The gram stain, also known as the Gram stain for Hans Christian Gram, is one of the first techniques budding microbiology students learn in their introductory lab courses. It’s even a good exercise for younger students (with proper supervision, of course), due to its simplicity and the colorful, beautifully stained cells that result from the procedure. The protocol is often taught in tandem with lessons on bacterial structure, since the differential staining helps determine whether an isolate is a gram-positive or gram-negative bacterium.
Because of its simplicity and the rapid time-to-result turnaround, gram staining also plays an important role in clinical microbiology. Learning the cell structure helps eliminate potential disease etiologies: learning an isolate is a gram-negative rod doesn’t tell you what the diagnosis is, but it helps eliminate what it isn’t. This information can also inform clinicians about the best-choice antimicrobial drug, at least until the isolate can be cultured and its drug-resistance profile characterized. While relatively technologically simple, gram staining has played a big role in diagnostics for decades.
But what if one technician’s gram-positive stain is another’s gram-negative? That’s the predicament outlined in a Journal of Clinical Microbiology publication in the June issue but available online now. The interpretation of a gram-stain slide is inherently subjective, and many factors can play a role in a correctly stained sample, including specimen fixation, staining protocol, and slide analysis. The study compared gram-stain techniques and results at four major U.S. clinical microbiology centers.
Standardized criteria were used to compare the abilities of technicians in different labs to correctly identify respiratory, fluid, biopsy/tissue, and wound cultures. Each site had hundreds of specimens screened for those that were “discrepant” – if a corresponding culture analysis grew bacteria not matching the designated gram stain, the original stain was reviewed by a senior lab member to determine whether the stain result was an error. The error could be either an incorrect stain, or an inaccurate assessment of bacterial presence.
All clinical labs had around 5% of their examined specimens flagged as discrepant, and the first hurdle was identified when trying to find the original slides: only 87% could be found due to disorganization. In examining the available slides, the researchers found only around a quarter of discrepancies were due to misread stains (positive mischaracterized as negative or vice versa). Most discrepant results were either smear negative/culture positive or smear positive/culture negative – meaning that the most common mistake was in determining whether there was a bacterial infection at all.
Differences in gram staining interpretations have been found in previous studies, but most studies have concentrated on the person-to-person variability within a single lab. This multi-center study was designed to emphasize the need for stronger standards across all microbiology labs, since these errors can majorly impact patient outcome. A small specimen size, low bacterial cell to tissue, or inadequate fixation can all lead to the false negatives, which could delay treatment. Fixation type (methanol versus heating) didn’t play a major impact, as the one site that used methanol fixation had comparable error rates to the three sites using heat fixation. An automated gram stainer may standardize the protocol, but it didn’t prevent the one site with this machine from interpretation-based errors.
To correct these mistakes, the research team recommends education: both education of doctors, to order the correct tests (unordered anaerobic growth tests led to stain positive/culture negative results) and education of technicians, to better prepare and analyze specimen slides. Taking a page from anatomic pathologists, the researchers also recommend tracking error by type and individual, which can reveal addressable patterns of errors to address. Standardization of this cornerstone diagnostic procedure will help minimize preventable medical errors.
Do you have experience in clinical microbiology? What changes would you propose to eliminate misread gram stains? Does the procedure need to be automated? Leave your thoughts and experiences in the comments!
-- Julie Wolf