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
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
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
* To study the immunophenotypic profile of hematogones: the
spectrum of antigen expression typical for normal evolution of B lineage
* 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
Patients were also separated according to clinical information.
* 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
* 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
* 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 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.
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
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
Clinical conditions with increased hematogones
In our study, the hematogones were abundant (>5% of bone marrow
cells) in several clinical conditions (Table 3).
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
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
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
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
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
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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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,
M Aouni is professor, Faculty of Pharmacy, University of Center,
M Kortas is professor, Faculty of Pharmacy, University of Center,
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
Volume 5 [micro]l dilution in
PBS at (1/5)
Table 2. Bone marrow hematogones by percent age group for
the 406 specimens.
(age, years) < 3 3-5 6-15 16-50 >50
All specimens 8 15 72 168 143
Number /positive 8 13 51 128 125
(intervals) [6-20] [5-15] [5-35] [1-27] [0,5-5]
(mean percent) 10,5 4,3 3,9 2,8 1,2
Table 3. Clinical conditions with increased hematogones for the
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
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).
TdT CD34 CD10 CD19 CD22 CD38
CD10 CD19 CD22 CD38
CD10 CD19 CD22 CD38 CD20 Slg *
(dim) (bright) (dim)
CD10 CD19 CD22 CD38 CD20 Slg *
Mature B Cells
CD19 CD22 CD38 CD20 Slg *
* The appearance of surface immunoglobulin is variable among
individual cells occurring from shortly before to after acquisition of
high level of CD20 expression.