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Less apoptosis in patients with 5q-syndrome than in patients with refractory anemia
Leukemia Research 26 (2002) 899–902
Less apoptosis in patients with 5q-syndrome than in patients with refractory anemia LaBaron T. Washington, Iman Jilani, Eli Estey, Maher Albitar∗ Department of Hematopathology, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, P.O. Box 72, Houston, TX 77030-4095, USA Received 10 August 2001; accepted 5 February 2002
Abstract The WHO classification of hematological malignancies includes 5q-syndrome as a separate category within myelodysplastic syndromes (MDS). Clinically, patients with 5q-syndrome have a milder disease than patients with other MDS. The basis for this difference is not known. Identifying 5q-syndrome can be difficult because some of its morphologic and cytogenetic features are similar to those of other MDS. We compared apoptosis between 5q-syndrome and other refractory anemias. We found lower levels of apoptosis in 5q-syndrome as detected by less disruption of mitochondrial potential (P = 0.008) and decreased annexin V positivity (P = 0.01). Our results suggest that lower apoptosis in 5q-syndrome may explain the milder clinical course of the disease and distinguish 5q-syndrome from other MDS. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Myelodysplasia; 5q-Syndrome; Apoptosis; Annexin V; Mitochondrial potential; Refractory anemia
1. Introduction The WHO classification of hematological malignancies includes the 5q-syndrome as a separate category within myelodysplastic syndromes (MDS). The chromosomal breakpoint in 5q-syndrome is on chromosome 5q but varies from 5q13 to 5q33 [4]. Patients with 5q-syndrome have distinct clinical and morphological characteristics. Clinically, these patients have a better prognosis than patients with other myelodysplastic syndromes [1]. Patients with 5q-syndrome usually have small, hypolobated megakaryocytes and less than 5% blasts in their bone marrow [2]. However, some cases of garden-variety MDS have the 5q-chromosome abnormality, and these cases must be distinguished from the more indolent cases of 5q-syndrome. Morphology and clinical presentation can usually help in making this distinction, but some cases of non-5q-syndrome MDS and acute myelogenous leukemia are morphologically similar to 5q-syndrome. Small, hypolobated megakaryocytes have been reported in patients with acute myeloid leukemia and with MDSs other than 5q-syndrome [2,3]. Also, a recent study suggested that there was no difference in survival between patients with ∗
Corresponding author. Tel.: +1-713-794-1292; fax: +1-713-794-1800. E-mail address: [email protected] (M. Albitar).
5q-syndrome and other MDS patients with <20% bone marrow blasts, casting doubt on whether 5q-syndrome is a distinct entity [4]. The biological basis for the milder course of 5q-syndrome is not known. However, the biological hallmark of MDS is ineffective hematopoiesis resulting from increased apoptosis in bone marrow [5,6]. In previous studies, apoptosis was detected by in situ end labeling of the fragmented apoptotic DNA and flow cytometric analysis. Flow cytometric analysis also allows quantification of changes in mitochondrial membrane potential, another event associated with apoptosis [7,8]. We investigated whether cases of refractory anemia (RA) could be differentiated from cases of 5q-syndrome by the degree of apoptosis as measured by flow cytometric analysis. Patients were considered to have 5q-syndrome if they had clusters of small, hypolobated megakaryocytes.
2. Materials and methods 2.1. Patients and morphologic classification Bone marrow aspirates were obtained from 40 previously untreated patients with MDS, including 32 with RA and 8 with 5q-syndrome. All samples were obtained under IRB approved protocol with concent form. All the RA cases
0145-2126/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 0 2 ) 0 0 0 3 9 - 5
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L.T. Washington et al. / Leukemia Research 26 (2002) 899–902
had dysplasia in two or more cell lineages and less than 5% bone marrow blasts. The 5q cases were defined based on the recent WHO classification. They all had increased hypolobulated megakaryocytes, anemia, cytogenetic evidence of isolated 5q and less than 5% blasts in bone marrow. 2.2. Measurement of apoptosis with annexin V Bone marrow samples (a minimum of 106 cells) were collected in EDTA tubes. The cells were isolated by density gradient centrifugation the both Histopaque 1119 and 1077. Polymorphonuclear leukocytes and mononuclear cells were mixed, washed, and stained with annexin V (fluorescein isothiocyanate) and propidium iodide as recommended by the manufacturer (Trevigen Inc., Gaithersberg, MD) and for CD 14 and CD 34. Briefly, cells washed in phosphate-buffered saline were incubated with propidium iodide and fluorescein isothiocyanate conjugated annexin V antibodies for 15 min, washed, processed and acquired with an FACSCalibur (Becton–Dickinson) within 5 min of staining. Events were analyzed by using CellQuest and Paint-a-Gate software (Becton–Dickinson). Percent positivity for annexin V was determined by comparison of the fluorescence distribution of annexin V in patients with either 5q-syndrome or RA to the distribution of annexin V
in control bone marrow samples obtained from individuals with solid tumors without marrow involvement. 2.3. Measurement of mitochondrial membrane potential (DePsipher assay) Bone marrow samples were collected in EDTA tubes and (minimum of 106 polymorphonuclear and mononuclear cells) were isolated using the double density gradient centrifugation technique described above. DePsipher reagent (5,5 ,6 ,6 -tetrachloro-1,1 ,3,3 - tetraethylbenzimidazolocarbocyanine iodide) (Trevigen, Inc., Gaithersburg, MD) was added and incubated at 37 ◦ C in 5% CO2 for 20–30 min. DePsipher is a dye that enters the mitochondria, polymerizes when the mitochondrial membrane potential is intact, and emits orange fluorescence. When the mitochondrial membrane potential is disturbed, the dye does not polymerize and emits green flourescence [9]. After staining with DePsipher, the cells were washed and analyzed immediately with an FACSCalibur. Events were analyzed by using CellQuest and Paint-a-Gate software (Becton–Dickinson). The degree of change in mitochondrial membrane potential was assessed by comparing the percentage of events with green fluoresence in patients with 5q-syndrome or RA with the percentage of events with green fluoresence in marrow samples obtained from patients without MDS.
Fig. 1. Representitive study of apoptosis in a patient with 5q-syndrome (C and D) and a patient with RA (A and B). The patient with 5q-syndrome had less disrupted mitochondrial membrane potential and lowered expression of annexin V and milder clinical features (hemoblobin: 7.3 g/dl; white cell count: 2800 l; platelets: 349,000 l−1 ) and presented with fatigue. The RA patient had 20q by cytogenetic and presented with fatigue and easy bruising (hemoblobin: 8.3 g/dl; white cell count: 2300 l−1 ; platelets: 51,000 l−1 ). Changes in mitochondrial membrane potential are revealed by the decreases in orange fluorescence on the granulocytes. FL-1 height represents green fluorescence. Granulocytes were chosen based on forward and side scatter properties. Quadrants were set based on cells from a normal control bone marrow. Panel A shows decreased orange fluorescence and thus, a disturbance in mitochondrial membrane potential and increased apoptosis. Panel B shows increased annexin V expression. Panel C shows relatively more orange fluorescence and thus less membrane potential disturbance and panel D shows a lower number of cells expression annexin V. Gating on the total cell population showed similar results.
L.T. Washington et al. / Leukemia Research 26 (2002) 899–902
2.4. Statistical analysis The Kruskal–Wallis test was used to compare categorical differences between the groups. 3. Results and discussion Morphologically, all 5q cases had increased numbers of small, hypolobated megakaryocytes. The granulocytes
901
showed mild dysplastic changes, including some pseudo p-Pelger-Huët cells (5–10%) with some hypogranulation. The erythroid cells were megaloblastoid in most cases. Apoptosis was measured by using two independent methods. Measurement of mitochondrial membrane potential and measurement of cell membrane changes with annexin V. All 40 patients were studied for mitochondrial membrane potential and 29 patients with RA and five with 5q-syndrome were studied for annexin V expression.
Fig. 2. The Kruskal–Wallis test was used to compare the mean percentages of annexin V expression (A) and between the RA and 5q groups (B). (A) Less disrupted mitochondrial membrane potential in patients with 5q-syndrome (P = 0.008). (B) Higher percentage of cells expressing annexin V in the 29 patients with RA than in the five cases of the 5q-syndrome (P = 0.01).
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L.T. Washington et al. / Leukemia Research 26 (2002) 899–902
Annexin V was evaluated in propidium iodine-negative cells only. As shown in Figs. 1B and D and 2A, flow cytometric analysis using annexin V as a measure of apoptosis showed a greater percentage of apoptotic cells in cases with RA (median: 9.3; range: 0.4–9.0) than in cases with 5q-syndrome (median: 1.5; range: 0.4–23.1) (P = 0.01). Flow cytometric analysis using DePsipher as a measure of disruption of mitochondrial potential also showed a greater percentage of apoptotic cells in patients with RA (median: 5.3; range: 0.4–53.6) than in patients with 5q-syndrome (median: 1.4; range: 0.1–9.0) (Fig. 1A and C), who had a statistically significant decrease in mitochondrial potential (P = 0.008). These findings are important because the major biological abnormality in MDS is believed to be increased apoptosis in bone marrow elements, leading to cytopenia despite increase in bone marrow cellularity. There was no significant difference in bone marrow cellularity between RA and 5q patients in our study group (P = 0.2). Patients with 5q-syndrome have significantly milder disease than other patients with MDS, and it has been suggested that it be considered distinct disease from other MDS, including some cases with chromosome 5q abnormality. Although the morphology of the megakaryocytes in 5q-syndrome is characteristic, frequently the diagnosis can be difficult. Our finding of relatively low levels of apoptosis in cases of 5q-syndrome provides a clue to the biology of this disease and MDS, and it can also be used to distinguish 5q-syndrome from other types of MDS. Further study is needed to dissect the genetic abnormality of 5q-syndrome and to determine whether apoptosis increases with disease progression. Acknowledgements L.T. Washington provided the concept, design, assembled the data, analyzed the data, and drafted the manuscript. I. Jilani contributed to the study design, provided technical
support, assembled the data, and helped with drafting the manuscript. E. Estey provided study materials, assisted with the data interpretation, provided statistical input, critical review of the manuscript, offered the necessary funding, and gave final approval. M. Albitar contributed extensively to all aspects of this study. References [1] Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman, J, et al. In: Proceedings of The World Health Organization classification of hematological malignancies report of the Clinical Advisory Committee Meeting. Airlie House, VA, November 1997. Mod. Pathol. 2000;13:193–207. [2] Van den Berghe H, Vermaelen K, Mecucci C, Barbieri D, Tricot G. The 5q-anomaly. Cancer Genet Cytogenet 1985;17:189–255. [3] Teerenhovi L. Specificity of haematological indicators for 5q-syndrome in patients with myelodysplastic syndromes. Euro J Haemat 1987;39:326–30. [4] Washington LT, Doherty D, Glassman A, Martins J, Ibrahim S, Lai R. 5q as a sole chromosome abnormality in myelodysplastic syndromes and acute myeloid leukemia (Abstract). Mod Pathol 2001;14:182A. [5] Raza A, Alvi S, Borok RZ, et al. Excessive proliferation matched by excessive apoptosis in myelodysplastic syndromes: the cause–effect relationship. Leuk Lymphoma 1997;27:111–8. [6] Merchant SH, Gonchoroff NJ, Hutchison RE. Apoptotic index by annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes. Cytometry 2001;46: 28–32. [7] Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993;197:40–5. [8] Katz E, Deehan MR, Seatter S, Lord C, Sturrock RD, Harnett MM. B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells. J Immunol 2001;166:137–47. [9] Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993;197(1):40–5.
Less apoptosis in patients with 5q-syndrome than in patients with refractory anemia LaBaron T. Washington, Iman Jilani, Eli Estey, Maher Albitar∗ Department of Hematopathology, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, P.O. Box 72, Houston, TX 77030-4095, USA Received 10 August 2001; accepted 5 February 2002
Abstract The WHO classification of hematological malignancies includes 5q-syndrome as a separate category within myelodysplastic syndromes (MDS). Clinically, patients with 5q-syndrome have a milder disease than patients with other MDS. The basis for this difference is not known. Identifying 5q-syndrome can be difficult because some of its morphologic and cytogenetic features are similar to those of other MDS. We compared apoptosis between 5q-syndrome and other refractory anemias. We found lower levels of apoptosis in 5q-syndrome as detected by less disruption of mitochondrial potential (P = 0.008) and decreased annexin V positivity (P = 0.01). Our results suggest that lower apoptosis in 5q-syndrome may explain the milder clinical course of the disease and distinguish 5q-syndrome from other MDS. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Myelodysplasia; 5q-Syndrome; Apoptosis; Annexin V; Mitochondrial potential; Refractory anemia
1. Introduction The WHO classification of hematological malignancies includes the 5q-syndrome as a separate category within myelodysplastic syndromes (MDS). The chromosomal breakpoint in 5q-syndrome is on chromosome 5q but varies from 5q13 to 5q33 [4]. Patients with 5q-syndrome have distinct clinical and morphological characteristics. Clinically, these patients have a better prognosis than patients with other myelodysplastic syndromes [1]. Patients with 5q-syndrome usually have small, hypolobated megakaryocytes and less than 5% blasts in their bone marrow [2]. However, some cases of garden-variety MDS have the 5q-chromosome abnormality, and these cases must be distinguished from the more indolent cases of 5q-syndrome. Morphology and clinical presentation can usually help in making this distinction, but some cases of non-5q-syndrome MDS and acute myelogenous leukemia are morphologically similar to 5q-syndrome. Small, hypolobated megakaryocytes have been reported in patients with acute myeloid leukemia and with MDSs other than 5q-syndrome [2,3]. Also, a recent study suggested that there was no difference in survival between patients with ∗
Corresponding author. Tel.: +1-713-794-1292; fax: +1-713-794-1800. E-mail address: [email protected] (M. Albitar).
5q-syndrome and other MDS patients with <20% bone marrow blasts, casting doubt on whether 5q-syndrome is a distinct entity [4]. The biological basis for the milder course of 5q-syndrome is not known. However, the biological hallmark of MDS is ineffective hematopoiesis resulting from increased apoptosis in bone marrow [5,6]. In previous studies, apoptosis was detected by in situ end labeling of the fragmented apoptotic DNA and flow cytometric analysis. Flow cytometric analysis also allows quantification of changes in mitochondrial membrane potential, another event associated with apoptosis [7,8]. We investigated whether cases of refractory anemia (RA) could be differentiated from cases of 5q-syndrome by the degree of apoptosis as measured by flow cytometric analysis. Patients were considered to have 5q-syndrome if they had clusters of small, hypolobated megakaryocytes.
2. Materials and methods 2.1. Patients and morphologic classification Bone marrow aspirates were obtained from 40 previously untreated patients with MDS, including 32 with RA and 8 with 5q-syndrome. All samples were obtained under IRB approved protocol with concent form. All the RA cases
0145-2126/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 0 2 ) 0 0 0 3 9 - 5
900
L.T. Washington et al. / Leukemia Research 26 (2002) 899–902
had dysplasia in two or more cell lineages and less than 5% bone marrow blasts. The 5q cases were defined based on the recent WHO classification. They all had increased hypolobulated megakaryocytes, anemia, cytogenetic evidence of isolated 5q and less than 5% blasts in bone marrow. 2.2. Measurement of apoptosis with annexin V Bone marrow samples (a minimum of 106 cells) were collected in EDTA tubes. The cells were isolated by density gradient centrifugation the both Histopaque 1119 and 1077. Polymorphonuclear leukocytes and mononuclear cells were mixed, washed, and stained with annexin V (fluorescein isothiocyanate) and propidium iodide as recommended by the manufacturer (Trevigen Inc., Gaithersberg, MD) and for CD 14 and CD 34. Briefly, cells washed in phosphate-buffered saline were incubated with propidium iodide and fluorescein isothiocyanate conjugated annexin V antibodies for 15 min, washed, processed and acquired with an FACSCalibur (Becton–Dickinson) within 5 min of staining. Events were analyzed by using CellQuest and Paint-a-Gate software (Becton–Dickinson). Percent positivity for annexin V was determined by comparison of the fluorescence distribution of annexin V in patients with either 5q-syndrome or RA to the distribution of annexin V
in control bone marrow samples obtained from individuals with solid tumors without marrow involvement. 2.3. Measurement of mitochondrial membrane potential (DePsipher assay) Bone marrow samples were collected in EDTA tubes and (minimum of 106 polymorphonuclear and mononuclear cells) were isolated using the double density gradient centrifugation technique described above. DePsipher reagent (5,5 ,6 ,6 -tetrachloro-1,1 ,3,3 - tetraethylbenzimidazolocarbocyanine iodide) (Trevigen, Inc., Gaithersburg, MD) was added and incubated at 37 ◦ C in 5% CO2 for 20–30 min. DePsipher is a dye that enters the mitochondria, polymerizes when the mitochondrial membrane potential is intact, and emits orange fluorescence. When the mitochondrial membrane potential is disturbed, the dye does not polymerize and emits green flourescence [9]. After staining with DePsipher, the cells were washed and analyzed immediately with an FACSCalibur. Events were analyzed by using CellQuest and Paint-a-Gate software (Becton–Dickinson). The degree of change in mitochondrial membrane potential was assessed by comparing the percentage of events with green fluoresence in patients with 5q-syndrome or RA with the percentage of events with green fluoresence in marrow samples obtained from patients without MDS.
Fig. 1. Representitive study of apoptosis in a patient with 5q-syndrome (C and D) and a patient with RA (A and B). The patient with 5q-syndrome had less disrupted mitochondrial membrane potential and lowered expression of annexin V and milder clinical features (hemoblobin: 7.3 g/dl; white cell count: 2800 l; platelets: 349,000 l−1 ) and presented with fatigue. The RA patient had 20q by cytogenetic and presented with fatigue and easy bruising (hemoblobin: 8.3 g/dl; white cell count: 2300 l−1 ; platelets: 51,000 l−1 ). Changes in mitochondrial membrane potential are revealed by the decreases in orange fluorescence on the granulocytes. FL-1 height represents green fluorescence. Granulocytes were chosen based on forward and side scatter properties. Quadrants were set based on cells from a normal control bone marrow. Panel A shows decreased orange fluorescence and thus, a disturbance in mitochondrial membrane potential and increased apoptosis. Panel B shows increased annexin V expression. Panel C shows relatively more orange fluorescence and thus less membrane potential disturbance and panel D shows a lower number of cells expression annexin V. Gating on the total cell population showed similar results.
L.T. Washington et al. / Leukemia Research 26 (2002) 899–902
2.4. Statistical analysis The Kruskal–Wallis test was used to compare categorical differences between the groups. 3. Results and discussion Morphologically, all 5q cases had increased numbers of small, hypolobated megakaryocytes. The granulocytes
901
showed mild dysplastic changes, including some pseudo p-Pelger-Huët cells (5–10%) with some hypogranulation. The erythroid cells were megaloblastoid in most cases. Apoptosis was measured by using two independent methods. Measurement of mitochondrial membrane potential and measurement of cell membrane changes with annexin V. All 40 patients were studied for mitochondrial membrane potential and 29 patients with RA and five with 5q-syndrome were studied for annexin V expression.
Fig. 2. The Kruskal–Wallis test was used to compare the mean percentages of annexin V expression (A) and between the RA and 5q groups (B). (A) Less disrupted mitochondrial membrane potential in patients with 5q-syndrome (P = 0.008). (B) Higher percentage of cells expressing annexin V in the 29 patients with RA than in the five cases of the 5q-syndrome (P = 0.01).
902
L.T. Washington et al. / Leukemia Research 26 (2002) 899–902
Annexin V was evaluated in propidium iodine-negative cells only. As shown in Figs. 1B and D and 2A, flow cytometric analysis using annexin V as a measure of apoptosis showed a greater percentage of apoptotic cells in cases with RA (median: 9.3; range: 0.4–9.0) than in cases with 5q-syndrome (median: 1.5; range: 0.4–23.1) (P = 0.01). Flow cytometric analysis using DePsipher as a measure of disruption of mitochondrial potential also showed a greater percentage of apoptotic cells in patients with RA (median: 5.3; range: 0.4–53.6) than in patients with 5q-syndrome (median: 1.4; range: 0.1–9.0) (Fig. 1A and C), who had a statistically significant decrease in mitochondrial potential (P = 0.008). These findings are important because the major biological abnormality in MDS is believed to be increased apoptosis in bone marrow elements, leading to cytopenia despite increase in bone marrow cellularity. There was no significant difference in bone marrow cellularity between RA and 5q patients in our study group (P = 0.2). Patients with 5q-syndrome have significantly milder disease than other patients with MDS, and it has been suggested that it be considered distinct disease from other MDS, including some cases with chromosome 5q abnormality. Although the morphology of the megakaryocytes in 5q-syndrome is characteristic, frequently the diagnosis can be difficult. Our finding of relatively low levels of apoptosis in cases of 5q-syndrome provides a clue to the biology of this disease and MDS, and it can also be used to distinguish 5q-syndrome from other types of MDS. Further study is needed to dissect the genetic abnormality of 5q-syndrome and to determine whether apoptosis increases with disease progression. Acknowledgements L.T. Washington provided the concept, design, assembled the data, analyzed the data, and drafted the manuscript. I. Jilani contributed to the study design, provided technical
support, assembled the data, and helped with drafting the manuscript. E. Estey provided study materials, assisted with the data interpretation, provided statistical input, critical review of the manuscript, offered the necessary funding, and gave final approval. M. Albitar contributed extensively to all aspects of this study. References [1] Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman, J, et al. In: Proceedings of The World Health Organization classification of hematological malignancies report of the Clinical Advisory Committee Meeting. Airlie House, VA, November 1997. Mod. Pathol. 2000;13:193–207. [2] Van den Berghe H, Vermaelen K, Mecucci C, Barbieri D, Tricot G. The 5q-anomaly. Cancer Genet Cytogenet 1985;17:189–255. [3] Teerenhovi L. Specificity of haematological indicators for 5q-syndrome in patients with myelodysplastic syndromes. Euro J Haemat 1987;39:326–30. [4] Washington LT, Doherty D, Glassman A, Martins J, Ibrahim S, Lai R. 5q as a sole chromosome abnormality in myelodysplastic syndromes and acute myeloid leukemia (Abstract). Mod Pathol 2001;14:182A. [5] Raza A, Alvi S, Borok RZ, et al. Excessive proliferation matched by excessive apoptosis in myelodysplastic syndromes: the cause–effect relationship. Leuk Lymphoma 1997;27:111–8. [6] Merchant SH, Gonchoroff NJ, Hutchison RE. Apoptotic index by annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes. Cytometry 2001;46: 28–32. [7] Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993;197:40–5. [8] Katz E, Deehan MR, Seatter S, Lord C, Sturrock RD, Harnett MM. B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells. J Immunol 2001;166:137–47. [9] Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993;197(1):40–5.