| Abstract|| |
Background: CD71 is a marker that has been usually used for identifying dysplasia in the erythroid series. We have tried to evaluate the expression of CD71 in various types of acute leukemias. Materials and Methods: We studied 48 patients of acute leukemia, of which 25 were acute myeloid leukemia (AML), 13 were precursor B-acute lymphoblastic leukemia (B-ALL), 8 were T-ALL, and 2 were mixed phenotype acute leukemia (T/myeloid) as per the WHO classification. Results: We found that the expression of CD71 was most prevalent in AMLs (84%), followed by T-ALL (50%) and least in B-ALL (30%). Conclusion: This finding clearly shows the higher expression of CD71 in AMLs compared to other common type of leukemias, such as B- and T-ALL. We suggest that the high expression of CD71 in AMLs could be used as a diagnostic marker and may also be used for minimal residual disease analysis after further studies in posttreatment scenario. This study is the first of its kind in the South Asian population.
Keywords: Acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, CD71, flow cytometry
|How to cite this article:|
Pande A, Dorwal P, Jain D, Tyagi N, Mehra S, Sachdev R, Raina V. Expression of CD71 by flow cytometry in acute leukemias: More often seen in acute myeloid leukemia. Indian J Pathol Microbiol 2016;59:310-3
|How to cite this URL:|
Pande A, Dorwal P, Jain D, Tyagi N, Mehra S, Sachdev R, Raina V. Expression of CD71 by flow cytometry in acute leukemias: More often seen in acute myeloid leukemia. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Jun 20];59:310-3. Available from: http://www.ijpmonline.org/text.asp?2016/59/3/310/188145
| Introduction|| |
Immunophenotyping of leukemic cells with monoclonal antibodies has become a universally accepted part of the workup of a patient with acute leukemia. The most widely used method nowadays is staining of cells in suspensions with fluorochrome-labeled antibodies, followed by flow cytometric analysis.
A number of CD markers are required for characterization and differentiation of leukemia. However, there will always be discrepancies to decide an optimal panel of antibodies. CD71 has been considered as one of the useful markers for evaluating immature cells in addition to other CD markers. CD71, the transferrin receptor 1, is a Type II transmembrane homodimeric glycoprotein (180 kDa) responsible mainly for cellular iron uptake via internalization of iron-loaded transferrin., Iron uptake occurs via the internalization of iron-loaded transferrin mediated by the interaction with the CD71, which helps in many metabolic processes for cell growth and proliferation, including DNA synthesis, electron transport, nitrogen fixation, and oxygen sensing., CD71 expression is increased on rapidly proliferating cells while expression is decreased or absent on nondividing cells. Some studies suggested that biologically aggressive tumors require large amounts of iron for active metabolism and rapid cell growth, and overexpression of CD71 has reported in different types of cancers such as glioma, pancreatic, and colon cancers.
Several studies have revealed that CD71 expression is decreased in dysplastic erythroid precursors. The significance of CD71 in MDS remains a subject of further investigation.,, Interestingly, lack of CD71 expression has been reported in Acute Erythroid Leukemia.
It has been reported and recommended that at least one or more B, T, myeloid, erythroid, and megakaryocytic reagents should be included in the essential CD marker panel for acute leukemia. In leukemias, we speculated that CD71 expression may be associated with the abnormal antigen expression. The level of expression for CD71 in different blast population may differ and therefore can be used as an aid for characterization of acute leukemia.
In the present study, we evaluated flow cytometric CD71 expression on blast population in different acute leukemias to assess the positivity and identify the associated level of expression with each different kind of acute leukemia, i.e., acute myeloid leukemia (AML), B-acute lymphoblastic leukemia (B-ALL), T-ALL, and mixed phenotype acute leukemia (MPAL) (B/myeloid).
| Materials and Methods|| |
Forty-eight bone marrow samples were collected from newly diagnosed patients with acute leukemia in our department. Twenty-five patients were diagnosed with AML, 13 patients had B-ALL, 8 patients had T-ALL, and 2 patients had MPAL (B/myeloid). Along with this, 5 normal bone marrow samples and 2 samples having increased number of hematogones were evaluated for CD71 expression.
CD71 surface expression was measured on bone marrow blast cells. Staining to identify blastic cells was chosen based on the routine bone marrow immunophenotyping performed for diagnostic purposes. The antibodies (surface and cytoplasmic) included anti-CD3 (cytoplasmic), CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD20, CD22 (cytoplasmic), CD33, CD34, CD45, CD56, CD61, CD64, CD71, CD79a (cytoplasmic), CD117, GlyA (CD235a), HLA-DR, and MPO (cytoplasmic). All antibodies used were from Becton Dickinson (BD) Biosciences, San Jose, CA, USA. CD71 (FITC, clone L01.1) was used for this study. Company recommended amount of respective antibodies, and 100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended. The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min. The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired.
The samples were then analyzed using a six-color flow cytometer (BD FACSVerse, Becton Biosciences, San Jose, CA, USA) with antibody panels against a variety of lymphoid, myelomonocytic, erythroid, and megakaryocytic antigens. List-mode data files were analyzed for each specimen using the FACSuite Software (Becton Biosciences, San Jose, CA, USA). Day-to-day fluorescence standardization of the flow cytometry was performed with BD Cytometer setup and tracking (CS&T) beads (Becton Biosciences, San Jose, CA, USA) by performing performance QC and assay and tube setting updates.
FACSuite software (Becton Biosciences, San Jose, CA, USA) was used for the analysis. A total of 20,000 cells were recorded per sample. Threshold gating was assigned based on forward scatter and side scatter (SSC) to exclude red blood cells, dead cells, and cell debris. A CD45/SSC gating strategy was used to gate the blasts cells. These cells were then plotted on CD71 histogram. The lymphocytes were taken as reference internal negative control. The staining intensity of CD71 on these cells was measured by the median fluorescence intensity (MFI). Because CD71 fluorescence and its respective control fluorescence were recorded using identical instrument settings, MFI values were independent of variations in signal amplification. CD71 was considered positive if the positive population comprised more than 20% of the cells. An unlabeled tube was run with each sample with only the gating marker (CD45) to set up the quadrant marker.
Statistical analysis was done with SPSS version 16.0 statistical software (IBM Corporation, Armonk, NY, USA). Variables were presented as mean, standard deviation, median, maximum, and minimum. Z-test was performed for comparison, and a P < 0.05 was considered to be statistically significant.
| Results|| |
Of the 48 acute leukemia patients evaluated, 25 had the diagnosis of AML while 13 were precursor B-ALLs, 8 were T-ALL, and 2 cases of MPAL (B/myeloid) were also studied. CD71 positivity was seen in 64.5% of all the leukemia cases (31 out of 48). Majority of AMLs showed positivity for CD71, being positive in 84% of the cases [Figure 1]. The CD71 positivity rate was only 30% in cases of B-ALL (4 out of 13 cases) [Figure 2]. T-ALLs have similar proportion of positive and negative cases (4 each), thereby giving a positivity of 50% [Table 1]. We can therefore infer that the CD71 positivity is seen maximum in cases of AML followed by T-ALL and least in cases of B-ALL. Z-test was performed to look for significant difference in the positivity in AML, B-ALL, and T-ALL cases. Significant difference was found between AML and B-ALL cases (P = 0.0009). However, no significant difference was observed between AML and T-ALL or B-ALL and T-ALL (P > 0.05).
|Figure 2: Lack of CD71 expression in B acute lymphoblastic leukemia case|
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|Table 1: Distribution of acute leukemia cases for CD71 expression and median fluorescence intensity of acute leukemia cases with CD71 positivity|
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Among the positive AML cases, the mean MFI of CD71 was found to be 524.50 while the mean MFI of positive B-ALL cases was found to be 378.56. The mean MFI of T-ALL with positive CD71 was 825.89. This shows that the T-lymphoid blasts have the brightest expression of CD71 followed by myeloid blasts and minimum with B-lymphoid blasts. The normal bone marrow samples gave the mean MFI of CD71 as 424.75, which was intermediate between B-ALL and AML. The hematogones were found to be negative for CD71 (mean MFI = 35). The erythroid population (CD45 negative, nonblast events) showed expression which was over and above all the leukemia cases with mean MFI of 3016.58.
| Discussion|| |
The present study also demonstrates that CD71 expression is seen maximum in cases of AML (84%) compared to B-ALL (30%) and T-ALL (50%). The difference in positivity of AML and B-ALL was found to be statistically significant.
The present findings of higher MFI of CD71 in the cases with T-ALL than the B-ALL correlate with the earlier studies done by Koehler et al. and Ploszynska et al. In our study, B-ALL and T-ALL patients show the CD71 positivity of 30% and 50%, respectively. In addition, the CD71 expression was highest seen in T-ALL followed by AML and the lowest expression in B-ALL was seen in the present study, which is similar to the earlier reports.,
It is well known that the erythroid cells express a higher level of CD71 than other lineage cells to facilitate the requirement of iron for hemoglobin synthesis. In our study, we found the CD71 expression of the erythroid population to be higher than all the leukemic blasts, which concurs with the earlier studies.
The importance of CD71 also reflects in the cases with remission and relapse where it may be helpful in the identification of the minimal residual disease, especially in cases of AML when its expression is more pronounced. However, this requires further studies at multiple stages in posttreatment scenario.
During flow cytometry analysis, we also observed that CD71 expression was not limited to erythroid precursor cells but was also found on other blasts, consistent with an earlier observation.
| Conclusion|| |
The present study demonstrates that the expression of CD71 is seen in majority of AML cases, followed by T-ALL and B-ALL cases. We could also demonstrate that the expression of CD71 (as MFI) is highest in cases of T-ALL followed by AML and least in cases of B-ALL. This is the first study of its kind in the Indian population.
We would like to thank Mr. Manish Singh for the support with statistics and Mr. Vijay Bahadur for logistical support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Pranav Dorwal
Department of Pathology and Laboratory Medicine, Medanta - The Medicity, Sector 38, Gurgaon - 122 001, Haryana
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]