Clinicohematological Study of Thrombocytosis

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1 Original Article Clinicohematological Study of Thrombocytosis Dinesh Yadav, Jagdish Chandra, Sunita Sharma 1 and Varinder Singh Departments of Pediatrics and 1 Pathology, Lady Hardinge Medical College and Associated Kalawati Saran Children s Hospital, New Delhi, India ABSTRACT Objective. To find out etiology and clinical course of thrombocytosis in Indian pediatric population. Methods. A total of 250 patients having thrombocytosis (defined as platelet count >500 x 10 9 /L) on haematological investigations were studied over one yr period. All patients were evaluated clinically and were subjected to investigations, including complete blood counts (CBC) with peripheral smear examination. To elucidate the possible role of inflammatory cytokines in pathogenesis of RT, levels of Interleukin-6 (IL-6) and C - reactive protein (CRP) were estimated. Results. Infants and young children (<2 yr age) were most common group, contributing 60% of total cases. Out of total 250 cases, only 3 (1.2%) cases were found to have primary thrombocytosis and remaining 98.8% cases were having RT. Among RT patients, infections (alone or in association with iron deficiency anemia) were most common cause, accounting for 65% cases, while iron deficiency anemia (IDA) was second most common cause accounting for 41.3% cases (12.6% IDA alone and 28.7 % in association with infections). Other causes included nutritional dimorphic anemia and patients on treatment for megaloblastic anemia, acute lymphoblastic leukemia (during treatment) and lymphoma. Among various groups of RT, IL-6 and CRP levels were higher in patients with infection with or without IDA than IDA alone. One child with essential thrombocytosis and one child with RT had thrombotic complications. On follow up, platelet counts normalized in most of the patients with treatment of underlying conditions. Conclusions. Results of this study suggest that essential thrombocytosis is extremely rare in children. Infections and IDA (alone or in association with infections) are common causes of RT. IL-6 and CRP levels are increased in patients with RT, to a higher level in patients with infections than in patients with IDA. Most patients with RT have uneventful recovery of platelet counts to normal range with treatment for underlying condition. [Indian J Pediatr 2010; 77 (6) : ] jchandra55@gmail.com Key words: Thrombocytosis; Iron deficiency anemia; Thromboembolic complications; Interleukin-6 Thrombocytosis, increase in the number of circulating platelets, is usually discovered as an incidental abnormality when complete blood counts are obtained for some unrelated reason. When found, however, it poses diagnostic dilemma to differentiate between essential and reactive thrombocytosis. Widespread use of electronic cell counters has led to a higher incidence of thrombocytosis being observed. Incidence of thrombocytosis in children attending the clinics varies from 6% to 15%. 1 Thrombocytosis is classified according to its origin into primary (or essential) and secondary (or reactive) forms. 2 While essential thrombocytosis is very rare in childhood, reactive thrombocytosis (RT) occurs frequently. Most of the cases of RT are associated Correspondence and Reprint requests : Dr Jagdish Chandra, Lecturer s Flat No 5, Lady Hardinge Medical College, New Delhi , India. [DOI /s ] [Received December 3, 2009; Accepted March 5, 2010] with infections. Unlike essential thrombocytosis, RT is usually not associated with thromboembolic and hemorrhagic complications and does not require antithrombotic prophylaxis, although cases of clinically significant thrombosis have been reported in patients with RT. 3-6 Inflammatory cytokine, Interleukin-6 (IL-6) plays a major role in the pathogenesis of RT, because of its prominent role in the acute phase response of inflammatory and neoplastic diseases. C-reactive protein (CRP) is also an acute phase reactant, whose hepatic synthesis may be mediated by IL-6. CRP levels are increased in acute infections and inflammatory conditions. 7-8 Most of the available studies on the thrombocytosis in children are from developed countries. There appears to be geographical variations in causes of RT, as Kawasaki disease was reported as a common cause of thrombocytosis in a study from Japan 9, though it was Indian Journal of Pediatrics, Volume 77 June,

2 Dinesh Yadav et al not replicated in other studies Therefore, it is probable that spectrum of causes may be different in India compared to developed countries. There is paucity of Indian literature on thrombocytosis, hence the present study was undertaken to ascertain etiology and clinical course of thrombocytosis. MATERIAL AND METHODS This prospective study was conducted in a tertiary health care centre for children. Children of both sexes up to the age of 18 yr having thrombocytosis (platelet count >500 x10 9 /L) 1 on investigations, were enrolled in the study following informed parental consent. Children with history of receiving blood transfusion in preceding one month without established clinical diagnosis, were excluded from the study. All patients were evaluated clinically and were subjected to investigations as follows: Complete blood counts (CBC) with peripheral smear examination, C - reactive protein (CRP) and Interleukin-6 (IL-6). Serum iron studies (serum Ferritin, Iron and TIBC) were done in patients having microcytic hypochromic anemia. Other relevant investigations were done according to the clinical history and the physical findings, to establish the diagnosis. IL-6 levels were done using DIACLONE kit by ELISA, CRP by Immunoturbidometric assay using RANDOX kit (CP 7950), ferritin by Microplate Immunoenzymometric Assay using BIOMEDA USA kit (product code DK0039), Iron by ferrozine calorimetric assay using LABKIT (Ref 30270) and TIBC by saturation- precipitation method using LABKIT (Ref 30340). Thrombocytosis was graded on the basis of platelet counts as follows 1 : (a) Mild - >500 to /L (b) Moderate - >700 to /L (c) Severe - >900 to /L (d) Extreme - >1000x10 9 /L Iron Deficiency Anemia (IDA) was defined by microcytic hypochromic picture on peripheral smear with s.ferritin <12 ng/ml or serum iron <51µg/dl and Total Iron Binding Capacity (TIBC) > 390 µg/dl. Cut offs for serum iron and TIBC were based on controls established in the laboratory. Thrombosis prophylaxis (low dose aspirin) was given to patients with severe/ extreme thrombocytosis and patients having moderate thrombocytosis having co-existing prothrombotic factors. 1 Results were analyzed using Windows SPSS software (version 12). For the comparison of means between different groups and means of two sets of readings within the same group, unpaired and paired Student s t-test were used, respectively. For comparison of proportions, Chi-square test was used. For correlation studies Carl-Pearson correlation coefficient (r) was used. The study was approved by institutional ethics committee. RESULTS A total of 250 cases upto 18 yr of age were included in the study, over one yr period. Infants and young children (<2 yr age) were most common group, contributing 60% of total cases. Twenty percent children were in 2-5 year age group, while children over 5 yr of age accounted for 20% of cases. Male-female ratio was similar in all age groups with males contributing more than half of cases (64% vs 36%). Table 1 shows etiology of thrombocytosis. Out of total 250 cases, only 3 (1.2%) cases were found to have primary thrombocytosis (2 CML and 1 essential thrombocytosis) and remaining 98.8% cases were having RT. Among patients with RT, infections were most common cause, accounting for 65% cases, with 35% cases having infection alone, while 29% had infections in association with IDA. Among infections, respiratory tract infections, gastro intestinal infections, CNS infections and tuberculosis were common causes (Table 1). Among various hematological disorders, iron deficiency anemia was most common cause, being responsible for 41.3% cases of RT (12.6% with IDA alone and 28.7% having various infections in association with IDA). Diamorphic anemia and patients of megaloblastic anemia during recovery accounted for thrombocytosis in 6.8% of the total cases with RT. Thirteen children with acute lymphoblastic leukaemia (ALL) developed thrombocytosis during later part of induction of remission phase of therapy. Similarly, one child of acute ITP had thrombocytosis during treatment with high dose dexamethasone therapy. All three cases with primary thrombocytosis had mild thrombocytosis. Out of total 247 cases of RT, mild, moderate and severe (or extreme) thrombocytosis was seen in 62%, 29% and 9.2% cases, respectively. Among 3 main groups in RT (Infections, Infections with IDA and IDA alone), proportion of cases having severe or extreme thrombocytosis was similar (p value 0.212). IL-6 and CRP levels were studied in all patients and correlated with severity of thrombocytosis. IL-6 had significant negative correlation with platelet count (r , p value 0.026), while correlation of CRP with platelet count was statistically not significant (r , p value 0.338). Mean IL-6 levels were significantly higher in patients with infection with or without IDA, than IDA alone (p value 0.025). Similar trend was observed in 644 Indian Journal of Pediatrics, Volume 77 June, 2010

3 Clinicohematological Study of Thrombocytosis TABLE 1. Etiology of Thrombocytosis Etiology No. of Patients (%) 1. Primary (clonal) thrombocytosis 3 (1.2) Chronic myeloid leukemia 2 (66.6) Essential thrombocytosis 1 (33.3) 2. Secondary (Reactive) thrombocytosis 247 (98.8) (A) Infections 86 (34.8) o Respiratory Tract Infections 43 (50) o GI Infections 17 (19.8) o CNS Infections 09 (10.5) o Urinary Tract Infections 05 (05.8) o Tuberculosis 05 (05.8) o Other infections 07 (08.1) (B) Infection with Iron Def Anemia 71 (28.7) o Respiratory Tract Infections 38 (53.5) o GI Infections 11 (15.5) o CNS Infections 08 (11.3) o Urinary Tract Infections 03 (04.2) o Tuberculosis 06 (08.4) o Other infections 05 (07.0) (C) Iron Deficiency Anemia alone 31 (12.6) (D) Other Hematological disorders 43 (17.4) o Diamorphic Anemia 14 (32.6) o Megaloblastic Anemia 03 (07.0) o Thalassemia 07 (16.3) o Acute Lymphoblastic Leukemia 13 (30.2) o Lymphoma (Hodgkin & Non 04 (09.3) Hodgkin) o Chronic ITP (steroid treatment) 01 (02.3) o Pure Red Cell Aplasia 01 (02.3) (E) Miscellaneous* 16 (06.4) *included seizure disorder (4), nephrotic syndrome (3), intrauterine infections (2), liver abscess (2), atypical Kawasaki disease (1), chronic pancreatitis (1), choledochal cyst (1), Tay Sach s disease (1) and glycogen storage disorder (1). mean CRP levels in 3 major groups in RT patients. Mean IL-6 and CRP levels were found to be maximum in patients presenting in first week of infection or inflammation (Table 2). This difference in mean IL-6 and CRP levels in patients presenting in first wk and second wk was highly significant (p value <0.001). Further, the difference in mean IL-6 levels in second wk and later was also highly significant (p value <0.0001). One of patients with essential thrombocytosis had Budd Chiari syndrome. This patient had mild thrombocytosis (platelet count /L) and antiphospholipid antibodies and received warfarin prophylaxis. Among RT patients, one patient with IDA had left posterior cerebral artery infarct (platelet count /L). Thirty three patients with RT having severe or extreme thrombocytosis or moderate thrombocytosis with associated prothrombotic conditions were given low dose aspirin prophylaxis. On follow up, platelet counts normalized in RT patients with treatment of underlying etiology. DISCUSSION Although infections accounted for most cases of RT, the most significant observation in present study is that IDA was the underlying cause in over 40% cases. This is in contrast to earlier studies, in which IDA accounted for upto 6% cases only Higher incidence of IDA in present cases was probably due to higher prevalence of nutritional anemia in our country (55.7% to 85.1% in different states in National Family Health Survey-III). 15 Most of earlier studies are from developed countries, where nutritional anemia is less prevalent In IDA, increased level of erythropoietin has been incriminated to have a possible role in stimulating megakaryopoiesis because of its structural homology with thrombopoietin. 16 Among other haematological causes, dimorphic anemia and megaloblastic anemia accounted for 40% cases in this subgroup. Children with dimorphic anemia had thrombocytosis at presentation, which may be due to concomitant iron deficiency, while cases with megaloblastic anemia developed thrombocytosis after starting B 12 and folic acid supplementation. Thrombocytosis during treatment for megaloblastic anemia has been reported earlier from the present centre and has been explained by increase in megakaryopoiesis due to sudden increase in availability of these micronutients. 17 Thirteen children with ALL developed thrombocytosis during the phase of marrow recovery, after completion of induction of remission phase. Three cases of Hodgkin s lymphoma and one case of non- Hodgkin lymphoma, also had thrombocytosis at diagnosis. Similar cases of rebound thrombocytosis have been reported in ALL and lymphoma by various workers , 18 One child with chronic ITP developed rebound thrombocytosis after starting steroid treatment. Injectable dexamethasone was started in this child in TABLE 2. IL-6 and CRP Levels Based on Duration of Illness in Patients with Infection with or Without IDA Duration < 1 week 1-2 weeks >2 weeks p value IL-6 (pg/ml) α β γ α/β=<0.001 α/γ=< β/γ=< CRP (mg/l) α β γ α/β=<0.001 α/γ=<0.001 β/γ= Indian Journal of Pediatrics, Volume 77 June,

4 Dinesh Yadav et al view of severe thrombocytopenia (platelet count 6 x10 9 / L). After 2 cycles of high dose dexamethasone therapy, he developed thrombocytosis (platelet count 534 x10 9 / L), following which further therapy was withheld. In ITP, megakaryopoiesis is increased in response to immune mediated hyperdestruction of platelets, thrombocytopenia being manifested only when peripheral destruction is more than what bone marrow can compensate for. With use of therapy, when peripheral destruction diminishes, platelet count gets normalized. At times, platelet overproduction in the phase of decreased or normalized platelet destruction may result in thrombocytosis. Yohannan et al also reported 3 cases with immune thrombocytopenia, who developed rebound thrombocytosis following steroid treatment. 9 Seven children with thalassemia (none had undergone splenectomy) had thrombocytosis. None of earlier pediatric studies have reported thalassemia causing RT. Increased erythropoietin levels, stimulating megakaryopoiesis is possibly responsible for thrombocytosis in thalassemia. Various systemic infections were most common cause of RT in the present series, a finding similar to previous studies However, tuberculosis was associated with RT in 4.5% cases. Higher incidence and prevalence of tuberculosis in this subcontinent is possibly responsible for higher occurrence of RT in association with tuberculosis. Tubercular infection has not been commonly reported to be a cause of RT, except in a study from Saudi Arabia. 10 IL-6 plays a major role in the pathogenesis of reactive thrombocytosis, as it stimulates megakaryopoiesis, both directly and indirectly, by stimulating hepatic thrombopoietin (Tpo) production. Ishiguro et al studied IL-6 and Tpo kinetics and reported that Tpo and IL-6 levels peaked in 1 st wk of infection when platelet levels were normal and declined in 2 nd and 3 rd wk, when platelet levels peaked. 19 In present series also, mean IL-6 and CRP levels were found to be maximum in patients presenting in first wk of infection or inflammation. Besides this, mean IL-6 and CRP levels were lower in patients with severe/ extreme thrombocytosis than mild thrombocytosis and both IL-6 and CRP had negative correlation with platelet count. Probable explanation for this observation could be that increase in IL-6 levels precedes the development of thrombocytosis and the peak in thrombocytosis probably occurs when IL-6 levels start falling. Among various groups of RT, IL-6 levels were higher in patients with infection with or without IDA than IDA alone. Similar trend was observed in mean CRP levels in 3 major groups in RT patients. Hollen et al 7 and Tefferi et al 8 studied IL-6 and CRP levels in patients with thrombocytosis and reported that IL-6 and CRP levels were raised in patients with RT with underlying inflammatory process and may be normal in patients with non inflammatory etiology of RT, though, they did not observe any significant correlation between platelet count and serum IL-6 levels. All three children with primary thrombocytosis had mild thrombocytosis; still one of them had a thrombotic complication. Similarly, a recent review on childhood thrombocytosis has reported that 30% children with primary thrombocytosis experience thromboembolic or hemorrhagic complications at the time of diagnosis or later, irrespective of platelet counts. 1 RT is usually a benign condition and platelet counts normalize rapidly with treatment of underlying etiology without causing any thromboembolic complications. No thromboembolic complications were observed in earlier pediatric series on thrombocytosis even in cases with severe and extreme thrombocytosis, suggesting that no active intervention is required for thrombocytosis in these children. However, patients having underlying iron deficiency state or other prothrombotic factors (hyperhomocystinemia, nephrotic syndrome, active tuberculosis, cases with Acute Lymphoblastic Leukemia receiving L- asparginase therapy, anti phospholipids antibodies, inherited thrombophilia etc.) can have thromboembolic complications and these patients may require antithrombotic prophylaxis. There are reports of increased incidence of thrombotic complications in association with IDA with or without thrombocytosis. 3-6 One of the present case also presented with iron deficiency anemia with extreme thrombocytosis and stroke. This increased incidence of thrombotic complications in IDA has been linked to various factors including thrombocytosis, increase in adhesive reticulocytes in circulation and associated infections leading to altered hemostasis. Maguire et al reviewed association between iron deficiency anemia and stroke in young children. They observed that IDA accounted for more than half of stroke cases in children without any other underlying illness, suggesting that iron deficiency anemia is itself a significant risk factor for stroke. 3 Dame and Sutor had also suggested that individually tailored thrombosis prophylaxis should be considered in patients with RT, if additional thrombotic risk factors are present. 1 Hence, all RT patients may not have benign course and antithrombotic prophylaxis should be considered in patients with severe/extreme thrombocytosis and patients with moderate thrombocytosis with associated prothrombotic factors. CONCLUSIONS Essential thrombocytosis is extremely rare in children. In most instances thrombocytosis is reactive in nature. 646 Indian Journal of Pediatrics, Volume 77 June, 2010

5 Clinicohematological Study of Thrombocytosis Infections and iron deficiency anemia are most common cause of secondary thrombocytosis. IL-6 and CRP levels are increased in patients with RT, to a higher level in patients with infections than in patients with IDA. Thromboembolic complications are extremely rare in patients with RT and most patients have uneventful recovery of platelet counts to normal range with treatment for underlying etiology for RT. Contributions: DY; collected and analysed the data, reviewed the literature and drafted the manuscript. JC; planned and supervised the study and helped in manuscript preparation. SS and VS; supervised the study. Conflict of Interest: None. Role of Funding Source: None. REFERENCES 1. Dame C, Sutor AH. Primary and secondary thrombocytosis in childhood. Br J Haematol 2005; 129: Schafer AI. Thrombocytosis. New Engl J Med 2004; 350: Maguire JL, deveber G, Parkin PC. Association between iron-deficiency anemia and stroke in young children. Pediatrics 2007; 120: Akins PT, Glenn S, Nemeth PM, Derdeyn CP. Carotid artery thrombus associated with severe iron-deficiency anemia and thrombocytosis. Stroke 1996; 27: Belman AL, Roque CT, Ancona R, Anand AK, Davis RP. Cerebral venous thrombosis in a child with iron deficiency anemia and thrombocytosis. Stroke 1990; 21: Sebire G, Tabarki B, Saunders DE, Leroy I, Leisner R, Martin SC, Husson B et al. Cerebral venous sinus thrombosis in children: risk factors, presentation, diagnosis and outcome. Brain 2005; 128: Hollen CW, Henthorn J, Koziol JA, Burstein SA. Elevated serum interleukin-6 levels in patients with reactive thrombocytosis. Br J Haematol 1991; 79: Tefferi A, Ho TC, Ahmann GJ, Katzmann JA, Greipp PR. Plasma interleukin-6 and C-reactive protein levels in reactive versus clonal thrombocytosis. Am J Med 1994; 94: Matsubara K, Fukaya T, Nigami H, Harigaya H, Hirata T, Nozaki H, baba K. Age-dependent changes in the incidence and etiology of childhood thrombocytosis. Acta Haematol 2004; 111: Chan KW, Kaikov Y, Wadsworth LD. Thrombocytosis in childhood: A survey of 94 patients. Pediatrics 1989; 84: Vora AJ, Lilleyman JS. Secondary thrombocytosis. Arch Dis Child 1993; 68: Yohannan MD, Higgy KE, Al-Mashhadani SA, Santhosh- Kumar CR. Thrombocytosis: Etiologic analysis of 663 patients. Clinic Pediatr 1994; 33: Gomber S, Kumar S, Rusia U, Gupta P, Agarwal KN, Sharma S. Prevalence and etiology of nutritional anemias in early childhood in an urban slum. Indian J Med Res 1998; 107: Lipschitz DA, Cook JD, Finch CA. A Clinical evaluation of serum ferritin as an index of iron stores. New Engl J Med 1974; 290: National Family Health Survey-3, International Institute of Population Sciences, Mumbai Bilic E. Amino acid sequence homology of thrombopoietin and erythropoietin may explain thrombocytosis in children with iron deficiency anemia. J Pediatr Hematol Oncol 2003; 25: Chandra J, Jain V, Narayan S et al. Tremors and thrombocytosis during treatment of megaloblastic anemia. Annals Trop Pediatr 2006; 26: Randi ML, Rossi C, Barbonr E, Pietrogrande F, Girolami A. Incidence of thrombocytosis in lymphomas. Leuk Lymphoma 1992; 7: Ishiguro A, Suzuki Y, Mito M et al. Elevation of serum thrombopoietin precedes thrombocytosis in acute infections. Br J Haematol 2002; 116: Indian Journal of Pediatrics, Volume 77 June,