Heterotopic ossification in patients with traumatic medullar injury: association with HLA-system antigens

Ossificação heterotópica em pacientes com lesão medular traumática: associação com antígenos do sistema HLA

ORIGINAL ARTICLE

Heterotopic ossification in patients with traumatic medullar injury: association with HLA-system antigens

Ossificação heterotópica em pacientes com lesão medular traumática: associação com antígenos do sistema HLA

Anita Weigand de Castro^I; Júlia Maria D'Andréa Greve^II

^IPhysiatrist

^IIAssociate Professor

^{Correspondence}

SUMMARY

The purpose of this study was to identify the prevalence of the human leukocyte antigen (HLA) class I and II on the spinal cord injury (SCI) patients and its relation with heterotopic ossification (HO). Fifty-four patients were studied (47 men and 7 women) with an average age of 33,5 ± 12,5 years (range 18 to 59 years) with SCI. Forty-four patients (81,5%) had complete and 10 had incomplete lesions. Twenty-three patients (42,6%) were tetraplegics, 31(57,4%) were paraplegics, 28 had thoracic injury and 3 had lumbar lesion. The tests used in the diagnosis of HO were bone scintigraphy, anteroposterior radiography of hip and knee in the paraplegics and also the shoulders in the tetraplegics. Computerized tomography was used when the others tests were unconcluded. The patients were divided in two groups: 28 patients with HO (52%) and 26 without HO (48%). The typing of HLA antigens class I and II was done in all patients. The results of the distribution of frequency of HLA antigens were assessed on both groups and it was not shown any difference with statistic significance. It was conclude that there was no association between HLA antigens class I and II with the OH development.

Key words: Spinal cord injuries; Ossification; Heterotopic; HLA antigens; Quadriplegia; Paraplegia.

RESUMO

O objetivo deste trabalho foi identificar a prevalência dos antígenos leucocitários humanos (HLA) de classe I e II nos pacientes com lesão medular traumática e sua relação com a ossificação heterotópica (OH). Foram estudados 54 pacientes (47 homens e 7 mulheres), idade de 33,5 ± 12,5 anos (18 a 59 anos), com lesão medular traumática. Quarenta e quatro pacientes (81,5%) apresentavam lesão medular completa e 10 pacientes tinham lesão incompleta; 23 pacientes (42,6%) eram tetraplégicos e 31 (57,4%) eram paraplégicos, sendo 28 com lesão torácica e 3 com lesão lombar. Para o diagnóstico de OH, os pacientes realizaram exames de imagem: cintilografia óssea, radiografias simples dos quadris e joelhos nos paraplégicos e também dos ombros nos tetraplégicos e tomografia computadorizada, quando os outros exames foram inconclusivos. Os pacientes foram divididos em dois grupos: 28 pacientes com OH (52%) e 26 sem OH (48%). Foi feita a tipificação dos antígenos HLA das classes I e II de todos os o pacientes. Os resultados da distribuição da freqüência dos antígenos HLA nos dois grupos foram avaliados e não mostraram diferença com significância estatística. Concluiu-se que não existe associação entre os antígenos HLA classes I e II e o desenvolvimento da ossificação heterotópica.

Descritores: Traumatismos da medula espinhal; Ossificação heterotópica; Antígenos HLA; Quadriplegia; Paraplegia.

INTRODUCTION

Heterotopic ossification (HO) is a metaplastic biological process with bone neoformation in soft tissues adjacent to large articulations where normally the bone tissue is not found. HO is frequent not only in patients with spinal cord injury, but also in patients that suffered cranioencephalic trauma (CET), or had severe burns or were submitted to surgical interventions such as total hip arthroplasty (THA), for instance.

HO is one of the most feared complications of spinal cord injury, since its course may be asymptomatic and surprise both the patient and the healthcare team.

In patients with medullar injury its incidence ranges from 13 to 81%, depending on the diagnostic method employed^(2,14). Only 10 to 20% of these patients present clinical manifestations. The evolution to ankylosis occurs in 5 to 10% of the cases, making it difficult for patients to change position and move by themselves, as well as to perform ordinary tasks, and interfering in the functional independence and professional activity⁽⁶⁾.

The ossification process may start early and be diagnosed already during the first weeks following the trauma ⁽¹⁰⁾. Usually it will occur between the first and fourth months (on the average) after the trauma, being more frequent during the second month after the injury, although it may occur up to one year after the event.

The first clinical manifestations are rigidity and limitation of the affected articulation, rise of local temperature, edema, hyperemia and, in some cases, systemic symptoms such as higher spasticity and mild fever. In HO, the most affected articulations following medullar injury are hip articulations, followed by knee and shoulder articulations and, rarely, elbow articulations^(2,6,18).

The etiology of HO is still unknown. Several theories advocate a traumatic, ischemic, metabolic and genetic origin. Studies show that when mesenchymal cells located in the bone marrow or adjacent to the periosteum and to the endosteum, they are activated by a specific stimulus and differentiate into osteoblasts. The osteoblasts then start producing and excreting the extra-cellular organic matrix, or osteoid, that is subsequently mineralized, forming hydroxy apatite crystals that characterize the bone tissue^(11,13).

HLA, the human leukocyte antigen, is the denomination in the human species of MHC (Major Histocompatibility Complex) and corresponds to a gene region located in the short arm of chromosome 6. The products from these genes are protein molecules occurring in the surfaces of various nucleate cells, including leukocytes. The chromosome segment corresponding to MHC is subdivided into regions that are responsible for Class I, II and III genes⁽¹⁾. Besides codifying Class I and II genes, MHC also codifies proteins of the complement system and TNF (tumoral necrosis factor), among others. Class I proteins are codified by genes A, B, C, E, F, G, H, J, K and L. Class I antigens are membrane glycoproteins occurring in all nucleate cells, HLA-A and HLA-B playing a major immune role, while HLA-C is less significant^(12,16). Class II antigens are also glycoproteins expressed on the surfaces of macrophages, monocytes, lymphocytes, B-cells, activated T-cells and some gamma-interferon- stimulated cells. Class II antigen products are involved in graft rejection and codified by three allelic series: DR, DQ and DP. The HLA-DP series is not serologically detected⁽¹⁶⁾.

Most studies involving the HLA system were performed 20 or 30 years ago, when only Class I HLA antigens were investigated, with controversial results. While some of these studies showed no statistically significant differences in the association of HLA-A and HLA-B antigens with HO in patients with medullar injury or cranio-encephalic trauma (CET)^(5,7,15,17), one of them has found higher prevalence of HLA-B18 antigens in patients with neurological injury and HO, although with no statistical significance⁽⁹⁾. Based on the association of HLA-B27 with seronegative arthropaties, one author found higher prevalence of this antigen in patients with medullar injury and HO, having reached the conclusion that it would influence the formation of bone tissue⁽⁸⁾.

Considering the advancements in the research of the HLA system, the purpose of this study was to identify the prevalence of Class I and II HLA antigens and their association with HO in patients with traumatic medullar injury.

CASES AND METHODS

This study included 54 patients [47 men (87%) and 7 women (13%)] with traumatic medullar injury, mean age of 33.5 ± 12.5 years (age group from 18 to 59 years). The prevailing cause of medullar injury was fall (38.9%), followed by traffic accidents (27.8%), wounds caused by guns (22.2%), diving (7.4%), and objects falling on the vertebral column (3.7%). Cervical injury levels were found in 23 patients; thoracic injury levels in 28 and lumbar injury levels in 3 patients; 42.6% of the patients were quadriplegic and 57.4% were paraplegic; 44 patients (81.5%) presented complete medullar injury and 10 (18.5%) had incomplete medullar injury.

All patients were submitted to diagnostic evaluation of HO and typing of antigens in the HLA system. For HO diagnosis the following examinations were performed (Figure 1):

1. Hip and knee standard X-rays in the paraplegic, plus shoulder X-ray in the quadriplegic, with patients in horizontal dorsal decubitus, neutral position.

2.Bone scintiscan using tecnecium 99m-labeled methylenediphosphonate (99mTc-MDP), with patients in horizontal dorsal decubitus, whole-body anterior and posterior projections. The images obtained in the 3^rd phase of the examination were used.

3. Computerized tomography performed in four patients where the results of other imaging examinations were inconclusive. During the examinations the focus was directed to the hip region.

The criterion used for HO diagnosis was to have at least one positive examination. Patients who did not have positive examinations for at least one year following the medullar injury were considered with negative diagnoses.

After the diagnostic evaluation, patients were divided into two groups – one including 28 patients with HO (52%) and one including 26 patients without HO (48%).

The typing of Class I HLA antigens of all patients was performed using the complement-mediated microly-mphotoxicity method, while Class II HLAs antigens were typed using the polymerase chain reaction-sequence specific primer (PCR-SSP) method.

To correlate the HLA antigen distribution between groups (with and without HO) the c² (chi-square) partition test was used. When at least one cell showed an expected frequency lower than 5, Fisher's exact test was used. The significance level for rejection of the null test was established as < 0.05 (a< 5%).

RESULTS

Data obtained for the frequency of Class I HLA antigens (20 HLA-A antigens and 29 HLA-B antigens) and Class II HLA antigens (14 HLA-DR antigens and 7 HLA-DQ antigens) as compared to the groups with HO and without HO, showed no statistically significant differences (Tables 1, 2, 3 and 4).

DISCUSSION

HO is a frequent complication of the spinal cord injury. Presenting discrete clinical manifestations in the early phase, it may evolve to severe ankylosis. Early diagnosis should be established, and treatment should start as early as possible. The incidence of HO among our patients was of 52%, which is similar to data found in the literature, where variations range from 13% to 81% ^(2,3,14). This large variation in incidence data may be attributed to the fact that in many studies the diagnosis or identification of HO was based only on the presence of a suggestive clinical picture, which is present in 10 to 20% of patients with HO. Most studies that showed higher incidences used more sensible diagnostic methods, such as bone scintiscan⁽⁴⁾, having found higher incidences of HO, as it was the case in our study; or, those studies investigated patients with complete injury only, having found HO in 81% of the cases⁽¹⁴⁾.

The rationale for the investigation of the frequency of Class I and II HLA antigens in patients with rachiomedullar trauma and its association with HO was that in previous investigations, when knowledge about the HLA system was still limited, Class II HLA antigens were not investigated.

For the typing of Class I HLA antigens the following antigens were analyzed: 20 HLA-A antigens and 29 HLA-B antigens; and, for Class II, 14 HLA-DR antigens plus 7 HLA-DQ antigens. A statistical analysis compared the individual frequency of each antigen in both groups (with and without HO), since the factor in common was traumatic medullar injury. HLA antigen frequency in the general population was not considered. No statistically significant differences were found between groups (with and without HO) as regards the presence of Class I HLA antigens, in agreement with the results found in literature^(7,15,17). HLA-B62 was more frequent in the group without HO (five patients) as compared with the group with HO (one patient), although with no statistical relevance.

Studies by Minaire et al⁽⁹⁾ found a statistically significant frequency of 25.6% for HLA-B18 in patients with HO. In our investigation we found a 7.1% frequency for HLA-B18 antigen in the group with HO and 7.7% in the group without HO.

Larson et al⁽⁸⁾ found a 24% frequency of HLA-B27 in patients with HO and 0% in patients without HO. In the present study HLA-B27 antigen was present in one patient of each group (with and without HO), with frequencies of 3.5% and 3.8%, respectively.

Garland et al⁽⁵⁾ found HLA-A2s in 60% of patients with medullar injuries and HO, although in the present study this frequency was of 39.2% in the HO group and 50% in the group without HO.

Also, the association of Class II antigens (HLA-DRs and HLA-DQs) with HO was not statistically relevant, and there are no similar studies in the literature for comparison.

Our conclusion is that individuals with traumatic medullary injury no association between HO and Class I and II antigens has occurred.

CONCLUSION

Despite of the fact that in this study we have not detected a possible genetic factor favoring the occurrence of HO or preventing it, one cannot discard the possibility that such factors are determinant for the formation of a heterotopic bone in the presence of spinal cord injury, thus leaving the field open to new investigations in the genetic area, as new genes of the HLA system and their products are identified, providing material for new studies on association with HO.

Work performed at Instituto de Ortopedia e Traumatologia of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - IOT HC - FMUSP

Trabalho recebido em 24/01/2003

Aprovado em 20/03/2003

• 1. Abbas AK, Lichtman AH, Pober JS. Complexo de histocompatibilidade principal. In: Imunologia celular e molecular. 3 ed. Rio de Janeiro: Revinter, 2000. p.97-115.
• 2. Bravo-Payno P, Esclarin A, Arzoz T, Arroyo O, Labarta AC. Incidence and risk factors in the appearance of heterotopic ossification in spinal cord injury. Paraplegia 30:740-745, 1992.
• 3. Colachis SC, Clinchot DM, Venesy D. Neurovascular complications of heterotopic ossification following spinal cord injury. Paraplegia 31:51-57, 1993.
• 4. Freed JH, Hahn H, Menter R, Dillon T. The use of the three-phase bone scan in the early diagnosis of heterotopic ossification (HO) and in the evaluation of didronel therapy. Paraplegia 20:208-216, 1982.
• 5. Garland DE, Alday B, Venos KG. Heterotopic ossification and HLA antigens. Arch Phys Med Rehabil 65:531-532, 1984.
• 6. Garland DE. A clinical perspective on common forms of acquired heterotopic ossification. Clin Orthop 263:13-29, 1991.
• 7. Hunter T, Dubo HIC, Hildahl CR, Smith NJ, Schroeder ML. Histocompatibility antigens in patients with spinal cord injury or cerebral damage complicated by heterotopic ossification. Rheumatol Rehabil 19:97-99, 1980.
• 8. Larson JM, Michalski JP, Collacott EA, Eltorai D, McCombs CC, Madorsky JB. Increased prevalence of HLA-B27 in patients with ectopic ossification following traumatic spinal cord injury. Rheumatol Rehabil 20:193-197, 1981.
• 9. Minaire P, Betuel H, Girard R, Pilonchery G. Neurological injuries, paraosteoarthropathies, and human leukocyte antigens. Arch Phys Med Rehabil 61:214-215, 1980.
• 10. Orzel JÁ, Rudd TG. Heterotopic bone formation: clinical, laboratory, and imaging correlation. J Nucl Med 26:125-132, 1985.
• 11. Otfinowski J. Heterotopic induction of osteogenesis in the course of neural injury. Patol Pol 44:133-168, 1993.
• 12. Panajotopoulos N, Guglielme L, Coelho V, Kalil J. Importância do sistema HLA em transplantes. In: Neumman J. ed. Transplantes de orgãos e tecidos. São Paulo: Sarvier, 1995. p.58-65.
• 13. Puzas JE, Miller MD, Rosier RN. Pathologic bone formation. Clin Orthop 245:269-281, 1989.
• 14. Renfree KJ, Banovac K, Hornicek FJ, Lebwohl NH, Villanueva PA, Nedd KJ. Evaluation of serum osteoblast mitogenic activity in spinal cord and head injury patients with acute heterotopic ossification. Spine 19:740-746, 1994.
• 15. Seignalet J, Moulin M, Pelissier J, Romain M, Bouffeard-Vercelli M, Lapinski H, Roquefeuil B. HLA and neurogenic paraosteoarthropathies. Tissue Antigens 21:268-269, 1983.
• 16. Weidebach WFS. Imunogenética da febre reumática: associação com os antígenos HLA-DR7 e DR53 definida pela técnica de microlinfocitotoxidade e pela análise do polimorfismo do comprimento dos fragmentos de restrição. [Dissertação], São Paulo: Instituto de Ciências Biomédicas da Universidade de São Paulo, 1992.
• 17. Weiss S, Grosswasser Z, Ohri A, Mizrachi Y, Orgad S, Efter T, Gazit E. Histocompatibility (HLA) antigens in heterotopic ossification associated with neurological injury. J Rheumatol 6:88-91, 1979.
• 18. Wittemberg RH, Perschke U, Bötel U. Heterotopic ossification after spinal cord injury. Epidemiology and risck factors. J Bone Joint Surg Br 24:215-218, 1992.

Publication Dates

• Publication in this collection
  03 June 2003
• Date of issue
  Apr 2003

History

• Accepted
  20 Mar 2003
• Received
  24 Jan 2003