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   Table of Contents      
ORIGINAL ARTICLE
Year : 2020  |  Volume : 6  |  Issue : 2  |  Page : 107-112

A Cross-Sectional Study Evaluating Inflammatory Markers in Hemophilia and Association with Chronic Complications


1 Department of Medicine, Lok Nayak Hospital, Maulana Azad Medical College, New Delhi, India
2 Department of Microbiology, Lok Nayak Hospital, Maulana Azad Medical College, New Delhi, India
3 Pulmonary Medicine, Safdarjung Hospital, New Delhi, India
4 Department of Gastroenterology, PGIMER, Chandigarh, India

Date of Submission27-Dec-2019
Date of Decision15-Jun-2020
Date of Acceptance27-Jun-2020
Date of Web Publication29-Aug-2020

Correspondence Address:
Senior Resident Jahnvi Dhar
Department of Gastroenterology, PGIMER, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mamcjms.mamcjms_95_19

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  Abstract 


Introduction: Inflammatory markers in hemophilia have been implicated in various bleeding-related complications such as hemarthrosis and muscle hematoma causing damage to the joints and muscle and overt bleeding episodes. Materials and Method: This cross-sectional study included 30 patients of hemophilia with acute bleeding episode (less than 48 hours) and 30 patients in non-bleeding group (more than 2 weeks). Hemoglobin, leucocyte count, platelet count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), serum albumin, serum fibrinogen, serum ferritin, serum lactate dehydrogenase (LDH), tumour necrosis factor alpha (TNF-α) and IL 1β levels in all the subjects was measured and a correlation was sought between the two groups and with the complications of the disease. Results: ESR, CRP, Fibrinogen, LDH, Ferritin, TNF-α and Interleukin (IL)-1β was higher in acute Bleeder group as compared to Non-bleeder group. The hemophilic patients with joint deformity had elevated ESR and IL-1β. Conclusion: In hemophilia patients, CRP, ESR, Fibrinogen, Ferritin, LDH, IL-1β and TNF-α may have a role as an acute bleeding marker. ESR and IL 1 beta may be markers of arthropathy in hemophilia suggesting that inflammation may have a vital role in hemophilic arthropathy.

Keywords: Acute bleeding, hemarthrosis, hemophilia, inflammatory markers


How to cite this article:
Aggarwal S, Kumar S, Gupta N, Garg S, Chakravarty A, Ish P, Dhar J. A Cross-Sectional Study Evaluating Inflammatory Markers in Hemophilia and Association with Chronic Complications. MAMC J Med Sci 2020;6:107-12

How to cite this URL:
Aggarwal S, Kumar S, Gupta N, Garg S, Chakravarty A, Ish P, Dhar J. A Cross-Sectional Study Evaluating Inflammatory Markers in Hemophilia and Association with Chronic Complications. MAMC J Med Sci [serial online] 2020 [cited 2020 Oct 27];6:107-12. Available from: https://www.mamcjms.in/text.asp?2020/6/2/107/293895




  Introduction Top


Hemophilia is a group of X-linked recessive hereditary disorders due to a mutation in factor VIII and factor IX gene. Males are mainly affected, while females remain asymptomatic. Hemophilia A is the most common (80% cases) with prevalence of 1 in 5,000–10,000 male births and Hemophilia B with a prevalence of 1 in 20,000–34,000 male births.[1] Hemophilia lowers blood plasma clotting factors. Thus, when a blood vessel is injured, a temporary scab is formed but the missing coagulation factors prevent fibrin formation.[1],[2]

Hemophiliac patients suffer from various bleeding related complications such as hemarthrosis, muscle hematoma causing damage to the joints and muscles and bleeding in the upper digestive tract[3] with mild, moderate and severe deficiency being the presence of 5%–40%, 1%–5% and <1% factor levels respectively.[4] The annual bleeding rate in severe hemophilic patients ranges from 9 to 18 to as high as a median of 44 per year.[4],[5]

Review of the recent literature suggests that bleeding in hemophilia is associated with an increase in inflammatory markers. After each bleeding episode, cytokines such as Interleukin (IL)-6, IL-1 and tumor necrosis factor (TNF)-α levels increase which in turn stimulate the liver to produce haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), α1-acid glycoprotein (AGP) and fibrinogen (Fb).[6],[7],[8],[9] In-vitro research by Rossendal et al.[7] demonstrated that harmful effects of joint bleeds on cartilage are independent of synovial inflammation. The presence of blood in joint cavity causes both direct and indirect cartilage damage through synovial inflammation and a single blood exposure of cartilage leads to persistent damage.

Hence, inflammatory, proliferative and proangiogenic changes occur out of proportion to underlying tissue injury in the blood-exposed joint occurring in a disordered manner leading to an extended pathology rather than repair. Standard therapy for a joint hemorrhage is to replace clotting factor until evidence of ongoing bleeding has stopped.[10],[11]

The present study is aimed to determine the presence of inflammatory markers in hemophiliac patients both in the acute bleeding state and non-bleeding state and then to study their association with chronic complications of hemophilia.


  Materials and Method Top


This cross-sectional study was conducted in the Department of Medicine and Hemophilia Day Care Centre (HDCC), Maulana Azad Medical College, New Delhi after being approved by the ethical committee of the institute and obtaining an informed consent from all patients over a period of two years. Patients more than 12 years of age diagnosed with hemophilia were included and those diagnosed with any autoimmune disorder, chronic infections like human immunodeficiency virus (HIV), Hepatitis B, C and critically ill patients were excluded. A sample size of convenience of 30 patients with an acute bleeding episode less than 48 hours and 30 patients without the acute bleeding episode in the past 2 weeks were enrolled in the study (total 60).

All the patients underwent the following investigations: hemogram with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), serum albumin, serum fibrinogen, HIV, HBsAg, Anti-HCV testing, serum ferritin, serum lactate dehydrogenase (LDH), tumour necrosis factor alpha (TNF-α) and Interleukin 1 (IL 1 β) levels. TNF-α and IL-1 levels were measured with an enzyme-linked immunosorbent asssay (ELISA) kit. The intensity of the produced colored complex produced was directly proportional to the concentration of TNFα and IL-1 present in the samples and standards (normal TNFα was 0–10 pg/ml and IL-1β was 0–15 pg/ml).

All the data were analyzed using statistical package of social service (SPSS) software 17 and Graph pad Prism software. Qualitative data were analyzed by Chi square and Fisher exact test. The quantitative variables were analyzed by using parametric (t-test and one-way ANOVA) and non-parametric test (Mann Whitney and Kruskal Wallis test) performed using the mean, standard deviation (SD), median and quartile. A comparison of quantitative variables between two groups was carried out using Chi-square test. A P-value of 0.05 was considered significant. The correlation between quantitative variables was performed using the Pearson correlation coefficient.


  Results Top


The mean age of the patient in Bleeder group was 21.90±6.7 years and in Non-Bleeder group 22.36±4.9 years with a maximum of 51.6 % in the age group of 13–20 years. All the 60 patients were suffering from severe hemophilia, in which 57 had hemophilia A and 3 had hemophilia B. The Annual joint bleeding rate (AJBR − number of bleeds in 1 year) was 3.766 in the bleeder group and 2.766 in the non-bleeder group with an average of 3.266.

All 30 patients of bleeder group presented with hemarthrosis, in which the most common joints involved were knee joint (63.30%) followed by elbow (16.70%), ankle (10%), right shoulder (6.70%) and right toe (3.30%). Non-bleeder patients presented with chronic pain, stiffness or joint deformity and decrease the range of motion (ROM) suggestive of arthropathy. Joint deformity was present in 37 out of 60 patients (61.66%), which included 20 patients (66.66%) from bleeder group and 17 patients (56.66%) from the non-bleeder group. The most common joint affected was the knee (78.37%) followed by elbow (8.1%), shoulder (5.4%), ankle (5.4%) and the toe (2.7%).

There was no significant statistical difference in hemoglobin (Hb) and total leucocyte count (TLC) levels in Bleeder Group and Non-Bleeder Group of hemophilia when compared to the normal reference range (Hb 12.0–15.5 g/dl, TLC 4000–11,000/cu.mm), with a P value of 0.07 and 0.74 [Table 1] and [Table 2].
Table 1 Comparison of inflammatory markers in the bleeder group and non-bleeder group

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Table 2 Inflammatory markers in hemophilic patients with and without joint deformity

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ESR was elevated in 53 (88.33%) out of 60 patients with severe hemophilia, including 26 (86.66%) from bleeder group and 27 (90%) from Non-Bleeder group, with mean value being 31.04±15.21 in Bleeder and 22.42±7.95 in Non-Bleeder Group (normal 0–10/hour) with significant P-value <0.05 [Table 1] and [Table 2]. Comparison between the two groups yielded positive outcome (P value 0.02) [Table 3]. The CRP was found to be positive in 27 (90%) patients in bleeder group (normally negative) and 5 patients (16.7%) in non-bleeder group (P value <0.0001). Serum Albumin (gm/dl) was 5.80±0.14 in bleeder and 3.90±0.36 in Non-Bleeder Group (normal 3.5-5.5 g/dl). There was a statistical difference in comparison between the two groups (P value <0.0001) but not with reference range. Serum fibrinogen (mg/dl) was 748.8±884.6 in bleeder and 251.6±170.5 in non-bleeder group. They were significantly elevated in bleeder group when compared to the normal (150–400 mg/dl) (P-value 0.003). Similarly, comparison of the two groups yielded a highly significant statistical result with P value of 0.0003. Serum Ferritin (ng/ml) was 440.6± 83.75 in bleeder and 426±69.91 in non-bleeder group, which were significantly higher than normal (24-336 ng/dl) (P-value <0.0001). But the comparison between the groups was not significant (p value 0.06) LDH (U/L) level was 454.3±188.3 in bleeder and 215.1±101.7 in non-bleeder group (normal 180-240 U/L). Comparison between the two groups had a significant P value of <0.0001 [Table 1].
Table 3 Comparison of various parameters in non-bleed subgroup stratified on the basis of joint deformity

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TNFα was found positive in 45 (75%) out of 60 patients which comprised of all 30 acute bleeders (100%) and 15 non-bleeders (50%). The mean value of TNFα (pg/ml) was 660.1±207 in bleeder and 105.6±130.9 in non-bleeder group, which was significant when compared to normal (0-15 pg/ml) with a P-value of 0.0001 and 0.0007 respectively. Also, comparison between the 2 groups was significant (P-value 0.0001) [Table 1].

The Level of IL-1β was found elevated in 38 (63.33%) out of 60 patients which included 22 (73.5%) acute bleeders and 16 (53.33%) non-bleeders. The mean IL-1β levels was 342.9±134.8 pg/ml in bleeder and 153.6+168.6 in non-bleeder group (normal 0–10 pg/ml), with significant p-value of <0.0001 and 0.0006 respectively. Similarly, the comparison between the two groups yielded a significant P value of 0.0007 [Table 1].

Comparison of Inflammatory markers in hemophilia patients with (n = 37 patients) and without (n = 23 patients) joint deformity

ESR, CRP, Ferritin, IL-1β, and TNF-α were elevated in 36(97.29%), 23(62.16%), 21(56.75%), 33(89.18%), 34(91.89%) patients with joint deformity, respectively and 20(86.95%), 10(43.47%), 11(47.82%), 10(43.47%), and 20(86.95%) patients without joint deformity, respectively. On comparing the 2 groups, we found that P-value of ESR and IL-1β was statistically significant (P value <0.0001) [Table 2].

Comparison of inflammatory markers in the non bleeder group with (n = 17 patients) and without (n = 13 patients) joint deformity

ESR, CRP, Ferritin, IL-1β and TNF-α were elevated in 16(94.11%), 4(29.41%), 9(52.94%), 16(94.11%) and 14(82.35%) patients with joint deformity respectively and 12(92.30%), 0%, 4(30.76%), 5(38.46%) and 10(76.12%) patients without joint deformity, respectively. Comparison yielded LDH and IL-1β to have a significant P-value (P value 0.03 and 0.001 respectively) [Table 3] suggesting a role of these inflammatory markers in hemophilic arthropathy irrespective of the current bleeding episode.


  Discussion Top


A total of 60 patients with hemophilia were enrolled in the study. The mean age of patients in this study was 22.13 years and maximum and minimum age was 13 and 53 years with maximum of 51.6 % in the age group of 13-20 years. All patients were suffering from severe hemophilia. In the current study, we found an average annual joint bleeding rate (AJBR) of 3.266. In the study by Fischer et al.[12] conducted on 42 patients with severe hemophilia, average AJBR was found to be 3.7. In another study by Van Dijk et al.[13] on 80 patients with severe hemophilia, the average AJBR was 2.1.

Most common presentation in our study was joint bleeding (Hemarthrosis) with knee joint (63.3%) being the most commonly affected. In a study by Roosendaal et al.[14] and Flood et al.[15], knee joint bleeding was the most common manifestation in severe hemophilia. The prevalence of joint deformity in our study was 61.66%. A study by Soucie et al.[16] demonstrated a prevalence of joint damage to a tune of 49.6 % in severe hemophilia. In the study, no statistically significant difference was observed in hemoglobin and TLC level between bleeder and non-bleeder group. It suggests that joint damage in hemophilia is a localized process of inflammation. To the best of our knowledge, no such study exists in literature comparing these parameters with the presence of joint damage.

In this study, ESR was found elevated in 53(88.33%) patients out of 60 patients with severe hemophilia including 26(86.66%) patients of acute bleed group and 27(90%) patients of non-bleed group. It was found elevated in 36(97.29%) patients with hemophilia with joint deformity (n = 37) as compared to 20(86.95%) patients with hemophilia without joint deformity. It was also found elevated in 16(94.11%) of patients of Non-Bleeder with joint deformity as compared to 12(92.30%) patients of Non-Bleeder without joint deformity, suggesting its role as a marker of chronic inflammation and arthropathy in hemophilia. In a review of the literature, no previous study was found to show the association of ESR with hemophilia to the best of our knowledge.

In our study, CRP was positive in 27 patients (90%) in bleeder and only in 5 (16.66%) out of the 30 patients in non-bleeder group. CRP was positive in 23(62.16%) of patients with joint deformity and 10(43.47%) without joint deformity. Non-bleeder patients with joint deformity had positive CRP in 4(29.41%) patients with none in non-bleeder without joint deformity. Thus, it suggests that CRP acts as a marker of acute bleeding and arthropathy in severe hemophilia. Similar findings were observed in a study by Karapnar et al.[17]

Serum albumin (g/L) was 5.80+0.14 in bleeder and 3.90±0.36 in non-bleeder group (normal 3.5-5.5g/l). This discrepancy in the result can be explained by the fact that blood samples of bleeder group were withdrawn within 24 hours of presentation and serum albumin starts declining after 16 to 24 hours of bleeding. There may be a role of serum albumin in chronic inflammation.

Fibrinogen level was found elevated in 43.33% patients with acute bleeding and only 10% in the non-bleed group (748.8±884.6 and 251.6±170.5 mg/dl respectively). The current study suggests that elevated fibrinogen levels may be associated with acute bleeding in hemophilia. There was no previous study documented to show the association of fibrinogen with hemophilia to the best of our knowledge.

Serum ferritin was found elevated in 33(55%) out of 60 patients with mean value of 440.6± 83.75 ng/dl in bleeder and 426±69.91 ng/dl in non-bleeder group, respectively, (normal 24-336 ng/dl). It was elevated in 9(52.94%) patients in the non-bleed group with arthropathy (n = 17) as compared to 4(30.76%) without arthropathy (n = 13). Thus, an elevated level of serum ferritin may be associated with hemophilic arthropathy. Similar findings were shown in a study by Karapnar et al.[17] LDH was found elevated in 35(58.33%) out of 60 patients, which includes 27(45%) patients in bleeder (454.3±188.3 U/L) and 8(13.33%) in non-bleeder (215.1±101.7 U/L) (normal 180-240 U/L). This study indicates that there is an association between LDH and hemophilia with acute bleeding. Thus, LDH may have a role as a marker of acute bleeding in severe hemophilia. There is no previous study available to compare the association of LDH in hemophilia.

In the study, TNF-α levels (normal 0-15pg/ml) were raised in bleeders (660.1+207 pg/ml) and non-bleeders (105.6+130.9 pg/ml). TNFα was elevated in 34 (91.89%) patients with joint deformity (n = 37) and 20(86.95%) without deformity (n = 23). It was also found elevated in 14(82.35%) of patients in non-bleeder group with arthropathy (n = 17) as compared to 10(76.92%) non-bleeders without arthropathy (n = 13). This study suggests that TNF-α may have a role as a marker of chronic inflammation and arthropathy in hemophilia. A study by Jansen et al.[18] described the beneficial effect of anti TNF-α to prevent joint damage in human articular tissue exposed to blood.

In the study, IL-1β levels (normal 0–10 pg/ml) were raised in both bleeders (342.9+134.8 pg/ml) and non-bleeder groups (153.6+168.6 pg/ml), with significant P values. IL-1β was elevated in 33(89.18%) patients with joint deformity 10(43.47%) without deformity. It was also found elevated in 16(70.58%) of patients in non-bleed group with arthropathy (n = 17) as compared to 5(38.46%) patients without arthropathy (n = 13) with significant P-value. This study suggests that IL-1β may have a role as a marker of chronic inflammation and arthropathy in hemophilia. A study by Sun et al.[19] showed that IL-1β increases in hemophilic mice presenting with hemarthrosis and use of anti-IL-1β prevents hemophilic joint damage.

We categorized the inflammatory markers in hemophilia in three patterns:
  1. Markers of acute bleeding: CRP, ESR, fibrinogen, ferritin, LDH, IL-1β, TNF-α
  2. Markers of chronic inflammation: ferritin, albumin, ESR, IL-1β and TNF-α
  3. Markers of arthropathy: ESR, IL-1β


However, further studies with larger sample size is needed to establish their role as markers of acute bleeding, ongoing inflammation and in arthropathy.


  Conclusions Top


Hence, we can conclude that inflammation plays a pivotal role both during acute bleeding and further complicating the course of established arthropathy. The prevalence of hemophilic arthropathy in this study in severe hemophilia was 61.66% which is higher than available literature.

CRP, ESR, serum fibrinogen, serum ferritin, LDH, IL-1β, TNF-α may have a role as acute Bleeding markers in hemophilia. Serum ferritin, serum albumin, ESR, IL-1β, TNF-α may have a role as markers of chronic inflammation in hemophilia and ESR, IL-1β may be markers of arthropathy in hemophilia. Similarly, anti-TNFα and anti-IL-1β may have a role in the prevention of hemophilic arthropathy and further studies are warranted for the same.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Van den Berg HM, Fischer K, Mauser-Bunschoten EP. Long-term outcome of individualized prophylactic treatment of children with severe hemophilia. Br J Haematol 2001;112:561-5.  Back to cited text no. 5
    
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Lafeber FPJG, Miossec P, Valentino LA. Physiopathology of hemophilic arthropathy. Haemophilia 2008;14:3-9.  Back to cited text no. 6
    
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McDonald A, Hoffman M, Hedner U, Roberts HR, Monroe DM. Restoring hemostatic thrombin generation at the time of cutaneous wounding does not normalize healing in hemophilia B. J Thromb Haemost 2007;5:1577-83.  Back to cited text no. 11
    
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Fischer K, Astermark J, van der Bom JG. Prophylactic treatment of severe hemophilia: comparison of an intermediate dose to a high-dose regimen. Haemophilia 2002;8:753-60.  Back to cited text no. 12
    
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Van Dijk K, Fischer K, van der Bom JG, Grobbee DE, van den Berg HM. Variability in the clinical phenotype of severe hemophilia: the role of the first joint bleed. Haemophilia 2005;11:438-43.  Back to cited text no. 13
    
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Roosendaal G, Lafeber FP. Blood-induced joint damage in hemophilia. Semin Thromb Hemost 2003;29:37-42  Back to cited text no. 14
    
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Flood E, Pocoski J, Michaels LA, McCoy A, Beusterien K, Sasane R. Patient-reported experience of bleeding events in hemophilia. Eur J Haematol 2014;93:19-28.  Back to cited text no. 15
    
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Soucie JM, Cianfrini C, Janco RL. Joint range-of-motion limitations among young males with hemophilia: prevalence and risk factors. Blood 2004;103:2467-73. Epub 2003 Nov 13.  Back to cited text no. 16
    
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Karapnar TH, Karadaş N, Özek G et al. The investigation of the relationship between joint findings and serum angiogenic and inflammatory factor levels in severe hemophilia a patients. Blood Coagul Fibrinolysis 2014;25:703-8.  Back to cited text no. 17
    
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Jansen NW, Roosendaal G, Hooiveld MJ. Interleukin-10 protects against blood induced joint damage. Br J Haematol 2008;142:953-61.  Back to cited text no. 18
    
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    Tables

  [Table 1], [Table 2], [Table 3]



 

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