CLBACT - Specimen Management in Microbiology

Preanalytical phase

Definition: everything before the test — ordering, collection, labeling, transport, storage, accessioning.

Preanalytical phase

Error share: seventy per cent of laboratory errors occur in the preanalytical phase.

Analytical phase

Definition: the test itself.

Post-analytical phase

Definition: reporting and interpretation.

Specimen integrity

The highest quality analytical techniques, from mass spectrometry to molecular assays, cannot salvage a compromised specimen, because downstream microbiological diagnosis is entirely dependent on upstream specimen integrity.

Uses of a specimen

• Cultivation (growth), identification, and antimicrobial susceptibility testing of microorganisms.
• Direct detection of infecting organisms by microscopy.
• Direct detection of specific organism products using chemical, immunologic or molecular techniques.
• Detection of patient antibodies produced in response to infection (serology).

Role of the microbiologist

To provide the clinician with instructions for collection, transport and storage.

Collection instructions

Must specify: • Safety considerations
• Anatomic site of specimen
• Collection instruction
• Mode of transport
• Labeling instructions
• Patient preparation
• Collection devices
• Minimal acceptable quality

Acute phase

The collection window. Collect while the pathogen load is climbing to its peak, and specifically before antibiotics are given. For viral investigations, collect within 2–3 days.

Tissue or needle aspirate

The gold standard specimen. Bypasses normal microbiota and captures anaerobes.

Fluid aspirates

The acceptable tier of the method hierarchy, below tissue or needle aspirates and above swabs.

Swabs

The last resort of the method hierarchy. Prone to drying out; limited volume recovery.

Cotton swabs

Strictly prohibited. Cotton fibers release toxic fatty acids that are actively bactericidal to fastidious organisms.

Dacron, Rayon, Calcium Alginate

The acceptable alternative swab materials to cotton.

General collection rules

• The correct anatomic site must be identified and selected for the collection.
• The site of collection must be properly disinfected before collection.
• The quantity collected must be sufficient for diagnostic testing.

Throat specimen

• Used to diagnose bacterial pharyngitis.
• The posterior pharynx, between the tonsillar pillars, should be firmly swabbed.
• The swab is placed into sterile transport media, or inoculated onto a blood agar plate immediately.

Nasopharynx specimen

Used to diagnose influenza, coronavirus and pertussis (whooping cough), or to detect carriers of Neisseria meningitidis and Staphylococcus aureus.

Nasopharynx swab technique

Cultured with a flexible, thin wire swab. The swab is guided gently into the nares along the floor of the nasal cavity, parallel to the palate, until resistance is met at the posterior nasopharynx.

Sputum specimen

• Used to diagnose pneumonia and tuberculosis.
• Deep cough secretions, not saliva.
• First morning specimen.

Bartlett's Classification

The system used to judge sputum suitability for culture, read on the purulent sections of the specimen. Also named as the Murray and Washington Sputum Grading System.

Acceptable sputum

More than 25 PMNs per LPF and fewer than 10 SECs per LPF.

Squamous epithelial cells

Indicate mouth contamination, so a high SEC count means the specimen is saliva rather than lower respiratory material.

Acceptable GI specimens

• Passed stools
• Washes
• Feces collected during endoscopy
• Rectal swabs
• Gastric aspirates
• Gastric biopsies

Stool container

A sterile wide-mouth container.

Rectal swabs

Go into enteric transport media.

Acceptable urine specimens

• Clean-catch midstream specimens
• Straight-catheterized urines
• Suprapubic aspirates
• Urine collected from a cystoscopy or other surgical method

Foley and bagged urine

Generally not acceptable urine specimens — Foley (indwelling) catheters, or a bagged pediatric collection of urine.

Urine specimen

Container: sterile, screw-cap container.

Urine specimen

Patient preparation: clean genitalia with soap and water. Special instructions: clean-catch, midstream.

Catheter specimen of urine

• Urine is obtained only from the sampling port on the tubing of the drainage bag.
• Clamp the catheter below the port so urine collects above it.
• Swab the port with an alcohol-impregnated swab.
• Withdraw with a syringe, then place in the specimen pot avoiding contact with the syringe.
• A catheter valve must be cleaned with an alcohol-impregnated swab first.

Blood culture container

A blood culture media set, aerobic and anaerobic.

Blood culture site preparation

Disinfect the site with 70 % alcohol, followed by a chlorhexidine or iodophor scrub.

Timing of a blood culture

Draw blood at the time of febrile episodes, and collect the specimen at 2 different sites.

Blood culture sets

Collect one set from the right arm and one set from the left arm per septic episode — 2 sites, 2 sets. Do not exceed 4 sets total in a 24-hour period.

Blood culture volume

20 mL or more for adults; 1–20 mL for pediatrics; or as the manufacturer's instructions require.

CSF collection site

The thecal sac that surrounds the spinal cord, with the needle entered between the L3 and L4 vertebrae.

CSF specimen

Container: sterile, screw-cap tube. Patient preparation: disinfect skin before aspiration. Special instructions: send to the laboratory immediately.

GC and chlamydia specimens

Male: urethral swab or urine. Female: cervical (endocervical) swab, vaginal swab, urethral swab, or urine.

Bacterial prostatitis specimen

Male: prostatic fluid, obtained by prostatic massage.

Specialized collection methods

Testing methods specific for the sexually transmitted diseases gonorrhea and chlamydia require them.

Wound specimen

Container: swabs for aerobic culture, or a sterile tube for aspirates. Patient preparation: wipe around the area with sterile saline or 70 % alcohol.

Wound swab site

Swab along the leading edge of the wound.

Shallow wound culture

Taken with a swab stick, as against a deep wound culture taken with a needle and syringe.

Deep wound culture

Taken with a needle and syringe, as against a shallow wound culture taken with a swab stick.

Test requisition

• Patient's name
• Hospital number
• DOB and gender
• Room number
• Specific anatomic site
• Date and time of specimen collection
• Diagnosis / relevant history
• Physician name and address
• Current antimicrobial therapy

Specimen label

• Patient's name
• Identification / hospital number
• Date of birth
• Date and time of collection
• Source
• Initials (who collected)

Requisition against label

Both carry the patient's name and identification number; only the requisition carries the physician, diagnosis, room number and current therapy, and only the label carries the initials of whoever collected the specimen.

Preservatives

Can be used when transport of the specimen is expected to be longer than 2 hours.

Boric acid

Maintains accurate urine colony counts (room temperature, 24 h).

Buffered formalin and PVA

Stool for ova and parasites; preserves trophozoite and cyst morphology.

Cary-Blair

Holds stool for enteric pathogens if the delay is more than about 2 h.

SPS

Sodium polyanethol sulfonate. The gold standard anticoagulant for blood cultures. Used at a strict 0.025 % concentration.

SPS above 0.025 %

Concentrations higher than 0.025 % will actively inhibit fastidious Neisseria species and anaerobic bacteria. The blood-to-SPS ratio is critical.

Heparin

Acceptable for viral cultures, but often inhibits the growth of gram-positive bacteria and yeast.

Citrate, EDTA and ACD

Efficacy not demonstrated for most organisms — not validated for microbiology use. ACD specifically is not appropriate for microbiology.

Transport window

Ideally the collected specimen is transported to the laboratory within 30 minutes, and not longer than 2 hours.

Transport packaging

Specimens are transported in leak-proof, sealable plastic bags marked with a biohazard label.

Transport safety

• Specimen and papers are kept separately.
• Specimens should not contain needles or sharps.
• Use of PPE.

CSF transport

Transport within 15 minutes after collection.

Anaerobic specimen transport

Transport not longer than 10 minutes; sensitive to the presence of oxygen.

Neisseria meningitidis

Sensitive to change in temperature during transport.

Shigella

Sensitive to change in pH during transport.

Transport immediately at RT

Body fluids; corneal scrapings; bone; gastric aspirate; prostatic samples; suprapubic aspirate; tissue.

Transport within 1 hour at RT

Gastric biopsy; stool.

Transport within 2 hours at RT

Abscess, lesions, wound; blood; BMA; genital tract specimens; respiratory tract specimens; urine.

Catheterized urine transport

30 minutes or less at RT if unpreserved, or 24 hours or less at RT if preserved. For catheterized urine, Foley or straight.

Transport within 24 hours at RT

With transport medium or preservative: stool; rectal swab; urine.

Transport within 72 hours at RT

Hair, nails, skin scrapings for fungal culture.

Stuart's medium

A transport or holding medium.

Amies medium

A transport or holding medium. Charcoal is added.

Cary-Blair transport medium

A transport or holding medium.

Transgrow

A transport or holding medium.

JEMBEC system

John E. Martin Biological Environmental Chamber. A transport or holding medium.

Bedside inoculation

Optimal for blood cultures; synovial and peritoneal fluid; N. gonorrhoeae (JEMBEC); B. pertussis (NP swab); and corneal scrapings.

Primary receptacle

Holds the culture; carries the biohazard label and the specimen identification label, and is sealed with waterproof tape.

Absorbent packing material

Packed around the primary receptacle in a triple-packaged specimen.

Secondary packaging

Encloses the primary receptacle and its absorbent packing material, and is closed with a cap.

Outer packaging

Carries the itemized list of contents, the infectious substance label, and the name, address and telephone number of the shipper.

Anaerobic specimen storage

Specimens suspected of containing anaerobic bacteria should never be stored in a refrigerator and must be kept at room temperature.

Storage temperatures

• 4 °C — refrigerator temperature
• 22–25 °C — ambient room temperature
• 35–37 °C — body temperature
• −20 °C — freezer temperature
• −70 °C — freezer temperature

Room temperature storage

CSF for bacteria; abscess, lesion, pustule; body fluids; stool (preserved); URT specimens (NP, nasal, throat swab); clean-voided midstream urine; tissue; genital specimens.

Refrigerator 4 °C storage

CSF for viruses; urine (24 h); stool (unpreserved); viral specimen; sputum; LRT specimens (BAL, BW, BB, endotracheal aspirate); foreign devices such as catheters.

Freezer storage

Serum for serology at −20 °C for 1 week or less; tissue for long-term storage at −70 °C.

CSF storage

CSF for bacteria is held at room temperature; CSF for viruses is refrigerated. The same fluid goes to two different places depending on what is being looked for.

Priority level 1

Critical / invasive. Amniotic fluid; blood; brain; CSF; heart valve; pericardial fluid.

Priority level 2

Unprotected. Body fluids (not level 1); bone; wound drainage; feces; sputum; tissue.

Priority level 3

Quantitation required. Catheter tip; urine; tissue for quantitation.

Priority level 4

Preserved. Feces or urine in preservative; swabs in holding medium, aerobic and anaerobic.

Insufficient specimen

If multiple requests are received but the specimen is insufficient, coordinate with the clinician on which tests are to be prioritized.

Date and time received

Always note the date and time the specimen was received in the laboratory.

Specimen rejection criteria

Labeling: the specimen label does not match the information on the requisition, or the specimen is not labeled at all.

Specimen rejection criteria

Transport: transported at the improper temperature; not transported in the proper medium; transport time exceeds the recommended post-collection to set-up time, or the specimen is not preserved.

Specimen rejection criteria

Integrity: the quantity of specimen is insufficient for testing; the specimen is leaking; the specimen is dried.

Specimen rejection criteria

Fixative: the specimen was received in a fixative (formalin).

Specimen rejection criteria

Site: the specimen has been received for anaerobic culture from a site known to have anaerobes as part of the normal microbiota — vagina, mouth.

Specimen rejection criteria

Value: processing the specimen would produce information of questionable medical value, for example a Foley catheter tip.

Judging adequacy

Was it the right site, collected the right way? • Correct anatomic site identified and selected.
• Site properly disinfected before collection.
• Collected before antibiotics were given, in the acute phase.
• Collected with an acceptable device.

Judging adequacy

Is there enough of it? Quantity sufficient for all the diagnostic testing requested. If several tests are requested and there is not enough specimen, the laboratory coordinates with the clinician on which tests to prioritize.

Judging adequacy

Is it the specimen, or the surface it came through? Sputum is graded before it is cultured; urine from a Foley catheter or a pediatric bag samples the device and the perineum rather than the bladder.

Judging adequacy

Did it survive the journey? Within the transport window for that specimen, at the right temperature, in the right medium; not leaking, not dried, not in fixative; labeled, and labeled consistently with the requisition.