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JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2007, p. 37543758 Vol. 45, No.

0095-1137/07/$08.000 doi:10.1128/JCM.01632-07
Copyright 2007, American Society for Microbiology. All Rights Reserved.

Mechanisms To Assess Gram Stain Interpretation Proficiency of
Technologists at Satellite Laboratories
Erik Munson,1,2* Timothy Block,1 Janice Basile,1 Jeanne E. Hryciuk,1 and Ronald F. Schell3,4,5
Wheaton Franciscan and Midwest Clinical Laboratories, Wauwatosa, Wisconsin 532261; College of Health Sciences, University of
WisconsinMilwaukee, Milwaukee, Wisconsin 532012; and Wisconsin State Laboratory of Hygiene3 and Departments of
Bacteriology4 and Medical Microbiology and Immunology,5 University of Wisconsin, Madison, Wisconsin 53706
Received 15 August 2007/Accepted 16 August 2007

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To address Gram stain interpretation proficiency in a satellite/centralized microbiology laboratory para-
digm, two programs were devised. In quality assurance program 1, nonmicrobiology technologists at satellite
laboratories were required to interpret standardized Gram-stained specimens of clinical material prepared by
an experienced microbiologist at a central laboratory. In quality assurance program 2, clinical Gram stains
prepared and read by the satellite laboratorians were reviewed by experienced microbiologists at the central
laboratory. Satisfactory performance (94%) was achieved in quality assurance program 1. In contrast, quality
assurance program 2 had a significantly lower overall performance (89%; P < 0.0001) due to poorer identi-
fication of host cells (93%) and bacteria (84%). A variety of intervention mechanisms, including continuous
monitoring, resulted in overall performance improvement (P < 0.006). While a technologist challenge has
educational merit, having a microbiologist review previously read slides is a better indicator of the technolo-
gists Gram stain interpretation proficiency.

Gram staining of primary clinical specimens has tremendous assessing the Gram stain interpretation competency of nonmi-
clinical utility. Studies have shown that Gram staining of spu- crobiology laboratorians and present a quality assurance pro-
tum can be predictive for several etiologies of lower respiratory gram for monitoring and increasing the proficiency of Gram
tract disease (1, 10, 11, 15, 16, 17, 19, 23). Gram stain data also staining.
influence the treatment of clinically significant bloodstream Full-spectrum clinical laboratories were present at three
infections (8, 13). In addition, the proper performance of the Milwaukee, Wisconsin, inpatient facilities until 1988, when two
primary Gram stain procedure can guide the processing of hospitals (B and C) consolidated their clinical microbiology
expectorated sputum specimens (14) and aid in the interpre- laboratories into a freestanding building (a central laboratory).
tation of cultured skin and soft tissue specimens (3, 26). Subsequently, the central laboratory assimilated the clinical
Bartlett (2) cited the virtual elimination of house staff microbiology services of a third hospital (A) in 1999. Algo-
laboratories (as an indirect result of the Clinical Laboratory rithms were devised in which the primary clinical specimens for
Improvement Amendments of 1988) and the outsourcing of routine bacteriology were processed onto appropriate media
primary specimens as two factors contributing to the decline in (22) and smeared for Gram staining and interpretation (25) by
clinical microbiology studies for patients with lower respiratory trained nonmicrobiology technologists at the satellite labora-
tract disease. Others have raised concerns over the clinical tories. Within 24 h, Gram-stained smears were forwarded to
diagnostic value of the Gram stain in terms of protocol stan- the central laboratory for confirmation of results.
dardization (5, 7, 18). As a result, many have discouraged the Three primary clinical specimens from a variety of sources
utilization of Gram-stained smears as predictors of certain were smeared and Gram stained quarterly by an experienced
clinical infections, particularly those involving the lower respi- microbiologist at the central laboratory (quality assurance pro-
ratory tract (10, 20, 21). gram 1). The number of cells (viewed at 100 magnification
Technologist competency has become of even greater im- for eukaryotic cells and 1,000 magnification for prokaryotic
portance with the centralization of microbiology testing and cells) was identified as rare (1 per field), few (1 to 5 per field),
the creation of satellite processing (rapid-response) laborato- moderate (5 to 20 per field), or abundant (20 per field).
ries staffed by nontraditional microbiologists (6). Moreover, Included within these primary clinical specimens were sputa to
accrediting agencies mandate the proficiency documentation be evaluated for culture processing acceptability, based on the
of all technologists who execute clinical Gram staining proto- criteria of Murray and Washington (14). Seventy satellite lab-
cols. In this report, we describe a mechanism for objectively oratory technologists were made aware of the specimen source
and were asked to provide Gram stain interpretation and semi-
quantitation of the eukaryotic and prokaryotic cells from the
* Corresponding author. Mailing address: Midwest Clinical Laborato-
ries, 11020 West Plank Court, Suite 100, Wauwatosa, WI 53226. Phone:
challenge slides. The results were forwarded to the central
(414) 256-1479. Fax: (414) 256-5566. E-mail: laboratory microbiologist. Furthermore, the central laboratory

Published ahead of print on 29 August 2007. microbiologists (on a rotating basis) daily selected a number of

VOL. 45, 2007 NOTES 3755

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FIG. 1. Gram stain interpretation proficiencies of three satellite laboratories, determined by quality assurance program 1 and delineated by
interpretation of bacteria (black bars) and host cells (white bars) and the sum of the two categories (shaded bars).

the satellite-laboratory-prepared smears that was commensu- interpreted with a higher success rate than bacteria (98% ver-
rate with the relative microbiology volume generated by that sus 89%; P 0.0001). When data from individual hospitals
hospital (quality assurance program 2). A descriptive Gram were examined (Fig. 1), personnel at hospital C showed the
stain report was documented by the microbiologist and for- lowest proficiency for host cell recognition and identification of
warded to the microbiology supervisor. bacteria. Upon the review of 2,517 randomly chosen Gram-
Reviews of the Gram-stained preparations in quality assur- stained preparations by quality assurance program 2, no sig-
ance programs 1 and 2 were documented using the following nificant differences in proficiency for the detection of host cells
system: 1 point was given for each correct Gram stain reaction, or bacteria were found among the laboratorians of the three
1 point for each bacterial morphology correctly identified, 1 satellite laboratories. Similar to the results for quality assur-
point for each host cell (squamous epithelial or inflammatory) ance program 1, greater proficiency in interpretation of host
morphology correctly identified, and 1 point for the quantity of cells than in bacterial observations was demonstrated by sat-
each host or bacterial cell type correctly identified. The accept- ellite laboratorians (P 0.001).
able range for quantitation was 1 gradation of the expected When the results of the two quality assurance programs were
value. Credit for quantity, Gram reaction, or cell type involved compared, the proficiency in interpretation of Gram stain re-
was not granted if a sputum specimen was rejected for culture; sults of quality assurance program 2 was 5.4% lower than that
the technologist received a slide value of 0. In addition, points of quality assurance program 1 (P 0.0001; Fig. 2). Analogous
were subtracted from a score when additional cell types or differences were detected between the programs for identifi-
bacteria were erroneously reported. Finally, point values for cation of bacteria and host cells (P 0.03). Upon delineation
host cells (quantity plus presence/absence) and bacteria (quan- of individual hospital data by cell type, the rates of proficiency
tity plus Gram stain result and morphology) were summed per noted for quality assurance program 1 were generally higher
preparation and expressed as a percentage of the expected than those for quality assurance program 2 (P 0.04).
value. The standard error of the mean was calculated for all The implementation of quality assurance programs 1 and 2
measures of Gram stain interpretation proficiency testing. caused a significant increase in Gram stain interpretation pro-
Comparisons of interlaboratory performances, cell types, and ficiency in the fourth quarter of 2006 (P 0.006; Fig. 3).
the two measures of Gram stain interpretation proficiency Improvements occurred in host cell and bacterial identification
testing were facilitated by the t test for independent samples. among the participants of quality assurance program 1 (P
Assessment of the temporal change (improvement) in labora- 0.01) and in host cell identification among the participants of
tory performance was facilitated by one-way analysis of vari- quality assurance program 2 (P 0.03). Among the hospitals,
ance (24). The alpha level was set at 0.05 before investigations the greatest improvement occurred with hospital C (P 0.005;
commenced, and all P values were two-tailed. Fig. 4A and B). The nonmicrobiology laboratorians at hospital
The overall Gram stain interpretation proficiency rate for 70 C performed consistently less skillfully than laboratorians at
satellite laboratory technologists, as determined by quality as- hospitals A and B prior to the implementation of the quality
surance program 1, was 94% (Fig. 1). However, host cells were assurance programs.

The diagnostic value of the Gram stain has been questioned

from a variety of perspectives. In a meta-analysis, Reed et al.
(21) reported Gram stain sensitivity of 15% to 100% for the
diagnosis of pneumococcal pneumonia, with specificity ranging
between 11% and 100%. In a regional proficiency testing pro-
gram, Church et al. (5) reported a significant lack of standard-
ization in reporting quantities of host cells and bacteria and
raised concerns about how this could impact patients over a
continuum of care. Cooper et al. (7) documented low inter-
technologist agreement rates for cell quantitation from Gram-
stained lower respiratory tract specimens. In a broad sense,
following a regional microbiology laboratory restructuring, a
number of subsequent proficiency challenges revealed that er-
ror rates increased in a number of laboratories, such as rural
facilities (noted to possess neither a technologist devoted to

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microbiology duties nor on-site pathologist or medical micro-
biologist oversight) that took on additional microbiology re-
sponsibilities (6). Combined with the clinical and bench-level
utility of the Gram stain procedure, the preceding factors pro-
vide an impetus for evaluating and maintaining the proficiency
of microbiologists and technologists who perform this process.
The central laboratory microbiologists who determined the
expected values for slides throughout these quality assurance
programs were subjected to in-house Gram stain interpreta-
tion proficiency testing. On a quarterly basis, all microbiolo-
gists reported findings for a randomly selected slide set. Data
were pooled, and the expected value for each slide was derived
from the majority of responses for each parameter. Unlike
quality assurance program 1, which granted credit for results
FIG. 2. Comparison of quality assurance programs 1 and 2 for within 1 gradation of an expected value for quantitation,
measure of overall Gram stain interpretation proficiency.
in-house proficiency testing used higher stringency (i.e., only
one acceptable value for quantitation). The microbiologist who
prepared the proficiency material and graded the responses for
quality assurance program 1 was one of two microbiologists

FIG. 3. Gram stain interpretation proficiency of nonmicrobiology laboratorians as determined by quality assurance programs 1 (squares) and
2 (diamonds) for four quarters of 2006.
VOL. 45, 2007 NOTES 3757

found in host cell analysis (93%) than in bacterial analysis

(84%). The rate of successful interpretation of host cells was
markedly higher than that observed in an intralaboratory study
(7). Cooper et al. (7) limited their study to lower respiratory
tract smears and exercised more stringency in their grading of
quantitative measures. However, the satellite technologists in
our study may have had more breadth of knowledge in hema-
tology and sterile body fluid analysis.
Quality assurance program 1 yielded higher rates of profi-
ciency than did quality assurance program 2. This was true for
each component of the monitoring and was realized at all three
satellite laboratories. While intralaboratory technologist col-
laboration may or may not have played a role in the increased
frequency of correct responses in quality assurance program 1,
one aspect that could have played a role was the nature of the

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Gram-stained smear itself. To ensure consistency in the survey,
specimens were preselected and smeared onto slides, with con-
tents stained, coverslipped, and previewed by the microbiolo-
gist at the central laboratory before presentation to the three
satellite laboratories. In contrast, Gram-stained smears en-
countered in quality assurance program 2 were prepared dur-
ing routine operations of the satellite laboratory. As such,
there may have been greater opportunity for microbiologists to
review overdecolorized gram-positive cocci and degenerated
leukocytescell types that may have been incorrectly reported
by satellite technologists.
A key component of any continuous quality assurance mon-
itoring program is ad libitum intervention (9). Throughout this
study, four major means of correction were implemented.
First, Gram-stained smears from either quality assurance pro-
gram 1 or 2 that yielded an egregiously incorrect response from
a satellite laboratorian were returned to a designated point
person from that laboratory. This individual personally re-
viewed the slide with the technologist and discussed any dis-
crepancies. Second, one-on-one interactions at a dual-headed
microscope took place between a central laboratory microbi-
ologist and the satellite laboratorian. Third, personnel from
the satellite laboratories and the central laboratory formed a
system-wide microbiology subcommittee, which provided a
conduit for the exchange of data, policies, and concerns. Fi-
nally, digital images of interesting findings from quality assur-
ance program 2 were disseminated via interlaboratory elec-
tronic mail in the format of a case presentation. Comprising
this document were the chart review, organism epidemiology,
FIG. 4. Gram stain interpretation proficiency of hospital C nonmi- and discussion of the relevance of the Gram-stained smear in
crobiology laboratorians, measured at the onset and later stages of the specific case. These mailings also provided a forum for
quality assurance programs 1 (A) and 2 (B). discussion of aberrant findings in Gram-stained preparations,
such as organisms partially treated with antimicrobial agents
(4, 12).
who performed at 100% of the expected value for in-house Analysis of variance data for the four quarters of quality
proficiency testing during the time frame of this investigation. assurance program 1 (Fig. 3) revealed an increase in Gram
The central laboratory microbiologists demonstrated a mean stain interpretation proficiency. Analogous improvement was
in-house proficiency rate of 95%. No significant differences seen via quality assurance program 2. However, neither hos-
existed between the average microbiologist proficiency score pital A nor hospital B demonstrated any improvement in qual-
and the mean demonstrated by the microbiology laboratory as ity assurance program 1 or 2 throughout the course of this
a whole (P 0.06). These data confirmed the validity of the monitoring period. It is likely that these two laboratories had
expected values. the most experience with satellite microbiology processing. In
Data from both quality assurance programs demonstrated further support of this statement, hospitals A and B possessed
the competency of satellite laboratorians in the interpretation the two highest proficiency rates (Fig. 1). In contrast, hospital
of Gram-stained material. However, greater proficiency was C showed significant improvement in total Gram stain inter-

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