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Clinical Pathology is responsible for quantitative and qualitative analysis and interpretation of patient specimens, including tissue, blood, urine, and other body fluids. Approximately 10 million clinical laboratory test results per year are performed in these laboratories.

Specimen Handling

Containers

Specimens are acceptable for processing only when collected and submitted in the appropriate container. Shipping containers, sterile specimen containers, transport media and swabs are available through Client Services. Specimen containers must be tightened securely to prevent leakage.

Labeling

Clearly print the patient’s complete name (as listed on the test request form) on the specimen container, culturette, or other specimen transport media. (Labeling of only the outside container is not acceptable).

Transport

Deliver specimens to the laboratory within 24 hours. Prompt processing minimizes loss in viability of potential pathogens and ensures a more accurate appraisal of flora present.

Specimen Rejection Criteria for Microbiology Specimens

Client Services will notify the ordering facility or healthcare provider of any test cancellations. Common causes for rejection of microbiology specimens are:

  • Inappropriate specimen for culture requested
  • Improper specimen container or transport media

Forms

General Microbiology Culture Types and Collection Instructions

The detection of microorganisms in blood has great diagnostic and prognostic importance. A completed test request form (particularly time of collection, diagnosis and antimicrobial therapy) is essential for timely and appropriate laboratory processing of the specimen.

All routine blood cultures are screened for aerobic and anaerobic bacterial and fungal isolates. All organisms isolated from blood cultures will be identified, susceptibility tests performed, if appropriate, and stock cultures made, unless the isolate is considered a skin contaminant. Stock cultures will be held for 30 days. Organisms that may be contaminants are coagulase-negative Staphylococcus species, Bacillus species, diphtheroid bacilli and viridans Streptococcus species.

Blood cultures from patients suspected of having Brucella must be specifically requested as special cultures. (See the individual culture listings of the specific organisms for further details).

Guidelines for Drawing Cultures

  1. In most adult infections, cultures of two to three separate venipunctures (20-30 mL per venipuncture) are sufficient. Studies have shown the following positive blood culture yields within a 24-hour period in patients without endocarditis:
    • First venipuncture (Set): 80%
    • First and second venipunctures (Set): 90%
    • First, second and third venipunctures (Set): 99%
  2. Based on these results, the laboratory will not accept more than four blood culture sets per 24 hours per patient.
  3. In suspected acute sepsis, meningitis, osteomyelitis, arthritis or acute untreated bacterial pneumonia, obtain two blood culture sets (from two separate venipuncture sites) before starting therapy.
  4. For suspected infective endocarditis.
    • Acute: Obtain three blood culture sets with three separate venipunctures during the first one to two hours of evaluation.
    • Subacute: Obtain three blood culture sets on Day 1 (15 minutes or more apart). If all three are negative at 24 hours, obtain three more.
  5. A direct correlation exists between the volume of blood cultured and the recovery rate of clinically significant organisms. For adult patients, each blood culture bottle should receive 10mL of blood. Isolators should receive 10mL as indicated on the tube. For pediatric patients, one to five mL of blood injected into one aerobic blood culture bottle is sufficient.

Specimen Collection

  1. After placing a tourniquet on the patient's arm, select a suitable vein. Once the vein has been found, cleanse the area with the series of 2% iodine, 70% alcohol, iodine and alcohol. Without touching the site of the venipuncture with the fingers, draw 20mL of blood into a sterile syringe. Inject 10mL of the specimen into each of two culture bottles after cleansing the stoppers with 2% iodine. Invert the containers several times to insure thorough mixture of the contents. (Blood culture systems are available through Client Services). For pediatric patients, write the volume of blood inoculated on the label.
  2. Blood cultures from patients suspected of having Histoplasma capsulatum, Brucella, Bartonella, Legionella or Mycobacterium must be specifically requested as special cultures and must be collected in a lysis-centrifugation (Isolatorâ„¢) tube. (See the individual culture listings of the specific organisms for further details.)

Complete a Test Request Form indicating the specific source of the specimen, diagnosis and organism(s) suspected. Label the specimen appropriately. Record the date and time the specimen was collected.

Submit specimens in urine culture transport tubes provided by Cleveland Clinic Laboratories. Indicate whether the specimen is from a clean catch or a catheter.

Indwelling Catheter Specimen Collection

Obtain the specimen with a needle and syringe. Select the puncture site 1-2 inches away from the catheter tube entry point. Cleanse the area to be punctured with 70% alcohol. Aspirate exactly 5mL of urine with a sterile needle and syringe. Disinfect the rubber stopper and aseptically transfer the specimen to the urine transport tube provided. Specimens obtained from the collection bag are not suitable for analysis. Foley tips will not be accepted.

Urine Culture Transport

Prevention of contamination by normal vaginal, perineal, and anterior urethral flora is the most important consideration for collection of a clinically relevant urine specimen. (See the Patient Collection Guidelines above for collection of a urine specimen). Unpreserved urine is an excellent growth medium for most bacteria. Unless urine is preserved during transport, bacteria may multiply, causing colony counts to be erroneously high. The maintenance medium in the transport kit prevents rapid multiplication of bacteria in the urine during shipment.

Instructions for use of urine culture transport system:

  1. Obtain the urine specimen from the patient in a clean cup.
  2. Open the pouch and remove the transfer device and tube.
  3. Submerge the pipette of the transfer device to the bottom of the urine container. The container may be tipped at an angle if the volume of the urine is limited.
  4. Place the transport tube in the holder portion of the transfer device and push it down as far as it will go, puncturing the stopper.
  5. Hold the tube and transfer device in position until the urine stops flowing into the tube.
  6. Remove the transport tube from the transfer device and shake the tube vigorously.
  7. Discard the transfer device and remaining cup of urine into appropriate biohazard disposal containers.

Precaution: The transport tube must be filled to the minimum line indicated on the tube. A tube that is not filled at least to this line is unacceptable for culture.

The following collection sites are considered respiratory specimens: mouth, nose, nasopharyngeal swabs, throat sputum, bronchial and tracheal aspirations, bronchoalveolar lavage (BAL) and Bartlett Fiberoptic Wimberly (BFW) brush. All specimens from these sites will be screened for fast-growing aerobic pathogenic organisms and for Beta Strep Group A.

The following organisms are examples of those not isolated by routine culture: Neisseria gonorrhoeae, Corynebacterium diphtheriae and Bordetella species. Specimens from the nose are the only respiratory specimens routinely screened for Haemophilus species. For information on culture of non-routine organisms, see the culture listing for the specific organism.

Refer to the Test Directory for collection information. Specimens must be submitted in transport media provided by the laboratory.

  • Specimens from normally sterile sites (such as body fluids, surgical specimens, joint fluids, etc.) will be cultured for aerobic pathogenic organisms. Anaerobic pathogens may be detected by routine cultures. However, be aware of the special requirements for the isolation of anaerobes. If an anaerobic organism is suspected, a specimen should be submitted for anaerobic culture.
  • Bone marrow, eye swabs, joint fluids and cerebrospinal fluids are routinely screened for fast-growing aerobic pathogenic bacteria including Haemophilus species and pathogenic Neisseria species.
  • Body fluids (exclusive of joint or cerebrospinal fluids) and surgical specimens will be routinely screened for fast-growing aerobic pathogens.
  • Genital tract specimens are tested for Neisseria gonorrhoeae and/or Chlamydia trachomatis by amplification. If Haemophilus ducreyi is suspected, a special request must be made. In cases of suspected toxic shock syndrome, the tentative diagnosis should be placed on the requisition form so that isolates of Staphylococcus aureus may be saved for TSST-1 testing. Vaginal specimens must be accompanied by a request for examination for a specific organism (i.e. group B streptococci, Candida, Trichomonias vaginalis) or for gram stain, which may be examined for the presence of clue cells, curved bacteria, relative proportion of gram-positive bacilli resembling lactobacilli vs. Gram-negative bacilli. Culture for Gardnerella vaginalis is neither sensitive nor specific for the diagnosis of bacterial vaginosis and will not be done.
  • Specimens from wounds, ulcers, and drainages will be screened for nonfastidious, fast-growing aerobic organisms. If an anaerobe is suspected, a specimen must be properly submitted and an anaerobic culture specifically ordered.

Nose or Nasopharyngeal Swabs

Collect in a manner to avoid contamination by adjacent structures.

Throat Specimens

Collect in a manner to avoid contamination by mouth and tongue. Throat specimens will be screened for Group A Strep only. Throat specimens for Neisseria ganorrohoeae, Bordetella pertussis or Corynebacterium diphtheriae will be screened only for that pathogen when specifically requested. Collect in a manner to avoid contamination by mouth and tongue.

Sputum Specimens

Collect by instructing the patient to remove dentures, rinse mouth, gargle with water and cough deeply, expectorating into appropriate collection container. All specimens labeled "sputum" that are consistent on cytologic examination with saliva or upper respiratory secretions will be rejected. Rejection or acceptance will be based on microscopic examination for the absence of/or minimal numbers of squamous epithelial cells.

Tracheal and Bronchial Aspiration

Collect by physician to avoid contamination with adjacent structures.

Fungus

Provide complete information on the Test Request Form to ensure that the specimen is cultured for all organisms (fungi/yeast) suspected. Additional patient history may be helpful. Include the patient occupation, history of travel or residence abroad, and any animal contacts. A single specimen may be cultured for both bacteria and fungi. Generally, a direct fungal smear exam will be performed on all specimens submitted for fungal cultures.

Yeast

Requirements are the same as for a fungal culture. Urine, vaginal, throat and stool requests are routinely processed for a yeast culture, even if ordered as a fungal culture.

Anaerobes

Cultures for fastidious anaerobes will not be performed on urine (nonsuprapubic aspirate) or specimens from body sites that have anaerobes as part of the normal flora (stool, skin, vagina/cervix, mouth, throat, septum, etc.). Any material collected from areas with no normal flora (body fluids, deep abscess exudate, tissue biopsies, transtracheal aspirates, etc.) is acceptable for anaerobic culture.

Specimens for anaerobic culture must be collected appropriately and transported in an anaerobic environment (anaerobe vial). Aspirated specimens, in general, are preferred. Swabs are unacceptable for anaerobe culture. Contact Client Services prior to collection of the specimen for consultation on collection procedures, containers, or transport.

Acid-Fast Bacillus

All cultures and stains for AFB are performed at Cleveland Clinic. Acid-fast stain results are generally available 24-48 hours from receipt of sample. Positive stains and/or culture results are called to the client. Transport specimens in sterile, leak-proof containers and place in appropriate shipping containers. Dry specimens or spilled-leaking specimens are not acceptable for AFB culture. Specimens submitted on swabs or sputums of low volume are discouraged for AFB culture because the yield is significantly decreased. Gastric contents must be neutralized with sodium carbonate or another alkaline buffer salt before transport.

Respiratory specimens may be submitted for Mycobacterium tuberculosis amplification. Only specimens that are AFB stain positive are appropriate for testing. An AFB culture and smear should be ordered in conjunction with this request. This test is specific for, but does not differentiate between, members of the M. tuberculosis complex, i.e. M. tuberculosis, M. bovis BCG, M. africanum and M. micrati. A negative test does not exclude the possibility of isolating M. tuberculosis complex from the AFB culture.