Correspondence
The Authors’ Reply We appreciate the comments by Juncà et al on our study1 of acute promyelocytic leukemia (APL) by flow cytometry. We agree that it is crucial to recognize exceptions and aberrancies so that all APL cases can be correctly identified. We also share their concern that “no single pattern is pathognomonic of a given disease.” However, we are surprised by their results. Compared with our results in which 0 of 149 APL cases had CD11b and CD11c expression, five (28%) of 18 cases were positive for CD11b in their series. In an attempt to explain the discrepancy, we offer the following comments. 1. We emphasized in our study that APL cells lacked CD11b in cells of both blast and granulocyte regions. While cells in the blast region varied in immunophenotypic features, a more consistent and often overlooked feature was the lack of CD11b expression by APL cells in the granulocyte region rather than CD11b– blasts per se. It is routinely presented as a distinct CD11b– population entirely isolated from the normal granulocytic counterpart (see Image 1 in Dong et al1). When there were doubts, the lack of both CD11b and CD11c expression in myeloid cells should raise the possibility of APL. 2. We were aware of the low frequency of CD11b expression by APL in the literature, as mentioned in our article. However, we were unable to reproduce that finding under stringent testing conditions, and we continue to validate this observation in current practice. We would like to point out that APL cells very often
display strong nonspecific staining for myeloid antigens, resulting in much higher background fluorescence intensity than lymphocytes in the same sample. Thus, there is no reliable internal negative control to ensure a positive reading. As a result, we rely on isotype-stained cells as the essential negative control for analyzing APL cases, so that the true myeloid antigen expression can be distinguished effectively from the fluorescence background based on the isotype control. In our experience, such nonspecific staining or false positivity is in particular more frequently seen in APL than other types of acute myeloid leukemia or lymphoid neoplasm. 3. We speculate that the specificity and cross-reactivity of antibody, the combination of antibodies and their fluorescent conjugates in analysis, and the particular fluorochrome conjugated to the anti-CD11b antibody may also play a role in the resulting fluorescence intensity. It is known that by virtue of fluorochrome selection, one can enhance or suppress the detection level of a particular antigen in flow cytometry analysis. However, one should expect that, under optimized test conditions, the general features of a disease be consistent, such as the lack of certain mature myeloid antigens in APL. Henry Y. Dong, MD, PhD Jia X. Kung, MD, PhD Vatsala Bhardwaj, MD, PhD Integrated Oncology New York, NY
Reference 1. Dong HY, Kung JX, Bhardwaj V, et al. Flow cytometry rapidly identifies all acute promyelocytic leukemias with high specificity independent of underlying cytogenetic abnormalities. Am J Clin Pathol. 2011;135:76-84.
© American Society for Clinical Pathology
Am J Clin Pathol 2014;141:900-901 901
Copyright of American Journal of Clinical Pathology is the property of American Society of Clinical Pathologists and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
The Authors’ Reply We appreciate the comments by Juncà et al on our study1 of acute promyelocytic leukemia (APL) by flow cytometry. We agree that it is crucial to recognize exceptions and aberrancies so that all APL cases can be correctly identified. We also share their concern that “no single pattern is pathognomonic of a given disease.” However, we are surprised by their results. Compared with our results in which 0 of 149 APL cases had CD11b and CD11c expression, five (28%) of 18 cases were positive for CD11b in their series. In an attempt to explain the discrepancy, we offer the following comments. 1. We emphasized in our study that APL cells lacked CD11b in cells of both blast and granulocyte regions. While cells in the blast region varied in immunophenotypic features, a more consistent and often overlooked feature was the lack of CD11b expression by APL cells in the granulocyte region rather than CD11b– blasts per se. It is routinely presented as a distinct CD11b– population entirely isolated from the normal granulocytic counterpart (see Image 1 in Dong et al1). When there were doubts, the lack of both CD11b and CD11c expression in myeloid cells should raise the possibility of APL. 2. We were aware of the low frequency of CD11b expression by APL in the literature, as mentioned in our article. However, we were unable to reproduce that finding under stringent testing conditions, and we continue to validate this observation in current practice. We would like to point out that APL cells very often
display strong nonspecific staining for myeloid antigens, resulting in much higher background fluorescence intensity than lymphocytes in the same sample. Thus, there is no reliable internal negative control to ensure a positive reading. As a result, we rely on isotype-stained cells as the essential negative control for analyzing APL cases, so that the true myeloid antigen expression can be distinguished effectively from the fluorescence background based on the isotype control. In our experience, such nonspecific staining or false positivity is in particular more frequently seen in APL than other types of acute myeloid leukemia or lymphoid neoplasm. 3. We speculate that the specificity and cross-reactivity of antibody, the combination of antibodies and their fluorescent conjugates in analysis, and the particular fluorochrome conjugated to the anti-CD11b antibody may also play a role in the resulting fluorescence intensity. It is known that by virtue of fluorochrome selection, one can enhance or suppress the detection level of a particular antigen in flow cytometry analysis. However, one should expect that, under optimized test conditions, the general features of a disease be consistent, such as the lack of certain mature myeloid antigens in APL. Henry Y. Dong, MD, PhD Jia X. Kung, MD, PhD Vatsala Bhardwaj, MD, PhD Integrated Oncology New York, NY
Reference 1. Dong HY, Kung JX, Bhardwaj V, et al. Flow cytometry rapidly identifies all acute promyelocytic leukemias with high specificity independent of underlying cytogenetic abnormalities. Am J Clin Pathol. 2011;135:76-84.
© American Society for Clinical Pathology
Am J Clin Pathol 2014;141:900-901 901
Copyright of American Journal of Clinical Pathology is the property of American Society of Clinical Pathologists and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
