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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2012  |  Volume : 55  |  Issue : 2  |  Page : 196-201
Philadelphia chromosome detection in chronic myeloid leukemia: Utility of phytohemagglutinin-stimulated peripheral blood culture


1 Department of Hematology, Postgraduate Institute of Medical Education & Research, Chandigarh, Punjab, India
2 Department of Internal Medicine, Postgraduate Institute of Medical Education & Research, Chandigarh, Punjab, India

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Date of Web Publication3-Jul-2012
 

   Abstract 

Background: The conventional cytogenetic approach to demonstrate Philadelphia (Ph) chromosome at times does not yield enough number of metaphases or are of suboptimal quality. Further, the rapid molecular tests have completely pushed this simple technique into disrepute. Aims: This study aimed to evaluate usefulness of phytohemagglutinin (PHA)-stimulated peripheral blood culture for detection of Ph chromosome in chronic myeloid leukemia (CML) patients. Materials and Methods: Fifty-six patients, including 11 newly diagnosed cases of CML and 45 patients of CML on imatinib therapy showing the presence of Ph chromosome in unstimulated samples, were included in the study. Cytogenetic analysis was done on unstimulated samples, i.e. bone marrow aspirate, 24- and 48-h peripheral blood culture, and compared with PHA-stimulated 72-h peripheral blood culture. Results: The preparations from PHA-stimulated peripheral blood culture samples in all 56 patients yielded high number of good-quality metaphases. All the 11 (100%) newly diagnosed patients and 39/45 (87%) of the patients on imatinib therapy showed the presence of Ph chromosome in PHA-stimulated samples. Addition of PHA-stimulated 72-h peripheral blood culture preparation can be of use for increasing the diagnostic yield in cases of CML with suboptimal results on conventional cytogenetics from bone marrow aspirate sample.

Keywords: Chronic myeloid leukemia, peripheral blood culture, Ph chromosome, PHA stimulation

How to cite this article:
Sachdeva MU, Varma N, Rana KS, Varma S. Philadelphia chromosome detection in chronic myeloid leukemia: Utility of phytohemagglutinin-stimulated peripheral blood culture. Indian J Pathol Microbiol 2012;55:196-201

How to cite this URL:
Sachdeva MU, Varma N, Rana KS, Varma S. Philadelphia chromosome detection in chronic myeloid leukemia: Utility of phytohemagglutinin-stimulated peripheral blood culture. Indian J Pathol Microbiol [serial online] 2012 [cited 2022 Aug 11];55:196-201. Available from: https://www.ijpmonline.org/text.asp?2012/55/2/196/97867



   Introduction Top


The Philadelphia (Ph) chromosome was the first chromosomal abnormality associated with a specific malignant disease in humans, namely chronic myeloid leukemia (CML). [1] Later, it was identified as one partner in a reciprocal translocation between chromosomes 9 and 22, referred to as t(9;22)(q34;q11). [2] Genomic sequences within the BCR gene on chromosome 22 are juxtaposed with those of the ABL tyrosine kinase gene on chromosome 9. This results in replacement of sequences encoded by the first exon of ABL with BCR-derived sequence and expression of a fusion protein BCR-ABL with deregulated tyrosine kinase activity. The t(9;22) is also found in 10-20% of adults and in 2-5% of children with acute lymphoblastic leukemia (ALL), as well as in occasional bona fide cases of acute myeloid leukemia (AML), lymphoma, and myeloma. [3],[4],[5],[6],[7],[8],[9] Therefore, t(9,22) can be regarded as a hallmark of CML only in the context of chronic myeloproliferative neoplasm.

Different methods are available for the determination of bcr-abl positive cells, which include conventional cytogenetics, fluorescent in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR)/nested RT-PCR, and real-time PCR. Comparative analysis of these techniques has shown significant correlation between all techniques, especially at the time of diagnosis. [10] WHO 2001 monograph made "CML-bcr/abl positive" a distinct entity, which, henceforth, made it necessary to demonstrate the presence of bcr/abl fusion gene by any of the above methodologies for diagnosing CML. [11] Conventional cytogenetics is able to demonstrate Ph chromosome in approximately 95% of cases of CML; however, rest of the cases require more sensitive molecular methods, which have completely pushed this simple and cost-effective technique into disrepute. [10] The molecular techniques are mostly available at tertiary care/referral centers, especially in developing countries. In addition, the conventional cytogenetic approach, although technically less demanding; many times does not yield satisfactory metaphases, and hence fails to pick Ph chromosome, documentation of which is indispensable in the therapeutic era of tyrosine kinase inhibitors (TKIs).

Simple technique of short time culture of peripheral blood mononuclear cells with stimulants like phytohemagglutinin (PHA) have been attempted in the past with mostly discouraging results, as PHA is known to stimulate normal T cells which eventually produce normal (Ph chromosome negative) metaphases. The authors have attempted to revive this simple technique which could be carried out even in less privileged diagnostic centers with some technical expertise. Therefore, this study was designed to evaluate the usefulness of PHA stimulated peripheral blood lymphocytes as a complementary technique to standard unstimulated bone marrow samples, done for evaluation of Ph chromosome to diagnose as well as follow-up CML patients on therapy.


   Materials and Methods Top


This study was conducted in the Department of Hematology in collaboration with the Department of Internal Medicine, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. All patients with clinical suspicion of a myeloproliferative neoplasm were referred to the Department of Hematology for laboratory confirmation and further sub-categorization. In addition, all patients of CML on TKI therapy, being followed up in the "Leukemia Clinic" of Department of Internal Medicine, PGIMER, were also referred for cytogenetic follow-up. The patients underwent peripheral blood sampling for complete hemogram, cytochemistry for leukocyte alkaline phosphatase (LAP) levels, and cytogenetic analysis. Bone marrow examination was simultaneously performed for morphological and cytogenetic evaluation.


   Cytogenetic Analysis Top


Three sterile tubes (labeled 1, 2, 3) containing 9 ml of RPMI 1640 (Sigma) (pH 7.2-7.4) supplemented with 10% fetal calf serum and antibiotics were set up for each patient. One milliliter of heparinized blood sample was added in each tube. PHA (Biological Industries, Israel) was added in tube No. 3 and all tubes were incubated at 37°C. After 24 h, 0.1 ml of colchicine (50 μg) and ethidium bromide (25 μg) was added in tube No. 1 and incubated at 37°C for 60 minutes. The tube was then centrifuged at 1100 rpm for 10 minutes, supernatant removed, and 5 ml of 0.075 M KCl was added. The tube was then incubated for 20 minutes at 37°C for hypotonic treatment of cells, followed by centrifugation at 1100 rpm for 10 minutes. The supernatant was discarded and 6-8 drops of freshly prepared cold fixative (3 parts methanol and 1 part acetic acid) were added slowly. Cells were resuspended in a small volume of the fixative, incubated for half an hour/overnight, and washed twice/thrice in the fixative to obtain a clear supernatant. Finally, cells were suspended in 3-4 drops of the fixative or depending upon the size of cell button. Slides were prepared by dropping a suspension of the pellet in the fixative onto pre-cleaned numbered glass slides and dried on a 40°C hot plate. Giemsa staining was done for examination under oil immersion on light microscope. This was followed by G-banding for analysis on karyotyping station (Leica, CW 4000). The slides were similarly prepared from tube No. 2 after 48 h and from tube No. 3 (PHA stimulated) after 72 h. [12]

A portion of bone marrow aspiration sample of each patient, obtained from posterior superior iliac spine, was taken in a citrated tube. Colchicine (50 μg) and ethidium bromide (10 μl/ ml) were added immediately. Further processing was done as for the peripheral blood sample and Giemsa-stained slides were examined under oil immersion on light microscope, followed by G-banding for microscopic analysis with Leica karyotyping station. [12]

The slides prepared from direct bone marrow aspirate were screened first for metaphases. In case satisfactory metaphases were not seen, slides prepared from 24-h unstimulated peripheral blood sample were screened and this was followed by screening of slides from 48-h unstimulated peripheral blood sample only in the absence of metaphases on 24-h sample. Slides prepared from PHA-stimulated peripheral blood samples were screened in all cases. The cytogenetic analyses of slides prepared from bone marrow samples, 24-h and 48-h peripheral blood cultures were clubbed together as unstimulated samples, whereas, the PHA-treated 72-h peripheral blood cultures were considered stimulated samples, for purpose of comparison. Patients showing the presence of Ph chromosome on conventional cytogenetics with unstimulated bone marrow or peripheral blood samples were included in the study. In the samples included for study, on average 20-30 good-quality metaphases could be screened from unstimulated samples, whereas 50 to >200 well-spread good-quality metaphases could always be screened from PHA-stimulated samples.


   Results Top


Fifty-six cases yielded satisfactory metaphases and presence of Ph chromosome in unstimulated samples. All these cases also had PHA-stimulated peripheral blood culture samples available for comparison. These 56 cases were selected for analysis of clinical data, peripheral blood findings, LAP score, bone marrow findings on light microscopy, and to compare the results of unstimulated and PHA-stimulated peripheral blood culture (72-h) samples. All the 56 cases yielded well-spread good-quality metaphases on PHA-stimulated peripheral blood samples. Forty-five out of these 56 patients were diagnosed cases of CML on TKI therapy and the remaining 11 patients were diagnosed as CML for the first time. The age of patients ranged from 15 to 63 years, with a mean age of 38.8 years. There were 26 males and 30 females in the study group. [Table 1] summarizes the clinical features, hemogram findings, LAP score, and result of cytogenetic analysis of newly diagnosed 11 patients of CML. All these 11 patients presented with splenomegaly. On light microscopic examination of bone marrow aspirate smears and trephine biopsies, nine were diagnosed as being consistent with CML in chronic phase, whereas two patients were in accelerated phase at the time of presentation. LAP score was low in all except one (normal range 35-110). The cytogenetic analysis showed the presence of Ph chromosome in all 11 patients in the slides prepared from PHA-stimulated cultures. Hence, there was a complete concordance for the presence of Ph chromosome between the unstimulated and PHA-stimulated cultures in the newly diagnosed 11 patients. The mean percentage of Ph-positive metaphases was 56.6% (range 40-100%) in the unstimulated samples in comparison to a mean of 26.8% (range 5-60%) in PHA-stimulated samples [Table 1]. However, it was easy to screen more than 50 good-quality metaphases in all PHA-stimulated samples, whereas in unstimulated sample preparations, multiple slides from each sample were required to be able to screen 20-30 satisfactory metaphases.
Table 1: Clinical details, hemogram findings, bone marrow examination report, LAP score, and Ph chromosome status in unstimulated and PHA-stimulated cultures of newly diagnosed patients

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able 2 summarizes the clinical features, hemogram findings, LAP score, and result of cytogenetic analysis of previously diagnosed 45 patients taking TKI therapy and on routine follow-up. Duration of follow-up on TKI treatment ranged from 0.1 to 8 years, with a median duration of 2.5 years. Splenomegaly was seen in five, hepatomegaly in three other patients, and hepatosplenomegaly in one patient. Bone marrow aspirate was reported as complete hematological remission (CHR) in 34 out of 45 (75.6%) patients and the rest of 11 (24.4%) patients were reported as not in CHR (NCHR). The analysis of slides prepared from PHA-stimulated samples of these 45 Ph chromosome positive cases revealed the presence of Ph chromosome in 39 (86.7%) patients. Remaining 6 (13.3%) cases were negative for Ph chromosome and showed a normal karyotype. Hence, there was concordance in the results of unstimulated and PHA-stimulated samples in approximately 87% of cases. PHA-stimulated samples again showed enough well-spread good-quality metaphases, making it easy to screen more than 50 plates in all cases, whereas multiple slides needed to be screened in order to study at least 20 satisfactory metaphases. The mean percentage of Ph chromosome positive metaphases in unstimulated samples was 23.4% (range 5-75%) and that of PHA-stimulated samples was 9.6% (range 0-33%) [Table 2].
Table 2: Clinical details, hemogram findings, bone marrow examination report, LAP score, and Ph chromosome status in unstimulated and PHA-stimulated cultures of patients on TKI therapy

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   Discussion Top


PHA stimulation of peripheral blood lymphocytes is a well-standardized and relatively simple technique used for cytogenetic studies. The karyotype of these stimulated lymphocytes is believed to represent the patient's constitutional chromosomal pattern, and therefore this technique has been frequently used for demonstrating constitutional chromosomal disorders and in chromosomal breakage studies. The authors have previously utilized PHA-stimulated peripheral blood lymphocyte culture effectively for chromosomal breakage studies in patients of bone marrow failure syndromes. [13],[14] Although Peter Nowell's [15] discovery of PHA, which stimulated T-lymphocytes to grow and divide, was a boon to clinical cytogenetics by eliminating the need for skin biopsy to obtain fibroblasts for culture, this had little or only a negative effect on leukemia cytogenetics. A normal karyotype in a 72-h PHA-stimulated blood culture from a leukemic patient could reflect dividing normal cells, not malignant cells. Such observations had completely discouraged the exploitation of this technique for leukemia cytogenetics.

Since the discovery of Ph chromosome five decades back, many attempts have been made to determine the precursor cell and hence the hematopoietic lineages involved in CML. Initial studies had categorically refuted the involvement of T-lymphocytes in the disease process and similar results were also seen in late nineties. [16],[17],[18],[19] Some studies on patients of CML blast crisis did reveal involvement of T-lymphocytes. [20],[21],[22],[23],[24] However, the direct evidence of T-lymphocyte lineage involvement in CML was shown by Haferlach et al.[25] using FICTION technique. An early hematopoietic stem cell origin and thus involvement of all hematopoietic cell lineages, including T-lymphocytes, in CML is well accepted now, which formed the basis of the present study. [26]

A 72-h in vitro culture of peripheral venous blood with PHA produced very good yield of well-spread metaphases. In addition, the morphology of chromosomes in metaphase spread was also much more satisfactory for analysis on G-banding as compared to the metaphases produced from bone marrow and unstimulated peripheral blood, which almost always yielded compact chromosomes, making the analysis much more laborious. In 10 patients (data not presented), in addition to the bone marrow sample and three peripheral blood culture tubes, a fourth culture tube without PHA stimulation was included. The cells from this tube were harvested at 72 h. These 72-h unstimulated cultures did not yield metaphases any better than other unstimulated samples used in the study. Therefore, PHA-stimulated 72-h cultures yielded the best metaphases for karyotyping.

The results of PHA-stimulated peripheral blood lymphocytes showed the presence of Ph chromosome in all newly diagnosed patients of CML. In other words, there was a complete concordance with the results of unstimulated samples in this group. There was also a high level of concordance (87%) between unstimulated and PHA-stimulated samples in the follow-up group of 45 patients. The percentage of Ph chromosome positive metaphases was less in PHA-stimulated samples when compared to that of unstimulated samples in the majority of cases [Table 1] and [Table 2]. This fall in percentage positivity is directly related to the fact that most metaphases in PHA-stimulated cultures are likely to arise from T lymphocytes, which is known to be the least affected lineage in CML. The percentage of Ph-positive metaphases thus varies in each case in accordance with strength of T-cell lineage involvement as part of CML clone. However, this fall in percentage positivity had a reduced overall impact because of high yield of good metaphases of PHA-stimulated samples which could be analyzed in much less time. The discordance of results seen in follow-up group, although not exclusively, was primarily limited to patients in "complete hematological remission" (five out of six discordant cases). One patient "not in complete hematological remission" with absence of Ph chromosome on PHA-stimulated samples had splenomegaly with anemia. His total leukocyte count and platelet counts were within normal limits. The mean percent of metaphases in unstimulated samples showing Ph chromosome for these six discordant cases was 9.3% (range 5-15%), although not statistically different (possibly due to less number of patients), which was lower than the overall mean percentage of 23.4% (range 5-75%). Since PHA-stimulated samples have shown overall lower percentage of Ph chromosome positive metaphases, screening of more number of metaphases is of help in demonstrating Ph chromosome, which is much easier than screening more slides of unstimulated sample, due to availability of large number of metaphases making the screening less time consuming as well as less labor intensive.

It becomes apparent from the results that PHA-stimulated 72-h culture of peripheral blood sample can complement the unstimulated samples for demonstration of Ph chromosome. In resource-limited peripheral laboratories, an additional tube of 72-h peripheral blood culture with PHA stimulation, besides conventional bone marrow aspirate sample, might add up to the diagnostic yield in patients of chronic myelogenous leukemia and to a lesser extent in the follow-up of patients on TKI therapy. The results also suggest T-lymphocytes to be part of neoplastic clone more often than reported in literature. However, a more sophisticated technique operating at single cell level would be required to confirm this observation. The comparative analysis in this study was limited to patients with Ph chromosome positivity in unstimulated samples. It would be interesting to compare the results of CML cases in which unstimulated samples did not yield satisfactory metaphases for demonstration of Ph chromosome but had good metaphases on PHA-stimulated samples. In view of the absence of demonstration of t(9,22) in unstimulated samples, such a study would require confirmation of diagnosis of CML in all these cases by FISH or any one of the available molecular methods in order to establish a gold standard for conventional cytogenetic analysis in PHA-stimulated cultures.


   Conclusion Top


PHA-stimulated 72-h peripheral blood culture yields well-spread good metaphases as compared to low yield in unstimulated samples and can be used along with conventional bone marrow aspirate cytogenetic analysis in the diagnosis and follow-up of CML patients. These results also suggest that T-lymphocytes may also show Ph chromosome in CML and possibly be part of neoplastic clone more often than reported in literature, which, however, needs confirmation by further studies.

 
   References Top

1.Nowell PC, Hungerford DA. A minute chromosome in human chronic granulocytic leukemia. Science 1960;132:1497-501.  Back to cited text no. 1
    
2.Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 1973;243:290-3.  Back to cited text no. 2
[PUBMED]    
3.Westbrook C, Hooberman A, Spino C, Dodge R, Larson R, Davey F, et al. Clinical significance of the BCR-ABL fusion gene in adult acute lymphoblastic leukemia: A Cancer and Leukemia Group B Study (8762). Blood 1992;80:2983-90.  Back to cited text no. 3
    
4.Suryanarayan K, Hunger SP, Kohler S, Carroll AJ, Crist W, Link MP, et al. Consistent involvement of the bcr gene by 9;22 breakpoints in pediatric acute leukemias. Blood 1991;77:324-30.  Back to cited text no. 4
    
5.Kurzrock R, Shtalrid M, Talpaz M, Kloetzer WS, Gutterman JU. Expression of c-ab1 in Philadelphia-positive acute myelogenous leukemia. Blood 1987;70:1584-8.  Back to cited text no. 5
[PUBMED]  [FULLTEXT]  
6.Preudhomme C, Lai JL, Plantier I, Demory JL, Zandecki M, Fenaux P. Cytogenetic and molecular remission in a case of acute myeloid leukaemia (AML) with inversion of chromosome 16 (Inv( 16)) and Philadelphia chromosome (Ph). Br J Haematol 1992;82:623-6.  Back to cited text no. 6
[PUBMED]    
7.Mitani K, Sato Y, Tojo A, Ishikawa F, Kobayashi Y, Miura Y, et al. Philadelphia chromosome positive B-cell type malignant lymphoma expressing an aberrant 190 kDa bcr-ab1 protein. Br J Haematol 1990;76:221-5.  Back to cited text no. 7
[PUBMED]    
8.Fujii H, Yashige H, Misawa S, Tanaka S, Urata Y, Matuyama F. Ph chromosome in a patient with non-leukemic non-Hodgkin B cell lymphoma. Am J Hematol 1990;35:213-5.  Back to cited text no. 8
[PUBMED]    
9.Martiat P, Mecucci C, Nizet Y, Stul M, Philippe M, Cassiman JJ, et al. P190 BCR/ABL transcript in a case of Philadelphia-positive multiple myeloma. Leukemia 1990;4:751-4.  Back to cited text no. 9
[PUBMED]    
10.Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger U, et al. Comparison of chromosome banding analysis, interphase- and hypermetaphase-fish, qualitative and quantitative pcr for diagnosis and for follow-up in chronic myeloid leukemia: A study on 350 cases. Leukemia 2002;16:53-9.  Back to cited text no. 10
[PUBMED]  [FULLTEXT]  
11.Jaffe ES, Harris NL, Stein H, Vardiman JW. World health organization classification of tumours of hematopoietic and lymphoid tissues. Iarc Press; Lyon, France: 2001. p. 1-351.  Back to cited text no. 11
    
12.Sandberg AA, Abe S. Cytogenetic techniques in hematology. Clin Haematol 1980;9:19-38.  Back to cited text no. 12
[PUBMED]    
13.Rowley JD. Editorial: The role of cytogenetics in hematology. Blood 1976;48:1-7.  Back to cited text no. 13
[PUBMED]  [FULLTEXT]  
14.Varma N, Varma S, Marwaha RK, Malhotra P, Bansal D, Malik K, et al. A study of multiple constitutional etiological factors in bone marrow failure syndrome patients from North India. Indian J Med Res 2006;124:51-6.  Back to cited text no. 14
[PUBMED]  Medknow Journal  
15.Nowell PC. Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res 1960;20:462-6.  Back to cited text no. 15
    
16.Whang J, Frei E, Tjio JH, Carbone PP, Brecher G. The distribution of the Philadelphia chromosome in patients with chronic myelogenous leukemia. Blood 1963;22:664-73.  Back to cited text no. 16
    
17.Nitta M, Kato Y, Strife A, Wachter M, Fried J, Perez A, et al. Incidence of involvement of the B and T lymphocyte lineages in chronic myelogenous leukemia. Blood 1985;66:1053-61.  Back to cited text no. 17
[PUBMED]  [FULLTEXT]  
18.Bartram CR, Raghavachar A, Anger B, Stain C, Bettelheim P. T lymphocytes lack rearrangement of the bcr gene in Philadelphia chromosome-positive chronic myelocytic leukemia. Blood 1987;69:1682-5.  Back to cited text no. 18
[PUBMED]  [FULLTEXT]  
19.Takahashi N, Miura I, Saitoh K, Miura AB. Lineage involvement of stem cells bearing the Philadelphia chromosome in chronic myeloid leukemia in the chronic phase as shown by a combination of fluorescence-activated cell sorting and fluorescence in situ hybridization. Blood 1998;92:4758-63.  Back to cited text no. 19
[PUBMED]  [FULLTEXT]  
20.Allouche M, Bourinbaiar A, Georgoulias V, Consolini R, Salvatore A, Auclair H, et al. T cell lineage involvement in lymphoid blast crisis of chronic myeloid leukemia. Blood 1985;66:1155-61.  Back to cited text no. 20
[PUBMED]  [FULLTEXT]  
21.Hernandez P, Carnot J, Cruz C. Chronic myeloid leukemia blast crisis with T-cell features. Br J Haematol 1982;51:175-80.  Back to cited text no. 21
[PUBMED]    
22.Griffin JD, Tantravahi R, Canellos GP, Wisch JS, Reinherz EL, Sherwood G, et al. T-cell surface antigens in a patient with blast crisis in chronic myeloid leukemia. Blood 1983;61:640-4.  Back to cited text no. 22
[PUBMED]  [FULLTEXT]  
23.Sun T, Susin M, Koduru P, Coffey EL, Dittmar K, Weiss R, et al. Extramedullary blast crisis in chronic myelogenous leukemia: Demonstration of T-cell lineage and Philadelphia chromosome in a paraspinal tumor. Cancer 1991;68:605-10.  Back to cited text no. 23
    
24.Kim AS, Goldstein SC, Luger S, Van Deerlin VM, Bagg A. Sudden extramedullary T-lymphoblastic blast crisis in chronic myelogenous leukemia: A nonrandom event associated with Imatinib?. Am J Clin Pathol 2008;129:639-48.  Back to cited text no. 24
[PUBMED]  [FULLTEXT]  
25.Haferlach T, Winkemann M, Nickenig C, Meeder M, Raam-Petersen M, Schoch R, et al. Which compartments are involved Philadelphia-chromosome positive chronic myeloid leukemia? An answer at the single cell level by combining May-Grunwald-Giemsa and fluorescent in situ hybridization British J Haematol 1997;97:99-106.  Back to cited text no. 25
    
26.Swerdlow SH, Campo E, Harris NL. Who classification of tumours of haematopoietic and lymphoid tissues. Lyon, France: Iarc Press; 2008. p. 1-367.  Back to cited text no. 26
    

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Correspondence Address:
Neelam Varma
Department of Hematology, Postgraduate Institute of Medical Education & Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.97867

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