Red cell morphology in leukemia, hypoplastic anemia and myelodysplastic syndrome

Pathophysiology 13 (2006) 217–225

Red cell morphology in leukemia, hypoplastic anemia and myelodysplastic syndrome Durjoy Majumder a , Debasis Banerjee b , Sarmila Chandra c , Subir Banerjee d , Abhijit Chakrabarti a,∗ a

Biophysics Division, Structural Genomics Section, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India b Department of Pathology, Ramakrishna Mission Seva Prathisthan, Kolkata 700026, India c Department of Hemato-Oncology, Kothari Medical Centre, Kolkata 700027, India d Department of Medicine, R.G. Kar Medical College, Kolkata 700037, India Received 3 June 2006; received in revised form 20 June 2006; accepted 23 June 2006

Abstract Leukemic patients of different classifications are associated with anemia. Such clinical conditions are often referred to as refractory anemia, paraoxymal nocturnal hemoglobinuria, hemolytic uremia and autoimmune hemolytic anemia, all of which could be categorized as the cancer cachexia. In the present work, we have studied the overall morphology of intact red cells in different leukemic patients along with patients of hypoplastic anemia (HPA) by scanning electron microscopy. We have also studied the ultrastructure of the red cell surface membranes by transmission electron microscopy. For all experiments, erythrocytes from normal individuals served as controls. We have shown direct evidence of the altered red cell (RBC) membrane morphology irrespective of the hemoglobin status of the patients which includes (1) presence of large central holes in RBCs of acute myeloid leukemia (AML), (2) presence of thorn- and horn-like structure in RBCs of acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) and (3) flaccid appearance of RBCs in chronic lymphocytic leukemia (CLL) patients. A mixture of the above mentioned structures were found in the red cells of patients suffering from myelodysplastic syndrome (MDS) and in case of patients of HPA the RBCs lost the normal biconcave structures. TEM studies revealed presence of pores with diameters ranging from 100 to 200 nm on the RBC membrane surface of myeloid leukemia with AML being the most prominent among others. Such pathophysiological alterations of the RBC morphology in leukemic patients could be identified as characteristic signature of the onset of anemia associated with the disease. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Electron microscopy; Red cells; Membrane pores; Anemia; Leukemia

1. Introduction Anemia is associated with all forms of hematological malignancies [1–3]. The clinical conditions are known as refractory anemia, paraoxymal nocturnal hemoglobinuria (PNH), hemolytic uremia and autoimmune hemolytic anemia [4–7]. Cell surface glycosylphosphatidylinositol linked CD55 and CD59 are often found in decreased amounts in patients of different forms of leukemia with PNH state ∗ Corresponding author. Tel.: +91 33 2337 5345–49; fax: +91 33 2337 4637. E-mail address: [email protected] (A. Chakrabarti).

0928-4680/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2006.06.002

[8–10]. Recent reports also indicate deficiencies of haem transport in a cell line of erythroleukemia, K562 [11]. Moreover, myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) are regarded as clonal stem cell disorders with partial differentiation indicating, therefore, all lineages of the hematopoietic system to be affected [12–16]. This may lead to leukemia induced anemia rather than deficiency of erythropoietin as suggested earlier [17]. Anemia is also associated with myeloproliferative disorder, CML, chronic lymphocytic leukemia (CLL), childhood acute leukemia and granular lymphoblastic leukemia [18–25]. In an earlier work, membrane abnormalities in terms loss of asymmetry and cross-linking of

218

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

skeletal proteins have been reported in the red cells of CML patients [26]. In a recent work, cell-specific cytograms were constructed from the data obtained from different leukemic patients using automated blood analyzer and artificial neural networking to identify different pathological lesions on the red cell cytogram [27]. The red cells isolated from childhood acute lymphoblastic leukemia patients, with low hemoglobin (Hb) level, indicated differences in membrane asymmetry and increased fragility compared to the normal cells [25]. Moreover, patients identified with hypoplastic anemia (HPA) are often found to get transformed into acute or chronic myeloid leukemia [28,29]. Therefore, a series of clinical reports are available in one hand indicating all types of leukemic patients experiencing anemia. On the other hand, there are no systematic studies or direct evidence on the identification of morphological alterations of red cells in leukemic patients experiencing anemia. In the present study, we have looked at the morphology of intact erythrocytes from bone marrow or peripheral blood samples of different leukemic patients to gain a direct evidence of hematopoietic system alteration under the state of hematological malignancy. For this, intact red cells by scanning electron microscopy (SEM), red cell membrane ultrastructural investigations were carried out by transmission electron microscopy (TEM) and flow cytometric (FACS) analysis for membrane asymmetry were done in several de novo patients of HPA, MDS, chronic and acute leukemia. The work presented here, shows distinct morphological differences in erythrocytes of leukemic patients from those of normal volunteers by SEM and porous structures on the membrane surface was evident by TEM analysis.

2. Materials and methods 2.1. Patients and normal individuals About 1 ml of peripheral blood (PBL) or bone marrow (BM) samples were collected at the time of diagnosis in a heparinized vial from different myeloid and lymphoid leukemia and hypoplastic anemia patients undergoing treatment in Kothari Medical Centre, Ramakrishna Mission Seva Prathisthan and R.G. Kar Medical College & Hospital, Kolkata, India. The diagnosis and morphological categorization of different types of leukemia were performed by light microscopy in accordance with the French–American–British (FAB) criteria. HPA were diagnosed by the bone marrow and physical examination. Out of the collected samples, we have investigated 21 AML patients (2 of them were M4, 1 was M3, 1 is M6 and rest of the samples were either M1or M2), 16 ALL patients (all were either L1 or L2 and either CD19+ or both CD19+ and CD20+ Blineage ALL), 15 CML patients (all were in chronic phase and are Ph+ ), 6 CLL patients (all were either CD19+ or both CD19+ and CD20+ B-lineage CLL), 11 MDS patients (7 were MDS-RA and 4 were MDS-AML) and 10 patients with HPA.

The clinical features of the individual patients are summarized in Table 1. For all the acute leukemia cases blast counts ranged from 50 to 85% and for all chronic leukemia blast counts were ≤5% with Hb levels between 10 and 13 g/dl; 7 and 8 g/dl for MDS-RA cases, and 8 and 9 g/dl for MDSAML patients. For HPA cases the bone marrow cellularity was ≤50% and Hb levels were between 9 and 10 g/dl. For all leukemic cases, only samples from newly diagnosed patients aged 6 months–90 years, who had not undergone any chemo/radio therapy (de novo), were included in this study. For HPA patients, the peripheral blood samples were collected at their second relapse of the disease. Peripheral blood was also collected from 10 normal healthy normal volunteers (NV). Patients, free from the clinically observable liver or kidney dysfunction who were without any treatment for more than 3 months before collection of the blood samples and whose serum electrolyte (Na+ , K+ and Ca2+ ) and SGOT and SGPT levels were within the normal values, were only considered for this study. Written consent as per institutional ethical guidelines was obtained from all patients and normal volunteers concerning the use of their samples for research purpose. Starting from blood collection to all steps in processing of the red blood cells, plastic wares were used to avoid any glass-induced morphological changes. 2.2. Isolation of red cells Collected blood samples were first diluted about four times with normal saline and then separated by Percoll (Sigma, St. Louis, USA) density gradient centrifugation at a density of 1.09 [30]. The upper layer contained white blood cells and was removed. Below the Percoll gradient thick band of red blood cells were collected, washed two times with Dulbecco’s phosphate buffer saline, pH 7.4 (PBS) supplemented with 1% d-glucose and used for further analysis. Red cell ghost membranes were prepared by hypotonic lysis for overnight at 4 ◦ C, of 106 freshly isolated red cells in 5 mM phosphate, 1 mM EDTA containing 20 ␮g/ml of PMSF at pH 8.0 following the procedure of Dodge et al. [31]. The membranes were washed and resuspended in PBS. 2.3. TEM analysis of red cell membrane The red cell membranes were applied on formver/carboncoated copper grids of 600 mesh and stained with 1% phosphotungstic acid before observing under the electron microscope (Hitachi H-600) operating at 75 kV accelerating voltage. 2.4. SEM analysis of intact red cells Freshly isolated red cells were fixed with 1.25% glutaraldehyde in PBS, followed by dehydration with graded alcohol and resuspended in acetone, applied on the glass cover slip mounted on the SEM grid and subjected to electron microscopic analysis (Quanta 200F, FEI, Switzerland).

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

219

Table 1 Representative clinical data of individual blood samples in leukemia, hypoplastic anemia and myelodysplastic syndrome Sample

Sex

Age (years)

Diagnosis

WBC × 109 l−1

Blast cells % in BM

Hb (g/dl)*

NV1 NV2 NV3 NV4 NV5 NV6 NV7 NV8 NV9 NV10 AML1 AML2 AML3 AML4 AML5 AML6 AML7 AML8 AML9 AML10 ALL1 ALL2 ALL3 ALL4 ALL5 ALL6 ALL7 ALL8 ALL9 ALL10 CML1 CML2 CML3 CML4 CML5 CML6 CML7 CML8 CML9 CML10 CLL1 CLL2 CLL3 CLL4 CLL5 CLL6 CLL7 CLL8 CLL9 CLL10 MDS1 MDS2 MDS3 MDS4 MDS5 MDS6 MDS7 MDS8 MDS9 MDS10 HPA1 HPA2 HPA3

M M F M M M M M M M M F M M M M M M M M M M M M M M M M M M M M F M F M M M M M M M M M M F M F M M F F M F F M F M M F M M F

26 27 26 25 35 39 44 35 48 52 38 32 44 55 60 38 45 36 49 52 0.8 2.5 12 0.6 4 3 6 14 2 1 55 61 56 52 51 58 65 32 39 31 55 66 38 70 68 72 58 82 75 65 22 25 68 75 76 70 72 70 72 92 16 18 21

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal AML-M2 AML-M2 AML-M1 AML-M1 AML-M1 AML-M4 AML-M2 AML-M2 AML-M2 AML-M6 ALL-L2-B ALL-L2-B ALL-L2-B ALL-L1-B ALL-L2-B ALL-L1-B ALL-L2-B ALL-L2-B ALL-L2-B ALL-L1-B Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ Chronic-Ph+ CLL-B CLL-B CLL-B CLL-B CLL-B CLL-B CLL-B CLL-B CLL-B CLL-B MDS-RA MDS-RA MDS-RA MDS-RA MDS-RA MDS-RA MDS-AML MDS-AML MDS-AML MDS-AML HPA HPA HPA

9.8 8.2 6.7 9.2 8.7 8.6 7.2 9.9 10.2 8.5 13.4 49.2 15.1 43 48 49.2 156 3 8 6.3 13.8 22 56 65.3 120 12 172.5 8.3 6.5 3 35 102 25 167 92 55 42 105 66 65 56 65.2 39 55.5 45 58 72 68 62 58.2 4.8 5.2 6 4.8 3.2 4.2 30 28.2 27.3 17.3 2.8 3.3 2.5

ND ND ND ND ND ND ND ND ND ND 48 83 56 62 53 76 90 92 88 57 52 65 50 59 92 53 58 88 78 56 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 22 35 32 28 ≤5, HPC ≤5, HPC ≤5, HPC

14.2 14.0 13.8 14.8 15.0 13.9 14.1 14.8 15.4 13.5 11.2 13.2 12.2 11.0 11.6 11.8 12.9 11.7 11.2 10.6 10.2 11.2 13.2 12.0 11.6 11.9 12.0 12.7 12.2 11.6 11.2 12.1 12.5 11.6 12.8 12.8 12.9 11.6 11.0 10.6 12.2 12.0 10.2 12.0 12.6 12.8 12.9 11.8 11.6 12.9 7.2 7.0 7.5 7.9 7.1 7.0 8.2 8.8 8.4 8.6 9.9 9.9 9.3

220

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

Table 1 (Continued ) Sample

Sex

Age (years)

Diagnosis

WBC × 109 l−1

Blast cells % in BM

Hb (g/dl)*

HPA4 HPA5 HPA6 HPA7 HPA8 HPA9 HPA10

M F M M M M M

25 36 22 40 32 24 31

HPA HPA HPA HPA HPA HPA HPA

1.2 2 0.9 3 1.8 2.6 2.7

≤5, HPC ≤5, HPC ≤5, HPC ≤5, HPC ≤5, HPC ≤5, HPC ≤5, HPC

9.5 9.8 9.9 9.8 9.5 9.2 9.0

NV: healthy normal volunteers, M: male; F: female; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; CML: chronic myeloid leukemia; Ph: Philadelphia chromosome; CLL: chronic lymphocytic leukemia; B: B cell leukemia; MDS: myelodysplastic syndrome; HPA: hypoplastic anemia; WBC: total white blood cells count in peripheral blood; BM: bone marrow, HPC: hypocellular marrow; * Hb: hemoglobin—as measured by sodium lauryl sulphate method using Sulphone (Sysmex, Singapore) and KX21 hematology analyzer (Sysmex, Singapore); ND: not done.

The image was viewed in low vacuum at 0.75 Torr, with a ˚ before the voltage of 7 kV, and the beam spot size was 21 A red cells were visualized and photographed at different magnifications. 2.5. Flow cytometric analysis The extent of binding of annexin V to study the red cell membrane asymmetry was determined by flow cytometry within 4–6 h of sample collection and after labeling with annexin V conjugated with phycoerythrin (AV-PE) as per manufacturer’s instruction (BD Parmingen, San Diego, USA). For labeling with AV-PE, RBCs were suspended in PBS (pH 7.4) supplemented with 2 mM CaCl2 to a final con-

centration of 1 × 106 cells/ml. AV-PE binding data were analyzed with the control unlabelled sample containing the same number of cells and the AV-PE in absence of Ca2+ . Both the labelled and unlabelled samples were incubated for 15 min in dark at room temperature. Flow cytometry of both unlabelled and AV-PE-labelled cells were done in FACSCalibur flow cytometer (Becton Dickinson, San Jose, USA) to measure one-color fluorescence (FL2) of AV-PE. The instrument was calibrated following standard protocols to achieve day-to-day reproducibility. The red cell population was defined by size in forward and side scatter plots. Events that correlated with intact erythrocytes were analyzed for fluorescence intensity using the same standard settings. For each sample, 30,000 events were acquired and analyzed by CellQuestProTM soft-

Fig. 1. Scanning electron micrograph of the isolated red blood cells, with magnifications shown below each micrograph, from chronic myeloid leukemia (CML, A), acute myeloid leukemia (AML, B), chronic lymphocytic leukemia (CLL, C) and acute lymphoblastic leukemia (ALL, D).

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

221

ware (Becton Dickinson, San Jose, USA). From the FSC and SSC clustered cell population was selected and analyzed for Fl2 histogram. Fluorescence intensities were expressed in logarithmic scale. The control sample, not treated with AV-PE, was used to set the region for positive fluorescence called the autofluorescence, such that the fraction of the cells with autofluorescence was lower than 0.2% of total events by setting a marker M1. The population of cells labelled with AV-PE above background was determined from the fraction of cells in this region in excess of that obtained with the unlabelled control. The marker M1 selection was set such that less than 0.2% of the cells were included in this region and data were expressed as percent binding of annexin V [32].

3. Results 3.1. SEM analysis of red cells Scanning electron microscopic observations revealed changes in the overall morphology of the red cells isolated from the leukemic patients and changes seemed to vary with the type of hematological malignancy (Fig. 1). In cases of HPA, there was loss of the biconcave shape of red cells with thinner and/or translucent appearance shown in Fig. 2. However, predominance of red cells with both thorn- and horn-like structures of acanthocytes and echinocytes were seen in MDS patients (Fig. 2). Distinct large central holes were seen in the red cells of AML samples without much change in the shape. In CML, MDS and ALL samples a predominance of acanthocytes and

Fig. 2. Scanning electron micrograph of the isolated red blood cells, with magnifications shown below each micrograph, from normal volunteers (normal, A), hypoplastic anemia (HPA, B) and myelodysplastic syndrome (MDS, C).

Fig. 3. Percentage of morphologically altered cells as detected by scanning electron micrograph of the red cell from healthy normal volunteers (NV), patients of hypoplastic anemia (HPA), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). Data presents as the mean and S.D. Bar shows the percentage of altered cells in the purified RBCs in different samples of a particular leukemia.

222

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

echinocytes were seen along with few cells exhibiting porous structures. In CLL samples, the red cell morphology looked distinctly thinner with low density flaccid appearance (Fig. 1). SEM analysis indicated the presence of a central hole in the intact red cells with lesser thorn- and horn-like structure in the red cells of AML patients compared to those of MDS and CML cases where a mixture of acanthocytes and echinocytes were observed. The numbers of the morphologically altered cells in the population for a particular type of leukemia are shown in Fig. 3. For such analysis, typically 100–150 cells were counted in each different RBC samples. 3.2. TEM analysis of red cells TEM analysis revealed that red cells isolated from leukemic patients showed porous structures on the membrane surface in comparison with the normal red cells or in cases of hypoplastic anemia (Fig. 4). These porous structures were mostly abundant in red cell membranes of myeloid

lineage leukemia—the most prominent being in AML following the order AML  MDS-AML > CML  HPA > ALL ≥ CLL. The pore diameter of the red cell membranes of AML patients ranged from 100 to 200 nm. Leukemia of lymphoid lineage showed the least or no porous structures on the red cell membrane surface even at a very high magnification. The cells of most of the MDS-RA cases could not re-associate after hypotonic lysis, as shown in Fig. 4C. 3.3. Annexin binding analyses by flow cytometry Table 2 summarizes the extent of binding of AV-PE to red cells in different cases of hematological malignancies. Red cells isolated from leukemic patients showed very low autofluorescence in the absence of AV-PE. Increase in binding of AV-PE compared to the normal indicated exposal of phosphatidylserine (PS) on the red cell surface for all different types of leukemic samples with preference for chronic leukemia.

Fig. 4. Transmission electron micrograph of the red cell membranes, with magnifications shown below each micrograph, from normal volunteers (normal, A), from a patient of hypoplastic anemia (HPA, B), myelodysplastic syndrome (MDS) with refractory anemia (MDS-RA, C), MDS with myeloid blasts (MDS-AML, D), of chronic myeloid leukemia (CML, E), acute myeloid leukemia (AML, F), chronic lymphocytic leukemia (CLL, G) and acute lymphoblastic leukemia (ALL, H). Arrows indicate the pores in the cell membrane.

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

223

Fig. 4. (Continued ) Table 2 Annexin V binding of red cells from normal subjects, in hypoplastic anemia (HPA) and in different cases of myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) Parameter

AV-PE% binding

Sample [mean ± S.E.M.] Normals (n = 10)

HPA (n = 5)

MDS (n = 2)

CML (n = 6)

AML (n = 10)

CLL (n = 2)

ALL (n = 10)

2.36 ± 0.34

3.2 ± 0.54, NS

0.45, ND

3.12 ± 1.39, P < 0.05

2.63 ± 0.23, NS

5.71, ND

2.90 ± 1.40, NS

n indicates the number of samples analyzed by flow cytometry; NS: not statistically significant; ND: not done.

4. Discussion The present study shows some characteristic features of red cell morphology and the degree of porosity in the red cell membrane in different types of leukemia—the most prominent in AML, following the order AML  MDSAML > CML  HPA > ALL ≥ CLL, the least in CLL. In an earlier study, the fetal-like feature was examined in red cells to establish the relationship of the type and phase of the leukemia in children with newly diagnosed ALL. Abnormal red cells were more frequently found at the time of diagnosis in acute nonlymphoblastic leukemia (ANLL) than in ALL [33]. A recent investigation on the erythrocyte features dur-

ing anemia in childhood ALL patients also showed altered characteristics of red cells [25]. Alterations in red cell morphology, in our opinion, could cause changes in hemoglobin stability. Under conditions such as oxidative stress, formation of hemichromes and other kinds of high molecular weight aggregates involving cytoskeletal proteins are favored leading to depletion of functional hemoglobins and early hemolysis of erythrocytes. Another possibility might be that the leukemia associated factors or small molecular weight toxic materials could lead to formation of membrane pores in the leukemic red cells [3,6]. This electron microscopic work is the first elaboration on the altered morphologic and ultrastructural features of the red

224

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

cells of HPA, MDS and of different leukemic patients. SEM images provide an authentic portrayal of red cell shape in vivo unlike in the traditional way where the red cell morphology may be distorted by spreading and drying of blood films. In this study, red cells were isolated from fresh blood or bone marrow samples only from newly diagnosed patients who had not undergone any chemo/radio therapy indicating the absence of any changes in the red cell ultrastructure and morphology due to aging or the treatment of any chemotherapeutic drugs. Moreover, the patients were free of any clinically observable liver or kidney dysfunction, indicating the altered morphology to be due to the leukemia induced changes in the erythroid lineage of the hematopoietic system. Scanning electron microscopic observations revealed gross changes in the overall morphology of the red cells with large central holes in the red cells of AML samples, presence of acanthocytes and echinocytes in MDS, CML and ALL samples along with the presence of thinner and low density red cells particularly in CLL cases (Fig. 1). Large pores of 100–200 nm diameters were seen in the TEM images of the cell surface membrane of leukemia with myeloid lineages, e.g. MDS-AML, CML and AML patients, respectively, shown in Fig. 4D–F. However, leukemia of lymphoid lineage showed the least or no such porous structures on their membrane surface (Fig. 4). Detailed analysis of different red cell morphology revealed that losses of normal biconcave shape were mostly found in HPA and were found in the order HPA  MDS ≡ CML > AML ≡ CLL > ALL. Cells with thorn- and horn-like formations were mostly found in ALL following the order ALL  MDS  CML > CLL ≡ AML and those with large central holes were found in AML and few cases of ALL. Some red cells appeared flaccid and were predominantly found in CLL and few in MDS cases. Therefore, in the myeloid lineages (from HPA to AML) the changes in RBC structure could take place through the following events of firstly losing the normal biconcave shape followed by appearance of thorn- and horn-like structures. On the other hand, in the lymphoid lineages (CLL to ALL), the changes might take place by losing the normal biconcave shape together with flaccid appearance followed by appearance of thorn- and horn-like structures. The binding of annexin V was increased in ALL and CML cases which was in agreement with the previous report in ALL [25,26]. The percent binding of annexin V also increased in all leukemic cases, significantly in case of CML patients (P < 0.05), indicating PS exposure in the red cell membrane without showing any correlation with other types of leukemia. The electron microscopic data indicate that there was a distinctive change in the red cells in different leukemic cases, even in between MDS and HPA. Taken together, the red cell morphological features could be used to identify leukemia induced pathophysiological alterations so that management can be taken earlier in patients without clinically observable anemia, a part of cancer cachexia which may be seen at a much later stage [3].

Acknowledgements Durjoy Majumder acknowledges University Grants Commision, India, for the award of a Senior Research Fellowship. We acknowledge help from Mr. Pulak Roy of the EM facility for assistance in the transmission electron microscopy. Thanks are due to Dr. M.K. Sanyal, Dr. T. Chini and Mr. S. Banerjee for helping us in the scanning electron microscopic measurements. References [1] H. Ludwig, Anemia of hematologic malignancies: what are the treatment options? Semin. Oncol. 29 (2002) 45–54. [2] T. Littlewood, F. Mandelli, The effects of anemia in hematologic malignancies: more than a symptom, Semin. Oncol. 29 (2002) 40–44. [3] H. Rubin, Cancer cachexia: its correlations and causes, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 5384–5389. [4] W.F. Rosse, Paraoxymal nocturnal hemoglobinuria, in: R. Hoffman, E.J. Benz Jr., S.J. Shantil, B. Furie, H.J. Cohen, L.E. Silberstein, P. McGlave (Eds.), Hematology: Basic Principles and Practice, third ed., Churchill Livingstone, New York/London/Philadelphia, 2000, pp. 331–342. [5] H. Hahn, I.S. Ha, H.S. Choi, H.Y. Shin, H.I. Cheong, H.S. Ahn, Y. Choi, Acute leukemia: an association with atypical hemolytic uremic syndrome, Pediatr. Nephrol. 18 (2003) 703–705. [6] M. Deutsch, S.P. Dourakis, K. Papanikolopoulos, M. Belegrati, T. Kalmantis, Autoimmune hemolytic anemia in a patient with acute myelocytic leukemia, Am. J. Hematol. 74 (2003) 147–156. [7] M.J. Kyasa, R.S. Parrish, S.A. Schichman, C.S. Zent, Autoimmune cytopenia does not predict poor prognosis in chronic lymphocytic leukemia/small lymphocytic lymphoma, Am. J. Hematol. 74 (2003) 1–8. [8] J. Meletis, E. Terpos, M. Samarkos, C. Meletis, E. Apostolidou, V. Komninaka, K. Korovesis, D. Mavrogianni, D. Boutsis, E. Variami, N. Viniou, K. Konstantopoulos, D. Loukopoulos, Detection of CD55− and/or CD59− deficient red cell populations in patients with lymphoproliferative syndromes, Hematol. J. 2 (2001) 33–37. [9] U. Oelschlaegel, I. Besson, C. Arnoulet, D. Sainty, R. Nowak, R. Naumann, Y. Bux, G. Ehninger, A standardized flow cytometric method for screening paroxysmal nocturnal haemoglobinuria (PNH) measuring CD55 and CD59 expression on erythrocytes and granulocytes, Clin. Lab. Haematol. 23 (2001) 81–90. [10] J. Meletis, E. Terpos, M. Samarkos, C. Meletis, E. Apostolidou, V. Komninaka, K. Anargyrou, K. Korovesis, D. Mavrogianni, E. Variami, N. Viniou, K. Konstantopoulos, Red cells with paroxysmal nocturnal hemoglobinuria-phenotype in patients with acute leukemia, Hematology 7 (2002) 69–74. [11] J.G. Quigley, Z. Yang, M.T. Worthington, J.D. Phillips, K.M. Sabo, D.E. Sabath, C.L. Berg, S. Sassa, B.L. Wood, J.L. Abkowitz, Identification of a human heme exporter that is essential for erythropoiesis, Cell 18 (2004) 757–766. [12] B. Mehrotra, T.I. George, K. Kavanau, H. Avet-Loiseau, D. Moore II, C.L. Willman, M.L. Slovak, S. Atwater, D.R. Head, M.G. Pallavicini, Cytogenetically aberrant cells in the stem cell compartment (CD34+ lin− ) in acute myeloid leukemia, Blood 86 (1995) 1139– 1147. [13] D. Hasse, M. Feuring-Buske, S. Konemann, C. Fonatsch, C. Troff, W. Verbeek, A. Pekrun, W. Hiddemann, B. Wormann, Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations, Blood 86 (1995) 2906–2912. [14] P.L. Greenberg, Myelodysplastic syndrome, in: R. Hoffman, E.J. Benz Jr., S.J. Shantil, B. Furie, H.J. Cohen, L.E. Silberstein, P. McGlave

D. Majumder et al. / Pathophysiology 13 (2006) 217–225

[15]

[16]

[17]

[18]

[19]

[20]

[21] [22]

[23]

(Eds.), Hematology: Basic Principles and Practice, third ed., Churchill Livingstone, New York/London/Philadelphia, 2000, pp. 1106– 1129. H. Enright, P. McGlave, Chronic myelogenous leukemia, in: R. Hoffman, E.J. Benz Jr., S.J. Shantil, B. Furie, H.J. Cohen, L.E. Silberstein, P. McGlave (Eds.), Hematology: Basic Principles and Practice, 3rd ed., Churchill Livingstone, New York/London/Philadelphia, 2000, pp. 1155–1171. W. Stock, M.J. Thirman, Pathobiology of acute myeloid leukemia, in: R. Hoffman, E.J. Benz Jr., S.J. Shantil, B. Furie, H.J. Cohen, L.E. Silberstein, P. McGlave (Eds.), Hematology: Basic Principles and Practice, third ed., Churchill Livingstone, New York/London/Philadelphia, 2000, pp. 979–999. M. Kim, J. Lee, C. Wu, S. Cho, K. Lee, Defective erythropoiesis in bone marrow is a mechanism of anemia in children with cancer, J. Kor. Med. Sci. 17 (2002) 337–340. R.S. Go, J.A. Lust, R.L. Phyliky, Aplastic anemia and pure red cell aplasia associated with large granular lymphocyte leukemia, Semin. Hematol. 40 (2003) 196–200. K.L. Mori, H. Furukawa, K. Hayashi, K.J. Sugimoto, K. Oshimi, Pure red cell aplasia associated with expansion of CD3+ CD8+ granular lymphocytes expressing cytotoxicity against HLA-E+ cells, Br. J. Haematol. 123 (2003) 147–153. A. Sternberg, H. Eagleton, N. Pillai, K. Leyden, S. Turner, D. Pearson, T. Littlewood, C. Hatton, Neutropenia and anaemia associated with T-cell large granular lymphocyte leukaemia responds to fludarabine with minimal toxicity, Br. J. Haematol. 120 (2003) 699– 701. J.G. Treleaven, N. Win, Hyperhaemolysis syndrome in a patient with myelofibrosis, Hematology 9 (2004) 147–149. M. Yasuyama, K. Kawauchi, K. Takei, T. Ogasawara, S. Ohkawa, Pure red cell aplasia occurring during the course of chronic myelogenous leukemia, Rinsho. Ketsueki. 45 (2004) 66–71 (Japanese). J. Hill, R.M. Walsh, S. McHam, F. Brody, M. Kalaycio, Laparoscopic splenectomy for autoimmune hemolytic anemia in patients with chronic

[24]

[25]

[26] [27] [28]

[29]

[30]

[31]

[32]

[33]

225

lymphocytic leukemia: a case series and review of the literature, Am. J. Hematol. 75 (2004) 134–138. H.A. El-Mahallawy, T. Mansour, S.E. El-Din, M. Hafez, S. Abd-elLatif, Parvovirus B19 infection as a cause of anemia in pediatric acute lymphoblastic leukemia patients during maintenance chemotherapy, J. Pediatr. Hematol. Oncol. 26 (2004) 403–406. S. Ghosh, S. Bandyopadhyay, D.K. Bhattacharya, C. Mandal, Altered erythrocyte membrane characteristics during anemia in childhood acute lymphoblastic leukemia, Ann. Hematol. 84 (2005) 76–84. A. Kumar, C.M. Gupta, Red cell membrane abnormalities in chronic myeloid leukaemia, Nature 303 (1983) 632–633. G. Zini, G. d’Onofrio, Neural network in hematopoietic malignancies, Clin. Chim. Acta 333 (2003) 195–201. S. Imashuku, S. Hibi, F. Bessho, M. Tsuchida, T. Nakahata, S. Miyazaki, I. Tsukimoto, N. Hamajima, Detection of myelodysplastic syndrome/acute myeloid leukemia evolving from aplastic anemia in children, treated with recombinant human G-CSF, Haematologica 88 (2003) ECR31. A. Taguchi, T. Tominaga, Y. Nakamori, M. Miyazaki, K. Shinohara, Two cases of acute myeloblastic leukemia evolving from aplastic anemia, Int. J. Hematol. 77 (2003) 471–475. H. Pertoft, T.C. Lakrent, Sedimentation of cells in colloidal silica (Percoll), in: T.G. Pretlow, T.P. Tretlow (Eds.), Cell Separation—Methods and Selected Application, vol. I, Academic Press, USA, 1982, pp. 115–152. J.T. Dodge, C. Mitchel, D.J. Henahen, The preparation and chemical characteristics of hemoglobin free ghosts of human erythrocytes, Arch. Biochem. Biophys. 100 (1963) 119–130. F.A. Kuypers, J. Yuan, R.A. Lewis, L.M. Snyder, C.R. Kiefer, A. Bunyaratvej, S. Fucharoen, L. Ma, L. Styles, K. de Jong, S.L. Schrier, Membrane phospholipid asymmetry in human thalassemia, Blood 91 (1998) 3044–3051. B.P. Alter, M.A. Weiner, M.B. Harris, Erythrocyte characteristics in childhood acute leukemia, Am. J. Pediatr. Hematol. Oncol. 11 (1989) 8–15.