Myeloperoxidase staining in the diagnosis of aggressive periodontitis.

AIMS: To evaluate neutrophil myeloperoxidase (MPO) staining procedure as a reliable, affordable and easily available diagnostic assay for aggressive periodontitis.
MATERIALS AND METHODS: Fifteen subjects were recruited in the study wherein five each were diagnosed as aggressive periodontitis and chronic periodontitis respectively, and five were periodontally healthy. Three millilitres (ml) of venous blood was collected using Vacutainers containing ethylene diamine tetra acetate (EDTA) and was subjected to MPO staining procedure. Histological picture was evaluated using a visual analogue scale (VAS).
RESULTS: MPO stained specimen of all the patients showed positive MPO staining of the neutrophils. The intensity of the stain of MPO granules was more in aggressive periodontitis specimen as compared to the chronic periodontitis patient specimen and healthy subject specimen. The staining characteristics were comparable for chronic periodontitis patients and healthy subject.
CONCLUSIONS: This study shows that there is a potential and probable place for MPO staining as an economical, relatively convenient and easily available assay in the diagnosis of aggressive periodontitis.

INTRODUCTION

Aggressive periodontitis was redefined as a complex disease exhibiting microbial alteration and cellular dysfunction that differentiate the underplying molecular mechanisms of its pathogenesis from chronic periodontal disease.[1]

Aggressive forms of periodontal disease have been defined based on the following primary features:[2]

Noncontributory medical history

Rapid attachment loss and bone destruction

Familial aggregation of cases

Secondary features that are considered to be generally, but not universally, present are:

Amounts of microbial deposits inconsistent with the severity of periodontal tissue destruction.

Elevated proportions of Actinobacillus actinomycetemcomitans and, in some populations, Porphyromonas gingivalis.

Phagocyte abnormalities.

Hyperresponsive macrophage phenotype, including elevated production of prostaglandins and interleukins in response to bacterial endotoxins.

Sub-classification of aggressive periodontitis includes:[2]

Localized aggressive.

Generalized aggressive.

The following three types were described by Mombelli et al.:[3]

Secure aggressive periodontitis, characterized by clinical loss of attachment of over 2 mm in under a year, or by a loss of over 2 mm before the age of 18 years.

Uncertain aggressive periodontitis, chara-cterized by clinical loss of attachment of over 2 mm, or by severe bone destruction before the age of 30 years.

Insecure aggressive periodontitis, characte-rized by attachment loss with an unclear rate of progression of around 2 mm in over a year.

Prevalence

On the basis of the epidemiological studies, aggressive periodontitis appears to occur in approximately one in 1000 adolescents, and seems to have a racial predisposition. Recent epidemiological studies using precise diagnostic criteria have reported an incidence of between 0.1 and 2.9%.[4-7]

Diagnosis

Clinical diagnosis

Microbiological diagnosis

Evaluation of host defences

Genetic diagnosis

Localized aggressive periodontitis has been associated with various abnormalities of host cell function. The polymorphonuclear leucocyte appears to play a pivotal role in aggressive periodontitis. Recent studies propose that the neutrophil abnormalities in localized aggressive periodontitis are the result of a chronic hyperactivated or primed state of the neutrophil.[8]

The neutrophil granules are of major importance for neutrophil function. When referring to phagocytes or leucocytes in general, the term granule is used more often than lysosome. The granules of neutrophils are generated during cell differentiation; they are produced for storage. On the basis of function and enzyme content, human neutrophil granules can be divided into three main types – azurophil, specific and small storage granules. Their function is not just to provide enzymes for hydrolytic substrate degradation – as in classical lysosomes – but also to kill ingested bacteria and, finally, to secrete their contents to regulate various physiological and pathological processes, including inflammation.[9]

Among azurophil granule constituents, myeloperoxidase (MPO) is a critical enzyme in the conversion of hydrogen peroxide to hypochlorous acid. Together with hydrogen peroxide and a halide cofactor, it forms the most effective microbicidal and cytotoxic mechanism of leucocytes – the MPO system.[9]

A study by Buchmann et al. has suggested that aggressive periodontitis patients present altered gingival crevicular fluid (GCF) MPO profiles and different MPO isoforms, reflecting the neutrophil stimulation in proportion to the neutrophil granule constituency.[10]

MPO is identified spectrophotometrically or by enzyme-linked immunosorbent assay (ELISA). These methods, although reliable and accurate, have a drawback of being expensive and of limited availability.

Objective of the study

The azurophil granules can be easily stained using a simple staining procedure. This study explored the neutrophil MPO staining profiles of patients with aggressive and chronic periodontitis in an attempt to find a reliable, affordable and easily available diagnostic assay for aggressive periodontitis.

The importance of neutrophil granules in inflammation is apparent from studies of several patients with congenital abnormalities of the granules. Patients with Chédiak-Higashi syndrome have a profound abnormality in the rate of establishment of an inflammatory response and have abnormally large lysosomal granules. MPO staining has been useful in demonstrating abnormal granules and in diagnosis.[9]

Rationale of the study

MPO staining can be a useful diagnostic tool in aggressive periodontitis. It has been proven that the MPO levels are elevated in GCF of patients with aggressive periodontitis. It has also been shown that the neutrophils from patients with aggressive periodontitis are hyperfunctional and hyperresponsive. This staining method has been utilized widely in patients with Chédiak-Higashi syndrome. For this reason, the MPO staining method has been extended to patients with aggressive periodontitis.

MATERIALS AND METHODS

Five patients with aggressive periodontitis, five patients with chronic periodontitis and five periodontally healthy subjects visiting the Department of Periodontics at J.S.S. Dental College and Hospital were recruited in the study. Informed consent was obtained from the subjects included in the study.

Inclusion criteria

Periodontally healthy patients with no clinical signs, symptoms and clinical findings of periodontal disease.

Patients diagnosed with chronic periodontitis and having probing pocket depth >5 mm with radiographic evidence of bone loss.

No history of systemic diseases.

No history of periodontal treatment.

No history of smoking.

No history of antibiotics/anti-inflammatories/other medications consumed in the past 6 months.

Method of investigation

Blood sampling

Three milliliters of venous blood was collected from the antecubital fossa using vacutainers (BD Vacutainer® , B.D. 1, Becton Drive, Franklin Lakes, NJ 07417, USA) containing ethylene diamine tetra acetate [Figure 1]. The samples were then appropriately transported to the Department of Pathology, J.S.S. Medical College and Hospital, for the staining procedure.

Figure 1

Collection of venous blood using vacutainer (containing ethylene diamine tetra acetate)

MPO staining procedure

This included the following steps:[11]

The smear was air-dried in 10 ml of 40% formalin and 90 ml of 95% ethanol for 30 s.

It was then washed under tap water for 30 s.

Two hundred and fifty milligrams (250 mg) of benzidine or ortho-toluidine in 6 ml of ethanol was dissolved and then diluted with 4 ml of distilled water.

Then, 0.02 ml of hydrogen peroxide was added. This solution was then completely poured on to the slide and allowed to remain for 7 min.

It was washed with tap water for 10 s and allowed to air dry.

This was counter-stained with Leishman's stain after 3 min.

RESULTS

For evaluation of the MPO staining characteristics, a visual analogue scale [Figure 2] was developed by the Department of General Pathology at J.S.S. Medical College and Hospital. The grading was as follows:

Figure 2

Visual analogue scale

Grade 0: No staining

Grade 1: Mild staining

Grade 2: Moderate staining

Grade 3: Intense staining

The MPO-stained specimen of all the patients showed positive MPO staining of the neutrophils.

The chronic periodontitis specimen and healthy specimen showed only grade 1 intensity [Figures 3 and 4].

Figure 3

Myeloperoxidase staining of the neutrophil in a chronic periodontitis patient

Figure 4

Myeloperoxidase staining of neutrophil in a periodontally healthy patient

The intensity of the stain of MPO granules was grade 2 or grade 3 in aggressive periodontitis specimen [Figure 5].

Figure 5

Myeloperoxidase staining of neutrophil in an aggressive periodontitis patient

The staining characteristics were comparable for chronic periodontitis patients and healthy subjects. The staining observed in the specimen of aggressive periodontitis patients indicated an increased intensity of MPO granule staining.

DISCUSSION

Periodontal inflammation may affect and worsen systemic conditions such as cardiovascular disease, preterm labour, diabetes mellitus and other clinical syndromes associated with leucocyte-mediated pathology. Several prominent neutrophil-specific molecules have been implicated as amplifiers of the inflammatory response.[1213] The MPO–hydrogen peroxide–chloride system of the neutrophil, as part of the innate host defence, possesses potent antimicrobial activity.[13] The liberation of the 150-kDa protein MPO, a potent oxidative enzyme from the azurophilic granules of neutrophils, in combination with reactive oxygen species, such as O2- , gives rise to hypochlorous acid (HOCl).[13]

The neutrophil granules are of major importance for neutrophil function. When referring to phagocytes or leucocytes in general, the term granule is used more often than lysosome. The terms are not fully equivalent; the term granules was originally derived from morphological observations whereas the term lysosomes is based on functional and biochemical characteristics of these cell organelles. Not all organelles that look like granules are necessarily typical lysosomes. The granules of neutrophils are generated during cell differentiation, which are produced for storage. On the basis of function and enzyme content, human neutrophil granules can be divided into three main types – azurophil, specific and small storage granules. Their function is not just to provide enzymes for hydrolytic substrate degradation – as in classical lysosomes – but also to kill ingested bacteria and, finally, to secrete their contents to regulate various physiological and pathological processes, including inflammation. Individual granule populations can be characterized morphologically (e.g., azurophil granules are larger and contain more electron-dense material than specific granules) or biochemically using enzyme markers or other substances. Neutrophil granules contain antimicrobial or cytotoxic substances, neutral proteinases, acid hydrolases and a pool of cytoplasmic membrane receptors. Among the azurophil granule constituents, MPO is a critical enzyme in the conversion of hydrogen peroxide to hypochlorous acid. The MPO system is formed together with hydrogen peroxide and a halide cofactor. It forms the most effective microbicidal and cytotoxic mechanism of leucocytes.[9]

The importance of neutrophil granules in inflammation is apparent from studies of several patients with congenital abnormalities of the granules. Patients with Chédiak-Higashi syndrome have a profound abnormality in the rate of establishment of an inflammatory response and have abnormally large lysosomal granules. The congenital syndrome of specific granule deficiency is an exceedingly rare disorder, characterized by diminished inflammatory responses and severe bacterial infections of the skin and deep tissues.[9]

Several studies have shown that the MPO levels are elevated in GCF in patients with aggressive periodontitis.[81012-14] Most studies use spectrophotometry to detect MPO levels. Azurophilic granules can be easily stained, and this test has been used extensively in Chédiak-Higashi syndrome. In this study, we have extended this staining procedure to aggressive periodontitis patients. The stained specimens show more intense staining of the neutrophil granules in aggressive periodontitis patients as compared with chronic periodontitis patients and periodontally healthy subjects. Further studies need to be undertaken to find out the difference between the staining profiles. This study shows that there is a potential and probable place for MPO staining as an economical, relatively convenient and easily available assay in the diagnosis of aggressive periodontitis.

CONCLUSION

MPO may be a reliable diagnostic tool in aggressive periodontitis. The sample size of this pilot study was small. The technique is simple and economical. Further studies are essential for validation. Improvizations on the VAS may be required in overcoming subjective discrepancies.