Flow cytometry: Immunophenotyping in 48 hairy cell leukemia cases and the relevance of fluorescence intensity in CDs expression for diagnosis Citometria de fluxo: Imunofenotipagem em 48 casos de leucemia de células cabeludas e a relevância das intensidades de fluorescências nas expressões dos ant...

May 30, 2017 | Autor: Sonia Nozawa | Categoria: Flow Cytometry, Morphological Analysis, Bone marrow, Monoclonal Antibody, Expression analysis
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Flow cytometry: Immunophenotyping in 48 hairy cell leukemia cases and the relevance of fluorescence intensity in CDs expression for diagnosis

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Original ARTICLE

Flow cytometry: Immunophenotyping in 48 hairy cell leukemia cases and the relevance of fluorescence intensity in CDs expression for diagnosis Citometria de fluxo: Imunofenotipagem em 48 casos de leucemia de células cabeludas e a relevância das intensidades de fluorescências nas expressões dos anticorpos monoclonais para o diagnóstico Nydia Strachman Bacal1, Eduardo Mantovani2, Silvia Grossl3, Sonia Tsukasa Nozawa4, Ruth Hissae Kanayama5, Ana Claudia Miranda Brito6, Claudio Ernesto Mendes Albers7, João Carlos de Campos Guerra8, Cristóvão Luis Pitangueira Mangueira9

ABSTRACT Objectives: To report the experience and the importance of flow cytometry immunophenotyping by measuring the positivity and antigenic expression intensity in the diagnosis of 48 patients with hairy cell leukemia (HCL). Methods: From November 1991 to June 2005, 4318 cases were analyzed by flow cytometry, 3556 (82.3%) of which were oncohematological diseases. Forty-eight cases of hairy cell leukemia (1.3%) were diagnosed. Morphological analysis was performed on slides stained with Grunwald Giemsa panchromatic dye, analyzed by two experienced professionals. The cytochemical analyses made were for acid phosphatase and tartrate-resistant acid phosphatase (TRAP). For antigenic expression analysis, the monoclonal antibodies used were: CD2, CD3, CD5, CD7, CD10, CD11c, CD19, CD20, CD22, CD23, CD25, CD38, HLA-DR, FMC-7, CD79b, CD103, IgM, IgG, IGD, kappa and lambda. Results: By analyzing positivity and monoclonal antibody expression intensity in the forward scatter vs. side scatter histograms (used between 1991 and 2001), and in CD19 vs. SSC histograms with sequential histograms (after 2001), it was possible to confirm this pathology and to discriminate residual cells after the specific therapy. Conclusion: Diagnostic confirmation of hairy cell leukemia by flow cytometry is a fast and accurate method that is useful in the clinical laboratory. The option for an initial CD19 vs. SSC histogram and an analysis of antigenic expression intensity in the bone marrow showed to be statistically more efficient.

Keywords: Leukemia, hairy cell; Flow cytometry; Immunophenotyping;  Antibodies, monoclonal

RESUMO Objetivo: Relatar a experiência e importância da imunofenotipagem por citometria de fluxo através da positividade e intensidade de expressões antigênicas no diagnóstico de 48 pacientes com leucemia de células cabeludas. Métodos: Entre novembro de 1991 e junho de 2005, 4.318 casos foram analisados por citometria de fluxo, sendo 3.556 pacientes com doenças oncoematológicas (82,3%) e diagnosticados 48 casos (1,3%) de leucemia de células cabeludas. As análises morfológicas foram realizadas em lâminas coradas pelo método pancromático May Grunwald Giemsa, por pelo menos dois experientes morfologistas. As análises citoquímicas realizadas foram fostatase ácida e fosfatase ácida inibida por tartarato (TRAP). Foram analisadas as expressões antigênicas dos anticorpos monoclonais: CD2, CD3, CD5, CD7, CD10, CD11c, CD19, CD20, CD22, CD23, CD25, CD38, HLA-DR, FMC-7, CD79b, CD103, IgM, IgG, IGD, Kappa e Lambda. Resultados: Através das análises dos porcentuais de positividade e intensidade de expressão dos anticorpos monoclonais encontrados nas regiões de células dos histogramas de volume e complexidade (utilizado de 1991 a outubro de 2001), e análise nas regiões de células CD19 positivas com histogramas seqüenciais, foi possível identificar essa patologia,

Study carried out at clinical labotatoy of Hopsital Israelita Albert Einstein HIAE - São Paulo (SP), Brazil. 1

Hematologist and Clinical Pathologist at the Hospital Israelita Albert Einstein - HIAE, São Paulo (SP), Brazil.

2

Biomedical Professional at the Laboratório Diagnósticos da América, São Paulo (SP), Brazil.

3

Biomedical Professional at the Laboratory of the Hospital Edmundo Vasconcelos, São Paulo (SP), Brazil.

4

Biomedical Professional at the Clinical Laboratory of the Hospital Israelita Albert Einstein - HIAE, São Paulo (SP), Brazil.

5

Pharmacist at the Clinical Laboratory of the Hospital Israelita Albert Einstein - HIAE, São Paulo (SP), Brazil.

6

Pharmacist at the Clinical Laboratory of the Hospital Israelita Albert Einstein - HIAE, São Paulo (SP), Brazil.

7

Biomedical Professional at the Clinical Laboratory of the Hospital Israelita Albert Einstein - HIAE, São Paulo (SP), Brazil.

8

Hematologist at the Hospital Israelita Albert Einstein - HIAE, São Paulo (SP), Brazil

MD; Coordinator of the Clinical Laboratory of the Hospital Israelita Albert Einstein - HIAE, São Paulo (SP), Brazil. Corresponding author: Nydia Strachman Bacal - Av. Albert Einstein, 627 - Morumbi - CEP 056551-901 - São Paulo (SP), Brazil - e-mail: [email protected]. Received on Sep 4, 2006 – Accepted on May 2, 2007 9

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Bacal NS, Mantovani E, Grossl S, Nozawa ST, Kanayama RH, Brito ACM, Albers CEM, Guerra JCC, Mangueira CLP

inclusive com discriminação de células residuais após terapêutica específica. Conclusão: A confirmação diagnóstica das leucemias de células cabeludas por citometria de fluxo é um método rápido, preciso e útil no laboratório clínico. A opção por um histograma inicial de CD19 x SSC e análise de intensidade de expressão antigênica na medula óssea mostrou-se estatisticamente mais eficiente. Descritores: Leucemia de células pilosas; Citometria de fluxo; Imunofenotipagem, Anticorpos monoclonais

INTRODUCTION The origin of hairy cell leukemia (HCL) has been controversial for many years. In an article of 1958, “Leukemic reticuloendotheliosis”, Bouroncle et al.(1) stated that it was a reticular cell neoplasia. In the debate on whether the origin of these mononuclear cells was in the phagocytic system, the opposing positions were based on their poor phagocytic capacity and on the presence of immunoglobulin Fc receptors on the cell surface. The demonstration that immunoglobulin is limited to the light chain indicates that HCL has its origin in a malignant B cell. It is a rare disease, with an incidence of 2% among leukemias, observed in adults, usually males (5:1 ratio), with a mean age of 55 years. The clinical picture of pancytopenia with splenomegaly associated with bone marrow aspiration, resulting from the reticulinic fibrosis, suggests the diagnosis of HCL(2-4). The characteristic cells are mononuclear with abundant cytoplasm, with thin projections that extend over the cell surface, round or oval nuclei, observed in the peripheral blood, infiltrating the bone marrow and in the red pulp of the spleen (figure 1).

SSC histograms, the region of these cells is typically next to the mature lymphocyte region (figure 2). Working with sequential histograms allows discriminating the pan-B cells (CD19, CD20), which improves the precision of the analysis (figure 3). A sequential histogram analysis is indicated mainly in bone marrow aspirates, since the medullary lymphoid population is composed of all B-cell ontogenesis.

Figure 2. Histograms analyzing B-lymphoid cells / hairy cells in bone marrow

A B Figure 1. A - Hairy cell in peripheral blood B - Tartrate resistant acid phosphatase

Figure 3. Characteristic histograms of hairy cell leukemia patients

The tumor cells present surface immunoglobulins IgM, IgD, IgG or IgA, and express B-cell lymphoid antigens (CD19, CD20, CD22, and CD79b). They are typically CD5-, CD10- and CD23-negative, and strongly express CD25, CD11c, and CD103(5,6). FMC-7 presents variable expression. HCL presents an intense expression of CD45, similar to other mature B-cell lymphoid leukemias. In FSC (forward scatter - volume) versus SSC (side scatter - granularity) histograms and in CD45 versus

The low specificity of monoclonal antibodies CD11c, CD22, CD25, and FMC-7 in the diagnosis of HCL can be overcome by the co-expression of these markers and by the very intense expression of CD20, CD103, CD19 itself, CD20 and CD22, and by the characteristic morphology and cytochemistry of the tartrate-resistant acid phosphatase (figure 1). TRAP is present in most cases, being very sensitive, but not very specific(7). It occurs in Epstein-Barr virosis, in Sézary syndrome,

einstein. 2007; 5(2):123-128

Flow cytometry: Immunophenotyping in 48 hairy cell leukemia cases and the relevance of fluorescence intensity in CDs expression for diagnosis

in prolymphocytic B-cell leukemia, and in chronic lymphocytic leukemia/diffuse small-cell lymphocytic lymphoma. The composition of isotypes of heavychain immunoglobulins IgG, IgA, IgM and IgD can be explained by functional studies which suggest that HCL represents a post-proliferative leukemia, in the last stages of normal B-cell ontogenesis. The strong antigenic expression of CD11c and CD103 is specific to HCL. CD25 presents low to moderate intensity. The variant HCL (HCLv) expresses pan-B antigens and surface immunoglobulin, such as CD11c and FMC7 (low intensity), and does not express CD25 and CD23. Of the characteristic antigens for HCL, one is almost always absent: either CD11c, or CD25, or CD103. Differentiation from HCL also occurs by lymphocytosis, the cell contains an evident nucleolus, and neutropenia is absent. TRAP is occasionally positive, but no strong labeling occurs. Differentiation from splenic marginal zone lymphoma is possible thanks to the weak expression intensity of CD103 and of the other immunological markers which are characteristic for HCL. Splenic lymphoma with villous lymphocytes (SLVL) presents with massive splenomegaly with lymphocytosis, and absence of reticulinic fibrosis in the bone marrow. The lymphocytes present cytoplasm projections which are similar to HCL, but only in one or two sites of the cell surface. Antibodies CD19, CD20 and CD22 and surface immunoglobulin present moderate to high intensity, the cell may express FMC-7 and CD11c, but co-expression of the three antigens characteristic for HCL (CD11c, CD25 and CD103) is not observed (table 1). Table 1. Immunophenotyping in B lymphoproliferative diseases

OBJECTIVE To demonstrate the importance of flow cytometry immunophenotyping by means of antigen positivity and expression intensity in confirming the diagnosis of HCL. METHODS Between November 1991 and June 2005, 48 new cases of HCL were diagnosed at the Clinical Laboratory of Hospital Israelita Albert Einstein, using 16 peripheral blood and 32 bone marrow samples; 39 patients were males and 9 were females. Morphological analyses were done by the May-Grunwald Giemsa panchromatic method, and 12 cytochemical tests for acid phosphatase and tartrate-resistant acid phosphatase (TRAP) were performed using the Goldberg and Barka method. The cytometers used were Profile II - Coulter until 1993, and thereafter XL-MCL-Coulter. The samples were prepared by separation of cells, initially only in Ficoll-Hypaque, then in Opti-Lyse C (Immunotech) or QPrep (Coulter); currently ammonium chloride is used. The monoclonal antibodies used were: CD2, CD3, CD5, CD7, CD10, CD11c, CD19, CD20, CD22, CD23, CD25, CD38, CD45, CD79b, CD103, HLA-DR, FMC-7, kappa and lambda light-chain immunoglobulins, heavychain IgM, IgG, IgD (surface and/or intracytoplasmatic) (table 2). Table 2. Monoclonal antibodies panels used nowadays with their respective fluorochromes and manufactures MoAb

Fluorochrome

Manufacturer

CD5

FITC

IMMUNOTECH

CD10

PE

IMMUNOTECH

CD11c

PE

IMMUNOTECH

MoAb

B-LL/SLL

B - PLL

HCL

HCLv

SLVL/SMZL

CD5

(++)

(+)

(-)

(+/-)

(+/-)

CD10

(-)

(-)

(-)

(+/-)

(-)

CD11c

(+/-)

(-)

(++)

(+/-)

(+/-) até (++)

CD19

PC5

IMMUNOTECH

CD23

(++)

(-)

(-)

(-)

(+/-)

CD20

RPE

DAKO CYTOMATION

CD25

(+/-)

(+/-)

(+)

(+/-)

(+/-)

CD22

FITC

IMMUNOTECH

(++)

(+/-)

(+/-)

(-)

(+/-) até (++)

CD23

RPE

DAKO CYTOMATION

CD25

RD1

CYTOSTAT/COULTER

(++) (+/-)

(++)

CD79b

PE

IMMUNOTECH

CD103

FITC

IQPRODUCTS

FMC-7

FITC

DAKO CYTOMATION

HLA-DR

FITC

IMMUNOTECH

KAPPA

FITC

DAKO CYTOMATION

LAMBDA

FITC

DAKO CYTOMATION

IgM

FITC

DAKO CYTOMATION

IgD

FITC

DAKO CYTOMATION

IgG

FITC

DAKO CYTOMATION

CD103 FMC-7

(+/-)

(++)

(++)

Ig H

IgM/D

IgM

IgG/A/M

Ig sup. TRAP

(+/-)

(++)

(++) (++)

Key: MoAb: Monoclonal Antibodies B-LL: B-lymphocytic leukemia; SLL: small cell lymphocytic lymphoma B-PLL: B-prolymphocytic leukemia; HCL: Hairy cell leukemia; HCLv: Hairy cell leukemia variant; SLVL: Splenic lymphoma with villous lymphocytes SMZL: Splenic marginal zone lymphoma TRAP: Tartrate resistant acid phosphatase Antigen expression: (-): negative; (+/-): weak; (+): moderate; (++): strong

Flow cytometry enables making the differential diagnosis from other conditions by means of the presence or absence of expression of specific antigens and by the expression intensity of these antigens.

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Key: MoAb: Monoclonal Antibodies

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Bacal NS, Mantovani E, Grossl S, Nozawa ST, Kanayama RH, Brito ACM, Albers CEM, Guerra JCC, Mangueira CLP

Until June 2000, the methodology employed to analyze hairy cells in histograms focused volume versus granularity (gate 1 - table 3) regions; then we started

working with identification of pan-B cells, CD19 positive vs. granularity (gate 2 - table 3), with greater cell discrimination sensitivity.

Table 3. Median percentage of positive cells and antigen expression of several monoclonal antibodies in bone marrow (BM) and peripheral blood (PB) by analysis in different regions GATE 1 - Forward Scatter vs. Side Scatter (Volume vs. Granularity) N/32

Med % BM

N/16

Med % PB

N/32

Med Exp. BM

N/16

Med Exp. PB

CD11c

11/32

59.00

10/16

51.00

11/32

8.26

10/16

3.34

CD20

14/32

55.50

13/16

55.00

14/32

44.50

13/16

12.90

CD22

14/32

54.80

13/16

48.00

14/32

8.67

13/16

6.32

CD25

14/32

43.55

13/16

52.00

14/32

4.10

13/16

2.48

CD103

1/32

52.80

1/16

82.10

1/32

2.63

1/16

1.06

FMC-7

12/32

46.30

10/16

48.50

12/32

4.31

10/16

4.70

Kappa

11/32

19.20

6/16

33.15

11/32

4.75

5/16

2.66

Lambda

8/32

22.60

8/16

45.00

8/32

3.90

8/16

6.67

IgM

6/32

33.10

1/16

87.20

6/32

3.68

1/16

3.11

IgD

5/32

22.00

1/16

75.20

5/32

8.45

1/16

8.92

IgG HLA-DR

1/32 13/32

10.60 67.00

1/16 13/16

5.70 58.00

1/32 13/32

4.88 15.50

1/16 13/16

5.02 10.10

GATE 2 - CD 19 vs. Side Scatter N/32

Med % BM

N/16

Med % PB

N/32

Med Exp. BM

N/16

Med Exp. PB

CD11c

17/32

95.48

2/16

73.15

17/32

29.18

2/16

14.35

CD20

17/32

99.45

2/16

99.05

17/32

63.45

2/16

50.20

CD22

17/32

96.00

2/16

94.15

17/32

16.45

2/16

10.16

CD25

17/32

93.75

1/16

97.9

17/32

10.91

1/16

5.16

CD103

17/32

88.50

2/16

93.95

17/32

16.00

2/16

5.77

FMC-7

17/32

85.45

2/16

48.90

17/32

17.38

2/16

18.95

Kappa

14/32

80.03

2/16

8.60

14/32

5.55

2/16

8.18

Lambda

14/32

19.55

2/16

87.35

14/32

5.45

2/16

11.06

IgM

17/32

22.80

1/16

14.20

17/32

6.13

1/16

2.10

IgD

16/32

37.80

1/16

0.80

16/32

8.61

1/16

2.25

IgG HLA-DR

16/32 17/32

76.90 98.82

1/16 2/16

97.40 94.10

16/32 16/32

7.72 14.80

1/16 2/16

9.48 11.64

For the statistical analyses, the quantitative variables are presented in tables containing the medians, maximum and minimum values by asymmetric distribution. The Shapiro-Wilks test(14) was applied for testing the normality of quantitative variables that were compared between two independent groups using Mann-Whitney’s nonparametric test(15-17). Statistical significance was assessed (two-tailed p values) and p values less than 0.05 were considered significant. The Minitab 14.1 software (State College, PA, USA) was used in statistical analysis of data.

RESULTS The frequency of HCL in our service was 1.3%, higher in males (4.3:1), and the mean age found was 53 years. In the 12 patients for whom TRAP was requested, it was positive, and the disease was confirmed by flow cytometry. einstein. 2007; 5(2):123-128

The hairy cell surface antigen found to have the most intense antigenic expression was CD20. On a logarithmic scale, the median of the expression channel was 17.45 in the peripheral blood samples (16 cases) and 55.60 in the bone marrow samples (32 cases), which can be seen in table 4 by different analyses. CD45 was the antigen with the second most intense expression. CD22 showed medium to high expression intensity, and FMC-7 showed variable expression. CD11c was the one with the most intense expression, followed by CD103 and CD25. The antigenic expression of CD79b showed moderate to high intensity. The absence of expression of CD5 and CD10 could be observed best in gate CD19 x Side Scatter, just as the other characteristics described above. In the comparative statistical analyses between antigenic expression intensities, we were able to observe that the monoclonal antibodies CD11c, CD20, CD22,

Flow cytometry: Immunophenotyping in 48 hairy cell leukemia cases and the relevance of fluorescence intensity in CDs expression for diagnosis

127

Table 4. Percentage and antigen expression of antibodies in 48 patients with presumptive diagnosis of hairy cell leukemia  

Bone marrow (32) %

EXP

Peripheral blood (16) %

MoAb CD5

N 32

median 14.13

N 32

median 7.91

N 14

median 46.00

N 14

median 7.35

CD10 CD11c

30 29

2.75 83.18

30 29

3.00 15.11

15 13

1.00 58.00

15 13

2.20 5.37

CD19

31

44.73

26

15.85

15

59.00

14

10.25

CD20

32

80.98

32

55.60

16

57.50

16

17.45

CD22 CD23

32 26

79.38 9.50

32 26

12.34 3.06

16 7

56.50 4.00

15 7

7.15 1.62

CD25 CD45

32 13

72.00 96.30

32 13

8.35 29.00

15 11

53.00 97.50

15 10

3.52 11.21

CD79b CD103

16 19

78.50 69.13

16 19

11.92 9.59

3 4

95.20 87.60

3 4

10.30 5.77

FMC7 HLA-DR

30 31

64.48 82.75

30 30

10.73 14.33

13 16

50.60 65.00

13 16

4.99 11.00

KAPPA LAMBDA

26 23

50.15 19.88

26 23

4.37 4.40

9 11

28.30 52.00

8 11

3.96 7.19

IGM IGG

24 22

27.43 35.25

24 22

5.45 8.10

2 2

50.70 38.00

2 2

2.61 5.59

IGD K INTRA

18 8

43.78 74.20

18 8

7.52 4.82

2 1

51.55 94.50

2 1

7.25 8.04

L INTRA

8

6.18

8

5.83

1

5.80

1

0.90

EXP

Key: MoAb: Monoclonal antibodies; %: Percentage; EXP: Antigen expression; N: Patients performed; INTRA: Intracitoplasmatic

     

Positivity (over 20%) Strong expression Moderate expression Weak expression

CD23, CD25, and FMC-7 were significantly less expressed in bone marrow than in peripheral blood (table 5). Analyzing the different cases, we observed four patients with absence of CD25 expression, but - taking into account that it was only after 1995 that we started testing for CD11c and FMC-7 and only after November 2000 for CD103 - three of these patients could not be analyzed adequately. In one case we had doubts about the labeling of CD25, since all the other antigenic expressions indicated HCL. CD10 was positive in 10% of patients.

DISCUSSION HCL is a rare disease, which was confirmed in our service, and the mean age found in our patients, as well as the predominance of males, showed to be in accordance with the literature(8). The surface antigen with the most intense antigenic expression in hairy cells was CD20, also similar to the findings in the literature(9). CD45 showed to be the antigen with the second highest expression intensity, which can also be observed in other mature lymphoid B-cell leukemias(10).

Table 5. Comparison of antigen expressions of monoclonal antibodies in peripheral blood (PB) and bone marrow (BM) CD 5 n CD 10 n CD 11c n CD 19 n CD 20 n CD 22 n CD 23 n CD 25 n CD 45 n CD 79b n CD 103 n FMC-7 n HLA-DR n Kappa n Lambda n

PB 7.35(1.46-51.40) 14 2.20(1.18-36) 15 5.37(1.84-45.8) 13 10.25(3.70-47.70) 14 17.45(1.81-99.10) 16 7.15(1.22-98.30) 15 1.62(0.4-2.70) 7 3.52(1.2-12.6) 15 11.21(3.27-53.30) 10 10.30(3.88-10.90) 3 5.77(1.06-11) 4 4.99(2.22-26.20) 13 11(4.25-27.90) 16 3.96(1.56-26.20) 8 7.19(1.94-25.50) 11

BM 7.84(1.68-274) 31 2.94(0-11) 19 11.50(2.70-86.10) 28 16.05(3.81-53.20) 24 49.10(5.14-276.50) 31 14.90(2.45-75.20) 31 3.37(1.59-13) 25 5.88(1.66-37.60) 31 29(2.57-60.50) 13 18.10(5.34-75.50) 15 16.55(2.41-78.50) 18 13.20(1.34-77.70) 29 14.70(2.71-73.80) 30 4.75(0.96-41.70) 25 4.23(0.88-23.30) 21

p 0.198 0.3596 0.0321 0.1265 0.0078 0.0126 0.0012 0.0339 0.2265 N/A N/A 0.0792 0.3871 0.6898 0.1532

N/A = not applicable Non-parametric Mann-Whitney test

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In our study, we observed that CD22 showed medium to high expression intensity and FMC-7 showed variable expression. Of the antigens which concomitantly characterize HCL, CD11c was the one with the most intense expression, followed by CD103 and by CD25, as reported in the literature(11). In the cases analyzed by us, differently from some reports in the literature(12-13), the antigenic expression intensity of CD79b was moderate to high. Ten percent of the patients analyzed were CD10positive, which is consistent with the data from the literature(18-19). The use of Ficoll-Hypaque for cell separation has not been recommended, in view of the possible loss of cells during the process. The use of Q-Prep and Opti-Lyse C reduces the specificity and requires a precise titration of the monoclonal antibodies; moreover, these methods are more expensive than the hemolytic buffer with ammonium chloride, which is the method we currently use. TRAP(7) used to be more frequently requested until the year 2000, because of the speed and accuracy of flow cytometry immunophenotyping, but currently the requests have become rarer. The methodological advances brought about by the modification of the FSC vs.SSC to sequential histograms, starting with CD19 vs. SSC, have permitted a greater accuracy in the analysis. The greater antigenic density observed by CD20, CD45 and CD22 is also found in other chronic lymphoproliferative B-cell diseases, but the concomitance of CD11c, CD25 and CD103 grants specificity to the diagnosis. Flow cytometry immunophenotyping proved to be a fast and accurate methodology that, with the availability of monoclonal antibodies and their combinations, is a useful complementary tool for a fast diagnosis of HCL. The highly intense antigenic expressions of the combined monoclonal pan-B and more specific antibodies make it possible to detect residual disease and to monitor the therapeutic.

CONCLUSIONS This study led us to the conclusion that we should choose to analyze CD19 vs. SSC histograms with sequential histograms in fixed gates of CD19, and separate upon analysis the population of normal lymphoid B-cells from the pathological cells in bone marrow. The antigenic expressions of several monoclonal antibodies were more

intense in the bone marrow cells than in the circulating peripheral blood cells.

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