mechanisms to assess gram stain proficiency of...

Mechanisms to Assess Gram Stain Proficiency of

Technologists at Satellite Laboratories

Erik Munson,1,2

* Timothy Block,1 Janice Basile,

1

Jeanne E. Hryciuk,1 and Ronald F. Schell

3,4,5

Wheaton Franciscan and Midwest Clinical Laboratories,1 Wauwatosa, Wisconsin 53226;

College of Health Sciences, University of Wisconsin--Milwaukee,2

Milwaukee, Wisconsin 53201; and Wisconsin State Laboratory of Hygiene3

and Departments of Bacteriology4 and Medical Microbiology and Immunology,

5

University of Wisconsin, Madison, Wisconsin 53706

Running title: Note

* -- Corresponding author Erik Munson

Midwest Clinical Laboratories

11020 West Plank Court

Suite 100

Wauwatosa, WI 53226

Telephone: (414) 256-1479

Facsimile: (414) 256-5566

Electronic mail: [email protected]

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01632-07 JCM Accepts, published online ahead of print on 29 August 2007

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ABSTRACT

To address Gram stain proficiency in a satellite/centralized microbiology laboratory paradigm,

non-microbiology technologists were required to interpret standardized Gram-stained

preparations of clinical material (quality assurance program 1). Clinical Gram stains prepared

and read by satellite laboratorians were reviewed by microbiologists (quality assurance program

2). Satisfactory performance was achieved (94%) in quality assurance program 1. In contrast,

quality assurance program 2 had a significantly lower overall performance (89%; P < 0.0001)

due to poorer identification of host cells (93%) and bacteria (84%). A variety of intervention

mechanisms, including continuous monitoring, resulted in overall performance improvement (P

≤ 0.006). While technologist challenge has educational merit, review of previously-read slides

is a better indicator of technologist Gram stain proficiency.

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Gram staining of primary clinical specimens has tremendous clinical utility. Studies have shown 1

that Gram staining sputum can be predictive for several etiologies of lower respiratory tract 2

disease [1,10,11,15,16,17,19,23]. Gram stain data also influence the treatment of clinically-3

significant bloodstream infection [8,13]. In addition, proper performance of the primary Gram 4

stain can guide the processing of expectorated sputum specimens [14] and aid in interpretation of 5

cultured skin and soft tissue specimens [3,26]. 6

7

Bartlett [2] cited virtual elimination of house staff laboratories (as an indirect result of the 8

Clinical Laboratory Improvement Amendments of 1988) and outsourcing of primary specimens 9

as two factors contributing to the decline in clinical microbiology studies for patients with lower 10

respiratory tract disease. Others have raised concerns over Gram stain clinical diagnostic value 11

in terms of protocol standardization [5,7,18]. As a result, many have discouraged utilization of 12

Gram-stained smears as predictors of certain clinical infections, particularly those involving the 13

lower respiratory tract [10,20,21]. 14

15

Technologist competency has become of even greater importance with the centralization of 16

microbiology testing and creation of satellite processing (rapid response) laboratories staffed by 17

non-traditional microbiologists [6]. Moreover, accrediting agencies mandate proficiency 18

documentation of all technologists who execute clinical Gram staining protocols. In this report, 19

we describe a mechanism for objectively assessing the Gram stain interpretation competency of 20

non-microbiology laboratorians and present a quality assurance program for monitoring and 21

increasing proficiency of Gram staining. 22

23

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Full-spectrum clinical laboratories were present at three Milwaukee, Wisconsin inpatient 24

facilities until 1988 when two hospitals (B and C) consolidated their clinical microbiology 25

laboratories to a free-standing building (central laboratory). Subsequently, the central laboratory 26

assimilated the clinical microbiology services of a third hospital (A) in 1999. Algorithms were 27

devised in which primary clinical specimens for routine bacteriology were processed to 28

appropriate media [22] and smeared for Gram staining and interpretation [25] by trained non-29

microbiology technologists at the satellite laboratories. Within 24 hours, Gram-stained smears 30

were forwarded to the central laboratory for confirmation of results. 31

32

Three primary clinical specimens from a variety of sources were smeared and Gram stained 33

quarterly by an experienced microbiologist at the central laboratory (quality assurance program 34

1). The number of cells (viewed with 100x magnification for eukaryotic cells and 1000x 35

magnification for prokaryotic cells) was identified as rare (<1 per field), few (1-5 per field), 36

moderate (5-20 per field), and abundant (>20 per field). Included within these primary clinical 37

specimens were sputa to be evaluated for culture processing acceptability, based on the criteria 38

of Murray and Washington [14]. Seventy satellite laboratory technologists were made aware of 39

the specimen source and asked to provide Gram stain interpretation and semi-quantitation of 40

eukaryotic and prokaryotic cells from the challenge slides. Results were forwarded to the central 41

laboratory microbiologist. Furthermore, central laboratory microbiologists (on a rotating basis) 42

daily selected a number of the satellite laboratory-prepared smears that was commensurate with 43

relative microbiology volume generated by that hospital (quality assurance program 2). A 44

descriptive Gram stain report was documented by the microbiologist and forwarded to the 45

microbiology supervisor. 46

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Review of the Gram-stained preparations in quality assurance program 1 and 2 was documented 48

using the following system: one point was given for each correct Gram stain reaction; one point 49

for each bacterial morphology present; one point for each host cell (squamous epithelial or 50

inflammatory) morphology present; and, one point for the quantity of each host or bacterial cell 51

type present. The acceptable range for quantitation was ± one gradation of the expected value. 52

Credit for quantity, Gram reaction, or cell type involved was not granted if a sputum specimen 53

was rejected; the technologist received a slide value of 0. In addition, points were subtracted 54

from the score when additional cell types or bacteria were reported. Finally, point values for 55

host cells (quantity plus presence/absence) and bacteria (quantity plus Gram and morphology) 56

were summed per preparation and expressed as percentage of expected value. Standard error of 57

the mean was calculated for all measures of Gram stain proficiency testing. Comparisons of 58

inter-laboratory performance, cell type, and the two measures of Gram stain proficiency testing 59

were facilitated by the t-test for independent samples. Assessment of temporal change 60

(improvement) in laboratory performance was facilitated by one-way analysis of variance [24]. 61

The alpha level was set at 0.05 before investigations commenced and all P values are two-tailed. 62

63

The overall Gram stain proficiency rate of 70 satellite laboratory technologists, as determined by 64

quality assurance program 1, was 94% (Fig. 1). However, host cells were interpreted with a 65

higher success rate than bacteria (98% versus 89%; P < 0.0001). When data were examined by 66

hospital (Fig. 1), personnel at hospital C showed the lowest proficiency for host cell recognition 67

and presence of bacteria. Upon quality assurance program 2 review of 2517 random Gram-68

stained preparations, no significant differences in proficiency for detection of host cells or 69

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bacteria were detected among the laboratorians of the three satellite laboratories. Similar to 70

quality assurance program 1, greater proficiency in the interpretation of host cells was 71

demonstrated by satellite laboratorians when compared to bacterial observations (P < 0.001). 72

73

When results from the two quality assurance programs were compared, quality assurance 74

program 2 was 5.4% lower than that determined by quality assurance program 1 (P < 0.0001; 75

Fig. 2). Analogous differences were detected between the programs for identifying bacteria and 76

host cells (P ≤ 0.03). Upon delineation of individual hospital data by cell type, higher rates of 77

proficiency were generally noted in quality assurance program 1 when compared to those of 78

quality assurance program 2 (P ≤ 0.04). 79

80

Implementation of quality assurance program 1 and 2 caused a significant increase in Gram stain 81

proficiency in the fourth quarter in 2006 (P ≤ 0.006; Fig. 3). Improvements occurred in host cell 82

and bacterial identification among participants of quality assurance program 1 (P ≤ 0.01) and 83

host cell indicators with participants of quality assurance program 2 (P = 0.03). Among the 84

hospitals, the greatest improvement occurred with hospital C (P ≤ 0.005; Fig. 4 A and B). Non-85

microbiology laboratorians at hospital C had consistently performed less skillfully than 86

laboratorians at hospital A and B prior to implementation of the quality assurance programs. 87

88

Gram stain diagnostic value has been questioned from a variety of perspectives. In a meta-89

analysis, Reed et al. [21] reported Gram stain sensitivity of 15-100% for diagnosis of 90

pneumococcal pneumonia, with specificity ranging between 11% and 100%. In a regional 91

proficiency testing program, Church et al. [5] reported a significant lack of standardization in 92

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reporting quantities of host cells and bacteria and raised concern how this could impact patients 93

over a continuum of care. Cooper et al. [7] documented low inter-technologist agreement rates 94

for cell quantitation from Gram-stained lower respiratory tract specimens. In a broad sense, 95

following a regional microbiology laboratory restructuring, a number of subsequent proficiency 96

challenges revealed that error rates increased in a number of laboratories, such as rural facilities 97

(noted to neither possess a technologist devoted to microbiology duties nor have on-site 98

pathologist or medical microbiologist oversight) that took on additional microbiology 99

responsibilities [6]. Taken together with the clinical and bench-level utility of the Gram stain 100

procedure, the preceding factors provide an impetus for evaluating and maintaining the 101

proficiency of microbiologists and technologists who perform this method. 102

103

Central laboratory microbiologists who determined expected values of slides throughout these 104

quality assurance programs were subjected to in-house Gram stain proficiency testing. On a 105

quarterly basis, all microbiologists reported findings on a randomly-selected slide set. Data were 106

pooled and the expected value of each slide was derived from the majority of responses for each 107

parameter. Unlike quality assurance program 1 that granted credit for ± one gradation of an 108

expected value for quantitation, in-house proficiency testing used higher stringency (i.e., only 109

one acceptable value for quantitation). The microbiologist who prepared proficiency material 110

and graded responses for quality assurance program 1 was one of two microbiologists who 111

performed at 100% of expected value for in-house proficiency testing during the timeframe of 112

this investigation. Central laboratory microbiologists demonstrated a mean in-house proficiency 113

rate of 95%. No significant differences existed between average microbiologist proficiency 114

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score and the mean demonstrated by the microbiology laboratory as a whole (P ≥ 0.06). These 115

data confirmed the validity of the expected values. 116

117

Data from both quality assurance programs demonstrated competency of satellite laboratorians in 118

the interpretation of Gram-stained material. However, greater proficiency was found in host cell 119

analysis (≥ 93%) than in bacteria analysis (≥ 84%). The rate of successful interpretation of host 120

cells was markedly higher than that observed in an intra-laboratory study [7]. Cooper et al. [7] 121

limited their study to lower respiratory tract smears and exercised more stringency in their 122

grading of quantitative measures. However, satellite technologists in our study may have had 123

more breadth of knowledge in hematology and sterile body fluid analysis. 124

125

Quality assurance program 1 yielded higher rates of proficiency than did quality assurance 126

program 2. This was true in each component of the monitor and was realized at all three satellite 127

laboratories. While intra-laboratory technologist collaboration may or may not have played a 128

role in the increased frequency of correct responses in quality assurance program 1, one aspect 129

that could have played a role was the nature of the Gram-stained smear itself. To ensure 130

consistency in the survey, specimens were pre-selected and smeared onto slides, with contents 131

stained, coverslipped, and previewed by the microbiologist at the central laboratory before 132

presentation to the three satellite laboratories. In contrast, Gram-stained smears encountered in 133

quality assurance program 2 were prepared during routine operations of the satellite laboratory. 134

As such, there may have been greater opportunity for microbiologists to review overdecolorized 135

Gram positive cocci and degenerated leukocytes--cell types that may have been incorrectly 136

reported by satellite technologists. 137

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138

A key component of any continuous quality assurance monitor is ad libitum intervention [9]. 139

Throughout this study, four major means of correction were implemented. First, Gram-stained 140

smears from either quality assurance program 1 or 2 that yielded an egregiously-incorrect 141

response from a satellite laboratorian were returned to a designated point person from that 142

laboratory. This individual personally reviewed the slide with the technologist and discussed any 143

discrepancies. Secondly, one-on-one interactions at a dual-headed microscope took place 144

between a central laboratory microbiologist and the satellite laboratorian. Third, personnel from 145

the satellite laboratories and the central laboratory formed a systemwide microbiology 146

subcommittee which provided a conduit for exchange of data, policy, and concerns. Finally, 147

digital images of interesting findings from quality assurance program 2 were disseminated via 148

inter-laboratory electronic mail in the format of a case presentation. Comprising this document 149

were chart review, organism epidemiology, and discussion of the relevance of the Gram-stained 150

smear in the specific case. These mailings also provide a forum for discussion of aberrant 151

findings in Gram-stained preparations, such as organisms partially treated with antimicrobial 152

agents [4,12]. 153

154

Analysis of variance data for four quarters of quality assurance program 1 (Fig. 3) revealed an 155

increase in Gram stain proficiency. Analogous improvement was seen via quality assurance 156

program 2. However, neither hospital A nor hospital B demonstrated any improvement in 157

quality assurance program 1 or 2 throughout the course of this monitor. It is likely that these two 158

laboratories had the most experience with satellite microbiology processing. In further support 159

of this statement, hospital A and B possessed the two highest proficiency rates (Fig. 1). In 160

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contrast, hospital C showed significant improvement in total Gram stain proficiency (Fig. 4) and 161

interpretation of bacteria in both quality assurance programs. 162

163

In conclusion, we describe a quantitative measure of Gram stain proficiency that can be applied 164

to challenges of satellite laboratorians and an off-site review of technologist performance. These 165

monitors can assess proficiency to the level of the laboratory, technologist, cell type, and 166

quantitative measure. Technologist challenge is best utilized as an educational tool. However, 167

off-site review is likely a better indicator of overall day-to-day Gram stain proficiency. When 168

combined, these measures enable a multi-site microbiology service to generate valid data both 169

for patient care and for practices of the clinical microbiology laboratory. 170

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Figure 1. Gram stain proficiency of three satellite laboratories, determined by quality

assurance program 1 and delineated by interpretation of bacteria (solid bars), host

cells (open bars), and sum of the two categories (shaded bars).

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Figure 2. Comparison of quality assurance program 1 and 2 for measure of overall Gram

stain proficiency.

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Figure 3. Non-microbiology laboratorian Gram stain proficiency, determined by quality

assurance program 1 (boxes) and 2 (diamonds), for four quarters of 2006.

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Figure 4. Gram stain proficiency of hospital C non-microbiology laboratorians, measured at

the onset and later stages of quality assurance program 1 (A) and 2 (B).

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