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Immunophenotypic analysis of bone marrow B lymphocyte precursors (hematogones) by flow cytometry.

INTRODUCTION

Hematogones were first described in the 1930s as lymphoid appearing cells on sternal marrow aspirates (1). Following the early morphologic descriptions, hematogones were generally regarded as undifferentiated stem cells but their real nature was unknown for more than half a century2. In the 1980s new information about their biologic significance began to emerge. Integration of indirect immune-fluorescence and immunoperoxidase staining with morphologic assessment and the terminal deoxynucleotidyl transferase (TdT) positive cells found in normal bone marrow were identified as hematogones3. Later it was revealed by flow cytometry that hematogones expressed other B cell precursor associated antigens3 (3,4).

Hematogones may morphologically resemble the neoplastic lymphoblasts of precursor B cells of acute lymphoblastic leukaemia (ALL) and their immune-phenotype also has features in common with neoplastic lymphoblasts (5,6).

Distinction in the bone marrow of benign B-lymphocyte precursors known as hematogones from neoplastic lymphoblasts of ALL is critical for disease management (in post-chemotherapy and post-bone marrow transplant regenerating marrow) (6,7).

The purposes of this prospective multiparametric flow study were:

* To quantify hematogones across age groups and a spectrum of clinical conditions.

* To study the immunophenotypic profile of hematogones: the spectrum of antigen expression typical for normal evolution of B lineage precursors.

* To compare their immunophenotype with that of neoplastic lymphoblasts reported in the literature.

MATERIALS AND METHODS

A prospective 4-color flow cytometry analysis of hematogones was performed during a 19-month period at the Faculty of Pharmacy, University of Center, Monastir. During this period 450 bone marrow specimens were submitted; 44 of these could not be assessed for hematogones for a variety of technical reasons, including lack of adequate numbers of viable cells in the sample.

Patients were separated by gender and age group of younger than 3 years, 3 to 5 years, 6 to 15 years, 16 to 50 years and 50 years and older.

Patients were also separated according to clinical information.

CYTOLOGICAL INVESTIGATION

* Aspiration of bone marrow: sternum punction in adults and iliac punction in children in ethylenediamine tetraacetic acid tube.

* Automatic staining of bone marrow smears by using the HEMATEK slide stainer (AMES company) and a HEMATEK bloc colorant stain pack (Bayer Diagnostica).

* Cytomorphologic examination of the bone marrow slides separately by 2 morphologists into healthy and pathological samples.

FLOW CYTOMETRY METHODS

Cell Isolation: Preparation

* Cell counts of the bone marrow specimens were first done on the Coulter MAXM blood cell counter.

* The cells were incubated for 15 minutes in the dark with each of the conjugated monoclonal antibodies.

* Erythrocyte lysis: erythrocytes were lysed using lysing solution (optilyse A 11895 Beckman Coulter) according to the manufacturer's instruction.

* Following the lysis step, the samples were washed two times with Phosphate buffered saline (PBS).

* Centrifuge for 5 minutes at 200x g, remove the supernatant by aspiration and shake the cell pellet carefully.

* The cell pellet was conserved at + 4[degrees]C.

* Cells were resuspended in PBS for acquisition.

Antibodies :

* Antibodies to the following antigens were used to specifically profile B cell

* precursors (Coulter- Immunotech):

* CD10 Fluorescein Isothiocyanate(FITC), CD34 (FITC), HLA DR (FITC), CD29 (FITC), CD38 R-Phycoerythrin, CD22 (PE), CD20 (PE), CD58 (PE), CD 19 Allophycocyanin (APC), CD45 R-Phycoerythrincyanin 5.1(PC 5).

* Four 4-color combinations were used in each case (Table 1).

* Data analysis was done using a FACS Calibur Flow cytometer (Becton Dickinson) with Cell Quest Pro Software (Becton Dickinson). For each experiment, 500,000 cells were analyzed.

Flow cytometry interpretation

Samples were acquired with a three color flow cytometer. Distinct cell populations (clusters) were identified based on any combination of forward and orthogonal light scatter properties and fluorescence intensity with various antibody combinations. Each specimen's event clusters were considered positive or negative when compared with the degree of the same specimen stained with the isotypic control antibody.

RESULTS

Morphologic features and distribution:

The specimens submitted for flow cytometry were systematically studied for morphology. Hematogones were frequently present in sufficient numbers to be recognized (1 to 13%). There was a spectrum of size and the exhibited features varied from mature lymphocytes to lymphoblasts of ALL.

They varied from 10 to 20p in diameter, with smaller cells predominating (Figure 1). The nucleus was round, oval with some indentation. The nuclear chromatin was condensed but homogenous. Nucleoli were absent or small and indistinct. There was scant or no discernible cytoplasm, but it was clearly seen in some of the cells. When present, the cytoplasm was moderately to deeply basophilic and devoid of inclusions, granules, or vacuoles. A relatively small percentage cells were indistinguishable morphologically from the lymphoblasts of ALL. Chromatin was fine and contained no obvious clumps. Some nucleoli were large and prominent. The presence of one or more nucleoli indicated immaturity.

[FIGURE 1 OMITTED]

Immunophenotypic features of hematogones:

A total of 406 bone marrow specimens were analyzed for hematogones using 4-color antibody combinations (Table 1).

In 325 (about 80%) of the 406 bone marrow specimens, hematogones were identified by flow cytometry. Selection of B precursors was done by characterization of CD19 + CD45 flow cells (Figure 2 and 3). In all instances the hematogone population exhibited a typical complex spectrum of antigens of B-lineage precursors. In our study the interpretation of flow cytometric data demonstrated that hematogone proliferations exhibited a complex spectrum of antigen expression that defineed the normal antigenic evolution of B cell precursors with predominance of intermediate and mature B lineage cells (Figure 4). The B cell subpopulation was defined by CD 19+ CD45 bright positivity and coexpression of other B lineage markers: CD20, CD22, C10, CD29, CD38 and CD58 in addition to HLADR and CD34.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Numerical variations of hematogones:

Associations of the percentage of bone marrow hematogones with age and sex were analyzed (Table 2).

A total of 406 bone marrow specimens from patients were analyzed. Sixty percent of the specimens were from males and forty percent from females. Ages ranged from 3 months to 89 years (mean 47 years, median 50 years).

Eight specimens were from patients aged less than 3 years, 15 from patients aged 3 to 5 years, 72 from patients aged 6 to 15 years, 168 from patients aged 16 to 50 years, and 143 from patients older than 50 years.

Clinical conditions with increased hematogones

In our study, the hematogones were abundant (>5% of bone marrow cells) in several clinical conditions (Table 3).

DISCUSSION

Hematogones were identified by 4-color flow cytometry using optimal antibody combinations in many bone marrow samples (3,6,7). Bone marrow hematogones were separately assessed as hematogone 1 populations of early stage and hematogones 2 of mid-stage precursor B cells, respectively. In some (about 30%) of the hematogones, a third type of hematogones could be assessed in the bone marrow samples (Figure 4) (6). Our study showed that intermediate hematogones predominated. Increased information about benign B lymphocyte precursors, especially the existence of a third type hematogones could provide a basis for better discrimination of B leukaemia cells even in very small amounts. In a multidisciplinary study, Rimsza, has demonstrated that hematogone-rich lymphoid proliferations exhibit a spectrum of B- lymphoid differentiation of antigen expression with predominance of intermediate and mature B lineage cells (8). Flow cytometry revealed in this study that intermediately differentiated cells (CD10+,CD19+) predominated and followed in frequency by CD20+ (8).

Hematogones may morphologically resemble the neoplastic lymphoblasts of precursor B ALL, and their immunophenotype also has features in common with neoplastic lymphoblasts. Thus, distinction of hematogones and neoplastic lymphoblasts of B cells present in bone marrow may cause diagnostic problems due to their morphologic and immunophenotypic similarities with neoplastic lymphoblasts of acute lymphoblastic leukaemia (5,6,9,10).

In the medical literature that we reviewed, the neoplastic lymphoblasts in precursor B ALL deviated from the normal B-lineage maturation spectrum and exhibited maturation arrest and over-, under-, and asynchronous expression of antigens observed on normal B-cell precursors. They often aberrantly expressed myeloid-associated antigens (5).

Hematogone populations always exhibit a continuous and complete maturation spectrum of antigen expression typical of the normal evolution of B-lineage precursors and they lack aberrant or asynchronous antigen expression (5). (Table 4) Hematogones are precursors which were defined by CD19 positivity and CD45 bright. The expression of antigen immaturity includes HLA DR and CD34, and the co-expression of the more mature markers CD19, CD20, CD22. These cells are blended and confused with those of mature B lymphocytes (CD10 negative) on CD45/SSC and could be better recognized on CD10 gating (6)

Leukemic cells can be distinguished from normal haematopoietic cells on the basis of morphology, of chromosomal or molecular abnormalities and immunophenotype. With flow cytometry using optimal antibodies in combination, the distinction can nearly always be made. However, we have to emphasize the difficulties in distinguishing these cells from residual marrow blasts after chemotherapy.9,10,11

Hematogones were identified by 4-color flow cytometry using optimal antibody combinations in most bone marrow specimens. They were more commonly found in higher numbers in children and there was a general decline in hematogones with increasing age12,13,14. They are often increased (> 5%) in regenerating marrow and in some clinical conditions (3,4,6).

In our study there was a significant decline in hematogones with increasing age, but a broad range was found at all ages, although, some adult's bone marrow contained relatively high numbers (Table 2 and 3).

In a study by Kallkury, flow cytometric analysis revealed 1% to 20% precursor B cells based on expression of 1 or more pan B cell antigens in addition to CD10, CD34 and terminal deoxynucleotidyl transferase (TDT) 11. In Caldwell's study hematogones were most commonly observed in young children, comprising up to 21% of marrow cells in normal infants 15.

It has been reported that the number of hematogones in bone marrow is variable; the hematogones are present in higher numbers in children and they are often increased in regenerating marrow and in some clinical conditions particularly in patients with cytopenias and neoplastic diseases (6,16,17). It has also been reported that there is a decline in the mean percentage of hematogones with increasing marrow involvement by neoplastic cells (17). The reason for the decline is uncertain but may relate to encroachment on the hematogone compartment by the neoplastic infiltrate: alteration of factors that regulate B lymphocytogenesis may also play a role. A study has shown that even though total hematogones may be decreased there is an increased proportion of stage 3 hematogones in marrow involved by lymphoma or leukaemia compared to un-infiltrated marrows (18).

Furthermore, hematogones are the predominant lymphoid population in the bone marrow of preterm infants (for10 to 60%; mean = 34%) of all cells. Flow cytometry revealed a level of 3.8 % of immature cells in a < 1 week- old neonate and 25.7% in a 19 week old infant 9.

They are reported to occur in large numbers in some healthy infants and young children and in a variety of diseases in both children and adults (12,13,14,19).

Hematogones may be particularly prominent in the regeneration phase following chemotherapy or bone marrow transplantation and in patients with autoimmune and congenital cytopenias, neoplasms, and acquired immunodeficiency syndromes. In some instances they constitute 5 % to more than 50 % of cells (6,20,21,22). Immune mediated thrombocytopenia is a clinical condition characterized by increased platelet destruction due to the sensitization of platelets by antibodies. A statistically significant increase in the percentage of hematogones was demonstrated in their bone marrow. An increased percentage of hematogones was demonstrated; with a mean of 18+/-15.2%, CD19+ with a mean of 27+/- 16.3% and CD 34+ with a mean of 18+/- 15.2%. This could be the sequence of an immunological response to the cause which determinates the disease, or the regeneration of the stem cell compartment following transient damage (23,24).

The presence of benign immature B cells has been noted to interfere with the flow cytometric analysis of cases of suspected acute lymphoblastic leukaemia because their immunophenotype (positive for CD19, CD10, CD34 and terminal deoxynucleotidyl transferase) is similar to that of pre B cells lymphoblasts and they simulate acute lymphoblastic leukaemia or lymphoma (20,21,25).

The percentage of marrow hematogones may fluctuate with disease status or persistent elevations may occur. Persistent elevations have been observed for 2 years following cessation of chemotherapy for ALL by one group of investigators and another group found elevations for more than a year following marrow transplantation (26,27,28).

The presence of hematogones in clinical samples should be recognized so as not to adversely influence prognostic studies 22' 25. Flow cytometry is reported to distinguish between these cell populations in nearly all instances

Identification of normal hematogones B contribute to better clarification of the detection of small numbers of blasts B of acute lymphoblastic leukaemia (29,30,31,32).

In conclusion, these findings suggest that it is important to continue this study by flow cytometric analysis of the lymphoblasts of ALL with the same 4 combinations of antibodies in order to clarify the optimal combination which clearly distinguishes B leukemic cell from hematogones. Such complementary investigations are necessary for the recognition of early relapsed ALL and disease progression. Thus these differences in hematogones and lymphoblasts of ALL would be very important and could be utilized for analysis of minimal residual disease after chemotherapy treatment of B ALL.

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REFERENCES

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(2.) Vogel P, Erf LA. Haematological observations on bone marrow obtained by sternal puncture. American Journal of Clinical Pathology. 1937, 7, 436-47.

(3.) Longacre TA, Foucar K, Crago S, Chen IM, et al. Hematogones: a multiparametric analysis of bone marrow precursor cells. Blood. 1989, 73, 543-52.

(4.) Vandersteenhoven AM, Williams JE, Borowitz MJ. Marrow B-cell precursors are increased in lymphomas or systemic diseases associated with B cell dysfunction. American Journal of Clinical Pathology. 1993, 100, 60-6.

(5.) Babusikova O, Zeleznikova T, Mlcakova A, Kusenda J, Stevulova L. The knowledge on the 3rd type hematogones could contribute to more precise detection of small numbers of precursor B-acute lymphoblastic leukaemia. Neoplasma, 2005, 52(6), 502-9.

(6.) McKenna RW, Asplund SL, Kroft SH. Immunophenotypic analysis of hematogones (B-lymphocyte precursors) and neoplastic lymphoblasts by 4- color flow cytometry. Leukaemia Lymphoma. 2004, 45(2), 277-85.

(7.) Riley RS, Massey D, Jackson-Cook C, Idowu M, Romagnoli G. Immunophenotypic analysis of acute lymphocytic leukaemia. Hematol Oncol Clin North Am. 2002, 16(2), 245-99.

(8.) Rimsza LM, Larson RS, Winter SS, Foucar K, et al. Benign hematogone-rich lymphoid proliferations can be distinguished from B lineage acute lymphoblastic leukaemia by integration of morphology, immunophenotype, adhesion molecule expression and architectural features. American Journal of Clinical Pathology. 2000, 114(1), 66-75.

(9.) Rimsza LM, Douglas VK, Tighe P, Saxonhouse MA, et al. Benign B-cell precursors (hematogones) are the predominant lymphoid population in the bone marrow of preterm infants. Biol Neonate. 2004, 86(4),247-53.

(10.) Chen W, Karandikar NJ, McKenna RW, Kroft SH. Stability of leukaemia- associated immunophenotypes in precursor Blymphoblastic leukaemia: a single institution experience. Am J Clin Pathol. 2007, 127(1), 39-46

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(12.) Lucio P, Parreira A, Van den Beemed MW, Van Lochem EG, et al. Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal disease in precursor-B-ALL. Leukemia. 1999, 13, 419-27.

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(14.) Loken MR, Slah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. Normal B lymphocyte development. Blood, 1987, 70, 1316-24.

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(17.) McKenna RW, Waschington LT, Aquino DB, Picker LJ, Kroft SH. Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4- color flow cytometry. Blood. 2001, 98(8), 2498-507.

(18.) Wright B, McKenna RW, Asplund SL, Kroft SH. Maturating B cell precursors in bone marrow: a detailed subset analysis of 141 cases by 4- color flow cytometry. Modern Pathology. 2002, 15: 270A.

(19.) Klupp N, Simonitsch I, Mannhalter C, Amann G. Emergence of unusual bone marrow precursor B-cell population in fatal Shwachman-Diamond Syndrome. Arch Pathol Lab Med. 2000, 124(9), 1379-81.

(20.) Vargas SO, Hasegawa SL, Dorfman DM. Hematogones as an internal control in flow cytometric analysis of suspected acute lymphoblastic leukemaia. Pediatr Dev Pathol. 2001, 4(5), 505-11.

(21.) Davis RE, Longacre, Cornbleet PJ. Hematogones in the bone marrow of adults. Immunophenotypic features, clinical settings, and differential diagnosis. American Journal of Clinical Pathology. 1994, 102(2), 201-11.

(22.) Davis BH, Scwartz M. ZAP-70 expression is low in normal precursor B cells or hematogones. Cytometry B clin cyto2006, 70B(4), 314-8.

(23.) Guiziry DE, E1 GW, Farhat N, Hassab H. Phenotypic analysis of bone marrow lymphocytes from children with acute thrombocytopenic purpura. Egypt J Immunol. 2005, 12(1), 9-14.

(24.) Fisgin T, Yarali N,Duru F, Kara A. CMV induced immune thrombocytopenia and excessive hematogones mimicking an acute B precursor lymphoblastic leukaemia. Leuk Res. 2003, 27(2), 193-6.

(25.) Hurford MT, Altman AJ, DiGiuseppe JA, Scherburne BJ, Rezuke WN. Unique pattern of nuclear TdT immunofluorescence distinguishes normal precursor B cells (Hematogones) from lymphoblasts of precursor B lymphoblastic leukaemia. Am J Clin Pathol. 2008, 129(5), 700-5.

(26.) Sandhaus LM, Chen TL, Ettinger LJ, Hirst Allen A, et al. Significance of CD10 positive cells in non malignant bone marrow of children. American Journal of Pediatric Hematology Oncology. 1993, 15, 65-70

(27.) Leitenberg D, Rappeport JM, Smith BR. B cell precursor bone marrow reconstitution after bone marrow transplantation. American Journal of Clinical Pathology. 1999, 102, 231-6.

(28.) Babusikova O, Zeleznikova T, Kirschnerova G, Kankuri E. Hematogones in acute leukaemia during and after therapy. Leuk Lymphoma, 2008, 49(10), 1935-44.

(29.) San Miguel JF, Vidriales MB, Lopez Berges C, Diaz Medivilla J, et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukaemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood. 2001, 98, 2456-62.

(30.) Coustan Smith E, Gajjar A, Hijiya N, Razzouk B1, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukaemia after relapse. Leukemia. 2004, 18, 499-504.

(31.) Coustan Smith E, Sancho J, Behm FG, Hancock ML, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukaemia. Blood. 2002, 100, 52-8.

(32.) Krampera M, Perbellini O, Vincenzi C, Zampieri F, et al. Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia. Haematologica. 2006, 91(8), 1109-12.

N Braham Jmili is professor, Faculty of Pharmacy, University of Center, Monastir, Tunisia.

S Nsaibia is a physician, Faculty of Pharmacy, University of Center, Monastir, Tunisia.

MC Jacob is a physician, EFS Rhone-Alpes, Department of Cellular Immunology, Grenoble, France.

H Omri is professor, Department of Clinical Haematology. Hospital Farhat Hached. Sousse. Tunisia.

MA Laatiri is professor, Department of Clinical Haematology. Hospital Farhat Hached. Sousse. Tunisia.

S Yacoub EFS is professor, Faculty of Pharmacy, University of Center, Monastir, Tunisia.

Y Braham is professor, Faculty of Pharmacy, University of Center, Monastir, Tunisia.

M Aouni is professor, Faculty of Pharmacy, University of Center, Monastir, Tunisia.

M Kortas is professor, Faculty of Pharmacy, University of Center, Monastir, Tunisia.

Address for Correspondence Braham: JMili Nejia, Laboratory of Haematology, HOSPITAL FARHAT HACHED, 4000 Sousse, Tunisia, Tel: 00216 98 68 52 08, Fax: 00216 73 22 67 02. Email: jmilinejia @yahoo.fr.
Table 1. Protocol of flow cytometry analysis.

              FITC            PE         APC
          fluorescein      R-Phyco-    Allophy-
         isothiocyanate    erythrin    cocyanin

1             IgG1           IgG1        CD19
2             CD10           CD38        CD19
3             CD34           CD22        CD19
4            HLA DR          CD20        CD19
5             CD29           CD58        CD19
Volume     5 [micro]l     5 [micro]l

                  PC5
             R- Phycoeryth-
             rincyanin 5.1

1                 CD45
2                 CD45
3                 CD45
4                 CD45
5                 CD45
Volume   5 [micro]l dilution in
              PBS at (1/5)

Table 2.  Bone marrow hematogones by percent age group for
the 406 specimens.

Patients
(age, years)          < 3      3-5     6-15    16-50       >50

Number /
All specimens           8       15      72       168       143

Number /positive        8       13      51       128       125

Hematogones
(intervals)        [6-20]   [5-15]   [5-35]   [1-27]   [0,5-5]

Hematogones
(mean percent)       10,5      4,3      3,9      2,8       1,2

Table 3.  Clinical conditions with increased hematogones for the
406 specimens.

Disease                                          Number of cases
Neoplastic disease:                               70/406 cases
Known myeloid leukaemia                                 50
Other neoplasias (Nonhaematopoietic neoplasms)          20
Cytopenia:                                       257/406 cases
Idiopathic thrombocytopenia purpura                     86
Megaloblastic anaemia                                   74
Others                                                  97
Infectious disease:                               79/406 cases

Table 4. Maturational sequence of bone marrow B cell
precursors (hematogones). Stage 1 hematogones
correspond to the least mature (top horizontal row).
Stage 2 includes middle rows and stage 3 the bottom
hematogone row. Mature marrow B lymphocytes are
shown for comparison(6).

Hematogones
Stage 1
  TdT            CD34   CD10   CD19   CD22    CD38
                                      (dim)   (bright)
Stage 2
                        CD10   CD19   CD22    CD38
                                      (dim)   (bright)

                        CD10   CD19   CD22    CD38       CD20 Slg *
                                      (dim)   (bright)   (dim)
Stage 3
                        CD10   CD19   CD22    CD38       CD20 Slg *
                                      (dim)   (bright)

Mature B Cells
                               CD19   CD22    CD38       CD20 Slg *
                                                         (bright
                                                         to negative)

* The appearance of surface immunoglobulin is variable among
individual cells occurring from shortly before to after acquisition of
high level of CD20 expression.
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Title Annotation:RESEARCH AND REPORTS
Author:Jmili, N. Braham; Nsaibia, S.; Jacob, M.C.; Omri, H.; Laatiri, M.A.; Yacoub, S.; Braham, Y.; Aouni,
Publication:Clinical Laboratory Science
Article Type:Clinical report
Date:Sep 22, 2009
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