Assessment of Circulating Red Cell and Platelet Microparticles Levels in Children with Non-Transfusion Dependent Beta-Thalassemia

doi:10.21203/rs.3.rs-5026230/v1

Background: Elevated circulating microparticles (MPs) have been hypothesized to be responsible for thromboembolic events (TEEs) in thalassemia patients. The aim of the study is to evaluate the circulating red cell and platelet microparticle levels in children with non-transfusion dependent Beta-thalassemia (NTDT) and its possible role in thromboembolic events.

Methods: Fifty NTDT children from Alexandria University Children's Hospital and 50 age- and sex-matched healthy children were enrolled in this study. CBC, LDH, serum ferritin, soluble transferrin receptor (sTfR), D-dimer. Serum levels of erythrocyte microparticles (EMPs) and platelet microparticles (PMPs) were measured by flow cytometry and serum B-type natriuretic peptide (NT- pro-BNP) levels were measured.

Results: Serum levels of EMPs and PMPs were significantly elevated in NTDT patients compared to healthy controls. Mean serum ferritin levels and mean sTfR levels were significantly higher in patients than in control. Twelve percent of our cases were splenectomized. Splenectomized patients had significantly higher platelet count, PMPs level, and serum ferritin level than non-splenectomized counterparts—no statistically significant difference in NT- proBNP level between patients and controls.

Conclusions: our study demonstrated the presence of elevated levels of EMPs and PMPs in NTDT patients compared to the control group. Splenectomized patients had higher platelet count, PMP levels, and serum ferritin levels. So, assessing EMPs and PMPs might provide efficacy in theearly detection of thromboembolic complications.

non transfusion

thalassemia

microparticles

Beta-thalassemia syndromes are a group of heterogeneous autosomal recessive hereditary anemia resulting from defective synthesis of β-globin chains of adult hemoglobin A, leading to a range of phenotypes from severe anemia to clinically asymptomatic individuals.[1, 2] There is a wide variety of clinically distinct thalassemia syndromes.[2], the Thalassemia International Federation guidelines (2022)[3] put a new classification for thalassemia based on clinical severity and transfusion requirements into two main groups: transfusion-dependent and non-transfusion-dependent.

Despite having high hemoglobin levels and being transfusion-independent, patients with NTDT experience most thalassemia-related complications more frequently than those with TDT. These patients often develop thromboembolic events such as deep vein thrombosis, portal vein thrombosis, pulmonary thromboembolism, and cerebral thrombosis, that are uncommon in TDT and typically manifest at an older age.[4] The prevalence of thrombotic events can reach up to 20% in NTDT patients compared with less than 1% in TDT patients.[5]

A hypercoagulable status has been identified in thalassemia, especially in NTDT. Hypercoagulability in thalassemia is a multifactorial condition recognizing platelet abnormalities and pathologic red blood cells as crucial factors responsible for thrombotic events. Thalassemic RBCs are associated with the expression of negatively charged phospholipids, which can increase thrombin generation.[6, 7]

Furthermore, damaged red blood cells and chronic platelet activation in β- thalassemia were associated with increased microparticles (MPs) release into the circulation. Increased levels of these RBCs and platelet MPs have been supposed to be responsible for the occurrence of thromboembolic events.[8, 9]

The risk of thrombosis is significantly increased by splenectomy in thalassemic patients.[10, 11] Splenectomy is associated with decreased clearance of damaged RBC, chronic platelet activation, and increased MP shedding.[12] Therefore, splenectomy, performed in NTDT patients, may result in severe thrombotic episodes and a rise of pro-thrombotic circulating microparticles (MP).[7]

Echocardiography is the gold standard method for the assessment of Pulmonary Hypertension (PHT). However, supplementary data that are useful in the evaluation of PHT in thalassemia patients include markers of right heart dysfunction, such as an amino-terminal fragment of proB-type natriuretic peptide (NT-proBNP)[13], which is a hormone released in response to cardiomyocyte stretching. In the early phases of cardiac involvement, the level of NT-pro BNP increases before an increase in diastolic pressure. Measurement of NT-proBNP provides diagnostic and mechanistic information concerning the development of PHT in thalassemia, and its level is a strong indicator of PHT in these patients.[14]

This study aimed to evaluate the levels of circulating EMPs and PMPs in NTDT patients and their possible role in thromboembolic events and to determine the serum level of NT-proBNP and its value as an indicator for PHT in NTDT patients.

This cross-sectional study included 50 children diagnosed with NTDT and 50 sex- and age-matched healthy children. Patients were recruited from regular attendants of the Pediatric Hematology Outpatient Clinic of a tertiary health care facility affiliated to a University Hospital, during the period from November 2022 to May 2023. Informed consent was obtained from the guardian of each patient and controlled before participation. This study was reviewed by the Ethics Committee for Human Resources in Alexandria Faculty of Medicine. All patients in the study were subjected to a thorough history-taking and physical examination. Laboratory investigations included complete blood count (CBC), LDH, D-dimer, and serum ferritin levels. Serum sTfR and serum NT- proBNP levels were measured by ELISA. A platelet aggregation test was performed using an aggregometer and an adenosine 5-diphosphate (ADP) agonist for the patient group. Serum levels of circulating EMPs and PMPs were measured by flow cytometry.

Statistical Methodology

Data analysis was carried out using the SPSS (Statistical Package for Social Science) program for statistical analysis (ver 25).[15] Shapiro–Wilk test of normality was used to assess the distribution of the quantitative variables.[16] Analysis of the normally distributed data was done using parametric statistics (mean ± SD, t-test), while of the not normally distributed data using a non-parametric test (median, Mann-Whitney U test).^[17] During sample size calculation, beta error was accepted up to 20%, with a power of study of 80%. An alpha level was set to 5% with a significance level of 95%. Statistical significance was tested at p-value < .05.[18]

The study included 50 NTDT patients, 32 males and 18 females, with an age range of 8–16 years, a mean of 10.42 ± 2.86, and a median of 9 years. Fifty age and sex-matched apparently healthy children were included as control. Six (12%) patients were splenectomized. None had clinical evidence of PHT or any apparent thromboembolic events or vascular disease.

Hemoglobin (Hb) level and Red Blood Cells (RBCs) count were significantly lower in NTDT patients than in the control group (p < .001). Reticulocyte count was significantly higher in patients than in control (p < .001). There was no significant difference between patients and controls regarding platelet count (p = .432). However, nine patients had thrombocytosis (platelet count ≥ 450 ×10³/µL).[19] Mean serum ferritin and mean sTfR levels were significantly higher in NTDT patients than control ((p < .001, p < .001; respectively) (Table 1).

In NTDT patients, the mean LDH level was 261.0 ± 74.69 U/L and 40 patients had elevated levels above normal value (100–190 U/L).[20]

D-dimer was near normal (less than 550 ug/L)[21]; however, 21 patients had higher levels above 550 µg/L, and 24% were splenectomized.

No statistically significant correlation was noticed between serum sTfR and ferritin levels (r = 0.094, p = .514).

Platelet aggregation was tested using an ADP agonist and was normal in all patients, ranging between 75–85% with a mean of 80.20 ± 3.61%.

EMPs and PMPs levels were significantly higher in the NTDT patients than in controls (p = < 0.00) (Table 2).

No significant correlation was encountered between EMPs and Hb Level, RBC count, hemolytic markers (reticulocyte count and LDH), and biochemical markers, including serum ferritin and sTfR level. Also, there was no significant correlation between PMPs level and platelet count or D-dimer level (Table 3).

The mean serum NT pro-BNP in NTDT patients was 98.48 ± 17.51pg/ml, while it was 91.82 ± 20.15 pg/ml among control without significant difference between them (p = .081). No correlation was noticed between serum NT- proBNP Level and different laboratory parameters, including Hb level, PLT count, D-dimer level, serum ferritin level, EMPs, and PMPs levels. There was no significant difference in NT- proBNP level between patients with normal PLT count (n = 41) and patients with thrombocytosis (n = 9) (p = .612). However, NT- proBNP level was significantly higher in patients with high D-dimer levels (n = 21) compared to patients with normal D-dimer levels (n = 29) (p = .047) (Table 4).

Six patients were splenectomized, they had significantly higher platelet count, serum ferritin level, and serum PMPs level compared to non-splenectomized patients (p = 0.005, 0.016, and 0.003)respectively. There was no statistically significant difference in D-dimer, EMPs, and NT-proBNP levels between splenectomized and non-splenectomized patients (Table 5).

There was a statistically significant positive correlation between PMPs and platelet count in splenectomized patients (r = 0.943, p = .005). Still, in non-splenectomized patients, there was no correlation (r = 0.046, p = .764), and no statistically significant correlation was noticed between PMPs level and D-dimer level in both splenectomized (r = 0.116, p = .827) and non-splenectomized patients (r=-0.038, p = .807).

Patients with thalassemia are at increased risk for Thrombo-Embolic Events (TEE) due to a hypercoagulable state, and this risk is known to be highest in patients with non-transfusion-dependent thalassemia.[6] The molecular and cellular mechanisms contributing to hypercoagulability are diverse.[22]

Pathological red blood cells and chronic platelet activation seem to be the key factors causing hypercoagulability.[7, 12, 22]

The procoagulant effect of these thalassemia RBCs is due to the increased surface expression of phosphatidylethanolamine (PE) and phosphatidylserine (PS) through the amplification of thrombin generation and initiation of platelet activation.[8, 23]

The high plasma levels of D-dimer can be taken as an indirect index of thrombin activity. D-dimer is a marker for fibrin formation and fibrinolysis and hence thrombotic risk in these patients.[24] In the present study the mean values of D-dimer were near normal (less than 550 µg/L) however, 21 patients had higher levels above 550 µg/L, and 24% of them were splenectomized.

Enhanced platelet aggregation and increased platelet activation markers are evidence of chronic platelet activation. In the present study, platelet aggregation was tested by ADP agonist and showed a normal range in all patients ranging between 75–85% with a mean of 80.20 ± 3.61%.[25]. None had hyperaggregation (more than 90%).[26] The results obtained from platelet aggregation vary in different studies and even among patients. Some studies have reported an increased[26, 27] platelet aggregation; on the other hand, others have reported either decreased[28] or even normal reactivity.[11, 29] Zahedpanah et al. (2018),[30] reported normal platelet aggregation by ADP agonist (80.4 ± 9.4) in 36 β‑TI young adult patients without significant difference compared to control (78.8 ± 10.4). They explained this by chronic platelet activation in β-TI, which might prevent the agonists' further stimulation of the platelets; actually, the activated platelets become refractory to additional stimulation. This variation in results in different studies can be attributed to difference in laboratory techniques due to disease characteristics or racial differences.[29]

Damaged RBC and chronic platelet activation are associated with increased microparticles released in circulation.

Microparticles (MPs) are shed submicrometric fragments harbouring negatively charged procoagulant phosphatidylethanolamine (PE) and phosphatidylserine (PS) in their extracellular membrane leaflet. PMPs constitute 70–90% of the total number of MPs found in circulation.[8, 9]

Increased levels of these RBCs and Platelet microparticles have been supposed to be responsible for the occurrence of thromboembolic events.[8]

In the present study, we evaluated the level of EMPs and PMPs in 50 NTDT patients and 50 age and sex-matched normal children. We found that the levels of EMPs were significantly higher in the thalassemic group compared to control. Our results agreed with several studies that reported elevation in EMPs in patients with thalassemia intermedia.[8, 9, 23, 31–33]

We studied the correlation between EMPs level with some studied parameters, including Hb level, RBCs count, hemolytic markers (reticulocytic count and LDH), and serum ferritin, and, no significant correlations could be elicited between the level of EMPs and previously mentioned parameters. Shahin et al.[23] reported a significant positive correlation between EMPs and ferritin levels and a significant negative correlation between EMPs and Hb levels. In contrast, no correlation was encountered between EMPs and RBC count or LDH level.

Similar findings were reported by Tantawy et al. (2013)[27] and Youssry et al. (2017)[8] A negative correlation between the Level of EMPs and Hb level was also reported by Mowafy et al. [31]; however, did not find a significant correlation between EMPs level and RBCs or serum ferritin level. The variability in correlation results can be explained by differences in the number, age, and/or clinical characteristics of the studied patients.

Platelet microparticles are procoagulant subcellular vesicles released from activated platelets and facilitate coagulation.[34] In the present study, we found that the mean PMP level was significantly higher among NTDT compared with the control group. Numerous studies showed that PMPs were significantly higher in β-thalassemia intermedia patients than healthy controls.[8, 9, 24, 33]

In the present study, there was no significant correlation between PMPs and platelets. Similarly to our results, Moawad et al. (2022)[34] reported no statistically significant correlation between PMPs level and platelet count. In agreement, Abdelaziz et al. (2022)[24] reported no statistically significant correlation between PMPs and platelet count in their study; however, they reported a positive correlation between PMPs level and platelet count in splenectomised patients. In our study, a significant positive correlation between PMPs and platelet count was elicited only in splenectomized patients who have significantly higher platelets count. This agrees with Tantawy et al.[27] who stated that this may indicate chronic platelet activation and thrombotic tendency in these patients.

There have been several reports of thromboembolic complications in thalassemia due to spontaneous platelet aggregation.[29]

The spectrum of thrombosis varies from subclinical derangement of hemostatic parameters up to manifest TE events such as pulmonary embolism and deep venous thrombosis.[24, 26, 35] Simultaneously, silent thrombosis can occur, as subclinical thrombi in the microvasculature of lungs and brain. PHT is a complication of disease progression in the absence of transfusion therapy. NTDT patients are five times more likely to have PHT than TDT patients.[36]

None of our patients showed clinical evidence of PHT. Echocardiography remains the cornerstone screening test for diagnosis of PHT. [13] However, Supplementary data useful in evaluating PHT in thalassemia patients include markers of right heart dysfunction, such as amino-terminal fragments of proB-type natriuretic peptide (NT-proBNP). Measurement of NT-proBNP provides diagnostic and mechanistic information concerning the development of PHT in thalassemia, and its level is a strong indicator of PHT in these patients.[14]

The hypercoagulable state in NTDT is associated with a high frequency of TEE complications. Splenectomy is considered an independent risk factor.[37] Clinical studies also confirmed that splenectomized TI patients have a higher incidence of TEE than non-splenectomized patients.[38–40] In the present study, we had six splenectomized patients (12%) and 44 non-splenectomized patients (88%). We compared different laboratory parameters between them and found that splenectomized patients had significantly higher platelet count (as the spleen is a scavenger for platelets) and serum ferritin levels than non-splenectomized patients. Splenectomy was a strong causative factor to the hypercoagulable state in thalassemia.[39] Similarly, Fayed et al. (2018)[35] reported statistically higher platelet count and serum ferritin levels among splenectomized TI children than non-splenectomized patients.

Regarding MPs, the level of circulating PMPs was significantly higher in splenectomized patients compared to non-splenectomized patients however we found no statistically significant difference in EMPs levels between splenectomized and non-splenectomized patients. In contrast, In agreement with us, Rady Moawad et al. [34] reported that splenectomized children (n = 13) had significantly higher PMPs compared with non-splenectomized counterparts (n = 27). In contrast to our result, the study by Shahin et al. (2005)[23] showed that the splenectomized TI children (7/20) had significantly higher EMP levels than non-splenectomized children. Youssry et al. (2017) [8], in their cross-sectional study on 87 thalassemia pediatric patients (39 β-TM and 48 TI), reported that significantly higher levels of both EMPs and PMPs were found in splenectomized thalassemic patients versus non-splenectomized patients which is considered a strong predisposing factor for thrombosis.

In our study, we could not elicit a correlation between PMPs and D-dimer levels in splenectomized patients which may be related to the limited number of studied cases. However, Tantawy et al reported a positive correlation between PMPs and D-dimer levels in their patients. This may be due to the higher number and older age of their studied patients (adolescents and adults).

In the present study, we assessed the level of NT- proBNP in patients and the control group, and we found that their level was higher in patients than control though the difference is not statistically significant between the two groups. Studies on NT-proBNP are relatively few and mostly on adults and mainly in thalassemia major.

Safniyat et al. (2020)[41] in a study on 82 young adult thalassemic patients reported mean serum NT-proBNP within the normal range, but the highest values were found in the TI group. On the contrary, Zimaity et al. (2019)[37] and Mokhtar et al. (2011)[42] reported significantly high NT-proBNP levels in adult thalassemia patients. Similarly Balkan et al. (2012)[43] reported significantly high NT-proBNP levels in patients than in control. The most important finding of their study was the increase of NT-proBNP levels in asymptomatic β-TM patients compared to controls which means that NT-proBNP secretion begins in the early phase of the disease. Voskaridou et al. (2007)[14] reported significantly elevated values of NT-proBNP in patients with pulmonary hypertension however even patients without pulmonary hypertension had elevated levels compared to control. Thus they concluded that serum NT-proBNP is a strong indicator of PHT in thalassemic patients.

Although, we didn’t elicit a correlation between NT-proBNP and D-dimer levels in the patients, however, when we compared NT proBNP levels between patients with normal D-dimer levels (n = 29) and patients with high D-dimer levels (n = 21), we found that NT- proBNP level was significantly higher in patients with high D-dimer level.

We couldn’t elicit clinical evidence of TE manifestations in our studied patients. This can be explained by the fact that the age of occurrence of TEE is 18 years. (Shash, 2022)

Our study demonstrated that EMPs and PMPs levels were significantly elevated in NTDT patients compared to healthy controls. PMPs level significantly correlated with platelet count in splenectomized patients. The thrombotic risk in NTDT patients, particularly after splenectomy, might be related to the increased circulating platelets MPs which may be associated with platelets activations, increased platelets aggregation, and hence thrombus formation. D-Dimer level is more in splenectomized patients, but not correlated with platelets MPs and this may be attributed to the limited number of the studied patients. Assessment of NT-proBNP in high-risk NTDT may be a strong indicator for the development of pulmonary hypertension in these patients.

RECOMMENDATIONS

Based on the results of the present study, the following are recommended: assessment of EMPs and PMPs might provide efficacy for early detection of thromboembolic complications. Regular cardiac evaluation including adequate evaluation of NT-pro BNP is required to detect early pulmonary hypertension. As splenectomized NTDT patients are at a high risk of developing TEE, so they should be followed regularly.

LIMITATIONS OF THE STUDY

Our study has some limitations, given the sample size, which was relatively small.

Abbreviation	Full term
ADP	Adenosine 5-Diphosphate
MPs	Circulating Micro-Particles
CBC	Complete Blood Count
EMPs	Erythrocyte Micro-Particles
Hb	Hemoglobin
NTDT	Non-Transfusion Dependent Beta-Thalassemia
PE	Phosphatidylethanolamine
PS	Phosphatidylserine
PMP	Platelet Micro-Particles
PHT	Pulmonary Hypertension
RBCs	Red Blood Cells
NT- Pro-BNP	Serum B-Type Natriuretic Peptide
sTfR	Soluble Transferrin Receptor
SPSS	Statistical Package For Social Science
TTE	Thrombo-Embolic Event
β-TM	β-Thalassemia Major

Ethics approval and consent to participate

Ethics approval

This research got ethics and research committee approval from Ethics Committee at the Faculty of Medicine, Alexandria University, Egypt, IRB No. 0001298, FWA No. 00018699. EC Serial Protocol Number 0201580 at 18 November 2021

Consent to participate

Each patient agreed and signed an informed consent form in Arabic and English.

Consent for publication: each patient agreed to publication.

Availability of data and materials: Data available on request from the authors

Competing Interests: The authors declare no conflicts of interest. The authors declare that they have no significant competing financial, professional, or personal interests that might have influenced the performance or presentation of the work described in this manuscript.

Funding: The authors received no specific funding for the conduction of this study.

Authors' contributions

B.El-D. and M.S. conceived of the presented idea. N.H. developed the theory.

N.H. and N.S. verified the analytical methods.

N.S. and H.D. responsible for the laboratory investigations.

N.H. encouraged W.I. to collect the findings of this work. All authors discussed the results and contributed to the final manuscript.

All authors discussed the results and contributed to the final manuscript.

Acknowledgments The authors acknowledge the statistical analysis team.

Origa R: beta-Thalassemia. Genet Med 2017, 19(6):609-619. doi: 10.1038/gim.2016.173.
Musallam KM, Rivella S, Vichinsky E, Rachmilewitz EA: Non-transfusion-dependent thalassemias. Haematologica 2013, 98(6):833-844. doi: 10.3324/haematol.2012.066845.
Farmakis D, Porter J, Taher A, Domenica Cappellini M, Angastiniotis M, Eleftheriou A: 2021 Thalassaemia International Federation Guidelines for the Management of Transfusion-dependent Thalassemia. Hemasphere 2022, 6(8):e732. doi: 10.1097/HS9.0000000000000732.
Shash H: Non-Transfusion-Dependent Thalassemia: A Panoramic Review. Medicina (Kaunas) 2022, 58(10):1496. doi: 10.3390/medicina58101496.
Taher AT, Musallam KM, Karimi M, El-Beshlawy A, Belhoul K, Daar S, Saned MS, El-Chafic AH, Fasulo MR, Cappellini MD: Overview on practices in thalassemia intermedia management aiming for lowering complication rates across a region of endemicity: the OPTIMAL CARE study. Blood 2010, 115(10):1886-1892. doi: 10.1182/blood-2009-09-243154.
Sirachainan N: Thalassemia and the hypercoagulable state. Thromb Res 2013, 132(6):637-641. doi: 10.1016/j.thromres.2013.09.029.
Cappellini MD, Motta I, Musallam KM, Taher AT: Redefining thalassemia as a hypercoagulable state. Annals of the New York Academy of Sciences 2010, 1202(1):231-236. doi:
Youssry I, Soliman N, Ghamrawy M, Samy RM, Nasr A, Abdel Mohsen M, ElShahaat M, Bou Fakhredin R, Taher A: Circulating microparticles and the risk of thromboembolic events in Egyptian beta thalassemia patients. Ann Hematol 2017, 96(4):597-603. doi: 10.1007/s00277-017-2925-x.
Habib A, Kunzelmann C, Shamseddeen W, Zobairi F, Freyssinet JM, Taher A: Elevated levels of circulating procoagulant microparticles in patients with beta-thalassemia intermedia. Haematologica 2008, 93(6):941-942. doi: 10.3324/haematol.12460.
Atichartakarn V, Angchaisuksiri P, Aryurachai K, Chuncharunee S, Thakkinstian A: In vivo platelet activation and hyperaggregation in hemoglobin E/beta-thalassemia: a consequence of splenectomy. Int J Hematol 2003, 77(3):299-303. doi: 10.1007/BF02983790.
Atichartakarn V, Chuncharunee S, Chandanamattha P, Likittanasombat K, Aryurachai K: Correction of hypercoagulability and amelioration of pulmonary arterial hypertension by chronic blood transfusion in an asplenic hemoglobin E/beta-thalassemia patient. Blood 2004, 103(7):2844-2846. doi: 10.1182/blood-2003-09-3094.
Klaihmon P, Phongpao K, Kheansaard W, Noulsri E, Khuhapinant A, Fucharoen S, Morales NP, Svasti S, Pattanapanyasat K, Chaichompoo P: Microparticles from splenectomized beta-thalassemia/HbE patients play roles on procoagulant activities with thrombotic potential. Ann Hematol 2017, 96(2):189-198. doi: 10.1007/s00277-016-2885-6.
Fraidenburg DR, Machado RF: Pulmonary hypertension associated with thalassemia syndromes. Ann N Y Acad Sci 2016, 1368(1):127-139. doi: 10.1111/nyas.13037.
Voskaridou E, Tsetsos G, Tsoutsias A, Spyropoulou E, Christoulas D, Terpos E: Pulmonary hypertension in patients with sickle cell/beta thalassemia: incidence and correlation with serum N-terminal pro-brain natriuretic peptide concentrations. Haematologica 2007, 92(6):738-743. doi: 10.3324/haematol.11136.
IBM Corp.: IBM SPSS Statistics for Windows, Version 25.0. In. Armonk, NY: IBM Corp.; Released 2017.
Shapiro S: The Shapiro-Wilk and related test for normality. Statistics (Ber) 2015. doi:
Field A: Discovering Statistics Using IBM SPSS Statistics, 4th edn. London, California, New Delhi: SAGE Publications Ltd; 2013.
Curran-Everett D: Evolution in statistics: P values, statistical significance, kayaks, and walking trees. In., vol. 44: American Physiological Society Bethesda, MD; 2020: 221-224.
Almanaseer A, Chin-Yee B, Ho J, Lazo-Langner A, Schenkel L, Bhai P, Sadikovic B, Chin-Yee IH, Hsia CC: An Approach to the Investigation of Thrombocytosis: Differentiating between Essential Thrombocythemia and Secondary Thrombocytosis. Advances in Hematology 2024, 2024(1):3056216. doi:
Motawea KR, Varney J, Talat NE, Rozan SS, Chébl P, Reyad SM: Lactate Dehydrogenase can be Considered a Predictive Marker of Severity and Mortality of Covid-19 in Diabetic and Non-Diabetic Patients. A Case Series. American Heart Journal 2022, 254:246-247. doi:
Chopra N, Doddamreddy P, Grewal H, Kumar PC: An elevated D-dimer value: a burden on our patients and hospitals. International Journal of General Medicine 2012:87-92. doi:
Cappellini MD, Musallam KM, Poggiali E, Taher AT: Hypercoagulability in non-transfusion-dependent thalassemia. Blood Rev 2012, 26 Suppl 1:S20-23. doi: 10.1016/S0268-960X(12)70007-3.
Shahin RS, Al-Habibi A-SM, Raga Abdel-Salam M, Gouda RM: Circulating Erythrocyte Derived Microparticles in Pediatric Thalassemia Intermedia Patients. The Journal of the Egyptian Society of Haematology & Research 2005, 13(1):1. doi:
Aziz HMA, El-Beih EA, Sayed DM, Afifi OA, Thabet AF, Elgammal S, Mansor SG, Moeen SM: Increased levels of circulating platelet microparticles as a risk of hypercoagulable state in β-thalassemia intermedia patients. The Egyptian Journal of Haematology 2022, 47(3):187-193. doi:
Abo-Elwafa HA, Youseff LM, Mahmoud RA, Elbadry MI, Tawfeek A, Aziz SP: Venous Thromboembolism Risk Assessment among Beta-thalassemia Patients. Journal of Applied Hematology 2023, 14(3):230-235. doi:
Bhattacharyya M, Kannan M, Chaudhry VP, Mahapatra M, Pati H, Saxena R: Hypercoagulable state in five thalassemia intermedia patients. Clin Appl Thromb Hemost 2007, 13(4):422-427. doi: 10.1177/1076029607303539.
Tantawy AA, Adly AA, Ismail EA, Habeeb NM: Flow cytometric assessment of circulating platelet and erythrocytes microparticles in young thalassemia major patients: relation to pulmonary hypertension and aortic wall stiffness. Eur J Haematol 2013, 90(6):508-518. doi: 10.1111/ejh.12108.
Chaudhary HT, Ahmad N: Frequency of platelet aggregation defects in children suffering fromg β-thalassemia. Saudi Journal for Health Sciences 2012, 1(2):92-98. doi:
Eldor A, Rachmilewitz EA: The hypercoagulable state in thalassemia. Blood 2002, 99(1):36-43. doi: 10.1182/blood.v99.1.36.
Zahedpanah M, Azarkeivan A, Ahmadinejad M, Tabatabaiee MR, Hajibeigi B, Maghsudlu M: Evaluation of platelet aggregation in splenectomized beta-thalassemia major and intermedia patients. Journal of Applied Hematology 2018, 9(4):126-130. doi:
Mohammed Mowafy N, Zaki Ali El Zohairy Y, Mahmoud Hussien S, Saleh Sadek Mohamed A: EVALUATION OF RED BLOOD CELL MICROPARTICLES IN THALASSEMIA. Al-Azhar Medical Journal 2016, 45(3):611-620. doi:
Ferru E, Pantaleo A, Carta F, Mannu F, Khadjavi A, Gallo V, Ronzoni L, Graziadei G, Cappellini MD, Turrini F: Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase. Haematologica 2014, 99(3):570-578. doi: 10.3324/haematol.2013.084533.
Chinsuwan J, Klaihmon P, Kadegasem P, Chuansumrit A, Soisamrong A, Pattanapanyasat K, Wongwerawattanakoon P, Sirachainan N: High Prevalence of Antiphospholipid Antibodies in Children with Non-Transfusion Dependent Thalassemia and Possible Correlations with Microparticles. Mediterr J Hematol Infect Dis 2020, 12(1):e2020071. doi: 10.4084/MJHID.2020.071.
Moawad MR, Eldash HH, Hussein SK, Eid MMA: Platelet derived micro particles and the risk of pulmonary hypertension in Egyptian patients with thalassemia major. Life Science Journal 2022, 19(12). doi:
Fayed MA, Abdel-Hady HE, Hafez MM, Salama OS, Al-Tonbary YA: Study of platelet activation, hypercoagulable state, and the association with pulmonary hypertension in children with beta-thalassemia. Hematol Oncol Stem Cell Ther 2018, 11(2):65-74. doi: 10.1016/j.hemonc.2017.05.028.
Sleiman J, Tarhini A, Bou-Fakhredin R, Saliba AN, Cappellini MD, Taher AT: Non-Transfusion-Dependent Thalassemia: An Update on Complications and Management. Int J Mol Sci 2018, 19(1):182. doi: 10.3390/ijms19010182.
El Zimaity MT, Abdelbary HM, Mohamed HS: Brain natriuretic peptide as a sensitive biomarker for early detection of cardiac affection in adult Egyptian patients with β-thalassemia. The Egyptian Journal of Haematology 2019, 44(1):34-39. doi:
Taher A, Isma’eel H, Mehio G, Bignamini D, Kattamis A, Rachmilewitz EA, Cappellini MD: Prevalence of thromboembolic events among 8,860 patients with thalassaemia major and intermedia in the Mediterranean area and Iran. Thrombosis and haemostasis 2006, 96(10):488-491. doi:
Taher AT, Musallam KM, Karimi M, El-Beshlawy A, Belhoul K, Daar S, Saned M, Cesaretti C, Cappellini MD: Splenectomy and thrombosis: the case of thalassemia intermedia. J Thromb Haemost 2010, 8(10):2152-2158. doi: 10.1111/j.1538-7836.2010.03940.x.
Cappellini MD, Robbiolo L, Bottasso BM, Coppola R, Fiorelli G, Mannucci AP: Venous thromboembolism and hypercoagulability in splenectomized patients with thalassaemia intermedia. Br J Haematol 2000, 111(2):467-473. doi: 10.1046/j.1365-2141.2000.02376.x.
Safniyat S, Shakibazad N, Haghpanah S, Amoozegar H, Karimi M, Safaei S, Safniyat S, Mohammadi H, Zekavat OR: Parameters of tissue iron overload and cardiac function in patients with thalassemia major and intermedia. Acta Haematologica Polonica 2020, 51(2):95-101. doi:
Mokhtar GM, Tantawy AA, Adly AA, Ismail EA: Clinicopathological and radiological study of Egyptian beta-thalassemia intermedia and beta-thalassemia major patients: relation to complications and response to therapy. Hemoglobin 2011, 35(4):382-405. doi: 10.3109/03630269.2011.598985.
Balkan C, Tuluce SY, Basol G, Tuluce K, Ay Y, Karapinar DY, Gurgun C, Bayindir O, Kavakli K: Relation between NT‐proBNP levels, iron overload, and early stage of myocardial dysfunction in β‐thalassemia major patients. Echocardiography 2012, 29(3):318-325. doi:

Table (1): Laboratory parameters in NTDT patients and control groups

Laboratory parameter	NTDT Patients (n = 50)	Control (n = 50)	P
Hemoglobin Level (g/dl)	7.51 ± 1.21	11.42 ± 0.58	<.001^*
Red blood cell count (x10⁶/Ul)	3.78 ± 0.68	4.92 ± 0.21	<.001*
Reticulocytes (%)	3.72 ± 1.41	0.55 ± 0.57	<.001*
Total leukocytic count (×10³/μL)	9.09 ± 5.32	7.41 ± 1.58	.159
Platelets count (×10³/μL)	330.1 ± 207.4	299.1 ± 78.1	.432
Ferritin (ng/ml)	441.4 ± 433.9	35.0 ± 12.07	<.001*
sTfR (μg/ml)	204.2 ± 60.39	31.37 ± 9.75	<.001*

*: Statistically significant at p<.05

Table (2): Circulating red cells, platelets micro-particles and NT-proBNP in NTDT patients and control groups

Laboratory parameter	NTDT Patients (n = 50)	Control (n = 50)	P
EMPs (%)	4.52 ± 4.67	1.90 ± 0.87	<.001*
PMPs (%)	6.09 ± 4.43	2.11 ± 0.73	<.001*
NT- proBNP (pg/ml)	98.48 ± 17.51	91.82 ± 20.15	.081

Table (3): Correlation between EMPs, PMPs, and NT-proBNP levels with different parameters in patients group (n = 50)

EMP vs.	r_s	p-value
Hemoglobin Level (g/dl)	-0.095	0.512
Red blood cell count (x10⁶/Ul)	-0.027	0.852
Reticulocytes (%)	-0.010	0.944
Lactate Dehydrogenase (U/L)	0.236	0.099
Ferritin (ng/ml)	-0.008	0.959
PMP vs.
Platelets count (×10³/µL)	0.193	0.180
D-dimer (µg/L)	0.078	0.590
NT-proBNP level vs.
Hemoglobin Level (g/dl)	-0.020	0.893
Platelets count (×10³/µL)	0.080	0.581
D-dimer level (µg/L)	0.137	0.344
Ferritin level (µg/ml)	0.051	0.724
EMPs level	0.012	0.936
PMPs level	0.230	0.108

r_s: Spearman correlation coefficient

Table (4): Comparison between patients with normal D-dimer level and patients with high D-dimer level according to NT- proBNP Level (n = 50)

	D-dimer level		p
	Normal (n = 29)	High (n = 21)	p
NT proBNP level
Mean ± SD.	94.32 ± 15.39	104.2 ± 18.97	0.047^*

Table (5): Comparison between Splenectomized and Non-Splenectomized according to some laboratory parameters

	Splenectomized (n = 6)	Non-Splenectomized (n = 44)	P
Platelets Count (×10³/μL)
Mean ± SD.	612.0 ± 362.7	291.7 ± 145.4	0.005^*
D-dimer level
Mean ± SD.	582.3 ± 147.8	477.9 ± 147.9	0.081
Ferritin level
Mean ± SD.	863.3 ± 649.4	383.9 ± 370.5	0.016^*
sTfR level
Mean ± SD.	223.1 ± 62.16	201.6 ± 60.41	0.590
EMPs
Mean ± SD.	2.58 ± 1.20	4.78 ± 4.91	0.311
PMPs
Mean ± SD.	11.90 ± 8.05	5.30 ± 3.07	0.003^*
NT_proBNP level Mean ± SD.	101.2 ± 18.71	98.11 ± 17.54	0.692

No competing interests reported.

Assessment of Circulating Red Cell and Platelet Microparticles Levels in Children with Non-Transfusion Dependent Beta-Thalassemia

Status:

Version 1

Abstract

BACKGROUND

METHODS

RESULTS

DISCUSSION

CONCLUSION

RECOMMENDATIONS

LIMITATIONS OF THE STUDY

Abbreviations

Declarations

References

Tables

Additional Declarations

Status:

Version 1