|Year : 2018 | Volume
| Issue : 1 | Page : 25-31
CD177 expression in β-thalassemia patients in Zagazig University
Maha R Abd El-Wahed1, Heba H Gawish1, Ashgan A Ghobashy2, Noha E Ismail1
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Pediatric Hematology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||12-Aug-2017|
|Date of Acceptance||29-Aug-2017|
|Date of Web Publication||3-Aug-2018|
Heba H Gawish
Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Sharkia, Zagazig, 44519
Source of Support: None, Conflict of Interest: None
Background Human neutrophil-specific antigen CD177 has an important role in various hematological, clinical, and immunological syndromes. Individuals with thalassemia were found to show an elevation in CD177 expression, which may be correlated with hematopoietic activity.
Patients and methods Our case–control study was carried out at Clinical Pathology and Pediatric Hematology Departments, Zagazig University Hospitals, Egypt. This study was conducted on 80 (68) patients divided into two groups − group 1 included 34 patients diagnosed with β-thalassemia major, and group 2 included 34 apparently healthy individuals (control). All were subjected to routine clinical, laboratory, and radiological examination, as well as special laboratory examination: CD177% and mean fluorescence intensity (MFI) of gated neutrophils by flow cytometry and soluble transferrin receptor (sTFR) by enzyme-linked immunosorbent assay technique.
Results There was a statistically significant difference as regards CD177 (MFI) and sTFR in thalassemia patients when compared with the control group (P<0.001). There was no significant correlation between CD177 (MFI) or percentage with both fetal hemoglobin and sTFR. Receiver operating characteristic curve analysis revealed that CD177 (MFI) is more sensitive but less specific than sTFR.
Conclusion CD177 on gated neutrophils was increased in thalassemia patients but not correlated with their erythropoietic activity.
Keywords: CD177, soluble transferrin receptor, thalassemia
|How to cite this article:|
Abd El-Wahed MR, Gawish HH, Ghobashy AA, Ismail NE. CD177 expression in β-thalassemia patients in Zagazig University. Egypt J Haematol 2018;43:25-31
|How to cite this URL:|
Abd El-Wahed MR, Gawish HH, Ghobashy AA, Ismail NE. CD177 expression in β-thalassemia patients in Zagazig University. Egypt J Haematol [serial online] 2018 [cited 2020 Feb 19];43:25-31. Available from: http://www.ehj.eg.net/text.asp?2018/43/1/25/238540
| Introduction|| |
Thalassemia is the typical model of highly expanded, ineffective erythropoiesis, and it is one of the most common disorders in the Mediterranean areas and other parts of the world. Because thalassemia genes are present in a very heterogeneous state, their simultaneous presence in both β-chain alleles gives a highly variable phenotypic expression. The gene defect presented clinically by variable degree of presentation, from thalassemia minor, intermedia, to the severe form of thalassemia major, which is dependent on regular transfusion ,.
The CD177 is an important gene on neutrophils that encodes the gene of neutrophil membrane glycoprotein (gp) NB1. CD177 antigen was given another name as human neutrophil 2a, and the gp carrying this antigen was called NB1 gp . The CD177 is a result of production and clusteration of monoclonal antibodies, which is specific for NB1 gp .
CD177 gene is highly similar to a gene called PRV-1 gene, which is highly expressed on neutrophils from patients with polycythemia rubra vera. It has been reported that overexpression of PRV-1 gene (CD177) is seen in most patients with polycythemia vera and some patients with other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis . Contrary to above, CD177 is not expressed in patients with acute myeloid leukemia, chronic myeloid leukemia, thrombocytosis, and secondary erythrocytosis .
In all these disorders, the degree of ineffective erythropoiesis is the main factor that affects severity of the disease. Two serum indicators, used in recent years, appear to indicate the erythropoietic activity and expansion of bone marrow in β-thalassemia: (a) soluble transferrin receptor (sTFR) concentration and (b) levels of erythropoietin (Epo). In that respect, it is important to know that serum Epo concentration mainly represents the hypoxia of tissue, which is not correlated to the degree of anemia in some cases; therefore, serum Epo is not an ideal marker to the activity of erythroid tissue caused by anemia .
| Aim|| |
The aim of this study was to evaluate the expression of CD177 in patients with thalassemia major disease and to investigate the probable correlations with serum sTFR levels, and therefore the possibility of using CD177 expression as an index for the degree of erythropoiesis and hence severity in β-thalassemia major patients.
| Patients and methods|| |
Our case–control study was carried out in Clinical Pathology and Pediatric Hematology Departments, Faculty of Medicine Zagazig University Hospitals, Egypt. Institutional Review Board and Ethics Committee of Faculty of Medicine, Zagazig University, approved this study.
Our study included 68 patients divided into two groups. Group 1 included 34 patients diagnosed with β-thalassemia major, selected from the Outpatient Hematology Clinic, Pediatrics Department, Zagazig University Hospitals, Egypt. There were 21 male and 13 female patients, and their ages ranged between 1 and 18 years. Those patients were diagnosed by clinical diagnosis, complete blood picture, and hemoglobin (Hb) electrophoresis. Group 2 included 34 apparently healthy individuals, 20 male and 14 female, as a control group; they were age-matched and sex-matched to the patient group. None of the control participants were related to the patients. Informed consent was obtained from all patients.
Patients with high neutrophil count and C-reactive protein level, patients presenting with current infections, painful situations, and patient refusing to sign a written consent were excluded.
| Methodology|| |
All patients in the study were subjected to history taking and clinical examination, radiograph on skull and extremities, and abdominal ultrasonography to evaluate the spleen status.
A volume of 7 ml of venous blood was taken from all patients included in this study.
The blood sample was divided into two parts as follows:
- A volume of 3.5 ml of blood was collected in a plain tube, left to clot, centrifuged, and serum was separated into two Eppendorf tubes − one of them was frozen at −20°C and used later to determine sTFR and the other was used immediately for liver function tests, kidney function tests, and serum ferritin.
- 3.5 ml of blood was collected in a vacutainer EDTA-containing tube used for complete blood count that was analyzed by Advia 120 (Siemens Healthcare Diagnostics, OH, USA), CD177 by flow cytometry, and Hb electrophoresis by capillary electrophoresis (Minicap; Sebia, Evry, France)
Flow cytometeric analysis of CD177 kit (FITC anti-human CD177) BioLegend (San Diego, California, USA).
CD177 is also known as neutrophil-specific antigen 1, NB1, and polycythemia rubra vera 1. It is a member of the uPAR family and is a GPI-linked cell surface gp with a molecular weight of 60 kD. It is thought to be involved in allogeneic and autoimmune responses to neutrophils ,.
Collected EDTA blood samples were processed within 24 h of collection (preserved at room temperature) and stained according to the manufacturer’s description.
Acquisition was done by flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) using Cell Quest software (BD Biosciences, San Jose, CA, USA).
Analysis and interpretation
Gatting on peripheral blood neutrophils was done based on their scatter characteristics in of FSC/SSC. Percentage of CD177-positive cells in gated neutrophil populations is presented by mean fluorescence intensity (MFI).
Human soluble transferrin receptor by enzyme-linked immunosorbent assay
Principle of the assay: This based on the standard sandwich enzyme-linked immunosorbent assay technology ,,.
Frozen samples were thawed and processed according to the manufacturer’s description.
Calculation and interpretation:
- Standard and sample were measured in duplicate.
- For calculation: the relative OD 450=the OD 450 of each well−the OD 450 of zero well.
- The standard curve was plotted as the relative OD 450 of each standard solution (Y) versus the respective concentration of the standard solution (ng/ml) (X).
- The human sTFR concentration of the samples was interpolated from the standard curve.
| Results|| |
Our study included 68 patients divided into two groups. Group 1 included 34 patients diagnosed with β-thalassemia major, selected from the Outpatient Pediatric Clinic, Zagazig University Hospitals. There were 21 (61.8%) male and 13 (38.2%) female patients. Their age ranged from 1 to 18 years with a mean±SD of 12.7±4. Group 2 included 34 apparently healthy individuals, 20 (58.8%) male and 14 (41.2%) female, as a control group. Their age ranged from 6 to 18 years with a mean±SD of 15.4±4.60.
In our study, there was a high statistical difference in the MFI of CD177 expression on the gated neutrophils and sTFR (ng/ml) in thalassemic patients when compared with the control group (P<0.001). There was no statistical difference as regards CD177 (%) (P>0.05) ([Table 1] and [Figure 1]).
|Table 1 Comparison between thalassemic group and control group as regards CD177 (mean fluorescence intensity and percentage) and soluble transferrin receptor (ng/ml)|
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|Figure 1 Comparison of CD177 expression (MFI) on the gated neutrophils between patient group and control group. MFI, mean fluorescence intensity.|
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The frequency of the occurrence of bone changes in thalassemic patients in the form of bossing skull with prominent frontal and parietal bone and enlarged maxilla was 28 (82.4%) patients and the frequency of absence is six (17.6%) patients. Five (14.7%) patients had normal spleen, whereas 12 (35.3%) patients had splenomegaly and 17 (50%) patients had splenectomy ([Table 2]). The mean±SD of HbA% is 49.8±31.7, the mean±SD of HbF% is 45.8±32.5, and the mean of HbA2% is 3.3±1.4. The mean±SD of serum ferritin (ng/ml) is 1731.1±854.2. The comparison between male and female patients as regards CD177 (MFI and percentage) and sTFR showed no statistical difference between males and females in the patient group as regards CD177 (MFI) and sTFR (P>0.05), whereas there was a statistical difference as regards CD177 (%) (P<0.05) ([Figure 2]).
|Table 2 Frequency of bone changes and spleen status in the patient group|
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|Figure 2 Comparison between males and females in the patient group as regards CD177 (%).|
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The study revealed no statistical difference between patients with and without bone changes as regards CD177 MFI and percentage (P>0.05). There was a statistical difference regarding sTFR (P<0.05) ([Table 3] and [Figure 3]).
|Table 3 Comparison between patients with bony changes and those without in the patient group as regards CD177 (mean fluorescence intensity and percentage) and soluble transferrin receptor (ng/ml)|
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|Figure 3 Comparison between those with bony changes and those without in the patient group as regards transferrin receptor (ng/ml). sTFR, soluble transferrin receptor.|
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We found no significant correlation between CD177 MFI and percentage expression with serum sTFR concentration (P>0.05) ([Table 4]). Correlation between CD177 MFI and percentage expression on the gated neutrophils with fetal Hb in the patient group revealed that there was no significant correlation between them (P>0.05) ([Table 5]). The receiver operating characteristic curve analysis for CD177 expression (CD177 MFI) revealed sensitivity and specificity of 82.4 and 64.7%, respectively, with 73.6% accuracy, whereas sTFR (ng/ml) showed sensitivity and specificity of 70.6 and 82.4%, respectively, with 76.5% accuracy ([Table 6] and [Figure 4],[Figure 5],[Figure 6]).
|Table 4 Correlation between CD177 (mean fluorescence intensity) expression and serum soluble transferrin receptor concentration (ng/ml) in the patient group (n=34)|
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|Table 5 Correlation of fetal hemoglobin (%) with CD177 (mean fluorescence intensity) in the patient group (n=34)|
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|Table 6 Receiver operating characteristic curve for CD177 expression (mean fluorescence intensity and percentage) on the gated neutrophils and soluble transferrin receptor (ng/ml)|
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|Figure 4 ROC curve for CD177 MFI. AUC, area under curve; MFI, mean fluorescence intensity; ROC, receiver operating characteristic.|
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|Figure 5 ROC curve for sTFR (ng/ml). AUC, area under curve; ROC, receiver operating characteristic; sTFR, soluble transferrin receptor.|
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|Figure 6 ROC curve for CD177 (%). AUC, area under curve; ROC, receiver operating characteristic.|
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| Discussion|| |
CD177 is an important neutrophil gene that encodes the neutrophil membrane gp NB1 . Previous studies have shown that the CD177 expression on neutrophils is increased in a number of physiologic and pathological states, including umbilical cord blood, pregnancy, anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, β-thalassemia, myeloproliferative neoplasm (MPNs), systemic lupus erythematosus, people with severe infections or burns, and in patients given granulocyte colony-stimulating factor .
Neutrophil CD177 expression is not increased in patients with chronic myelogenous leukemia or acute myelogenous leukemia . Our study has focused on CD177 expression in individuals with β-thalassemia major. In such cases, the severity of the disease depends mainly on the amount of ineffective erythropoiesis. TFR and sTFR play an important role in iron hemostasis and many associated conditions. In recent decades, the link between sTFR and a large number of hematological disorders has been investigated. Many conditions are associated with changes in sTFR concentration. Increased amounts of sTFR have been reported in autoimmune hemolytic anemia, hereditary spherocytosis, α-thalassemia and β-thalassemia/HbE, HbH disease, sickle-cell anemia, polycythemia vera (all characterized by increased erythroid proliferation), and in iron-deficiency anemia (decreased tissue iron stores) .
Idiopathic myelofibrosis, myelodysplastic syndrome, and chronic lymphocytic leukemia were associated with normal or increased sTFR levels .
Normal sTFR concentrations have been shown in hemochromatosis, acute and chronic myeloid leukemia, and solid tumors. Patients with chronic renal failure and aplastic anemia, as well as those post bone marrow transplantation, have decreased TFR concentrations ,.
Our study showed a highly significant increase in MFI of CD177 expression on the gated neutrophil in thalassemic patients compared with the controls, which may be linked to their Epo-dependent expanded erythropoietic activity. These results were similar to results of Zoi et al. , who quantified CD177 mRNA by real-time quantitative PCR and reported that patients with thalassemia had increased expression of CD177 mRNA.
Taniguchi et al.  reported that CD177% expression was greater in females (70.2±18.3%) than in males (66.8±15.0%). Matsuo et al.  noted the difference in human neutrophil 2a expression by sex. This was compatible with results of our study in the patient group that revealed a significant difference between male and female children regarding CD177% expression on the gated neutrophils .
El Nawawy et al.  stated that sTFR in regularly transfused thalassemic patients could be used as an accurate and reliable indicator of successful erythroid marrow suppression and for the determination of optimal pretransfusion Hb levels in thalassemia on an individual basis, with sTFR being the most sensitive indicator. Zoi et al.  and Ricchi et al.  reported higher levels of serum sTFR in patients with β-thalassemia major syndromes compared with the healthy controls. All of them reported a highly significant increase in serum sTFR in the thalassemic patients when compared with controls.
In contrast to the results of the study by Zoi et al. , we found no significant correlation between MFI and percentage of CD177 expression on the gated neutrophils and serum sTFR concentration. This could be explained by the different age group and methodology.
Our study revealed no significant correlation between Hb concentration and neither CD177 expression (MFI and percentage) on the gated neutrophils (P=0.48, 0.88) nor serum sTFR concentration (P=0.48) in the thalassemia group.
In agreement with our work, researchers who studied thalassemia intermedia reported that no clear relationship was observed between Hb concentration and serum sTFR concentration . Tancabelic et al.  stated that sTFR levels were only slightly elevated in thalassemic cases (1.5 times controls). This can be explained by the fact that total erythropoietic expansion in thalassemia is not only Epo-related but also dependent on genetic capability of the erythroid cell to produce HbF. The relationships between anemia, HbF, and total erythropoiesis in thalassemia are more complex than expected; hypertransfusion regimen used in those patients with higher pretransfusion Hb levels can tremendously affect the extent of erythropoietic expansion.
On the other hand, investigators found that there was a significant inverse correlation between Hb and sTFR in thalassemia major ,,.
A work by Cazzola et al.  studied the relationship between transfusion regimen and suppression of erythropoiesis in β-thalassemia major; they stated that sTFR was the parameter more closely related to mean pretransfusion Hb.
Montaser et al.  found that Hb and sTFR levels showed a strong negative correlation in β-thalassemic cases (inverse correlation).
We found no significant correlation between absolute neutrophil count and CD177 expression on the gated neutrophil (percentage and MFI). This reflects that the increased CD177 expression in thalassemic patients was not related to increased neutrophil count, and most probably reflects the increase in erythopoietic activity in thalassemic patients.
Our work showed that sTFR levels have no differences observed with age range, but Allen et al.  reported that levels are somewhat lower in fetuses and newborns compared with adults. The difference in hormonal level might be responsible for the increased neutrophil expression of CD177 in adults than in children .
In the early postnatal period, sTFR levels are comparable to those of adults with marrow suppression, but they soon increase again so that infants and adolescents tend to have slightly higher TFR levels than adults .
Receiver operating characteristic curve analysis of sTFR and CD177 expression on the gated neutrophils (MFI) revealed that CD177 expression on the gated neutrophils was more sensitive but less specific than sTFR in detection of erythropoietic activity in thalassemic patients.
| Conclusion|| |
CD177 expression on the gated neutrophils was increased in thalassemic patients but not correlated with their erythropoietic activity assessed by the measurement of serum sTFR levels as a simple serum marker for the erythropoietic activity. Higher-scale studies might be needed to evaluate both markers as regards their role in detecting the erythropoietic activity of thalassemic patients and whether the combining of the two markers is useful or not.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]