROSPINAL FLUID (CSF) a. Collection and Handling ü inserting a needle into the subarachnoid space at the level of the lumbar spine ü3 TO 4 TUBES Tube 1 Chemical and Serologic Tests Tube 2 Microbiology Tube 3 Cell Count üViral studies refrigerated up to 24hours or frozen at -70°C b. Processing üSpecimens are centrifuged and decanted, using the sediments for smear and culture Examples of Serologic Tests: a. Counter Current Immune Electrophoresis b. Coagglutination Test PHADEBACT c. Latex Agglutination Test Culture Media a. Enrichment Broth (TSB of THIO) b. CAP Gram-negative cocci c. BAP Gram-positive cocci d. EMB, MAC Gram-negative bacilli e. Fungal media f. Tissue culture MAJOR LABORATORY RESULTS FOR THE DIFFERENTIAL DIAGNOSIS OF MENINGITIS BACTERIAL TUBERCULAR VIRAL FUNGAL ü Elevated WBC count ü Elevated WBC count ü Elevated WBC count ü Elevated WBC count ü Neutrophils present ü Lymphocytes and ü Lymphocytes present ü Lymphocytes and ü Marked protein monocytes present ü Moderate protein monocytes present elevation ü Moderate to marked elevation ü Moderate to marked ü Markedly decreased protein elevation ü Normal glucose level protein elevation glucose level ü Decreased glucose level ü Normal lactate level ü Decreased glucose level ü Lactate level >35 mg/dL ü Lactate level >25 mg/dL ü Lactate level >25 mg/dL ü Positive Gram stain and ü Pellicle formation ü Positive India Ink with C. bacterial culture neoformans ü Positive Limulus lysate ü Positive immunologic test result with gram test for C. neoformans negative organisms 3. THROAT AND NASOPHARYNGEAL SPECIMENS A. Throat Swab - S. pyogenes - Alpha Streptococcus Should be diagnose with the following precautions: 1. Posterior pharynx, tonsils and any areas of purulence, inflammation and ulceration 2. Inoculated to Soybean-Casein Digest Agar containing 5% sheep blood 3. Bacitracin Disk Test 4. Incubated at 35°C for 18-24 hours with reincubation of 1 day üPatient throat should be swab from the uvula to the tonsil on both sides of the throat üS w a b i s m o i s t e n e d w i t h S t u a r t ’ s a n d A m i e s medium üAdequate for the recovery of: Adenovirus Herpes Virus C. diphteriae Chlamydia Mycoplasma Yeast Haemophilus spp. B. Nasopharyngeal Swab recommended for the isolation and detection of carriers specimen of choice for the recovery of B. pertusis, Respiratory Syncytial Virus, Parainfluenza Virus and Viruses causing rhinitis üC. diphtheria cultured on Loeffler’s Agar slant and Cystein-Tellurite Agar Plate üB. pertussis Charcoal Cephalexin Medium or Bordet-Gengou Agar üNeisseria meningitidis and Neisseria gonorrhoeae Modified Thayer-Marin or Martin-Lewis Agar 4. SPUTUM üFirst morning specimen ü2 to 3 consecutive early-morning sputum üMethods: ØDeep Cough (expectorated sputum) ØAerosol-Induced (induced sputum) Other means of obtaining sputum: a. Gastric Aspiration b. Transtracheal Aspiration c. Transthroracic Needle Biopsy d. Bronchoscopy with Aspiration or Biopsy e. Thoracentesis f. Open Lung Biopsy g. Bronchoalveolar Lavage (BAL) h. Protected Catheter Bronchial Brushing Culture üCultured on MAC, BAP, CAP Anaerobic culture obtained by percutaneous aspiration and protected bronchial brush Gram’s and Acid Fast Stain  done on direct smear 5. GASTROINTESTINAL TRACT a. Stool Collection and Handling üWalnut-sized specimen üDelivered within 1 hour Staining of Smears Gram Staining a. Many clumps of gram(+) cocci  Staphylococcal infection b. Many thin, comma-shaped gram(-) bacilli  Campylobacter and Vibrio c. Large numbers of large and thin gram(+) bacilli  C. difficile Acid-Fast Staining detect Crypstosporidium spp. and Mycobacteria Culture a. General supportive media yeast spp., staphylococci, enterococci and gram(-) bacilli b. Moderately selective media MAC, EMB Enterobacteriaceae, Vibrios and other pathogen HEA, XLD, SSA Salmonella and Shigella to be detected c. Highly selective agar Brilliant Green, Bismuth Sulfite Salmonella spp. Campy-Blood Agar Campylobacter spp. TCBS Vibrio cholera CIN Yersinia enterocolitica Cycloserine Cefoxitin Egg Fructose Agar (CCFA) C. difficile üIncubated at 35-37°C and examined at 24 and 48 hours for colonies except: - Campy-BAP - TCBS - CIN Agar - CCFA d. Enrichment broth Selenite F, Hajna Gram Negative, Tetrathionate  Salmonella, Shigella and Campylobacter b. Gastric Aspirate best specimen for infants used for examination of AFB must be neutralized within one hour of collection c. Gastric Biopsy recommended for the detection of and isolation of Helicobacter pylori d. Rectal Swab for bacterial and viral culture Cary-Blair or Stuart’s Transport Medium 2.5 cm swab- anal sphincter Methylene Blue- fecal leukocoytes 6. URINE Collection üClean-catch midstream urine üSuprapubic Aspiration üCatheterization a. Straight Catheter b. Indwelling Catheter(Foley Catheter) Handling üCultured within 2 hours after collection üRefrigeration at 4°C for 24 hours Gram Stain of Uncentrifuged Specimen simplest and most rapid method for detecting significant bacteriuria  pre s e n c e o f 1 o rg a n is m /OI F c o rre la t e s w it h significant bacteriuria (>105 CFU/ml) Culture a. 5% Sheep BAP b. MAC c. Columbia Colistin-Nalidixic Agar (CNA) d. PEA Colony Count a. Pour Plate Method b. Calibrated Loop Method (0.01 or 0.001ml) Interpretation a. Colony count >105 CFU/ml (100, 000 CFU/ml) for 1 or 2 bacterial species b. Colony count >105 CFU/ml for 3 or more bacterial spp. c. Colony count >103 /ml of a bacterial specie from midstream urine of a male d. Colony count 102 − 103 /ml of 1 or 2 bacterial spp. from females with lower urinary tract symptoms e. Colony count < 5,000 CFU/ml f. Presence of organism in any quantity obtained by suprapubic aspiration 7. ABSCESS (LESION, WOUND PUSTULE, ULCER) needle and syringe Superficial Abscess swab along the leading edge of the wound Deep Abscess aspirated from the wall of the abscess or the advancing margin of the lesion 8. BODY FLUIDS Amniotic, Abdominal, Ascites/Peritoneal, Bile, Synovial, Pericardial, Pleural) 9. BONE specimen may require homogenization 10. EAR Inner ear clean ear canal with mild soap, aspirate fluid with needle if eardrum intact; use swab if eardrum ruptured Outer ear remove debris or crust from ear canal with saline-moistened swab; rotate swab in outer canal 11. FOREIGN BODIES a. Intrauterine Device (IUD) usually cultured for the detection of Actinomyces spp. b. Catheters and Prosthetic Valves Maki Roll Technique M o r e t h a n 1 5 c o l o n i e s a r e r e q u i r e d t o p e r f o r m identification and susceptibility test Whole Foley catheter should not be cultured 12. GENITAL TRACT Cervix and Vagina  swab endocervical canal or vaginal mucosa Urethra flexible swab inserted 2-4 cm into urethra for 2-3 s or collect discharge Prostate gland secretions on the swab or in the tube should be collected 13. TISSUE disinfect skin; do not allow tissue to dry out CRITICAL (PANIC) VALUES 1. Positive blood cultures 2. Positive CSF Gram stain or culture 3. S t r e p t o c o c c u s p y o g e n e s ( g r o u p A Streptococcus) in a surgical wound 4. Gram stain suggestive of gas gangrene 5. Positive acid-fast stain 6. Positive antibiotic-resistant bacteria (e.g., Vancomycin-resistant S. aureus) 7. Positive for Legionella, Francisella and Brucella