«EVALUATION OF ORAL NEUTROPHIL LEVELS AS A QUANTITATIVE MEASURE OF PERIODONTAL INFLAMMATORY LOAD IN PATIENTS WITH SPECIAL NEEDS By Anita Moosani BSc, ...»
As well, it is not clear that the currently available tests are accurate enough to provide reliable information regarding disease activity and progression, and so the potential benefit to patients is not clear (Kaufman & Lamster, 2000). Hence, the diagnostic value of these tests has not been sufficient enough to be incorporated widely into the dental practice setting, nor are they easy enough to be used as a routine chair-side test. Moreover, it is also important to point out that performing tests that rely on assessment of GCF are simply not possible on uncooperative patients with special needs.
Evaluation of Oral Neutrophils in Periodontal Inflammation The orogranulocytic migratory rate (OMR) index was developed by Klinkhamer (1968), as an objective method for quantifying one aspect of the inflammatory process in the periodontium. This test is based predominantly on the rate of leukocyte migration from the gingival sulcus to the oral cavity. It was proposed that the increase in vascularity and cell migration, as in cases of periodontal inflammation, could be measured by the OMR index.
When an inflammatory reaction occurs in periodontal tissues, the rate of cell migration is increased, leading to increased formation of cellular infiltrate and increased granulocyte migration (Klinkhamer & Zimmerman, 1969). Woolweaver, Koch, Crawford, and Lundblad (1972) demonstrated that the predominant leukocyte in oral saline rinses was the PMN, which appeared to retain its vitality and function. The OMR was also shown to increase in relation to increased gingival inflammation, and also correlated with increased pocket depths (Klinkhamer & Zimmerman, 1969). Hence, the OMR index was proposed as a reliable indicator of the severity of periodontal disease, and most importantly, it did not require „clinical judgment‟ for interpretation (Klinkhamer, 1968). However, the OMR index was determined by counting the number of oral granulocytes present in a series of twelve sequential oral mouthwashes, lasting 30 seconds each, making it difficult to apply to the clinical setting. Again, this is rather time-consuming and difficult for cooperative patients and when it comes to uncooperative special needs patients, it is virtually impossible to carry out reliably and accurately.
Oral Neutrophil Quantification to Assess Periodontal Disease Status Recognition that the quantity of crevicular leukocytes could be used as a potential indicator for gingival inflammation was described in 1970 (Attstrom, 1970). It was first proposed in 1978 to quantify oral PMN levels in order to assess the status of periodontal disease as well as responses to treatment in patients (Raeste & Aura, 1978). As also discussed above, it has been shown repeatedly that the quantity of oral PMNs varies directly with the extent of gingival inflammation (Attstrom, 1970; Schiött & Löe, 1970). In addition, oral PMN counts correlate positively with increasing pocket depths and the gingival index (Woolweaver, Koch, Crawford, & Lundblad, 1972). In developing a simple method for oral neutrophil quantification, a non-invasive oral rinse assay modified from Wright, Meierovics, & Foxley (1986), was validated at the University of Toronto‟s Department of Periodontology in 2006.
Using this approach, patients with chronic periodontal disease provided oral rinses before and after phase I periodontal treatment. Samples were then processed in the lab and PMNs were counted, and it was found that following initial treatment, there were clear reductions in the levels of oral PMNs. Given this finding, it was anticipated that such an assay could be used to monitor disease activity, severity, and progression (Bender, Thang, & Glogauer, 2006).
The oral rinse assay has also been used successfully in the medical field to monitor PMN delivery in extracellular tissues (Wright, Meierovics, & Foxley, 1986; Akpek, Knight, & Wright, 2003; Cheretakis, Dror, & Glogauer, 2005). Wright, Meierovics, and Foxley (1986) have shown that acute neutropenia following chemotherapy translated to reduced oral PMN levels. Investigators have also demonstrated a direct correlation between oral and blood PMNs in adults undergoing myelosuppressive chemotherapy (Akpek, Knight, & Wright, 2003). Cheretakis, Dror, and Glogauer (2005) observed PMN recovery in myeloid engraftment haematopoietic stem cell transplantation. It is important to note that the changes in oral PMN levels in both studies occurred prior to detection of changes in blood PMNs (Wright, Meierovics, & Foxley, 1986; Akpek, Knight, & Wright, 2003). Thus, it has been suggested that the presence of PMNs in tissues could be a better indicator of susceptibility to infection than circulating white blood cell counts, and might actually predict marrow engraftment better than tests of peripheral levels of PMNs (Wright, Meierovics, & Foxley, 1986).
Limitations in Diagnosis with the Oral Rinse Assay Advantages of the oral rinse assay include large volumes available for analyses and ease of sample collection. However, the oral rinse assay cannot be applied to the clinical setting yet, due to the reliance on lab-based techniques (i.e. PMN counting using the microscope). In addition, salivary constituents arise from many sources, including serum and host factors in GCF such as epithelial cells, inflammatory cells, cell mediators, subgingival and supragingival bacteria, oral epithelial cells that have sloughed, and foreign substances such as food and oral hygiene products (Lamster & Grbic, 1995). Moreover, cells that are collected in the salivary samples are non-specific, and include cells from the entire oral cavity, which could be influenced by the presence of other infective or inflammatory lesions involving the oral mucosa. The oral rinse also dilutes the cells that are acquired from the main area of interest – the gingival sulcus. Most importantly for the purposes of this investigation, the use of the oral rinse assay is impractical if not outright impossible in patients who lack the cooperation and/or coordination required to rinse and expectorate reliably.
Relation of Myeloperoxidase to Periodontal Disease Myeloperoxidases (MPO) are neutrophil specific enzymes that are involved in PMN bacterial killing mechanisms via the oxidation of chlorine ion as described earlier. MPO is a heme containing protein and the unique ability of this enzyme to oxidize chlorine is recognized to be vital for the host defense system by killing invading pathogens (Gaut et al., 2001). Thus, MPOs act as a specific marker of primary PMN granules released in response to offending stimuli (Gessler, Pfenninger, Pfammatter, Carrel, & Dahinden, 2002). MPO measurement has shown to be a good model for estimating PMN quantities in inflamed tissues (Bradley, Priebat, Christensen, & Rothstein, 1982). MPO has been found at higher levels in patients with periodontal disease (Smith, Hinrichs, & Melnyk, 1986), and at varying concentrations that correlates with the clinical severity of periodontitis (Wolff et al., 1988). Studies have also shown an increased level of activity and quantity of MPOs at the sulcus as a result of inflammation at periodontitis sites (Cao & Smith, 1989; Gomes et al., 2009). Decreased MPO activity has also been demonstrated following periodontal treatment (Smith, Hinrichs, & Melnyk, 1986; Wolff et al., 1988). MPO measurement offers a simple and less technical procedure for estimating PMN presence in the gingival crevice compared to PMN counting (Cao & Smith, 1989). Since the quantity of PMNs can be taken as a measure of the intensity of the disease process, MPOs can be used as a marker of PMN content, and would prove useful in both the diagnosis and evaluation of treatment responses geared towards resolving inflammatory conditions (Bradley, Priebat, Christensen, & Rothstein, 1982). Animal models have revealed PMN MPO as a good indicator of systemic rather than local inflammation in a chronic inflammatory condition (Queiroz-Junior et al., 2009). Furthermore, measurement of MPO has already been applied in the medical field successfully to monitor PMN activity after cardiopulmonary bypass in paediatric cardiac patients (Gessler, Pfenninger, Pfammatter, Carrel, & Dahinden, 2002). Though MPOs are not a specific marker of periodontitis, it is postulated that increased levels of MPOs from the GCF may reflect the number of PMNs present in the gingival sulcus, and further may reveal the extent of gingival inflammation (Cao & Smith, 1989).
Oral Neutrophil Quantification – A Novel Approach Recognition of the aforementioned limitations has led to the development of a novel technique that quantifies PMNs, using an oral swab or Q-tip™, which is traced along the buccal surfaces of the upper and lower dental arches, along the tooth crown and gingival interface. GCF is absorbed onto the swab head and the swab is then dipped in sterile saline to make a solution containing the lysed PMNs and their contents, including MPO. This technique uses a colourimetric reaction to measure oral PMNs present on the swab via MPO activity. Hydrogen peroxide is added to each PMN-containing solution in order to activate a chromogenic reagent called 2, 2‟ – azinobis (3-ethylbenzothiazoline-6-sulphonic acid), also known as ABTS. In turn, ABTS contains diammonium salts that react with MPOs present in PMN granules and this reaction leads to a visual colour change (blue). The intensity or optical density of the blue stain (i.e. the darkness of the blue color), correlates with increased numbers of PMNs in the sample (Landzberg, 2009). Previous tests have confirmed that the colourimetric reaction does not occur with a control or sterile swab alone (M. Glogauer, personal communication, July 7, 2010). The colourimetric reaction is measured by comparing the resulting blue sample colour change by eye, mechanically to a standard curve produced using blood neutrophils, or using ultraviolet light to measure the sample‟s absorbance (with the FLUOstar Optima microplate reader). The PMN assay using the oral swab technique has been validated for accuracy and reproducibility, and correlates well to the PMN counts derived from the oral rinse technique (M. Glogauer, personal communication, July 7, 2010). Thus, oral PMNs in the sample can be quantified to provide an objective measure of gingival and periodontal inflammation, thereby providing valuable diagnostic information about gingival health status, and may identify early signs of inflammation that may lead to future tissue breakdown. The PMN assay may also provide a standardized method for clinical measurement of periodontal inflammation in patients who cannot be examined using routine methods; that is, the uncooperative special needs patient population.
This would help to overcome the problem of subjectivity met with conventional measures of periodontal disease in all populations, and particularly in the special needs population, and would be extremely helpful insofar as assessment of treatment outcomes are concerned.
RESEARCH OBJECTIVES To assess the baseline gingival health status of uncooperative patients with special needs attending Toronto‟s Mount Sinai Hospital Dental Program for Persons with Disabilities using the PMN assay and conventional methods To validate the PMN assay by correlating levels of gingival inflammation as assessed by PMN counts collected from the oral swab technique with conventional clinical parameters in patients with special needs receiving comprehensive dental care under GA, and at subsequent recall examination To monitor responses to periodontal treatment by comparing gingival inflammation as assessed by the PMN assay and conventional methods pre-treatment under GA and at the subsequent follow-up examination To assess the usefulness and feasibility of the PMN assay as a quantitative measure of periodontal inflammatory disease in the uncooperative special needs patient
1. All of the uncooperative patients with special needs who are assessed in this cohort will demonstrate a high prevalence of gingival inflammation, regardless of what diagnostic methods are used.
2. Oral PMN levels derived from oral swabs will correlate with traditional measures of gingival inflammation.
3. The PMN assay will allow for monitoring of gingival health status and responses to treatment when comparing pre- and post- treatment evaluations.
4. It will be feasible to use the PMN assay using the oral swab technique to assess gingival inflammation in the uncooperative special needs population.
Ethics Review Prior to study commencement, the research protocol for this project was approved by the Mount Sinai Hospital Research Ethics Board and the Office of Research Ethics at the University of Toronto, Toronto, Ontario, Canada.
Sample Size Calculation Sample size calculations were performed prior to study commencement using sample size calculation software (Faul, Erdfelder, Lang, & Buchner, 2007). The required sample size to detect a correlation of at least 0.4 (ρsmallest) or greater between changes in probing depths and neutrophil counts at an α = 0.05 significance level, with 80% power (1-β = 0.80), using a two-sided test (Ho: ρ = 0 versus H1: ρ ≠ 0), was calculated as 44 patients.
Funding Funding for this research project was provided by the Department of Dentistry at Mount Sinai Hospital, Toronto, Ontario, Canada.
Conflict of Interest The investigators of this study have no conflicts of interest. The dental care and treatment of the patients involved was not affected in any way, regardless of their participation. The investigators did not receive any sponsorship.
Examiner Training and Calibration A multi-examiner approach was inherent to this study due to the operating room and staffing schedules at the Mount Sinai Hospital Dental Clinic. The evaluators in this study consisted of Mount Sinai Hospital General Dental Residents and University of Toronto Paediatric Dental Graduate Residents. The oral swab and periodontal examination data that was collected in the operating room and on the follow-up examination was performed by residents who were trained according to the definitions and format specific to this study, to ensure standardization.