Cytometry Part B (Clinical Cytometry) 86B:371–372 (2014)
Issue Highlights
Issue Highlights—November 2014 This issue of Cytometry Part B: Clinical Cytometry contains a number of exciting articles on a wide variety of topics. Included among these is an article by Peijers and colleagues concerning the harmonization of immunophenotyping. Both harmonization and standardization of immunophenotyping are topics of keen interest to cytometrists. Whereas standardization requires strict adherence to an accepted practice or marker set, harmonization permits more flexibility while still achieving highly consistent results. Standardization of various clinical assays has been the topic of several key manuscripts in this journal over the past several years (1–3). For example, efforts towards the standardization of detection of clones in paroxysmal nocturnal hemoglobinuria (PNH) have been presented by Sutherland and colleagues (1) as well as by Fletcher and coworkers (2). In recent years harmonization inflow cytometry has been the subject of high visibility papers such as those by Maecker and colleagues (4,5) as it has been increasingly recognized that there needs to be a high degree of consistency among laboratories while still permitting a certain amount of flexibility in the performance of assays by individual laboratories. In this issue, Preijers and coworkers (6) compare the use of alternative antibody clones and cytometers to those used in the original standardized protocol (7). By demonstrating a high degree of comparability between the standard reagents and their own, Preijers et al have made this protocol more ‘agnostic’ about the use of a particular brand of reagents or instrument, and thus harmonizing data collection among a potentially wider group of laboratories. Also in this issue, Krishnan and coworkers take a close look at analytical variables such as anticoagulants, age, and gender in the measurement of cytokines and chemokines from the peripheral circulation of healthy donors (8). Increasingly, these variables are being recognized as significantly impacting data from both multiplex bead cytokine assays (9) as well as immunophenotyping (10). In the current study Krishnan and colleagues show profound anti-coagulant and age-dependent differences in the measurement of various cytokines and chemokines using a 23-plex bead array assay. A key ‘take home’ message from this paper is the proper design and implementation of clinical protocols for cytokine measurements. The clinic must take care to collect all specimens in only the one anticoagulant specified in the protocol and never substitute serum for plasma, or vice versa. Doing so could lead to artifacts within the data and lead to spurious conclusions in the study. The agerelated differences observed in the levels of several cytokines indicate the need to carefully match the ages of any control cohort to the subjects of the study.
C 2014 International Clinical Cytometry Society V
Perspectives from J. Philip McCoy, Jr.
Chronic lymphocytic leukemia (CLL) being the most common leukemia and showing marked differences in progression among patients is often the topic of reports in this journal. Chief among these reports are those dealing with better methods or approaches for defining biomarkers for disease progression in CLL. ZAP-70 (Zetachain-associated protein kinase 70), a T cell signaling protein, has been associated with an adverse outcome in CLL when expressed by increased numbers of B cells, and many recent reports deal with strategies to improve the detection and assessment of Zap-70 (11–14). In this issue Witkowska et al (15) examine in vitro apoptosis of de novo CLL cells in the context of disease progression. Here they demonstrate that increased apoptosis is associated with stable disease whereas low levels of in vitro apoptosis after 48 hours of culture correlated with poor responses to chemotherapy. They further note that the level of bcl-2 expression was higher in cells with lower in vitro apoptosis – a finding that ties in nicely with a recent report in this journal by Degheidy and coworkers reporting bcl-2 levels as a biomarker for 13q14 deletion in CLL (16).
*Correspondence to: J. Philip McCoy, Jr., PhD. E-mail: mccoyjp@ mail.nih.gov Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.21186
372
McCoy
Finally, in this issue, cytometry goes to the dogs: we have a contribution from the field of veterinary medicine. Drs Papakonstantinou and O’Brien present a series of eleven samples of canine lymphoma which have been studied using high content imaging for immunophenotyping and morphometric analyses (17). A quick Pub Med search for immunophenotyping of canine lymphoma reveals less than two dozen reports in the literature, so one can easily see that this is a relatively new field with limited numbers of monoclonal antibodies. Needless to say, the state of the art in using immunophenotyping to characterize human lymphomas is far more mature as evidenced by several recent studies in this journal (18–20). Hopefully, reading the current study will foster more interactions with our colleagues in veterinary medicine and help to advance the diagnostic efforts in that field.
8.
9.
10.
11.
12.
J. Philip McCoy, Jr., PhD* NHLBI, National Institutes of Health Bethesda, MD 20892
13.
LITERATURE CITED
14.
1. Fletcher M, Sutherland DR, Whitby L, Whitby A, Richards SJ, Acton E, Keeney M, Borowitz M, Illingworth A, Reilly JT , Barnett D. Standardizing leucocyte PNH clone detection: An international study. Cytometry Part B Clin Cytom 2014;86B:311–318. 2. Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry Part B Clin Cytom 2012;82B:195–208. 3. Patkar N, Alex AA, Bargavi B, Ahmed R, Abraham A, George B, Vishwabandya A, Srivas-tava A, Mathews V. Standardizing minimal residual disease by flow cytometry for precursor B lineage acute lymphoblastic leukemia in a developing country. Cytometry Part B Clin Cytom 2012;82B:252–258. 4. Maecker HT, McCoy JP Jr; FOCIS Human Immunophenotyping Consortium. A model for harmonizing flow cytometry in clinical trials. Nat Immunol 2010;11:975–978. 5. Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol 2012; 12:191–200. 6. Preijer FWMB, Huys E, Favre C, Moshaver B. Establishment of harmonization in immunophenotyping: A comparative study of a standardized one-tube lymphocyte-screening panel. Cytometry Part B Clin Cytom 2014;86B:418–425. 7. van Dongen JJM, Lhermitte l, B€ ottcher S, Almeida J, van der Velden VHJ, Flores Montero J, Rawstron A, Asnafi V, Lecrevisse Q, Lucio P, Mejstrikova E, Szczepanski T, Kalina T, de Tute R, Br€ uggemann M,
15.
16.
17.
18. 19.
20.
Sedek L, Cullen M, Langerak AW, Mendonc¸a A, Macintyre E, MartinAyuso M, Hrusak O, Vidriales MB, Orfao A on behalf of the EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708) EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 2012;26:1908–1975. Krishnan VV, Ravindran R, Wun T, Luciw PA, Khan IH, Janatpour K. Multiplexed measurements of immunomodulator levels in peripheral blood of healthy subjects: Effects of analytical variables based on anticoagulants, age, and gender. Cytometry Part B Clin Cytom 2014; 86B:426–435. Biancotto A, Feng X, Langweiler M, Young NS, McCoy JP. Effect of anticoagulants on multiplexed measurement of cytokine/chemokines in healthy subjects. Cytokine 2012;60:438–446. Santiago MI, Stetler-Stevenson M, Yuan C, Ramesh P. The effect of different anticoagulants on specimen stability and leukocyte subsets. Cytometry Part B Clin Cytom (P35) 2012;82B:339. Adams RL, Cheung C, Banh R, Saal R, Cross D, Gill D, Self M, Klein K, Mollee P. Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry. Cytometry Part B Clin Cytom 2014;86: 80–90. Kern W, Bacher U, Haferlach C, Alpermann T, Dicker F, Schnittger S, Haferlach T. Frequency and prognostic impact of the aberrant CD8 expression in 5,523 patients with chronic lymphocytic leukemia. Cytometry Part B Clin Cytom 2012;82:145–150. Preobrazhensky SN, Szankasi P, Bahler DW. Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic control antibodies. Cytometry Part B Clin Cytom 2012;82:78–84. Wiggers TG, Westra G, Westers TM, Abbes AP, Strunk A, KuiperKramer E, Poddighe P, van de Loosdrecht AA, Chamuleau ME. ZAP70 in B-CLL cells related to the expression in NK cells is a surrogate marker for mutational status. Cytometry Part B Clin Cytom 2014;86:280–287. Witkowska M, Nowak W, Cebula-Obrzut B, Majchrzak A, Medra A, Robak T, Smolewski P. Spontaneous in vitro apoptosis of de novo chronic lymphocytic leukemia cells correlates with risk of the disease progression. Cytometry Part B Clin Cytom 2014;86B:410–417. Degheidy HA, Gadalla SM, Farooqui MZ, Abbasi F, Arthur DC, Bauer SR, Wilson WH, Wiestner A, Stetler-Stevenson MA, Marti GE. Bcl-2 level as a biomarker for 13q14 deletion in CLL. Cytometry Part B Clin Cytom 2013;84:237–247. Papakonstantinou S, O’Brien PJ. High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas. Cytometry Part B Clin Cytom 2014;86B:373–382. Craig FE, Brinkman RR, Ten Eyck S, Aghaeepour N. Computational analysis optimizes the flow cytometric evaluation for lymphoma. Cytometry Part B Clin Cytom 2014;86:18–24. Demurtas A, Stacchini A, Aliberti S, Chiusa L, Chiarle R, Novero D. Tissue flow cytometry immunophenotyping in the diagnosis and classification of non-Hodgkin’s lymphomas: A retrospective evaluation of 1,792 cases. Cytometry Part B Clin Cytom 2013;84:82–95. Song JY, Filie AC, Venzon D, Stetler-Stevenson M, Yuan CM. Flow cytometry increases the sensitivity of detection of leukemia and lymphoma cells in bronchoalveolar lavage specimens. Cytometry Part B Clin Cytom 2012;82:305–312.
Cytometry Part B: Clinical Cytometry
Issue Highlights
Issue Highlights—November 2014 This issue of Cytometry Part B: Clinical Cytometry contains a number of exciting articles on a wide variety of topics. Included among these is an article by Peijers and colleagues concerning the harmonization of immunophenotyping. Both harmonization and standardization of immunophenotyping are topics of keen interest to cytometrists. Whereas standardization requires strict adherence to an accepted practice or marker set, harmonization permits more flexibility while still achieving highly consistent results. Standardization of various clinical assays has been the topic of several key manuscripts in this journal over the past several years (1–3). For example, efforts towards the standardization of detection of clones in paroxysmal nocturnal hemoglobinuria (PNH) have been presented by Sutherland and colleagues (1) as well as by Fletcher and coworkers (2). In recent years harmonization inflow cytometry has been the subject of high visibility papers such as those by Maecker and colleagues (4,5) as it has been increasingly recognized that there needs to be a high degree of consistency among laboratories while still permitting a certain amount of flexibility in the performance of assays by individual laboratories. In this issue, Preijers and coworkers (6) compare the use of alternative antibody clones and cytometers to those used in the original standardized protocol (7). By demonstrating a high degree of comparability between the standard reagents and their own, Preijers et al have made this protocol more ‘agnostic’ about the use of a particular brand of reagents or instrument, and thus harmonizing data collection among a potentially wider group of laboratories. Also in this issue, Krishnan and coworkers take a close look at analytical variables such as anticoagulants, age, and gender in the measurement of cytokines and chemokines from the peripheral circulation of healthy donors (8). Increasingly, these variables are being recognized as significantly impacting data from both multiplex bead cytokine assays (9) as well as immunophenotyping (10). In the current study Krishnan and colleagues show profound anti-coagulant and age-dependent differences in the measurement of various cytokines and chemokines using a 23-plex bead array assay. A key ‘take home’ message from this paper is the proper design and implementation of clinical protocols for cytokine measurements. The clinic must take care to collect all specimens in only the one anticoagulant specified in the protocol and never substitute serum for plasma, or vice versa. Doing so could lead to artifacts within the data and lead to spurious conclusions in the study. The agerelated differences observed in the levels of several cytokines indicate the need to carefully match the ages of any control cohort to the subjects of the study.
C 2014 International Clinical Cytometry Society V
Perspectives from J. Philip McCoy, Jr.
Chronic lymphocytic leukemia (CLL) being the most common leukemia and showing marked differences in progression among patients is often the topic of reports in this journal. Chief among these reports are those dealing with better methods or approaches for defining biomarkers for disease progression in CLL. ZAP-70 (Zetachain-associated protein kinase 70), a T cell signaling protein, has been associated with an adverse outcome in CLL when expressed by increased numbers of B cells, and many recent reports deal with strategies to improve the detection and assessment of Zap-70 (11–14). In this issue Witkowska et al (15) examine in vitro apoptosis of de novo CLL cells in the context of disease progression. Here they demonstrate that increased apoptosis is associated with stable disease whereas low levels of in vitro apoptosis after 48 hours of culture correlated with poor responses to chemotherapy. They further note that the level of bcl-2 expression was higher in cells with lower in vitro apoptosis – a finding that ties in nicely with a recent report in this journal by Degheidy and coworkers reporting bcl-2 levels as a biomarker for 13q14 deletion in CLL (16).
*Correspondence to: J. Philip McCoy, Jr., PhD. E-mail: mccoyjp@ mail.nih.gov Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.21186
372
McCoy
Finally, in this issue, cytometry goes to the dogs: we have a contribution from the field of veterinary medicine. Drs Papakonstantinou and O’Brien present a series of eleven samples of canine lymphoma which have been studied using high content imaging for immunophenotyping and morphometric analyses (17). A quick Pub Med search for immunophenotyping of canine lymphoma reveals less than two dozen reports in the literature, so one can easily see that this is a relatively new field with limited numbers of monoclonal antibodies. Needless to say, the state of the art in using immunophenotyping to characterize human lymphomas is far more mature as evidenced by several recent studies in this journal (18–20). Hopefully, reading the current study will foster more interactions with our colleagues in veterinary medicine and help to advance the diagnostic efforts in that field.
8.
9.
10.
11.
12.
J. Philip McCoy, Jr., PhD* NHLBI, National Institutes of Health Bethesda, MD 20892
13.
LITERATURE CITED
14.
1. Fletcher M, Sutherland DR, Whitby L, Whitby A, Richards SJ, Acton E, Keeney M, Borowitz M, Illingworth A, Reilly JT , Barnett D. Standardizing leucocyte PNH clone detection: An international study. Cytometry Part B Clin Cytom 2014;86B:311–318. 2. Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry Part B Clin Cytom 2012;82B:195–208. 3. Patkar N, Alex AA, Bargavi B, Ahmed R, Abraham A, George B, Vishwabandya A, Srivas-tava A, Mathews V. Standardizing minimal residual disease by flow cytometry for precursor B lineage acute lymphoblastic leukemia in a developing country. Cytometry Part B Clin Cytom 2012;82B:252–258. 4. Maecker HT, McCoy JP Jr; FOCIS Human Immunophenotyping Consortium. A model for harmonizing flow cytometry in clinical trials. Nat Immunol 2010;11:975–978. 5. Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol 2012; 12:191–200. 6. Preijer FWMB, Huys E, Favre C, Moshaver B. Establishment of harmonization in immunophenotyping: A comparative study of a standardized one-tube lymphocyte-screening panel. Cytometry Part B Clin Cytom 2014;86B:418–425. 7. van Dongen JJM, Lhermitte l, B€ ottcher S, Almeida J, van der Velden VHJ, Flores Montero J, Rawstron A, Asnafi V, Lecrevisse Q, Lucio P, Mejstrikova E, Szczepanski T, Kalina T, de Tute R, Br€ uggemann M,
15.
16.
17.
18. 19.
20.
Sedek L, Cullen M, Langerak AW, Mendonc¸a A, Macintyre E, MartinAyuso M, Hrusak O, Vidriales MB, Orfao A on behalf of the EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708) EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 2012;26:1908–1975. Krishnan VV, Ravindran R, Wun T, Luciw PA, Khan IH, Janatpour K. Multiplexed measurements of immunomodulator levels in peripheral blood of healthy subjects: Effects of analytical variables based on anticoagulants, age, and gender. Cytometry Part B Clin Cytom 2014; 86B:426–435. Biancotto A, Feng X, Langweiler M, Young NS, McCoy JP. Effect of anticoagulants on multiplexed measurement of cytokine/chemokines in healthy subjects. Cytokine 2012;60:438–446. Santiago MI, Stetler-Stevenson M, Yuan C, Ramesh P. The effect of different anticoagulants on specimen stability and leukocyte subsets. Cytometry Part B Clin Cytom (P35) 2012;82B:339. Adams RL, Cheung C, Banh R, Saal R, Cross D, Gill D, Self M, Klein K, Mollee P. Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry. Cytometry Part B Clin Cytom 2014;86: 80–90. Kern W, Bacher U, Haferlach C, Alpermann T, Dicker F, Schnittger S, Haferlach T. Frequency and prognostic impact of the aberrant CD8 expression in 5,523 patients with chronic lymphocytic leukemia. Cytometry Part B Clin Cytom 2012;82:145–150. Preobrazhensky SN, Szankasi P, Bahler DW. Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic control antibodies. Cytometry Part B Clin Cytom 2012;82:78–84. Wiggers TG, Westra G, Westers TM, Abbes AP, Strunk A, KuiperKramer E, Poddighe P, van de Loosdrecht AA, Chamuleau ME. ZAP70 in B-CLL cells related to the expression in NK cells is a surrogate marker for mutational status. Cytometry Part B Clin Cytom 2014;86:280–287. Witkowska M, Nowak W, Cebula-Obrzut B, Majchrzak A, Medra A, Robak T, Smolewski P. Spontaneous in vitro apoptosis of de novo chronic lymphocytic leukemia cells correlates with risk of the disease progression. Cytometry Part B Clin Cytom 2014;86B:410–417. Degheidy HA, Gadalla SM, Farooqui MZ, Abbasi F, Arthur DC, Bauer SR, Wilson WH, Wiestner A, Stetler-Stevenson MA, Marti GE. Bcl-2 level as a biomarker for 13q14 deletion in CLL. Cytometry Part B Clin Cytom 2013;84:237–247. Papakonstantinou S, O’Brien PJ. High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas. Cytometry Part B Clin Cytom 2014;86B:373–382. Craig FE, Brinkman RR, Ten Eyck S, Aghaeepour N. Computational analysis optimizes the flow cytometric evaluation for lymphoma. Cytometry Part B Clin Cytom 2014;86:18–24. Demurtas A, Stacchini A, Aliberti S, Chiusa L, Chiarle R, Novero D. Tissue flow cytometry immunophenotyping in the diagnosis and classification of non-Hodgkin’s lymphomas: A retrospective evaluation of 1,792 cases. Cytometry Part B Clin Cytom 2013;84:82–95. Song JY, Filie AC, Venzon D, Stetler-Stevenson M, Yuan CM. Flow cytometry increases the sensitivity of detection of leukemia and lymphoma cells in bronchoalveolar lavage specimens. Cytometry Part B Clin Cytom 2012;82:305–312.
Cytometry Part B: Clinical Cytometry
