Pulmonary complications are a major cause of morbidity

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1 Pulmonary Thrombotic Arteriopathy in Patients With Sickle Cell Disease Moses O. Adedeji, MD; Julio Cespedes, MD; Kay Allen, MD; Charu Subramony, MD; Michael D. Hughson, MD Background. Shortened life expectancy due to pulmonary hypertension (PH) is seen in 5% to 10% of patients with sickle cell disease. The principal factors suspected of causing PH are pulmonary thromboemboli (PE) and in situ arterial thrombosis. Objective. To investigate the possible role that PE or in situ arterial thrombosis play in the development of PH in sickle cell disease. Methods. Autopsies of 12 patients with sickle cell disease were correlated with clinical data from medical records. Results. Right ventricular hypertrophy was present in 9 of 12 patients. Six patients with right ventricular hypertrophy had in large elastic pulmonary arteries. All patients with elastic artery had fresh or in small muscular pulmonary arteries. Hypertensive small arterial changes were present in 5 of these 6 patients. Six patients showed no in elastic arteries. Among these 6 patients, 3 had right ventricular hypertrophy and recent and, as well as hypertensive changes in small arteries. One of these 3 patients demonstrated plexiform-like lesions and fibrinoid necrosis of small arteries. Three patients without right ventricular hypertrophy had pneumonia or pulmonary edema with no identifiable pulmonary artery pathology. Conclusions. Arterial thrombosis with PH and cor pulmonale was regarded as the cause of death among most of these patients. Elastic artery are pulmonary thromboemboli, but pulmonary thromboemboli are always associated with widespread thrombosis of small arteries. Widespread thrombosis of small arteries alone was associated with PH in some cases. This finding suggests that pulmonary thromboemboli may be a late complication of PH and cor pulmonale and that an in situ thrombotic arteriopathy underlies the development of PH in most patients with sickle cell disease. (Arch Pathol Lab Med. 2001;125: ) Pulmonary complications are a major cause of morbidity and mortality in patients with sickle cell disease (SCD). Acute pulmonary complications consist of the acute chest syndrome that presents a differential diagnosis between pneumonia, pulmonary infarction, fat embolism from infarcted bone marrow, and parvovirus infection. 1 5 Chronic pulmonary complications are thought to be the result of repeated episodes of the acute chest syndrome and include pulmonary, pulmonary hypertension (PH), and cor pulmonale. 6 8 Most deaths among SCD patients with chronic lung disease are the result of PH and cor pulmonale. 6,9,10 The pathogenetic mechanisms underlying the PH in SCD are poorly understood. 6,9,10 It has been suggested that PH is the result of arterial occlusion secondary to pulmonary thromboemboli (PE) or in situ thrombosis, but it has been noted that the pathologic findings in SCD are often indistinguishable from those seen in primary PH Other possibilities include increased intravascular shear forces, as a result of the adherence of sickled erythrocytes or injury by Accepted for publication June 8, From the Department of Pathology, University of Mississippi Medical Center, Jackson. Presented at the annual meeting of the United States and Canadian Academy of Pathologists, Atlanta, Ga, March 5, Reprints: Michael D. Hughson, MD, Department of Pathology, University of Mississippi Medical Center, 2500 N State St, Jackson, MS ( mhughson@pathology.umsmed.edu). free fatty acids released from infarcted bone marrow. Such factors may produce vasospasm and elevated pulmonary blood pressures with the subsequent development of hypertensive vascular changes. 10 The present study investigates the pulmonary pathology of patients dying with SCD. This study was undertaken to determine whether a pathologic analysis of the pulmonary arteries may discriminate between these pathophysiologic considerations and contribute to understanding the pathogenesis of PH in SCD. MATERIALS AND METHODS Autopsies were performed on 12 patients with SCD between 1991 and 2001 at the Department of Pathology, University of Mississippi Medical Center, Jackson. The clinical records of these patients were reviewed for the following information: age, sex, hemoglobin electrophoresis, and clinical complications of SCD, including stroke, acute chest syndrome, and bone pain crisis. Lungs were examined for pneumonia,, and infarcts and for,, and intraluminal fibrous webs in large pulmonary arteries. Hearts were examined for right ventricular hypertrophy. Right ventricular hypertrophy was diagnosed on the basis of gross autopsy findings when the right ventricle was more than 0.4 cm thick and there was notable thickening of the right ventricular trabecular muscles. Six to 10 blocks of tissue from each lung were cut in a series of 4- m sections and stained with hematoxylin-eosin, Masson trichrome, and Verhoeff van Gieson stains. The pulmonary arteries were divided into elastic ( 1000 m) and small muscular arteries (100 to 1000 m) on the basis of cross-sectional diameter. 12 The elastic arteries were examined for recent and and for Arch Pathol Lab Med Vol 125, November 2001 Sickle Cell Pulmonary Arteriopathy Adedeji et al

2 Figure 1. Patient 6. A recent pulmonary embolus is shown in a large pulmonary artery in a 46-year-old woman with homozygous hemoglobin SS (HbSS) disease (hematoxylin-eosin, original magnification 40). Figure 2. Patient 6. This small pulmonary artery reveals multiple recanalized lumina and the fibrointimal proliferation of a thrombotic arteriopathy (hematoxylin-eosin, original magnification 100). Figure 3. Patient 6. This small pulmonary artery shows grade III (Heath and Edwards) pulmonary hypertensive changes consisting of concentric intimal (hematoxylin-eosin, original magnification 200). Figure 4. Patient 4. A, This gross photograph of lung shows old infarcts consisting of wedge-shaped areas of subpleural (large arrows) together with a fibrous web in an elastic pulmonary artery (small arrow) of a 15-year-old with HbSS disease. B, The histologic section of the fibrous arterial web shows thick connective tissue bands crossing the lumen of the artery (hematoxylin-eosin, original magnification 40). Figure 5. Patient 7. A small artery contains an organizing thrombus and recanalized lumina. Only small artery were found in this 22-year-old patient (hematoxylin-eosin, original magnification 200). The muscular arteries were evaluated for the changes of PH, as described by Heath and Edwards, 13 and for a thrombotic arteriopathy as defined by Katzenstein. 12 Fat emboli were identified as intravascular marrow fragments containing fat cells causing luminal occlusion or the distension of arterioles and alveolar capillaries by large clear fat vacuoles. Immunohistochemical stains were performed to evaluate the cellular components of fresh and organizing small artery. The antibodies employed were the endothelial markers factor VIII-related antigen (Signet Laboratories, Dedham, Mass) and CD34 (BioGenex, San Ramon, Calif); smooth muscle specific actin (Dako Corporation, Carpinteria, Calif); and inflammatory cell markers myeloperoxidase (Dako), leukocyte common antigen (Ventana Medical Systems Inc, Tucson, Ariz), and CD68 (Dako) for macrophages. Immunostaining was performed using the Ventana 320 automated immunostainer using biotinylated secondary antibodies followed by avidin-peroxidase. Color was developed using diaminobenzidine as substrate. Negative controls consisted of the SCD lung sections with the primary antibody omitted. RESULTS The clinical and pathologic findings of this study are summarized in the Table. The ages of the patients ranged from 8 to 47 years (mean 27 years). There were 7 males and 5 females. Nine patients had homozygous hemoglobin SS (HbSS) disease and 3 had sickle cell and hemoglobin C (HbSC) disease. Eleven patients had episodes of pain crisis before death, and 10 died at the time of a pain crisis. A clinical diagnosis of PH was made in 1 patient (patient 1). One patient (patient 7) was diagnosed as having PE after hip replacement surgery several months prior to death, but evidence for deep leg vein thrombosis was not Arch Pathol Lab Med Vol 125, November 2001 Sickle Cell Pulmonary Arteriopathy Adedeji et al 1437

3 Case Age, y/sex Hb Summary of Clinical and Pathologic Findings in 12 Sickle Cell Disease Patients* Clinical Diagnosis at Time of Death 1 8/M SS Pain crisis, CHF Other Clinical Diagnosis CVA, pulmonary hypertension 2 34/M SC Pain crisis Systemic hypertension 3 47/M SC Pain crisis, ACS Muscular Arteries RVT, cm Elastic Arteries Hypertensive Thrombotic With Large Artery Thrombi 0.8 Recent and, 0.6 Recent and, Schizophrenia 0.5 Organized, fibrous webs, 4 15/F SS Pain crisis None 0.4 Recent and, fibrous webs Organized, luminal webs Recent and Marrow Emboli No Many Organized No Organized No 5 21/M SS Pain crisis Cerebral palsy 0.6 Recent None Recent and 6 46/F SS Pain crisis, CVA, pneumoninized Recent and orga- ACS 7 22/M SS Pain crisis, ACS, CHF Pulmonary embolism 0.5 Recent and, Without Large Artery Thrombi 0.6 Atherosclerositimal Concentric in- 8 21/F SS Pain crisis None 0.5 No change Recent and Recent and No Rare Rare No Other Lung Findings Infarct,, edema, extensive sickling Edema, infarct Fibrosis, pneumonia Edema, extensive sickling, Infarct, edema Infarct,, edema, pneumonia Edema Edema, extensive sickling, alveolar wall necrosis, pneumonia Edema, extensive sickling, alveolar wall necrosis 9 23/M SS Pain crisis, None 0.6 Atherosclerositimal Concentric in- Recent and orga- Rare ACS, CHF fibronized sis, plexiform-like lesions, fibrinoid necrosis 10 26/F SS Pain crisis None 0.3 No change Focal intimal None No Extensive sickling 11 45/M SC Septicemia, Pneumonia, 0.3 No change None None No Pneumonia DIC CVA 12 18/F SS Pain crisis CVA 0.3 No change None None No Edema * HbSS indicates homozygous hemoglobin SS; HbSC, sickle cell and hemoglobin C; CHF, congestive heart failure; ACS, acute chest syndrome; DIC, disseminated intravascular coagulation; CVA, ischemic stroke; and RVT, right ventricular thickness. investigated clinically. Four patients were diagnosed clinically as having an acute chest syndrome. Six patients had recent or in the proximal elastic pulmonary arteries (Figure 1). Only 2 patients in this group had an acute chest syndrome, and none were clinically diagnosed as having PE. Deep leg vein thrombosis was found at autopsy in the 1 patient (patient 6) in whom it was investigated. In all patients with elastic artery, widespread recent and or alone were found in small muscular arteries (Figure 2). was present in the small arteries of 5 of these patients (Figure 3). Pulmonary infarcts were found in 4 patients with large artery. Wedge-shaped areas of subpleural pulmonary from scarred infarcts were observed in 4 patients, all of whom had large artery (Figure 4). Of the 6 patients who did not have in elastic pulmonary arteries, 3 had widespread recent and in small muscular arteries (Figure 5). These 3 patients also had concentric intimal hyperplasia of the small arteries. Plexiform-like lesions and fibrinoid necrosis were found at branch points of a few small arteries in 1 of these 3 cases (case 9) (Figure 6). In this clinical setting, the plexiform-like lesions could not be clearly discriminated from recanalized, 14 therefore they were not regarded as being identical to the plexiform lesions of primary PH. Two of the patients with small artery, including the patient with plexiform-like lesions, showed extensive intravascular sickling and alveolar wall necrosis 1438 Arch Pathol Lab Med Vol 125, November 2001 Sickle Cell Pulmonary Arteriopathy Adedeji et al

4 (Figure 7). This alveolar wall necrosis was different from pulmonary infarcts in that it was patchy and multicentric rather than wedge-shaped and subpleural. Patient 7, who was diagnosed clinically as having postoperative PE, did not have large artery at autopsy. In cases with in muscular pulmonary arteries, including those with large artery, sickled erythrocytes were frequently enmeshed in and adherent to the small arterial. Three of the patients who did not have large artery also did not have hypertensive changes or in muscular arteries. In one of these patients, there was extensive pulmonary intravascular sickling that was associated clinically with a pain crisis, but there was no necrosis of alveolar walls, no fat emboli, and no discernible pathology other than a marked collection of sickled erythrocytes in the lung vasculature. Right ventricular hypertrophy was seen in 9 patients (Figure 8), including the 6 patients with large artery and the 3 patients with only small artery. The patients who did not have any pulmonary arterial did not have right ventricular hypertrophy. Fat emboli were found in a few small muscular arteries or arterioles in 4 of the 12 patients. In patient 2, who had large artery, fat emboli containing the cellular elements of bone marrow were widespread and were found in small arteries and arterioles in which there were fresh (Figure 9). Fresh associated with erythrocyte sickling or fat emboli showed large numbers of myeloperoxidase-, CD45-, and CD68-positive inflammatory cells enmeshed in and surrounding the developing thrombus (Figure 10). Organized showed recanalized vessels lined by factor VIII-related antigen and CD34- positive cells, and proliferative fibrointimal tissue containing smooth muscle actin positive cells. CD45-positive cells were rarely present, and CD68-positive cells were not seen in. COMMENT The most common finding among these SCD cases was a thrombotic arteriopathy of small pulmonary arteries. Such arteriopathy was found in 9 of the 12 autopsies and consisted of widespread recent or, eccentric intimal, multiple recanalized lumina, or intravascular webs. 12 All patients with this thrombotic arteriopathy had right ventricular hypertrophy, and the small arterial changes of PH were seen in 8 of the 9 cases. The within large elastic pulmonary arteries are most certain to be PE and are designated as such throughout this article. Deep leg vein thrombosis as a source of PE was found in the 1 autopsy where it was sought. All of the 6 patients with PE had a small artery thrombotic arteriopathy. In addition to a thrombotic arteriopathy, all patients with PE had chronic cor pulmonale and hypertensive changes of small pulmonary arteries. This included 1 patient in whom only fresh were found in elastic arteries. These findings indicate that PE are likely to be a late complication of PH and cor pulmonale, and that the predominant cause of PH in patients with SCD is widespread small arterial thrombosis. The small artery may be derived from recurrent small PE. Histologically, however, fresh in the small arteries commonly demonstrated sickled erythrocytes enmeshed within the fibrin of developing clots. Together with the absence of clinical histories suspicious of emboli, the findings suggest that rather than PE, the thrombotic arteriopathy is the result of in situ thrombosis promoted by erythrocyte sickling. 6,10,12 In 1 patient, who died in a pain crisis, fresh in small arteries contained fat emboli, providing evidence that in some cases the embolization of infarcted bone marrow causes the small artery thrombosis. Our findings support the hypothesis of Powars et al, 6 who emphasized the local rather than the embolic nature of the process and proposed that the PH of SCD is caused primarily by an obstructive arteriopathy resulting from in situ thrombosis. This point of view was also held by Collins and Orringer, 7 who performed bilateral venograms on 2 SCD patients. Although old and recent PE were found in 1 case, the patient had severe small artery disease, and the authors thought that PE developed because of advanced cor pulmonale due to microvascular occlusion. The pathogenetic mechanisms underlying the vascular occlusions are thought to be multifactorial and to be a long-term consequence of the sickling disorder. 11 The rigidity of the sickled erythrocytes and increased adherence of erythrocytes to each other and to endothelium have been considered principal factors leading to acute vasoocclusion. 11 The release into the lungs of free fatty acids from infarcted bone marrow is thought to be an additional cause of vascular damage. 11 Although 11 of our patients were in pain crisis prior to death, fat emboli associated with microvascular thrombosis extensive enough to have contributed to death were found in only 1 case. This would suggest that fat emboli are a relatively uncommon cause of the thrombotic arteriopathy. If, however, free fatty acids could initiate microvascular thrombosis in the absence of histologically demonstrable fat emboli, our method of study would make little contribution toward evaluating this pathophysiologic process. Normal blood flow may be altered by other factors. Coagulation is activated in the microvasculature of SCD patients, and the cytokine activation of platelets and endothelium promotes clotting, vasoconstriction, and inflammatory cell adherence. 15 By immunohistochemistry, we find large numbers of inflammatory cells in the fresh associated with both fat emboli and erythrocyte sickling, which supports the concept that inflammatory cell adherence is important in the development of these acute lesions. Only 1 of the patients in our study was clinically diagnosed as having PH, representing an underdiagnosis of PH in SCD patients that has been observed clinically. 6,10 This underscores the fact that PH is silent until late in its course and frequently requires pulmonary arterial catheterization to be adequately evaluated. Less invasive techniques may also be used. By echocardiography, Sutton et al 10 evaluated right ventricular dysfunction in 60 patients with SCD as a means of diagnosing PH and found evidence for PH in 12 patients (20%), although it was suspected in only 6 (10%). Chronic lung disease is reported in approximately 5% of patients with SCD. 6,7 It tends to occur in patients at an average age of 25 to 33 years and consists of perfusion and diffusion defects, indicating that pulmonary as well as vascular occlusion contribute to the disorder. 6,7 The range and average age of the patients in our study were nearly identical to data reported in clinical series of sickle cell lung disease. We found that significant pulmo- Arch Pathol Lab Med Vol 125, November 2001 Sickle Cell Pulmonary Arteriopathy Adedeji et al 1439

5 Figure 6. Patient 9. A, This small muscular artery demonstrates concentric intimal hyperplasia and the early development of multiple vascular channels resembling a plexiform lesion at a branch point (hematoxylin-eosin, original magnification 200). B, Evidence of severe pulmonary hypertension is found in this 23-year-old patient with HbSS disease. Fibrinoid necrosis is seen at the branch point of a small artery (hematoxylineosin, original magnification 200) Arch Pathol Lab Med Vol 125, November 2001 Sickle Cell Pulmonary Arteriopathy Adedeji et al

6 nary occurred only in patients with PE and that most of the areas of were old infarcts. While our study supports in situ small artery thrombosis as underlying the development of PH in SCD, it also shows that thromboemboli contribute substantially to the morbidity and mortality of chronic sickle cell lung disease. Autopsy studies, while valuable, usually capture this illness in its final stages and certainly do not unequivocally resolve the question of the role that thromboembolism may play in the initiation of PH. The definitive studies will probably require an investigation of PH earlier in its course. The evaluation of patients for deep vein thrombosis and pulmonary perfusion defects in the beginning stages of PH would help clarify whether PE or an in situ thrombotic arteriopathy is the cause of PH in SCD. Illustrations were prepared by John Coleman PhD, MD. References 1. Kirkpatrick MB, Bass JB. Pulmonary complications in adults with sickle cell disease. Pulm Perspect. 1989;6: Charache S, Scott JC, Charache P. Acute chest syndrome in adults with sickle cell anemia. Arch Intern Med. 1979;139: Barrett-Conner E. Pneumonia and pulmonary infarction in sickle cell anemia. JAMA. 1973;244: Haynes J Jr, Allison RC. Pulmonary edema: complication in the management of sickle cell pain crisis. Am J Med. 1986;80: Johnson CS, Verdgem TD. Pulmonary complications of SCD. Semin Respir Med. 1988;9: Powars D, Weidman JA, Odom-Maryon T, Niland JC, Johnson C. Sickle cell chronic lung disease: prior morbidity and the risk of pulmonary failure. Medicine. 1988;67: Collins FS, Orringer EP. Pulmonary hypertension and cor pulmonale in sickle hemoglobinopathies. Am J Med. 1982;73: Francis RB. Large vessel occlusions in sickle cell disease: pathogenesis, clinical consequences and therapeutic implications. Med Hypotheses. 1991;35: Gokhale S, Adegboyega P, Veasey S, Haque AK. Pulmonary changes in sickle cell hemoglobinopathy: an autopsy study. Mod Pathol. 2000;13:5A. 10. Sutton LL, Castro O, Cross DJ, Spencer JE, Lewis JF. Pulmonary hypertension in sickle cell disease. Am J Cardiol. 1994;74: Weil JV, Castro O, Malik AB, Rodgers G, Bonds DR, Jacobs TP. Pathogenesis of lung disease in sickle hemoglobinopathies. Am Rev Respir Dis. 1993;148: Katzenstein AA. Pulmonary hypertension and other vascular disorders. In: Katzenstein and Askin s Surgical Pathology of Non-Neoplastic Lung Disease. 3rd ed. Philadelphia, Pa: WB Saunders Co; 1997: Major Problems in Pathology. Vol Heath D, Edwards JE. The pathology of hypertensive pulmonary vascular disease: a description of six grades of structural changes in the pulmonary arteries with special reference to congenital cardiac septal defects. Circulation. 1958;18: Moser KM, Bloor CM. Pulmonary vascular lesions occurring in patients with chronic major vessel thromboembolic pulmonary hypertension. Chest. 1993; 103: Francis RB, Johnson CS. Vascular occlusion in sickle cell disease: current concepts and unanswered questions. Blood. 1991;77: Figure 7. Patient 9. Severe erythrocyte sickling is present in the lung vasculature that is associated with alveolar wall necrosis (hematoxylin-eosin, original magnification 400). Figure 8. Patient 4. This cross-section of the heart of a 15-year-old boy shows hypertrophy of the right ventricle. Secondary endocardial sclerosis is found on the hypertrophied trabecular muscles of the right ventricle. Figure 9. Patient 2. This patient, who had both pulmonary emboli and a thrombotic arteriopathy of small arteries, died in a pain crisis. This photomicrograph shows one of the large number of fresh small artery containing fat emboli (hematoxylin-eosin, original magnification 200). Figure 10. Patient 8. A small artery shows large numbers of CD68-positive cells in a fresh thrombus. The immunohistochemical stains for myeloperoxidase and leukocyte common antigen showed the same clustering of inflammatory cells ( 200). Arch Pathol Lab Med Vol 125, November 2001 Sickle Cell Pulmonary Arteriopathy Adedeji et al 1441