|Year : 2015 | Volume
| Issue : 1 | Page : 19-26
Value of CD11a and CD18 in flow cytometric immunophenotypic diagnosis of acute promyelocytic leukemia
Azza M S El Danasoury1, Abeer A Saad El Dein1, Mervat A Al-Feky1, Seham M Ezzat2, Mohamed T Sallam1, Gihan M Kamal3, Ghada Gohary3, Rania I El-Meehy1
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical Pathology, Tanta Cancer Center, Tanta, Egypt
3 Department of Internal Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Web Publication||15-Apr-2015|
King Faisal Specialist Hospital and Research Center, P. O. Box 3354, MBC: 64, Riyadh 11211
Source of Support: None, Conflict of Interest: None
As acute promyelocytic leukemia (APL) has the highest curability among different acute amyeloid leukemia (AML) subtypes and requires a unique form of treatment, it is important to quickly establish or exclude the diagnosis of this subtype of leukemia. Furthermore, accurate and rapid diagnosis of APL is critical due to its association with disseminated intravascular coagulation. Patients and methods: This study was conducted on 100 newly diagnosed AML M0-M3 patients and a comprehensive flow cytometric immunophenotypic (FCM IPT) analysis of their peripheral blood (PB) and/or bone marrow samples with the inclusion of CD18 and CD11a monoclonal antibodies was done. Results: Cases of AML-M3 did not express human leukocyte antigen (HLA)-DR, CD11a and CD18, a highly significant different finding compared to non M3 cases (P < 0.001). The positive predictive value (PPV), negative predictive value (NPV), specificity and sensitivity of HLA-DR, and CD34 negativity (alone and in combination) were lower than the PPV, NPV, specificity and sensitivity of the combined negativity HLA-DR, CD11a, and CD18 antigens. Conclusion: Testing for CD18 and CD11a in conjunction with routine diagnostic FCM IPT panel will append extra diagnostic power to FCM IPT in the differentiation of difficult cases of AML-M3 from AML-(M0-M2).
Keywords: Acute promyelocytic leukemia, CD11a, CD18
|How to cite this article:|
El Danasoury AM, Saad El Dein AA, Al-Feky MA, Ezzat SM, Sallam MT, Kamal GM, Gohary G, El-Meehy RI. Value of CD11a and CD18 in flow cytometric immunophenotypic diagnosis of acute promyelocytic leukemia. J Appl Hematol 2015;6:19-26
|How to cite this URL:|
El Danasoury AM, Saad El Dein AA, Al-Feky MA, Ezzat SM, Sallam MT, Kamal GM, Gohary G, El-Meehy RI. Value of CD11a and CD18 in flow cytometric immunophenotypic diagnosis of acute promyelocytic leukemia. J Appl Hematol [serial online] 2015 [cited 2022 Dec 3];6:19-26. Available from: https://www.jahjournal.org/text.asp?2015/6/1/19/155180
| Introduction|| |
Acute amyeloid leukemia (AML) is a highly aggressive malignant disease resulting from genetic alterations in hematopoietic progenitor cells.  However, due to the nature of hematopoiesis, there are numerous points along the differentiation process of myeloid cells where the expansion of leukemia cells of various maturation stages is possible.  In 1976, the French-American-British cooperative group (FAB) published guidelines for the classification of AML which were based on both differentiation and maturation pattern of leukemia cells. 
This classification has been revised to include proposals of M0-M7 groups.  Though separate entities themselves, M0-M3 all demonstrate some degree of differentiation which primarily affects granulocytes and they differ mainly in their respective maturation stages along the granulocytic pathway. 
Clinically, useful subgrouping focuses increasingly on prognostically significant antigenic features and/or phenotypes that determine therapy. Moreover, recognition of antigen expression patterns that reflect genotypes has gained interest. 
Acute promyelocytic leukemia (APL) or AML M3 according to FAB was related to translocation (15;17) (q22;q21). By morphology, APL is characterized by abnormal promyelocytes often containing Auer rods More Details and coarse azurophilic granules. However, in approximately 20% of cases, the promyelocytes are small and hypogranular.  The prompt and correct diagnosis of APL is especially important due to the dangerous association with disseminated intravascular coagulation. 
The single-cell declaration makes flow cytometry extremely different from any other tool utilized in diagnostic hematopathology. 
Flow cytometry immunophenotyping can serve as a screening test for APL before the results of cytogenetic or molecular testing for t(15;17)(q22;q21)/PML-RARα.  The immunophenotype of the leukemic cells in patients with APL associated with t(15;17) (q22;q21) has long been considered highly specific. However, other APL-like phenotypes, presumably lacking sensitivity to treatment with ATRA have been reported and must be reliably distinguished from ATRA sensitive PML/RAR pos APL, given the availability of this genotype-specific therapy. ,,
CD11a/CD18 is expressed on early hematopoietic progenitor cells and on all mature leukocytes.  CD11a/CD18 (beta-integrin family) is a member of integrins which are major cell adhesion molecules, mediating cell-cell and cell-extracellular matrix interactions.  The integrins have been organized into eight distinct subfamilies based on β-subunit associations. The integrin β2 subunit CD18 forms heterodimers with each of four CD11 α-subunits - CD11a (αL), CD11b (αM), CD11c (αX), and CD11d (αD) - to form a subclass of integrins known as β2 integrins, or CD11-CD18. ,, CD11a/CD18 (αLβ2, LFA-1) is the most broadly expressed leukocytes integrins. 
Aim of the Study
This study aims to fully characterize flow cytometric (FCM) immunophenotype of AML M0-M3 using a comprehensive panel of monoclonal antibodies with the inclusion of CD11a and CD18. Data will be analyzed to resolve the immunophenotypic (IPT) overlap and define the possible role of FCM in the precise diagnosis and differentiation of AML-M3 associated with t(15;17) from AML (M0-M2).
| Patients and methods|| |
This study was conducted on one hundred newly diagnosed adult AML patients FAB (M0-M3) attending the Hematology/Oncology unit of Ain Shams University hospitals, during the period from September 2008 to December 2010.
All patients were subjected to the following:
Complete history taking, thorough clinical examination, abdominal ultrasonography, and diagnostic workup for AML cases including the following:
Final diagnosis and classification of AML patients was according to FAB and WHO classifications of acute myeloid neoplasms. Suspected AMl-M3 cases and suspected AML-M2 cases were tested for t(15;17) and t(8;21) respectively by fluorescence in situ hybridization (FISH). Only positive cases were included in this study.
- Complete blood counts (CBC) on Coulter LH-750 cell counter (Coulter Electronics, Hialeah, FL, USA) with examination of PB smears stained with Leishman stain
- Bone marrow (BM) aspiration with morphological examination of Leishman stained smears
- Cytochemical studies for myeloperoxidase (MPO)
- FCM IPT: Performed on PB or BM samples using an extended panel of MoAb on Coulter Epics XL 3-color flow cytometer (Coulter Electronics, Hialeah, FL, USA).
Flow Cytometric Immunophenotyping
- Two milliliter PB samples were obtained on ethylenediamine tetra-acetic acid, dipotassium salt (K 2 -EDTA) in vacutainer tubes (final concentration of 1.5 mg/mL) for CBC and FCM IPT
- Half to one milliliter BM aspirate was obtained; smears were prepared and stained with Leishman stain for morphological examination and cytochemical staining
- One milliliter BM aspirate was obtained on K 2 -EDTA in vacutainer tubes for FCM IPT
- One milliliter BM aspirate was obtained on lithium heparin in vacutainer tubes for FISH analysis. Samples were referred on the same day of sampling to Cytogenetic Unit at Ain Shams University Hospitals.
- A panel of MoAbs was used including antibodies against progenitor antigens (CD34 and human leukocyte antigen [HLA]-DR), myeloid markers (CD13, CD33, CD14 and cytoplasmic MPO), B-cell markers (CD10, CD20, CD19, and CD79a) and T-cell markers (CD2, CD3, CD5 and CD7). In addition to CD11a and CD18 which were tested for their pattern of expression in AML cases; all MoAbs were supplied by Beckman Coulter, Fullerton, CA, USA. All antibodies were fluorescein isothiocyanate (FITC) labeled except CD34, CD14, CD10, CD7, and CD18, which were labeled by the phycoerythrin (PE)
- Phycoerythrin-cyanine 5 labeled CD45 was used for gating on cells of interest
- Specific isotypic controls for FITC and PE-conjugated monoclonal antibodies were used
- Phosphate-buffered saline (PBS): 120 mMNaCl, 2.7 mMKCl, 10 mM phosphate buffer, pH 7.4; commercially available from Sigma, St. Louis, MO
- Ammonium chloride-based erythrocyte lysing solution: 8.29 g (0.15M) NH 4 Cl, 1 g (10 mM) KHCO 3 , 0.037 g (0.1 mM) EDTA, 1 L distilled water, adjusted to pH 7.3.
- PB or BM aspirate samples were processed on the same day of sample collection. They were counted using coulter cell counter, and the total leukocytic count was adjusted to be around 5 × 10 9 /L using PBS
- 100 μL of adjusted sample was aliquoted in the control tube as well as each test tube
- 20 μL of CD45 was added to each tube (control and test tubes)
- 20 μL of each MoAb or isotypic control was added into the corresponding tubes
- The control and test tubes were incubated for 15 min at room temperature, protected from light
- After incubation, 1-2 mL of ammonium chloride-based erythrocyte lysing solution was added to every tube
- Tubes were vortexed then analyzed using Coulter Epics XL flow cytometer (Coulter Electronics, Hialeah, FL, USA).
*Samples were considered positive for a certain marker when ≥20% of cells were expressing it, except for CD34 and MPO where expression by 10% of cells was sufficient to confer positivity (Matutes et al., 2006).
Statistical analysis of the data was performed using SPSS 15 software package under Windows XP operating system.
The utility of the four markers (CD34, HLA-DR, CD11a, and CD18) in the differentiation of M3 from non-M3 AML was evaluated by the calculation of the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).
Since the aim was to evaluate the four markers for diagnosis of M3 carrying t(15;17), the truly positive cases were those diagnosed as M3 positive for t(15;17) by FISH.
| Results|| |
The study included 100 AML patients who were admitted at the Hematology and Oncology Unit in Ain Shams University. These patients were classified into two groups: M3 group and non M3 group (M0-M2) according to FAB and WHO classifications of acute myeloid neoplasms. ,
Acute Amyeloid Leukemia-M3 Group
This group included 20 classic M3 cases. They were 12 males and 8 females with a male to female ratio 1.5:1. Their age ranged from 22 to 46 years with a mean of 34.3 ± 6.8. Diagnosis of AML-M3 cases was based on clinical PB and BM findings and was confirmed by positivity for t(15;17) using FISH in all cases [Figure 1].
|Figure 1: Typical case of acute amyeloid leukemia-M3; positive for t(15;17) by fluorescence in situ hybridization|
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This group included 80 patients, 46 males and 34 females with male to female ratio 1.4:1. Their age ranged from 29 to 63 years with a mean of 44.2 ± 9.5. They included 3 subgroups; M0 (8 cases), M1 (44 cases) and M2 (28 cases).
The clinical findings of M3 and non M3 groups are summarized in [Figure 2].
|Figure 2: Frequency of positive clinical findings in M3 and non M3 groups|
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Immunophenotypic Differences Between M3 Group and Non-M3 Group (M0-M2)
[Table 1] summarizes the comparison between the expressions of different markers in the two groups and [Figure 3] and [Figure 4] illustrate immunophenotyping of typical M3 and M2 cases respectively.
|Table 1: Comparison between the expression of different markers in M3 and non M3 patients |
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Utility of the Four Markers (CD34, Human Leukocyte Antigen-DR, CD11a, and CD18) in Distinguishing M3 from Non-M3 Cases
[Table 2] shows the sensitivities of the negativity of each the four markers: CD34, HLA-DR, CD11a and CD18 for diagnosis of M3 to be 95%, 100%, 100%, and 100%, respectively. The sensitivity of double negativity for CD34 and HLA-DR and the sensitivity of the negativity for the triad HLA-DR, CD11a and CD18 was 100%. In this study, we have taken the cytogenetic findings as a reference for both positivity and negativity as seen in [Table 3].
|Table 2: CD34, HLA-DR, CD11a, and CD18 expression in M3 and non-M3 patients |
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|Table 3: Performance of the four relevant markers (single or in combination) in the diagnosis of M3 t(15;17) |
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The specificities of the negativity of each of the four markers: CD34, HLA-DR, CD11a, and CD18 were 81.2%, 90%, 98.7%, and 93.7%, respectively. The specificity of double negativity for CD34 and HLA-DR (93.8%) was lower than that of the negativity of the triad HLA-DR, CD11a, and CD18 (100%), [Figure 5] and [Table 3].
|Figure 5: The diagnostic performance of negativity to (human leukocyte antigen [HLA]-DR and CD34) versus (HLA-DR, CD11a, and CD18) as regard the sensitivity, specificity, positive predictive value, and negative predictive value in M3-acute amyeloid leukemia|
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| Discussion|| |
Flow cytometry is a valuable tool for the diagnosis of acute leukemia and is now replacing cytochemistry in most routine laboratories worldwide. , Due to the great degree of heterogeneity which is displayed by AML, the practice of immunophenotyping in AML is somewhat more difficult, and interpretation is less clear than in other hematological malignancies such as ALL. However, immunophenotyping of AML by flow cytometry is of value with the utilization of monoclonal antibodies which are specific for both different paths of differentiation and for different stages of maturation and thus the differentiation and maturation of the subclass of AML. 
Acute promyelocytic leukemia is characterized by leukemic cells blocked at the promyelocytic stage of granulocytic differentiation.  Accurate and rapid diagnosis of APL is critical due to its association with disseminated intravascular coagulation.  Also, because APL has the highest curability and requires a unique form of treatment, it is important to quickly establish or exclude the diagnosis of this subtype of leukemia.  This is made possible by recognizing the characteristic morphologic features and the typical immunophenotype of APL, with confirmation by molecular and cytogenetic testing for the presence of t(15;17). The diagnosis becomes more difficult when APL with documented t(15;17) falls into the morphologic spectrum of other subtypes of AML in the FAB classification or when a case of AML with an immunophenotype characteristic of APL has no detectable molecular or cytogenetic evidence of the t(15;17). ,
The aim of this study was to perform a comprehensive FCM IPT analysis of acute myeloid leukemia FAB (M0-M3) with the use of an extended panel of monoclonal antibodies in an attempt to better define the IPT characteristics of cells in AML-M3 patients with t(15,17). This would strengthen the role of FCM in distinguishing ATRA sensitive APL from other APL-like phenotype.
This study was conducted on one hundred newly diagnosed AML patients attending the Hematology/Oncology unit of Ain Shams University hospitals, during the period from September 2008 to December 2010.
Diagnosis was based on a thorough history taking and clinical examination together with laboratory investigations including CBC, BM aspiration together with immunophenotyping. FISH studies were performed to diagnose t(15;17) positive, ATRA sensitive M3 and t(8;21) positive M2. All 100 cases were tested for the expression of CD11a and CD18 on PB or BM samples.
In the current study, 20 patients were diagnosed as having APL. The provisional diagnosis of APL was considered based on FCM IPT of leukemic cells. Absence of CD34 and HLA-DR expression together with an expression of CD13, CD33, and strong MPO expression were considered characteristic of APL. Diagnosis was confirmed in all cases by FISH analysis for t(15;17).
One of the most striking features of APL is its age-associated incidence rate. Its incidence increases steadily during the teen years, reaches a plateau during early adulthood, and remains constant until it decreases after the age of 60 years.  This is in concordance with our study in which the age was significantly lower in M3 patients (22-46 years with a mean age of 34.3 ± 6.8) compared to non-M3 patients (29-63 years with a mean age of 44.2 ± 9.5), P < 0.05.
In this study, CD13, CD33, and MPO were positive in 70%, 100% and 100% of cases in the M3 group compared to 65%, 95%, and 90% in non-M3 group, respectively. This is consistent with previous report. 
As regards aberrant expression of B- and T-cells antigens in the studied AML cases, CD7 was aberrantly expressed in 2 out of 20 AML-M3 cases (10%) in comparison to 30 out of 80 non-M3 AML cases (37.5%). Aberrant CD7 expression is a common finding in the context of FCM IFT of AML (M0-M2). Kaleem et al., and Tiftik et al., reported CD7 expression in AML M0-M2 that ranged from 24% to 80% and in up to 6% of AML-M3 patients.
None of the 100 cases in this study expressed CD2, CD3, CD5, CD10, CD19 or CD20. In comparison to other T- and B-cells markers, CD2 has been detected more frequently in cases of AML-M3 (approximately 25%), than in other FAB subtypes of AML (approximately 5%) and according to Lin et al., and Albano et al., ; it has been advocated to high relapse rate and poor clinical outcome. In a study by Albano et al.,  , a strong association was found between CD34 and CD2 expression among his 136 APL cases a finding not reproduced in our cases.
Grimwade et al. in 2002,  demonstrated that the majority of the examined APL cases expressing CD2 belonged to the hypogranular variant form of APL.
Groczyca  demonstrated four IPT pattern in APL cases. The second most common type, corresponding to the hypogranular (microgranular) variant of M3-AML (M3 v) differed from classical APL by low SSC and frequent co-expression of CD2 and CD34. As the M3 cases in the current study are all classical cases, this might explain the lack of CD2 co-expression in our cases.
As regards CD34 and HLA-DR, the milestone markers for differentiation between M3 and non-M3, they were both expressed in 100% of AML-M0 and M1 cases. On the other hand, CD34 was expressed in 53.5% of AML-M2 cases and HLA-DR in 71.4% (20 cases), and both antigens were expressed in 76.3% of cases. Only 4 AML-M2 cases (5%) showed simultaneous lack of HLA-DR and CD34. Promsuwicha and Auewarakul  studied 99 cases of AML-M2 for HLA-DR and CD34 expression. Among their AML cases with t(8;21), HLA-DR, CD34 or both antigens were expressed in the majority of cases (90.5%, 80.8%, and 79.8%, respectively). Higher incidence of HLA-DR negativity in non APL-AML was reported by Zhou et al. . They detected HLA-DR negativity in 15/33 (45%) of non M3-AML patients.
All the AML-M3 cases in the current study were negative for HLA-DR expression while only one case (5%) was positive for CD34. 64 APL cases were studied by Promsuwicha and Auewarakul  for HLA-DR and CD34 expression. They found that in AML with t(15;17), HLA-DR was expressed in 4.7% and CD34 was expressed in 15.6% of cases, and none of the cases expressed both HLA-DR and CD34. Several reports describe CD34 expression in APL as a unique clinical feature associated with leukocytosis and hypogranular morphology and are associated with a poor clinical outcome. , One author went further and suggested the use of CD34 expression as a reliable marker to distinguish between M3 and classic APL. 
To better resolve the overlap between AML-M3 and non-M3 cases where CD34 and HLA-DR status of expression is not conclusive, we tested the expression of CD11a and CD18 in M3-AML and non M3-AML cases. In non-M3 group, CD11a and CD18 were expressed in 75/80 (93.8%) and 79/80 (98.8%), respectively. It is worth noting that all non-M3 cases expressed at least one of the two markers. None of the M3 cases expressed these markers. This is consistent with Chan and Watt,  who found that APL cells uniquely lack CD11a and CD18 expression. However, Zhou et al.;  demonstrated negativity for either marker to be 92% (33/36) for CD11a and CD18 in M3-AML cases and 39% (13/33) for CD11a and 45% (15/33) for CD18 in non M3-AML cases. This difference may be due to including microgranular M3-AML cases and different categories in non M3-AML group (AML cases with maturation, with multilineage dysplasia, with monocytic differentiation, and AML not further classified) in their study. In addition, they used a cut-off value of 30% for CD11a, CD18 positivity.
In the current study, CD34 negativity had a PPV of 55.8%, NPV of 98.4%, sensitivity of 95%, and specificity of 81.2%. HLA-DR negativity had a PPV of 71.4%, NPV of 100%, sensitivity of 100%, and specificity of 90% while CD34 and HLA-DR double negativity had a PPV of 82, 6%, NPV of 100%, sensitivity of 100%, and specificity of 93.8%. These figures are very close to those reported by Promsuwicha and Auewarakul,  who demonstrated that CD34-negativity and HLA-DR-negativity had the highest PPV and NPV (85% and 100%, respectively) as well as the highest sensitivity and specificity (100% and 90.8%, respectively) for differentiating t(15;17) from t(8;21) positive AML.
In our study, CD11a negativity had a PPV of 95.2%, NPV of 100%, sensitivity of 100%, and specificity of 98.7%. CD18 negativity had a PPV of 80%, NPV of 100%, sensitivity of 100%, and specificity of 93.7%. From these results, the most powerful discriminant marker in our study was CD11a, followed by HLA-DR then CD18. According to Paietta  and Paietta et al., HLA-DR was the most powerful and next in line was CD11a followed by CD18.
Moreover, (HLA-DR, CD11a, and CD18) were not expressed in any of our APL patients. So, negativity of these three antigens showed the highest PPV, NPV as well as sensitivity and specificity (100%). Zhou et al., demonstrated that (HLA-DR, CD11a, and CD18) negativity was 81% sensitive and 88% specific for the diagnosis of M3-AML.
According to different researches, APL could be categorized immunophenotypically as being characterized by consistent absence or very low expression of CD15, CD11a, CD11b, CD11c, CD18, CD66b, and CD66c. At the same time, the blasts show CD13 heterogeneous expression (broad histogram), whereas CD33 expression is homogeneous (sharp histogram). CD34 and HLA-DR are frequently absent. This characteristic immunophenotype, in practised hands, has been reported to show 100% sensitivity and 99% specificity for predicting APL molecular rearrangement Orfao et al. in  Falini and Mason  Paietta  Cullen et al. 
The value added by testing for CD11a and CD18 expression was evident in three of our cases. In fact, in our study, we identified 2 cases of AML-M2 with negative HLA-DR and CD34 expression in one case and negative CD34 in the other. This is unusual of AML with maturation (FAB-M2) where these markers are reported to be typically expressed. At this point of FCM IPT, the diagnosis of AML-M3 would have been more appropriate, especially that both cases presented with pancytopenia in the PB and one case showed a blast morphology not typical for AML-M2 blasts with abundant basophilic cytoplasm, containing fine azurophilic granules. However, the expression of CD11a and CD18 in both cases helped in the diagnosis of AML-M2 which was confirmed later by the presence of t(8,21) and absence of t(15;17) in both cases.
Another case (Case no. 11) presented with pancytopenia in the PB with the presence of large irregular promyelocytes with scanty basophilic cytoplasm and fine scanty granules in the PB, and BM stained smears. The FCM IPT was not conclusive for APL though HLA-DR was not expressed by the leukemic cells; but CD34 was expressed on 28% of the cells. Rapid diagnosis of APL in this case was not possible, and recommendation of FISH for t(15;17) was mandatory. However, the use of CD11a and CD18 which were both negative together of application of the negativity triad (HLA-DR, CD11a, and CD18) made the diagnosis of AML-M3 more solid and the patient started ATRA even before the result of FISH for t(15;17) confirmed the diagnosis.
| Conclusion and recommendations|| |
From this study, we conclude that testing for CD18 and CD11a expression in conjunction with routine diagnostic FCM IPT panel will append extra diagnostic power to FCM IPT in the differentiation of difficult cases of AML-M3 from AML-M2. Adding CD18 and CD11a to the routine immunophenotyping panel for AML is therefore recommended.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]