Pre-analytical parameters are an important compo- nent of microbiology testing. High-quality results from
the clinical microbiology laboratory are directly related to specimen quality. Pre-analytical steps in clinical microbiology testing include selection and proper ordering of the appropriate test method (appropriate test usage), selection and collection of high-quality spe- cimens that are representative of the disease process, and timely transport of the specimen to the laboratory [1]. Problems with these pre-analytical steps in the testing process can result in laboratory errors. Pre- analytical errors include those related to specimen selec- tion, specimen collection and transport, and storage, as well as test ordering. Often, errors that occur down- stream in the testing process can be traced back to inap- propriate test selection or problems with specimens.
Errors Related to Specimens
Many of the errors that occur in clinical microbiology are related to specimen quality. Selection, collection, and transport of good-quality and appropriate speci- mens are critical components of obtaining accurate, meaningful results in clinical microbiology. Selection of an appropriate specimen is critical to ensure isolation and identification of the pathogenic agent [2]. The fol- lowing sections discuss general categories in which errors in specimen selection, collection, transport, and storage occur that can give misleading or erroneous microbiology results.
Specimens Collected from the Wrong Anatomic Site Specimens must reflect the disease process; speci- mens that are collected from the wrong site can yield misleading results. A common example is wound spe- cimens that are collected by swabbing the surface of the lesion. The pathogen causing the infection is unlikely to be recovered from these samples. Rather, a
sample collected from deep within the wound is pref- erable. These samples are more likely to reveal the pathogen involved in the infectious process, and they are less likely to be contaminated with commensal organisms from the skin.
Specimens are Contaminated with Endogenous Flora
Specimens that must be collected through sites that contain endogenous flora (e.g., sputum and urine) must be collected using methods that avoid or mini- mize contamination. Growth of normal flora may inhibit growth and identification of the pathogen, resulting in a false-negative culture. Cultures that are overgrown with contaminating flora can be difficult to interpret, delaying the reporting of culture results. The presence of contaminating organisms may also result in workup (identification and susceptibility testing) of organisms that are not involved in the disease process (false positive).
Specimens Were Collected after Administration of Antimicrobial Agents
Many pathogens are highly susceptible to antimicro- bial therapy and are rapidly killed following antimi- crobial administration. For example, in patients with meningococcal meningitis, cerebrospinal fluid (CSF) may be sterilized within 30 min of administration of antimicrobials. In cases in which a bacterial pathogen is suspected but culture results are negative, the possi- bility of antimicrobial administration prior to specimen collection should be considered. Failure to isolate and identify the pathogen can lead to incorrect patient management.
Suboptimal Volume of Sample was Submitted for Culture
A specimen may be subjected to a concentration procedure prior to analysis due to low bacterial or viral burden (e.g., CSF) in the specimen. A common cause of false-negative culture results, especially in the mycobacteriology laboratory, is insufficient volume.
Sterile body fluids such as pleural fluid, peritoneal fluids, or CSF are usually concentrated by the labora- tory as part of the initial processing procedure. An insufficient volume of specimen may lead to false- negative culture results. Most laboratories have mini- mal recommended and required volumes; adherence to these guidelines can ensure optimal sensitivity of the test. Correct volume of specimen is especially important in the clinical mycobacteriology laboratory.
Respiratory specimens being cultured for isolation of Mycobacterium tuberculosisare concentrated to optimize recovery of the organism.
An Inappropriate Transport Device Has Been Used The wide variety of tests and methods used in microbiology means that many different collection and transport devices are used. Transport devices are intended to stabilize the specimen, protect the organ- isms from degradation, maintain an anaerobic environ- ment (for anaerobic culture requests), and prevent overgrowth of contaminating flora. The microbiology laboratory should have specific transport devices that are intended for specific groups of organisms (e.g., anaerobic transports) or specific tests.
Prolonged Transport Time
If specimens are collected but not transported to the laboratory for processing quickly, fastidious organisms and anaerobes can quickly be lost. For specimens that may be delayed in transport to the laboratory, special collection devices with transport media should be used. Cultures for anaerobes (e.g., abscess material, tis- sues, and biopsies) are especially subject to loss of via- bility unless the material is transported in appropriate anaerobic transport media. Anaerobes are rapidly killed upon exposure to oxygen; cultures submitted to the laboratory for anaerobes should be processed with- out a delay.
Storage of Specimen at Inappropriate Temperature If the microbiology laboratory does not process spe- cimens on every shift, specimens that arrive at the lab- oratory may need to be stored temporarily; most specimens can be stored at 4C to maintain organism integrity and suppress the growth of contaminants.
However, certain organisms are very sensitive to cold temperatures, and cultures for these pathogens should be performed immediately after specimens are col- lected. Examples of organisms that do not survive well during prolonged transport or in a refrigerated storage are Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, and Haemophilus influenzae.
Specimens should be transported to the laboratory without delay and not refrigerated.
Errors Related to Microbiology Test Ordering
The diversity of microorganisms and the wide vari- ety of test methods available can be a challenge for laboratory customers. With rapidly changing technolo- gies, microbiology laboratories are regularly updating and improving their testing profile. Customers are not always aware of the methods that are used by their lab- oratory. Good communication to laboratory customers is necessary to ensure that physicians are aware of the recommendations for testing and that they know what is or is not included in a certain test. Inappropriate tests
are ordered when physicians are not aware of what a culture- or polymerase chain reaction (PCR)-based test includes. Failure to order the correct test can result in failure to isolate or detect a pathogen. Some of the more common occurrences are described here.
Routine Culture Does Not Include Organism of Interest
Stool cultures for enteric pathogens are one of the most commonly ordered cultures in the clinical micro- biology. Routine stool cultures may not include special methods for isolation of uncommon pathogens such as Yersinia enterocoliticaorVibriospp. To prevent this type of common ordering error, physicians should consult the laboratory user guide to determine what is included in the test they have ordered.
Special Methods are Needed for Isolation of Organism
Inappropriate testing may also occur when special methods are needed to identify certain pathogens. For example, some organisms cannot be isolated in routine cultures. Routine lower respiratory cultures generally do not include methods for isolation and identification of fastidious respiratory pathogens such as Legionella and Bordetella. Special collection and transport meth- ods as well as culture methods or PCR testing should be performed for these organisms. The laboratory must make physicians aware of the diagnostic strategy used in the facility for unusual pathogens to prevent test ordering and interpretation errors. The laboratory should have guidelines available for diagnosis of infec- tions due to these difficult-to-culture organisms. For many of these uncultivable organisms, molecular test- ing or serological testing may be the most appropriate method to diagnose them.
Multiple Methods are Available but the Wrong Method is Ordered
With the advent of more sensitive and specific test- ing methodologies, there may be several options avail- able for testing. Two important examples are herpes simplex virus (HSV) encephalitis and cryptococcal meningitis. Less sensitive methods to diagnose these illnesses are still available (e.g., “viral culture” or “fun- gal culture”). For both of these organisms, highly sen- sitive, rapid methods are available, and the old methods are obsolete for diagnosis. Health care provi- ders should ensure that the appropriate test is ordered.
For HSV encephalitis, the recommended test is a molecular assay such as PCR rather than culture. For cryptococcal meningitis, a cryptococcal antigen test is recommended rather than CSF fungal culture or the obsolete India ink test. Good communication with the laboratory is critical, and health care providers should
consult the laboratory’s guidelines to ensure that the correct test is ordered[3].
Errors in Blood Culture Collections
Pre-analytical errors associated with drawing speci- mens for blood cultures can have significant effects on the results of the culture. Several commonly encoun- tered errors may occur with blood cultures submitted to the microbiology laboratory, including submission of a single set of blood cultures; inadequate volume of blood; drawing blood cultures through a catheter; and inadequate preparation of the draw site, resulting in contamination. These errors have significant impact on the laboratory as well as on patient care.
Single Blood Culture Sets
Submitting a single set (e.g., one aerobic and one anaerobic bottle) or a single bottle for the diagnosis of bacteremia has several disadvantages that can lead to errors in culture workup and interpretation of results.
Most important, the sensitivity of the blood culture is reduced. Many studies have shown that optimal sensi- tivity is achieved with two or three sets of blood cul- tures [4,5]; current clinical practice guidelines for diagnosis of sepsis recommend that two blood culture sets be obtained within 24 hr of each other. When soli- tary bottles or sets are submitted, the likelihood of iso- lating a pathogen is reduced because the total volume of blood that is cultured is less than the optimal vol- ume, effectively reducing the sensitivity of the culture.
There are recommended benchmarks for solitary blood culture rates; an ideal rate is less than 5% [6].
Laboratories can monitor solitary blood culture rates as part of their quality management program and improve their solitary collection rate by implementing changes such as ensuring a blood culture order includes recom- mendations for two sets and re-educating phlebotomy or other staff who collect blood cultures. In a College of American Pathologists Q-Probes study performed in 2001, the lowest rate of solitary blood cultures was seen in institutions that had implemented several practices:
having phlebotomists draw cultures, instituting a policy requiring two sets per order, and monitoring of the soli- tary collection rate[6].
Single bottles or single sets also present a challenge in interpretation when organisms that may represent contaminants are isolated (e.g., coagulase-negative staphylococci and streptococci). Interpretation of blood cultures that grow organisms that are potential con- taminants can be difficult when the culture consists of a single set[7]. With certain organisms, it can be diffi- cult to determine whether the organism represents a pathogen or a contaminant[8].
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ISSUES WITH PRE-ANALYTICAL ERRORS
Inadequate Volume of Blood Collected
Commercially available blood culture bottles require a certain defined blood volume to be drawn into the bottles. For adults, the recommendation is 8 10 mL of blood per bottle. The correct volume of blood is impor- tant for two reasons: (1) Optimal sensitivity is achieved when the correct volume of blood is cultured, and (2) anticoagulants such as sodium polyanetholsulfo- nate that are included in the culture media are diluted to the level at which they will not inhibit the growth of fastidious microorganisms. For pediatric patients, blood volumes are generally calculated based on the weight of the patient.
Drawing Blood Cultures through a Catheter
Recommendations for blood cultures are to draw through a venipuncture. Drawing blood cultures through a catheter can result in several confounding problems, including culture and workup of colonizing organisms and dilution of blood by fluids in the catheter line.
Drawing blood cultures through a vascular catheter should be discouraged[8 10]. Discarding an initial ali- quot of blood when using a catheter to obtain the blood specimen does not reduce the rate of contamination[11].
Inadequate Preparation of the Draw Site
Preparation of the venipuncture site to inactivate skin organisms is necessary to prevent growth of con- taminants. The isolation and identification of blood culture contaminants is a significant problem for clini- cal microbiology laboratories and for patient care pro- viders. Workup of contaminating organisms adds extra, unnecessary work for the laboratory and, impor- tantly, may provide misleading results to clinicians, resulting in unnecessary antimicrobial therapy for patients. Careful adherence to the laboratory’s proce- dure for preparation of the draw site for blood cultures can reduce the growth of microbial contaminants [9,10]. A policy for workup of positive blood cultures based on the organism identification and number of bottles may help prevent excess work and misleading reports from the laboratory.
Delayed Entry of Blood Culture Bottles into Automated Instruments
Blood cultures should be placed into automated instruments immediately after collection (ideally, within 1 hr). In reality, delays in bottle entry often occur, especially when the microbiology laboratory is located off-site, such as in a central laboratory or at a referral laboratory. In these cases, delays of up to 24 hr are not uncommon. The storage conditions and the length of time for blood cultures can result in reduced recovery of organisms[12].
Pre-Analytical Error: Microbiology Cultures Not Ordered on Surgical Specimens Submitted to the Pathology Laboratory
A common occurrence is failure to order microbio- logical testing potential for an infectious process when specimens are surgically obtained for submission to pathology. Often, these specimens are submitted in formalin, therefore obviating the possibility of cultur- ing any live organisms.
Suggestions for Reduction in Pre-Analytical Errors in the Clinical Microbiology Laboratory
Good communication between the laboratory and the physicians using the laboratory is the best way to prevent pre-analytical errors. The laboratory should routinely check and update its specimen collection and test ordering guidelines. A program of electronic updates to test menus is ideal. The laboratory supervi- sors and directors should also be available for consul- tation and guidance on test selection, ordering, and interpretation.