THROMBOPHILIC RISK FACTORS ARE UNCOMMON IN YOUNG PATIENTS WITH RETINAL VEIN OCCLUSION JASMINA AHLUWALIA, MD,* SANDEEP RAO, MD,* SUBHASH VARMA, MD,† AMOD GUPTA, MS,‡ SUNIL BOSE, MSC,* JOSEPH MASIH, MSC,* REENA DAS, MD, DNB,* NARENDER KUMAR, MD,* SHANO NASEEM, MD,* PRASHANT SHARMA, MD, DM,* MAN UPDESH SINGH SACHDEVA, MD,* NEELAM VARMA, MD* Purpose: To study the thrombotic factors, namely deficiencies of plasma proteins C, S, and antithrombin, factor V Leiden mutation, and positivity for antiphospholipid antibodies in young patients with retinal vein occlusion. Methods: The thrombophilia parameters listed above were analyzed from the laboratory records of 50 patients with the clinical diagnosis of retinal vein occlusion, aged less than 50 years. Results: A single prothrombotic factor was seen in 2 (4%) cases. The highest positivity was for the antiphospholipid antibodies (lupus anticoagulant in 6%, anticardiolipin antibodies in 2%, and anti-b 2 glycoprotein 1 in 10% cases). Other than one case where antiphospholipid syndrome was confirmed, these were transient. One patient had antithrombin deficiency. Protein C and protein S deficiency and factor V Leiden mutation were not seen in this group. Conclusion: Our data suggest that these thrombophilia risk factors are not commonly associated with retinal vein occlusion, and there is a need for studies on other factors that contribute to the development of this condition. RETINA 35:715–719, 2015
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antiphospholipid antibodies—lupus anticoagulant, anticardiolipin (ACA), and anti-b 2 glycoprotein 1 (anti-b2 GP1) antibodies—constitute an important etiological group of acquired risk factors in patients with thrombosis. Individuals with genetic thrombophilia present at early age with recurrent thrombosis or with thrombosis at unusual sites. With ready access to most of the tests for genetically determined prothombotic factors in the big cities of developing countries, there is increasing demand for thrombophilia testing in patients who have thrombosis at various sites. Ethnic variation is known to exist in prevalence of some of these factors. Studies have shown that some of the thrombophilic genetic risk factors such as the prothrombin gene mutation, which is prevalent among the white population, are almost nonexistent in our part of the continent.2 A study from South India reported hyperhomocysteinemia as risk factor in central retinal vein occlusion (CRVO),3 but, to the best of our knowledge, there is a paucity of data regarding the frequency of the common thrombophilic parameters in young patients with RVO. In this study, we present the laboratory data from a cohort of patients with
etinal vein occlusion (RVO) due to thrombosis is a cause of visual loss. Though its exact prevalence in India is not known, a recent study on a rural agrarian cohort from Central India has reported a prevalence of 0.8%.1 It may recur or, at times, become bilateral. The well-known risk factors are hypertension and diabetes. Though common in older patients, this condition is also seen in younger persons lacking the conventional risk factors prompting a search for thrombophilic factors in RVO. Genetically inherited factors such as factor V Leiden mutation, prothrombin gene G20210A mutation, and deficiency of protein C (PC), protein S (PS), and antithrombin have an increased risk of thrombosis. Also, From the Departments of *Hematology and †Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India; and ‡Department of Ophthalmology, Advanced Eye Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India. None of the authors have any financial/conflicting interests to disclose. Reprint requests: Jasmina Ahluwalia, MD, Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India; e-mail: [email protected]
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RVO, aged less than 50 years who were screened for these predisposing risk factors.
Materials and Methods Patients Patients younger than 50 years from North India with a diagnosis of RVO after an ophthalmologic examination and who were referred to coagulation laboratory for thrombophilia workup were included in this study. Anticoagulant therapy, liver disease, recent onset of thrombosis, and pregnancy are known to interfere with some of the clot-based functional assays of thrombophilia testing; therefore, a brief history pertaining to this was routinely obtained from patients before drawing samples. Sampling was deferred in patients with recent onset of symptoms in the last 6 weeks. Patients with liver dysfunction or those who were on anticoagulant therapy for other reasons were excluded from the study. Venous samples were collected and aliquoted for PC, PS, antithrombin, lupus anticoagulant, ACA antibody, antib2 GP1 antibody, and factor V Leiden mutation studies. Functional PC and protein S activity levels were measured by clot-based assays, and antithrombin level was determined by a synthetic chromogenic substrate method in the STA Compact Coagulation analyzer (Diagnostica Stago, Asnieres, France) using the following kits: STA Staclot PC, STA Staclot Protein S, and STA Stachrom ATIII. The activity levels of PC, PS, and antithrombin in test plasma were expressed as percentages of the standard plasma. Abnormal results were repeated on fresh samples. Previously established reference ranges of PC, PS, and antithrombin functional assays for our population, determined by screening 130 normal individuals were used to establish the deficiency states. Testing for lupus anticoagulant was performed using commercial diluted Russell’s viper venom test and diluted Russell’s viper venom confirm assays. Immunometric enzyme assay was performed for the quantitative determination of ACA and anti-b2 GP1 antibodies using commercial kits (Orgentec Diagnostika GmbH, Germany). Both IgG and IgM isotypes were assayed. For ACA, IgG levels .10 G Phospholipid U/mL and IgM levels .7 M Phospholipid U/mL were considered positive. For anti-b2 GP1 antibodies, values of IgG or IgM .5.0 U/mL were considered as positive. Positive tests were repeated after 12 weeks as per the recommendations of the ISTH.4 Coagulation and antibody assays of the laboratory are under regular appraisal in the World Health Organization International External Quality Assessment Scheme for coagulation and immunochemistry, respectively.
Factor V Leiden testing was performed on genomic DNA from peripheral blood collected in sodium ethylenediaminetetra acetic acid by standard procedures. Amplification of a 224-bp fragment of the factor V gene was performed using polymerase chain reaction as previously described.5 Digestion of the polymerase chain reaction products with MnlI restriction enzyme yielded 3 fragments of 104, 83, and 37 bp in the normal gene, whereas 141 and 83 bp in the mutant gene carrying the G-A mutation at position 1,691 (factor V Leiden) as this mutation abolishes one of the cleavage sites. Thus, subjects could be classified as normal (two normal factor V alleles), heterozygous for factor V Leiden, or homozygous for factor V Leiden. Samples from healthy volunteers without any other comorbidity were used as controls, tested for the presence of prothrombotic risk factors, and compared with the patients. Cases and controls showing the presence of prothrombotic factors were compared by Fisher’s exact test to look for significant associations between prothrombotic factors and RVO. P values of ,0.05 were considered significant.
Results Fifty North Indian patients aged less than 50 years and presenting with RVO were analyzed in this study. Of these, 37 had CRVO, whereas the remaining 13 had branch retinal vein occlusion. Three patients with CRVO had concomitant central retinal artery occlusion. There was history of recurrence in one case of branch retinal vein occlusion. There were no comorbid conditions in any of the 50 patients. The median age was 32 years (range, 11–47 years). There were 3 children (aged less than 18 years) in the study sample. There was no sex predilection, and the male to female ratio was 1.2:1. Low functional antithrombin levels were seen on two separate occasions in a single case with CRVO. Positivity for lupus anticoagulant by diluted Russell’s viper venom test was found in 3 patients with CRVO. However, only one female patient continued to remain positive for LA on repeat testing after 12 weeks. This patient had no history of previous thrombosis or recurrent fetal losses. Anticardiolipin antibody levels were elevated transiently in a single patient and control. Testing for factor V Leiden showed normal pattern in all the individuals (cases and controls). Protein C and PS deficiency were not seen in any of the patients in our study. Anti-b 2 glycoprotein 1 testing was available for only 38 patients because this test was introduced in our laboratory in 2008. Though positivity for this antibody was
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THROMBOPHILIA IN RETINAL VEIN OCCLUSION AHLUWALIA ET AL
the highest (10%) in the number of cases tested, this positivity was transient, and therefore a diagnosis of antiphospholipid syndrome was not made. At least one prothrombotic factor could be identified in 8 of 50 (16%) cases. However, only in 2 (4%) cases, a female with CRVO and antithrombin deficiency and another with persistently positive LA leading to antiphospholipid syndrome, a thrombophilic determinant could be demonstrated on repeat testing. None of the patients showed positivity for more than one thrombophilic factor. Pediatric patients and patients with branch retinal vein occlusion did not test positive for this panel of thrombophilic factors. The frequency of various genetic and acquired thrombophilic risk factors in our study population is summarized in the Table 1. During this period, seven cases of central retinal artery and one case of branch retinal artery occlusion were also referred for testing. The median age of these cases was 24 years (range, 10–35 years). There were four males and four females. Two patients had a history of stroke, and one was pregnant at the time of developing central retinal artery occlusion. The latter had low PS levels at the time of testing. Although repeat testing was advised, it was not performed because the patient was lost to follow-up. Transient LA positivity was seen in a single case. The results of thrombophilia workup of patients with central retinal artery occlusion are shown in Table 2. A single control sample tested positive for ACA antibodies but was negative on repeat testing. Statistically, no significant difference was observed between the patient and controls for any of the prothrombotic determinants evaluated. Discussion Continued research efforts have significantly contributed to the identification of a number of genetic
and acquired risk factors in thrombotic disorders. Data regarding prevalence of these risk factors in patients with venous thrombosis are in contrast with variable prevalence in different population and in different conditions including RVO. In this study, we have analyzed the frequency of the inherited and acquired thrombophilic risk factors in our patients with retinal vein occlusion. Hereditary and acquired deficiencies of PC, PS, and antithrombin are reported to be important risk factors in young patients with RVO.6 In our study, functional PC and protein S deficiencies were not seen and in the single case with antithrombin deficiency, and family screening for antithrombin deficiency was negative. Though individual studies have reported significant association with PC deficiency and RVO,7 a metaanalysis of the published studies by Rehak et al8 has failed to establish association with PC, PS, or antithrombin III deficiency. Antiphospholipid syndrome is an acquired thrombophilia and autoimmune disease characterized by antiphospholipid antibodies and at least one clinical criterion, the most common being venous or arterial thrombosis or recurrent fetal loss.9,10 As in thrombosis at other sites, transiently positive antiphospholipid antibodies were seen; however, persistent positivity was rare and seen in only a single case. The role of antiphospholipid antibodies in the pathogenesis of RVO has been under study for many years. However, the results of individual studies are often contradictory, probably because of small sample sizes. In the meta-analysis of the case– control studies by Rehak et al,11 prevalence of antiphospholipid antibodies in patients with RVO was significantly higher than in control subjects. Our patient population showed low positivity for antiphospholipid antibodies.
Table 1. Prevalence of Prothombotic Risk Factors in Individuals and Controls With RVO
Age (median), years M:F ratio PC functional deficiency PS functional deficiency Antithrombin III deficiency, n (%) Lupus anticoagulant positivity, n (%) ACA antibody positivity, n (%) Anti-b2 GP1 antibody (n = 38), n (%) Factor V Leiden
CRVO (n = 37)
BRVO (n = 13)
Combined CRVO and BRVO (n = 50)
Controls (n = 56)
30 1:1.05 Nil Nil 1 (2.7) 3 (8.1)† 1 (2.7)‡ 3/25 (12)‡ Nil
36 2.3:1 Nil Nil Nil Nil Nil 0/13 (0) Nil
32 1.2:1 Nil Nil 1 (2) 3 (6) 1 (2) 3 (10) Nil
35 2.1:1 Nil Nil Nil Nil 1 (1.7) Nil Nil
*P , 0.05: significant. †One patient tested positive again on repeat testing, the others were transiently positive. ‡All patients were transiently positive. BRVO, branch retinal vein occlusion.
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Table 2. Prevalence of Prothrombotic Risk Factors in Individuals With Retinal Artery or Branch Retinal Artery Occlusion Parameter
n=8
Age (median), years M:F ratio PC functional deficiency PS functional deficiency, n (%) Antithrombin III deficiency Lupus anticoagulant positivity, n (%) ACA antibody positivity Anti-b2 GP1 antibody Factor V Leiden mutation
24 1:1 Nil 1 (14)* Nil 1 (14)† Nil 0 of 4 tested Nil
*Patient tested during pregnancy. †Transiently positive.
Two meta-analyses8,12 have shown moderately higher prevalence of factor V Leiden in patients with RVO than in controls. However, there is marked racial variation in the prevalence of this mutation that is uncommon in Indians. The prevalence of heterozygosity for this mutation in our region of the country is 3.5% with an allele frequency of 1.58%.13 All patients in our study showed normal pattern for factor V Leiden mutation analysis. Central retinal vein occlusion is a rare condition in pediatric age group with most of the cases described in the literature attributable secondary to ocular pathology. The various underlying conditions reported include cyanotic congenital heart disease, infective endocarditis, sarcoidosis, and exanthema subitum associated with human herpes virus 6. Only one case in the literature showed occurrence of antiphospholipid antibody in a 6-year-old child.14 In our study population, none of the pediatric patients had any underlying ocular pathology. Screening for thrombophilia to decide for initiation of antiplatelet or anticoagulation is debatable. Use of antiplatelet therapy to improve visual acuity and neovascularization is not recommended at present. Therefore, the benefits of screening for thrombophilia with the data available are not clear.15 Significantly, CRVO has also been reported in individuals who have been receiving therapeutic anticoagulation for other indications.16,17 The narrow therapeutic margin of oral anticoagulants demands the presence of clear-cut indications for starting this form of therapy. To date, the benefit of anticoagulation for visual prognosis in patients with RVO positive for thrombophilic disorders has not been investigated. None of our patients received anticoagulation for RVO. When patients test positive for a heritable thrombophilic risk factor, there remains the issue whether or not to screen other family members. Though indiscriminate screening is neither economical nor justified, for RVO, presently, there are no data with respect to selective screening of family members at risk for thrombosis at other sites, for example, a pregnant sister
or a relative likely to be immobilized for long periods. Family screening for antithrombin deficiency in a single case that tested positive for antithrombin deficiency failed to reveal deficiency in a sister and the father who consented for testing. Thrombophilic factors have a synergistic role in causing venous thrombosis. Some like factor V Leiden may by themselves have low risk for thrombosis; however, the presence of another thrombophilic factor may increase the odds for thrombosis. In our study, no patient showed persistent positivity for more than one risk factor. None of the patients had a history or family history suggestive of early venous thrombosis. Currently, patients with RVO are investigated for dyslipidemias and diabetes. Both of these conditions are commoner in the elderly; however, with the altered lifestyles of the modern age, the incidence of these systemic disorders among the younger population is on the rise, and it would be important to screen young patients for these two important factors. A complete blood count, erythrocyte sedimentation rate, urea and creatinine estimation, and serum protein electrophoresis would help to exclude systemic disorders.18 Additional tests such as the measurement of plasma homocysteine levels and tests for thrombophilic factors may unmask these uncommon risk factors in young patients with RVO. An adequately large sample size needs to be tested to draw reasonable conclusions. The limitation of our study remains the sample size, but as a pilot effort, our data show that prothrombotic factors are present in a minority of patients with RVO. In the patients who had a thrombophilic factor, there was no history of recurrent thrombosis till date. These data suggest that screening for prothrombotic risk factors in patients with CRVO would be of low priority in our region and is partly in consonance with the recent guidelines, which no longer recommends screening for thrombophilia in patients with retinal vessel occlusion.19 The need for screening at-risk family members of patients with RVO and thrombophilia remains unclear. There is a need for larger prospective randomized controlled study to determine recurrence rates and the benefits of anticoagulation in patients with retinal vessel occlusion. There also remains the need to search for other etiological factors for RVO especially in younger patients. Key words: retinal vein occlusion, thrombophilia, protein C, protein S, antithrombin, factor V Leiden. References 1. Jonas JB, Nangia V, Khare A, et al. Prevalence and associations of retinal vein occlusions: the Central India Eye and Medical Study. Retina 2013;33:152–159.
THROMBOPHILIA IN RETINAL VEIN OCCLUSION AHLUWALIA ET AL 2. Garewal G, Das R, Ahluwalia J, et al. Prothrombin G20210A is not prevalent in North India. J Thromb Haemost 2003;10: 2253–2254. 3. Narayanasamy A, Subramaniam B, Karunakaran C, et al. Hyperhomocysteinemia and low methionine stress are risk factors for central retinal venous occlusion in an Indian population. Invest Ophthalmol Vis Sci 2007;48:1441–1446. 4. Miyakis S, Lockshin MD, Atsumi D, et al. International consensus statement on an update of the preliminary classification criteria for antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. 5. Ridker PM, Miletich JP, Stampfer MJ, et al. Leiden and risks of recurrent idiopathic venous thromboembolism. Circulation 1995;92:2800–2802. 6. Bertram B, Remky A, Arend O, et al. Protein C, protein S, and antithrombin III in acute ocular occlusive diseases. Ger J Ophthalmol 1995;4:332–335. 7. Tekeli O, Gursel E, Buyurgan H. Protein C, protein S and antithrombin III deficiencies in retinal vein occlusion. Acta Ophthalmol Scand 1999;77:628–630. 8. Rehak M, Rehak J, Muller M, et al. The prevalence of activated protein C (APC) resistance and factor V Leiden is significantly higher in patients with retinal vein occlusion without general risk factors. Case–control study and meta-analysis. Thromb Haemost 2008;99:925–929. 9. Pengo V, Ruffatti A, Legnani C, et al. Clinical course of high risk patients diagnosed with antiphospholipid syndrome (APS). J Thromb Haemost 2010;8:234–236. 10. Tripodi A. Testing for lupus anticoagulants: all that a clinician should know. Lupus 2009;18:291–298.
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11. Rehak M, Muller M, Scholz M, et al. Antiphospholipid syndrome and retinal vein occlusion. Meta-analysis of published studies [in German]. Ophthalmologe 2009;106:427– 434. 12. Janssen MC, den Heijer M, Cruysberg JR, et al. Retinal vein occlusion: a form of venous thrombosis or a complication of atherosclerosis? A meta-analysis of thrombophilic factors. Thromb Haemost 2005;93:1021–1026. 13. Garewal G, Das R, Varma S, et al. Heterogeneous distribution of Factor V Leiden in patients from north India with venous thromboembolism. J Thromb Haemost 2003;1: 1329–1330. 14. Hartnett ME, Laposata M, Van Cott E. Antiphospholipid antibody syndrome in a six-year-old female patient. Am J Ophthalmol 2003;135:542–544. 15. Squizzato A, Manfredi E, Bozzato S, et al. Antithrombotic and fibrinolytic drugs for retinal vein occlusion: a systematic review and a call for action. Thromb Haemost 2010;103: 271–276. 16. Browning DJ, Fraser CM. Retinal vein occlusions in patients taking warfarin. Ophthalmology 2004;111:1196–1200. 17. Mruthyunjaya P, Wirostko WJ, Chandrashekhar R, et al. Central retinal vein occlusion in patients treated with long-term warfarin sodium (Coumadin) for anticoagulation. Retina 2006;26:285–291. 18. Wong TY, Scott IU. Retinal vein occlusion. N Engl J Med 2010;363:2135–2144. 19. Baglin T, Gray E, Greaves M, et al. Clinical guidelines for testing for heritable thrombophilia. Br J Haematol 2010;149: 209–220.
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antiphospholipid antibodies—lupus anticoagulant, anticardiolipin (ACA), and anti-b 2 glycoprotein 1 (anti-b2 GP1) antibodies—constitute an important etiological group of acquired risk factors in patients with thrombosis. Individuals with genetic thrombophilia present at early age with recurrent thrombosis or with thrombosis at unusual sites. With ready access to most of the tests for genetically determined prothombotic factors in the big cities of developing countries, there is increasing demand for thrombophilia testing in patients who have thrombosis at various sites. Ethnic variation is known to exist in prevalence of some of these factors. Studies have shown that some of the thrombophilic genetic risk factors such as the prothrombin gene mutation, which is prevalent among the white population, are almost nonexistent in our part of the continent.2 A study from South India reported hyperhomocysteinemia as risk factor in central retinal vein occlusion (CRVO),3 but, to the best of our knowledge, there is a paucity of data regarding the frequency of the common thrombophilic parameters in young patients with RVO. In this study, we present the laboratory data from a cohort of patients with
etinal vein occlusion (RVO) due to thrombosis is a cause of visual loss. Though its exact prevalence in India is not known, a recent study on a rural agrarian cohort from Central India has reported a prevalence of 0.8%.1 It may recur or, at times, become bilateral. The well-known risk factors are hypertension and diabetes. Though common in older patients, this condition is also seen in younger persons lacking the conventional risk factors prompting a search for thrombophilic factors in RVO. Genetically inherited factors such as factor V Leiden mutation, prothrombin gene G20210A mutation, and deficiency of protein C (PC), protein S (PS), and antithrombin have an increased risk of thrombosis. Also, From the Departments of *Hematology and †Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India; and ‡Department of Ophthalmology, Advanced Eye Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India. None of the authors have any financial/conflicting interests to disclose. Reprint requests: Jasmina Ahluwalia, MD, Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India; e-mail: [email protected]
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RVO, aged less than 50 years who were screened for these predisposing risk factors.
Materials and Methods Patients Patients younger than 50 years from North India with a diagnosis of RVO after an ophthalmologic examination and who were referred to coagulation laboratory for thrombophilia workup were included in this study. Anticoagulant therapy, liver disease, recent onset of thrombosis, and pregnancy are known to interfere with some of the clot-based functional assays of thrombophilia testing; therefore, a brief history pertaining to this was routinely obtained from patients before drawing samples. Sampling was deferred in patients with recent onset of symptoms in the last 6 weeks. Patients with liver dysfunction or those who were on anticoagulant therapy for other reasons were excluded from the study. Venous samples were collected and aliquoted for PC, PS, antithrombin, lupus anticoagulant, ACA antibody, antib2 GP1 antibody, and factor V Leiden mutation studies. Functional PC and protein S activity levels were measured by clot-based assays, and antithrombin level was determined by a synthetic chromogenic substrate method in the STA Compact Coagulation analyzer (Diagnostica Stago, Asnieres, France) using the following kits: STA Staclot PC, STA Staclot Protein S, and STA Stachrom ATIII. The activity levels of PC, PS, and antithrombin in test plasma were expressed as percentages of the standard plasma. Abnormal results were repeated on fresh samples. Previously established reference ranges of PC, PS, and antithrombin functional assays for our population, determined by screening 130 normal individuals were used to establish the deficiency states. Testing for lupus anticoagulant was performed using commercial diluted Russell’s viper venom test and diluted Russell’s viper venom confirm assays. Immunometric enzyme assay was performed for the quantitative determination of ACA and anti-b2 GP1 antibodies using commercial kits (Orgentec Diagnostika GmbH, Germany). Both IgG and IgM isotypes were assayed. For ACA, IgG levels .10 G Phospholipid U/mL and IgM levels .7 M Phospholipid U/mL were considered positive. For anti-b2 GP1 antibodies, values of IgG or IgM .5.0 U/mL were considered as positive. Positive tests were repeated after 12 weeks as per the recommendations of the ISTH.4 Coagulation and antibody assays of the laboratory are under regular appraisal in the World Health Organization International External Quality Assessment Scheme for coagulation and immunochemistry, respectively.
Factor V Leiden testing was performed on genomic DNA from peripheral blood collected in sodium ethylenediaminetetra acetic acid by standard procedures. Amplification of a 224-bp fragment of the factor V gene was performed using polymerase chain reaction as previously described.5 Digestion of the polymerase chain reaction products with MnlI restriction enzyme yielded 3 fragments of 104, 83, and 37 bp in the normal gene, whereas 141 and 83 bp in the mutant gene carrying the G-A mutation at position 1,691 (factor V Leiden) as this mutation abolishes one of the cleavage sites. Thus, subjects could be classified as normal (two normal factor V alleles), heterozygous for factor V Leiden, or homozygous for factor V Leiden. Samples from healthy volunteers without any other comorbidity were used as controls, tested for the presence of prothrombotic risk factors, and compared with the patients. Cases and controls showing the presence of prothrombotic factors were compared by Fisher’s exact test to look for significant associations between prothrombotic factors and RVO. P values of ,0.05 were considered significant.
Results Fifty North Indian patients aged less than 50 years and presenting with RVO were analyzed in this study. Of these, 37 had CRVO, whereas the remaining 13 had branch retinal vein occlusion. Three patients with CRVO had concomitant central retinal artery occlusion. There was history of recurrence in one case of branch retinal vein occlusion. There were no comorbid conditions in any of the 50 patients. The median age was 32 years (range, 11–47 years). There were 3 children (aged less than 18 years) in the study sample. There was no sex predilection, and the male to female ratio was 1.2:1. Low functional antithrombin levels were seen on two separate occasions in a single case with CRVO. Positivity for lupus anticoagulant by diluted Russell’s viper venom test was found in 3 patients with CRVO. However, only one female patient continued to remain positive for LA on repeat testing after 12 weeks. This patient had no history of previous thrombosis or recurrent fetal losses. Anticardiolipin antibody levels were elevated transiently in a single patient and control. Testing for factor V Leiden showed normal pattern in all the individuals (cases and controls). Protein C and PS deficiency were not seen in any of the patients in our study. Anti-b 2 glycoprotein 1 testing was available for only 38 patients because this test was introduced in our laboratory in 2008. Though positivity for this antibody was
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THROMBOPHILIA IN RETINAL VEIN OCCLUSION AHLUWALIA ET AL
the highest (10%) in the number of cases tested, this positivity was transient, and therefore a diagnosis of antiphospholipid syndrome was not made. At least one prothrombotic factor could be identified in 8 of 50 (16%) cases. However, only in 2 (4%) cases, a female with CRVO and antithrombin deficiency and another with persistently positive LA leading to antiphospholipid syndrome, a thrombophilic determinant could be demonstrated on repeat testing. None of the patients showed positivity for more than one thrombophilic factor. Pediatric patients and patients with branch retinal vein occlusion did not test positive for this panel of thrombophilic factors. The frequency of various genetic and acquired thrombophilic risk factors in our study population is summarized in the Table 1. During this period, seven cases of central retinal artery and one case of branch retinal artery occlusion were also referred for testing. The median age of these cases was 24 years (range, 10–35 years). There were four males and four females. Two patients had a history of stroke, and one was pregnant at the time of developing central retinal artery occlusion. The latter had low PS levels at the time of testing. Although repeat testing was advised, it was not performed because the patient was lost to follow-up. Transient LA positivity was seen in a single case. The results of thrombophilia workup of patients with central retinal artery occlusion are shown in Table 2. A single control sample tested positive for ACA antibodies but was negative on repeat testing. Statistically, no significant difference was observed between the patient and controls for any of the prothrombotic determinants evaluated. Discussion Continued research efforts have significantly contributed to the identification of a number of genetic
and acquired risk factors in thrombotic disorders. Data regarding prevalence of these risk factors in patients with venous thrombosis are in contrast with variable prevalence in different population and in different conditions including RVO. In this study, we have analyzed the frequency of the inherited and acquired thrombophilic risk factors in our patients with retinal vein occlusion. Hereditary and acquired deficiencies of PC, PS, and antithrombin are reported to be important risk factors in young patients with RVO.6 In our study, functional PC and protein S deficiencies were not seen and in the single case with antithrombin deficiency, and family screening for antithrombin deficiency was negative. Though individual studies have reported significant association with PC deficiency and RVO,7 a metaanalysis of the published studies by Rehak et al8 has failed to establish association with PC, PS, or antithrombin III deficiency. Antiphospholipid syndrome is an acquired thrombophilia and autoimmune disease characterized by antiphospholipid antibodies and at least one clinical criterion, the most common being venous or arterial thrombosis or recurrent fetal loss.9,10 As in thrombosis at other sites, transiently positive antiphospholipid antibodies were seen; however, persistent positivity was rare and seen in only a single case. The role of antiphospholipid antibodies in the pathogenesis of RVO has been under study for many years. However, the results of individual studies are often contradictory, probably because of small sample sizes. In the meta-analysis of the case– control studies by Rehak et al,11 prevalence of antiphospholipid antibodies in patients with RVO was significantly higher than in control subjects. Our patient population showed low positivity for antiphospholipid antibodies.
Table 1. Prevalence of Prothombotic Risk Factors in Individuals and Controls With RVO
Age (median), years M:F ratio PC functional deficiency PS functional deficiency Antithrombin III deficiency, n (%) Lupus anticoagulant positivity, n (%) ACA antibody positivity, n (%) Anti-b2 GP1 antibody (n = 38), n (%) Factor V Leiden
CRVO (n = 37)
BRVO (n = 13)
Combined CRVO and BRVO (n = 50)
Controls (n = 56)
30 1:1.05 Nil Nil 1 (2.7) 3 (8.1)† 1 (2.7)‡ 3/25 (12)‡ Nil
36 2.3:1 Nil Nil Nil Nil Nil 0/13 (0) Nil
32 1.2:1 Nil Nil 1 (2) 3 (6) 1 (2) 3 (10) Nil
35 2.1:1 Nil Nil Nil Nil 1 (1.7) Nil Nil
*P , 0.05: significant. †One patient tested positive again on repeat testing, the others were transiently positive. ‡All patients were transiently positive. BRVO, branch retinal vein occlusion.
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Table 2. Prevalence of Prothrombotic Risk Factors in Individuals With Retinal Artery or Branch Retinal Artery Occlusion Parameter
n=8
Age (median), years M:F ratio PC functional deficiency PS functional deficiency, n (%) Antithrombin III deficiency Lupus anticoagulant positivity, n (%) ACA antibody positivity Anti-b2 GP1 antibody Factor V Leiden mutation
24 1:1 Nil 1 (14)* Nil 1 (14)† Nil 0 of 4 tested Nil
*Patient tested during pregnancy. †Transiently positive.
Two meta-analyses8,12 have shown moderately higher prevalence of factor V Leiden in patients with RVO than in controls. However, there is marked racial variation in the prevalence of this mutation that is uncommon in Indians. The prevalence of heterozygosity for this mutation in our region of the country is 3.5% with an allele frequency of 1.58%.13 All patients in our study showed normal pattern for factor V Leiden mutation analysis. Central retinal vein occlusion is a rare condition in pediatric age group with most of the cases described in the literature attributable secondary to ocular pathology. The various underlying conditions reported include cyanotic congenital heart disease, infective endocarditis, sarcoidosis, and exanthema subitum associated with human herpes virus 6. Only one case in the literature showed occurrence of antiphospholipid antibody in a 6-year-old child.14 In our study population, none of the pediatric patients had any underlying ocular pathology. Screening for thrombophilia to decide for initiation of antiplatelet or anticoagulation is debatable. Use of antiplatelet therapy to improve visual acuity and neovascularization is not recommended at present. Therefore, the benefits of screening for thrombophilia with the data available are not clear.15 Significantly, CRVO has also been reported in individuals who have been receiving therapeutic anticoagulation for other indications.16,17 The narrow therapeutic margin of oral anticoagulants demands the presence of clear-cut indications for starting this form of therapy. To date, the benefit of anticoagulation for visual prognosis in patients with RVO positive for thrombophilic disorders has not been investigated. None of our patients received anticoagulation for RVO. When patients test positive for a heritable thrombophilic risk factor, there remains the issue whether or not to screen other family members. Though indiscriminate screening is neither economical nor justified, for RVO, presently, there are no data with respect to selective screening of family members at risk for thrombosis at other sites, for example, a pregnant sister
or a relative likely to be immobilized for long periods. Family screening for antithrombin deficiency in a single case that tested positive for antithrombin deficiency failed to reveal deficiency in a sister and the father who consented for testing. Thrombophilic factors have a synergistic role in causing venous thrombosis. Some like factor V Leiden may by themselves have low risk for thrombosis; however, the presence of another thrombophilic factor may increase the odds for thrombosis. In our study, no patient showed persistent positivity for more than one risk factor. None of the patients had a history or family history suggestive of early venous thrombosis. Currently, patients with RVO are investigated for dyslipidemias and diabetes. Both of these conditions are commoner in the elderly; however, with the altered lifestyles of the modern age, the incidence of these systemic disorders among the younger population is on the rise, and it would be important to screen young patients for these two important factors. A complete blood count, erythrocyte sedimentation rate, urea and creatinine estimation, and serum protein electrophoresis would help to exclude systemic disorders.18 Additional tests such as the measurement of plasma homocysteine levels and tests for thrombophilic factors may unmask these uncommon risk factors in young patients with RVO. An adequately large sample size needs to be tested to draw reasonable conclusions. The limitation of our study remains the sample size, but as a pilot effort, our data show that prothrombotic factors are present in a minority of patients with RVO. In the patients who had a thrombophilic factor, there was no history of recurrent thrombosis till date. These data suggest that screening for prothrombotic risk factors in patients with CRVO would be of low priority in our region and is partly in consonance with the recent guidelines, which no longer recommends screening for thrombophilia in patients with retinal vessel occlusion.19 The need for screening at-risk family members of patients with RVO and thrombophilia remains unclear. There is a need for larger prospective randomized controlled study to determine recurrence rates and the benefits of anticoagulation in patients with retinal vessel occlusion. There also remains the need to search for other etiological factors for RVO especially in younger patients. Key words: retinal vein occlusion, thrombophilia, protein C, protein S, antithrombin, factor V Leiden. References 1. Jonas JB, Nangia V, Khare A, et al. Prevalence and associations of retinal vein occlusions: the Central India Eye and Medical Study. Retina 2013;33:152–159.
THROMBOPHILIA IN RETINAL VEIN OCCLUSION AHLUWALIA ET AL 2. Garewal G, Das R, Ahluwalia J, et al. Prothrombin G20210A is not prevalent in North India. J Thromb Haemost 2003;10: 2253–2254. 3. Narayanasamy A, Subramaniam B, Karunakaran C, et al. Hyperhomocysteinemia and low methionine stress are risk factors for central retinal venous occlusion in an Indian population. Invest Ophthalmol Vis Sci 2007;48:1441–1446. 4. Miyakis S, Lockshin MD, Atsumi D, et al. International consensus statement on an update of the preliminary classification criteria for antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. 5. Ridker PM, Miletich JP, Stampfer MJ, et al. Leiden and risks of recurrent idiopathic venous thromboembolism. Circulation 1995;92:2800–2802. 6. Bertram B, Remky A, Arend O, et al. Protein C, protein S, and antithrombin III in acute ocular occlusive diseases. Ger J Ophthalmol 1995;4:332–335. 7. Tekeli O, Gursel E, Buyurgan H. Protein C, protein S and antithrombin III deficiencies in retinal vein occlusion. Acta Ophthalmol Scand 1999;77:628–630. 8. Rehak M, Rehak J, Muller M, et al. The prevalence of activated protein C (APC) resistance and factor V Leiden is significantly higher in patients with retinal vein occlusion without general risk factors. Case–control study and meta-analysis. Thromb Haemost 2008;99:925–929. 9. Pengo V, Ruffatti A, Legnani C, et al. Clinical course of high risk patients diagnosed with antiphospholipid syndrome (APS). J Thromb Haemost 2010;8:234–236. 10. Tripodi A. Testing for lupus anticoagulants: all that a clinician should know. Lupus 2009;18:291–298.
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11. Rehak M, Muller M, Scholz M, et al. Antiphospholipid syndrome and retinal vein occlusion. Meta-analysis of published studies [in German]. Ophthalmologe 2009;106:427– 434. 12. Janssen MC, den Heijer M, Cruysberg JR, et al. Retinal vein occlusion: a form of venous thrombosis or a complication of atherosclerosis? A meta-analysis of thrombophilic factors. Thromb Haemost 2005;93:1021–1026. 13. Garewal G, Das R, Varma S, et al. Heterogeneous distribution of Factor V Leiden in patients from north India with venous thromboembolism. J Thromb Haemost 2003;1: 1329–1330. 14. Hartnett ME, Laposata M, Van Cott E. Antiphospholipid antibody syndrome in a six-year-old female patient. Am J Ophthalmol 2003;135:542–544. 15. Squizzato A, Manfredi E, Bozzato S, et al. Antithrombotic and fibrinolytic drugs for retinal vein occlusion: a systematic review and a call for action. Thromb Haemost 2010;103: 271–276. 16. Browning DJ, Fraser CM. Retinal vein occlusions in patients taking warfarin. Ophthalmology 2004;111:1196–1200. 17. Mruthyunjaya P, Wirostko WJ, Chandrashekhar R, et al. Central retinal vein occlusion in patients treated with long-term warfarin sodium (Coumadin) for anticoagulation. Retina 2006;26:285–291. 18. Wong TY, Scott IU. Retinal vein occlusion. N Engl J Med 2010;363:2135–2144. 19. Baglin T, Gray E, Greaves M, et al. Clinical guidelines for testing for heritable thrombophilia. Br J Haematol 2010;149: 209–220.
