Delivering the goods to microbiology labs
Transporting fluids, blood cultures, and more
Forget what you’ve learned before: There is such a thing as being too good in the laboratory.
So says J. Michael Miller, PhD, D(ABMM), chief of the laboratory response branch of the Biopreparedness Program at the Centers for Disease Control and Prevention. Speaking in a panel last May at the annual meeting of the American Society for Microbiology, Dr. Miller said that a specimen that is inappropriately collected, stored, or transported will be counterproductive for the laboratory and clinician. "We can take that bad specimen and we can do exhaustive bacteriology-but it’s going to produce invalid information that can mislead physicians."
Dr. Miller, author of a leading textbook on specimen management, stressed that "from the standpoint of effectiveness of the laboratory, nothing is more important than the adequacy and condition of the specimen received for examination." His message: If specimens are not properly collected or handled, the laboratory can contribute little or nothing to any investigation or to the welfare of the patient.
Microbial specimen transport devices by and large have not evolved as fast as the demands placed on them by the centralization of labs and the establishment of core labs, said panel convener Paul Bourbeau, PhD, D(ABMM), director of the microbiology laboratory at Geisinger Medical Center, Danville, Pa. "As recently as 1992, over 50 percent of the samples tested in our laboratory were collected in our hospital. Last year, more than 80 percent came in a car. Consequently, we don’t get many specimens that are cultured within four hours of collection."
"Where we used to worry about getting specimens in two hours, now it’s two days," he said. "And historically quality control has been generally absent from these devices. It’s only in the last year and a half that the first document, M-40, dealing with quality control of specimen transport has been developed by the NCCLS."
Cost-cutting measures throughout health care have relegated many purchasing decisions to nonlaboratorians, but microbial transport devices should not be seen as commodities, Dr. Bourbeau warns. All swabs are not equal in their ability to collect specimens for culture. There are various ways to transport tissues, fluids, or specimens that don’t come on swabs. We shouldn’t automatically trust the quality control of the transport devices on the market.
Specimen management is the key to accurate laboratory diagnosis. "Without it," Dr. Miller stressed, "we’re not going to have much luck at the bench. Appropriate specimen management influences patient care decisions because we need to get the correct organism off of the transport device, and it impacts hospital infection control, patient length of stay, cost control, and laboratory efficiency. To me, that looks like a pretty important topic-yet most physicians learn about the subject on the floor from people who have also never heard any lectures on specimen management."
Dr. Miller sees four key challenges, not only for the laboratory but also for the manufacturer of the transport system:
Survival of the organism in courier transport.
- Safety-whether or not the device is going to open and spill.
- Security-who has access to these specimens.
- Storage-the temperatures and devices needed to maintain viability of the organisms.
There is a big difference between what the physician needs and what the laboratory needs, he points out. "The doctor needs to know three things: Is my patient’s illness caused by microbes; if it is, what is it, and how do I treat it? One thing we need to communicate is that we need a specimen representative of disease process. Every day you get a specimen that you know is not really representative of the disease process. Yet you worked it up anyway," Dr. Miller said.
One classic example is a specimen labeled "wound." Taking a swab, rubbing it over the surface of a wound, and sending it to the laboratory to culture is not an uncommon practice. "But that’s not representative of the disease process," he said. "The disease is going on at the advancing margin of that lesion, and that’s what needs to be tested-not the debris in the wound."
Another example is the swab labeled "ear." "Ninety-five percent of the time the diagnosis of that swab was acute otitis media, but that disease occurs behind the eardrum, and the physician is rarely going to run a swab back there where the disease process is. So we get a swab of external ear flora."
Selecting the appropriate specimen, using the correct collection method and system, and knowing whether to transport a specimen cold or at ambient temperature are critical parts of the preanalytical component, he said. "It is not just ’stick the swab in and send it to the lab.’ There are many specimens for which swabs are not appropriate." The etiologic agents must also survive transport containers, conditions, and couriers, because "things that cause disease need to be alive when you get them," he noted.
Pathologist Raymond Bartlett, MD, was known to say that the most laboratory work and the greatest cost are usually associated with specimens of the least clinical value. "Most of us can understand this," Dr. Miller said, "because we know we see some really nasty specimens that probably should not have been taken. We spend a lot of money, but it’s not useful because they’re inappropriately selected, collected, and transported. Whatever we do with that specimen in the laboratory regarding further workup, we need to note on the report to the physician the potentially compromised nature of the specimen.
"There are a number of culture requests we can actually say no to, and the literature will back us up," he added. Examples are using careful Gram stain evaluation to reject some sputum specimens, many wound cultures, endotracheal cultures, and contaminated urine cultures. Vaginal cultures can be rejected because Gram stains are used to diagnose and correlate vaginosis and vaginitis.
Getting the specimen to the laboratory involves using proper collection devices and containers, and getting it to the lab in a hurry. "At that point you can begin to do your magic and identify virtually anything that grows," he said. Ironically, however, that means the success of the laboratory may depend on a person who has just been told to collect a specimen but who may know the least about microbiology.
While we may take it on faith that commercially available laboratory transport devices will perform to a certain standard, swab transport devices in particular can have many inhibitory properties, Dr. Bourbeau said. Components include the stick, the glue used to attach the fiber, the fiber itself, the tube to hold the device (which may be gas permeable or impermeable), a gel media or liquid media in a sponge, and the packaging material.
Sticks are made of copolymers of butadiene and polystyrene, which is the same material used for Petri dishes. "You may say a stick is a stick, but if you make it too stiff and put it in the throat of the patient, it might break off, while if it’s too soft you’ll have trouble wringing them out," Dr. Bourbeau said. Wooden sticks, because of the way wood is processed, can also have toxic compounds and should not be used for microbiology testing. Glues are generally polyvinyl compounds, which can be antibacterial. There have been reported cases of the glue producing toxicity, he said.
The fibers for swabs may be cotton, rayon, Dacron (a brand made by Du Pont that is the basis of polyester), or alginate, which comes from the ocean. "Cotton makes great clothing but can be both a growth inhibitor and growth promoter of various strains of bacteria, because there are unsaturated long-chain fatty acids that diffuse out of cotton fibers in the presence of liquids," Dr. Bourbeau explained. Because it’s a natural product, lot-to-lot variations in cotton should be suspected, he noted.
Cellulosic fibers, derived from trees or plants, include rayon, acetate, and lyocell. Synthetic fibers, on the other hand, are made from chemicals from refined petroleum or natural gas. The main synthetics are polyester, nylon, polyolefin, acrylic, and spandex.
Rayon is described by the textile industry as highly absorbent, soft, comfortable, and versatile, and it dyes easily and drapes well, Dr. Bourbeau said. "But when you look for a swab transport for your laboratory, are you concerned about good drapeability? The manufacturer who’s out there making a biological transport device is competing for or buying the same product that 99.9 percent of the world uses for another purpose. It’s incumbent on manufacturers to use a product appropriate for our narrow use."
One difference between Dacron and rayon is that it’s necessary to add surfactants to Dacron because it is naturally hydrophobic. The surfactants that will make it able to take up liquids generally are antibacterial. In fact, the reason Dacron never works quite as well as rayon for culture is because of surfactants, Dr. Bourbeau said. "If you try to get rid of the surfactants, Dacron won’t take up the liquid, and if you leave the surfactants there, it can be really difficult to find lots that are without at least some antibacterial properties."
"Then when you’re done with that fiber, when you’ve wound it around the end of the stick, you have to add something to it so it will stick, so the ’bud’ doesn’t come apart," he said. "You don’t want fiber coming off in the wound. Rayon swabs are sprayed with methylcellulose or something similar, and Dacron swabs are heated in a process called thermal attraction that is used to firm up the bud. Some hydropolymers applied to rayon have been shown to interfere with rapid antigen tests such as group A strep.
"So you have to be careful you don’t get false-positive reactions simply because of the methylcellulose put on swabs. It’s important to appreciate that a manufacturer may make a product for a specific use-for example, culture-and the end-user may use it for a different use-for example, a rapid antigen test." The user has to verify that the collection device is compatible with the specific test that’s being performed, Dr. Bourbeau advised. Lastly, "both Dacron and rayon are bleached, so you have to be sure whiteners don’t add antibacterial properties," he said.
Industry’s priorities may also be different than those of laboratories. An industry article noted that commercially manufactured cotton wool is finished to a pH of 4.5 to give a high degree of "crunch," a physical characteristic known to be attractive to purchasers. "Now they were talking about Q-tips, but if you were using something in your laboratory with a pH of 4.5 , you would obviate a lot of problems with specimen transport because you’d kill the organisms before they got to your laboratory," Dr. Bourbeau said.
"In the ideal world, a swab would have great absorption and great release; however, you don’t usually get both," he said. Fiber swabs like Dacron and rayon absorb better than foam swabs, but foam swabs release much better than fiber swabs, and one of the reasons is that to keep the fiber from coming off the tip of the stick, the end of the bud must be tight, which can make it difficult to release liquid.
Even something as innocent as the sponge has to be screened for toxicity. A particular cellulose sponge, which is no longer used for specimen transport, was associated with a dramatic die-off of the organisms that had been exposed to it. The inhibitory properties of cellulose sponges, possibly due to residual sulfur from the process that breaks down the wood, are well known and well documented, Dr. Bourbeau said. "Some sponges also have quaternary ammonium compounds added as a preservative, and I think many of you realize these are disinfectants used around your hospital."
Charcoal, which absorbs pollutants out of the air, can absorb and therefore
detoxify free fatty acids produced by microorganisms and can then prevent photochemical
oxidation reactions, detoxifying media of reduced forms of oxygen. "It’s a scavenger
for products that can be potentially toxic. The effects of charcoal are to reduce
the production of potentially toxic hydrogen peroxide and free radical compounds
and trap free radicals that are present," Dr. Bourbeau said. "And it’s not coincidental
that organisms like Legionella and Bordetella media all have charcoal
Hydrogen peroxide, too, can be toxic to organisms like N. gonorrhoeae, L.
pneumophila, C. jejuni, and T. pallidum, although E. coli can
tolerate relatively high concentrations of hydrogen peroxide.
"Once the swab has the right glue, right stick, the right medium,
and the right everything else, we have to sterilize it," Dr. Bourbeau continued.
He listed three methods: steam sterilization, ethylene oxide, and irradiation.
Steam sterilization is associated with thermal denaturation and oxidation of proteins and lipids; it inactivates enzymes and destroys metabolic and reproductive mechanisms, he explained. "It works really well, but it can’t be used where residual moisture would be a problem-for example, maybe you have a product wrapped in a paper wrapper. Some plastics will also melt under the exposure to steam." Lastly, it’s not adaptable to mass production. "I suspect many of you use it if you prepare a small amount of media in your lab, or you’re doing research applications with homemade media," he said.
Ethylene oxide works well by causing alkalization of groups of nucleic acids, leading to cell death, but ethylene oxide cannot be used in devices that contain liquid media that would come in contact with it, because residual ethylene oxide would kill the microorganisms being transported.
"So if you have a transport device like the old Culturette, which had an ampule inside, you can use ethylene oxide because it doesn’t come in contact with the media. But if you have a CulturePlus or a Copan product where there’s a sponge with liquid media, if you were to use ethylene oxide, it would remain in the media after you finished sterilization and would be very toxic to microorganisms," Dr. Bourbeau said.
The third sterilization alternative, one that is widely used for medical devices, is irradiation with gamma rays or an electron beam that ionizes key cellular components, especially nucleic acids, leading to cell death.
"Industry puts pallets of material, swabs and other medical devices used in your hospital into chambers that are the size of tractor-trailers. There they are subjected to gamma irradiation. It works well and it works fast, but the amount of radiation that’s used varies widely and it’s not clear how much is actually necessary."
"My impression from talking to manufacturers is if you’re not sure how much radiation to use, you use a little bit more," he said. "But it’s clear that radiation can damage the polymers that are associated with the target microorganisms, fibers, polyethylenes, and other natural and synthetic polymers. The residual effect of irradiation is creation of free radicals and superoxides, hydrogen low-molecular-weight hydrocarbons, discoloration, and oxidation." There have been reports of implantable devices that have actually turned yellow from the effects of irradiation, he added.
He compared gamma radiation to a pinball in a vacuum. "Once you start those molecules moving, they just keep moving, they don’t stop, so the free radicals that are created just keep reacting and reacting, and one of the things that can be toxic to bacteria, which charcoal can negate, is this free radical superoxide and hydrogen low-molecular-weight hydrocarbons."
"So the practical effect of gamma radiation is you can produce a lot of compounds that are potentially toxic for microorganisms, and the manufacturer must attempt to minimize the effect of gamma radiation. You start out with the best product you can possibly get, but then you subject your product to gamma radiation, so you create toxic molecules and potentially damage the product."
Creativity is required to minimize the total radiation, he said, and since the amount of radiation needed is proportional to the bioburden of the product, "if you can get as sterile a product to begin with as possible, you can minimize the radiation. And then you try to create an environment and add chemicals that negate the effects of irradiation, because you don’t want to end up with a swab that glows in the dark," he said.
To stem interference from packaging, some manufacturers use a tube that’s gas-impermeable, allowing them to gas the packaging with nitrogen.
"They’ll basically evacuate and add nitrogen, because the free radicals, the oxygen radicals and peroxide, come from the presence of oxygen, so if you have nitrogen inside the product that you’re exposing to the gamma radiation, if you remove as much oxygen as possible, you can minimize production of free radicals and peroxides."
Laboratories rely on reputable manufacturers to provide a high-quality commercial specimen collection and transport media system, which they in turn distribute to their physician clients, Dr. Bourbeau said. "The challenge for manufacturers is to produce a transport device that controls for multiple variables, including raw materials in processing, to make a consistently functioning biological device. The vagaries of the process indicate the finished product is far more than a commodity, and strict quality control is obligatory."
Until recently, there wasn’t an accepted standard for manufacturers to follow, but now the lab community is considering the proposed M-40 standard developed by NCCLS. The potential harm that specimen transport can cause, however, makes it crucial in the meantime for laboratories to take control of the quality of the products they purchase.
Anne Paxton is a writer in Seattle.