From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Simultaneous Genotypic and Immunophenotypic Analysis of Interphase Cells
Using Dual-Color Fluorescence: A Demonstration of Lineage Involvement in
Poly cyth emia Vera
By Cathy M. Price, Edward J. Kanfer, Susan M. Colman, Nigel Westwood, A. John Barrett, and Melvyn F. Greaves
Fluorescent in situ hybridization has become a useful technique by which chromosomal abnormalitiesmay be shown in
interphase cells. We present a dual-fluorescence method
whereby a chromosomal and immunophenotypic marker can
be visualized simultaneously in the same interphase cell.
Two patients with the myeloproliferativedisorder polycythemia vera and trisomy for chromosome 8 have been studied
using this technique and selective involvement of the my-
eloid and erythrocyte lineages has been shown by the
detection of the trisomy in immunophenotypedcells. Simultaneous analysis of genotype and immunophenotype in
individual cells from patients with myeloproliferative disorders or leukemia may help identify the developmental and
lineage status of cells in which molecular alterations have
resulted in clonal advantage.
o 1992by The American Society of Hematology.
R
type (fluorescein isothiocyanate [FITC]: green fluorescence) simultaneously in the same interphase cell. We
describe the application of this dual-fluorescence technique
to lineage analysis in two patients with the myeloproliferative disease polycythemia vera (PV) using trisomy of chromosome 8 as a marker of the abnormal clone. Trisomy 8 is a
nonrandom chromosome abnormality in PV occurring in
approximately 16% of karyotypically abnormal cases and,
like other chromosomal changes [eg, del(20q) and +9], it
may occur alone or as a secondary event.1°
ECENT ADVANCES in interphase cytogenetics using
fluorescent in situ hybridization (FISH) of chromosome- or gene-specific probes have facilitated the detection
of chromosomal abnormalities in a variety of hematologic
This technique is particularly useful in
cases in which conventional cytogenetics have provided
evidence for a clonal chromosomal marker but there are
few dividing cells. The correlation of cell type and chromosomal abnormality can provide additional information relating to lineage involvement in leukemia and the developmental level of the “target” cell for clonal abn~rmalities.~
Dual
classification of this type, however, is not feasible with
normal interphase in situ methods because a prerequisite of
the technique is the removal of both the cytoplasm and the
cytoplasmic membrane of the cells to allow more efficient
nuclear hybridization. This problem has, in part, been
overcome by the use of sequential morphologic staining of
blood or bone marrow smears to locate a specific cell type
followed by in situ hybridization with a relevant probe to
identify the chromosomal abnormality. This approach has
been used successfully to assess minimal residual disease in
patients with acute lymphoblastic leukemia in apparent
complete remi~sion.~
However, in many hematologic malignancies it may be difficult to determine cell type by
morphology alone and in such cases immunophenotypic
analysis can be a useful complementary investigation. The
MACISH (morphology, antibody, chromosome, in situ
hybridization) technique has been used to identify clonal
chromosomal abnormalities in immunophenotyped metaphase and interphase cell^,^,^ but removal of the cytoplasm
before in situ hybridization makes simultaneous analysis of
both genotype and immunophenotype difficult. Studies to
determine lineage involvement of specific cell types with a
clonal chromosomal abnormality using these techniques are
usually based, therefore, on the analysis of the percentage
of cells showing a particular immunophenotype in comparison with those having the chromosomal marker rather than
a cell by cell
In this report, we have used the recent observation that
the alkaline phosphatase-Fast Red reaction product of the
alkaline phosphatase-anti-alkaline phosphatase (APAAP)
immunophenotyping method produces a bright red fluorescence that is visible using both fluorescein and rhodamine
filter^.^ It is possible, therefore, when using FISH with a
fluorescein-labeled chromosomal probe, to visualize both
immunophenotype (APAAP: red fluorescence) and genoBlood, Vol80, No4 (August 15). 1992: pp 1033-1038
MATERIALS AND METHODS
Brief details of the two patients used in this study are given
below. Conventional cytogenetic analysis showed that the peripheral blood from both patients was chimeric for normal and
abnormal karyotypes. In both patients the abnormal clone or
clones had trisomy of chromosome 8 in addition to other abnormalities. This trisomy was used as a marker for the malignant cells.
Patient 1. This 67-year-old man presented in October 1991with
a scrotal hydrocele. He was noted to have a raised hemoglobin and
further investigations, including the production of erythropoietinindependent erythroid colony growth, confirmed the diagnosis of
PV.” Cytogenetic analysis showed a normal male karyotype and
the presence of two hyperdiploid clones, both with trisomy 8 in
addition to other chromosomal abnormalities: 46,XY/47,XY, +8,
+9, - 12,t(6;12)/48,XY, +8, +9, - 12,t(6;12),del( 12q). Peripheral
blood samples were obtained from this patient at this time. Shortly
after presentation, he required emergency surgery for an ischaemic
lower limb and sustained a fatal cerebro-vascular event postoperatively.
~
~
~
~
From the Leukaemia Research Fund Centre, Institute of Cancer
Research; the Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital; and the Department of Haematology, United Medical and Dental Schools, St Thomas’s Campus,
London, UK.
Submitted Februaly 4,1992; accepted April 30, 1992.
Supported by the Leukaemia Research Fund of Great Britain, the
Kay Kendal Leukaemia Fund, and an EEC Concerted Action Grant
(Biomed I).
Address reprint request to Cathy M. Price, PhD, Leukaemia
Research Fund Centre, Institute of Cancer Research, Chester Beatty
Laboratories, 237 Fulham Rd, London SW3 4JB, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971l92l8004-0006$3.00/0
1033
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1034
Patient 2. This 60-year-old woman presented with PV in 1978,
from which time she received intermittent therapy with busulphan
and venesections. In 1989, her blood film was noted to show red
blood cell morphology consistent with marrow fibrosis and this was
confirmed with marrow biopsy. Six months before this study, a
repeat marrow examination showed progressive fibrosis and also
featuressuggestive of developing leukemic transformation. Cytogenetic analysis showed a normal female karyotype and a hyperdiploid clone with trisomy 8, in addition to other abnormalities:
46,XX/48,XX,+8,+del(l)(qter-p2l),t(l;21)(ql2;pll).Peripheral
blood samples were obtained from the patient for this study in June
1991 when the presence of the same hyperdiploid clone was
confirmed. In August 1991, she died after a short illness with
hemorrhagic features.
Cell material. Peripheral blood samples from both patients and
a normal female control were obtained by venesection and placed
in preservative-free heparin. Whole peripheral blood was cultured
from 2 to 48 hours and harvested for routine cytogenetic analysis.
The remainder of the sample was centrifuged over Lymphoprep
(Nycomed, Oslo, Norway) to obtain a mononuclear cell (MNC)
fraction. The MNC were used in hematopoietic colony assays,
cytocentrifuged on to glass slides, or stored in liquid nitrogen.
Colony assay. MNC were cultured at a concentration of 2 X
105/mL in Iscove’s medium containing 40% fetal calf serum, 2%
mol/L 2-mercaptoethanol, 1%
bovine serum albumin (BSA),
methylcellulose, 5 U of erythropoietin, and 30 U granulocytemacrophage colony-stimulating factor (GM-CSF). Cultures were
incubated at 37°C in a humidified 5% COz atmosphere for 14 days.
Individual erythroid (burst-forming unit-erythroid [BFU-E]) and
granulocyte/macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) colonies were removed using a micropipette,
suspended in phosphate-buffered saline (PBS), and cytocentrifuged onto glass microscope slides. The slides were either fixed in
methanol and Giemsa stained to confirm morphologic cell type or
air dried, wrapped in foil, and stored at -20°C for later staining
and FISH.
Slide preparation. Fresh MNC from patient 1 and a normal
control, and cryopreserved MNC from patient 2 were resuspended
in PBS to a final concentration of 106/mL.One hundred microliters
of cell suspension was cytocentrifuged onto glass slides (cytospins),
air dried, and stored wrapped in foil at -20°C.
Immunophenotype analysis. Cytospins of MNC were stained
with a panel of murine monoclonal antibodies to determine cell
lineage using a three-stage (APAAP) unlabeled bridge method for
immunophenotyping.12 Briefly, slides stored at -20°C were defrosted at room temperature before being unwrapped, fixed in
acetone, and air dried. After rehydration in Tris-buffered saline
(TBS), the slides were incubated in a moist chamber with 20 kL of
normal human serum (NHS), diluted 1:lO in TBS, at room
temperature for 5 minutes. Ten microliters of the appropriate
primary antibody (murine monoclonal antibody) was added and
the slides incubated as above for 1 hour. The slides were washed in
TBS for 20 minutes and 20 p,L of the second layer, an unlabelled
rabbit antimouse antibody (Dako Ltd, High Wycombe, UK), was
added and the slides incubated for 45 minutes in the humid
chamber. The slides were washed in TBS as above. Twenty
microliters of antirabbit APAAP complexes (Dako Ltd) was then
added and the slides were again incubated at room temperature for
45 minutes. The slides were washed as before and the alkaline
phosphate Fast Red substrate was added. Color development was
monitored using a light microscope. Finally, the slides were washed
in TBS for 10 minutes and the cells counterstained in hematoxylin.
The slides were then air dried, mounted in an aqueous mounting
medium Glycergel (Dako Ltd), and scored for positive cells using a
light microscope.
PRICE ET AL
Fig 1. A confocal image of an interphase nucleus from patient 2
counterstained with propidium iodide (red fluorescence) showing
three chromosome 8 signals (FITC; green fluorescence)
Antibodies. The lymphoid-associated antibody for T cells was
CD3 (T28; a gift from Dr P. Beverley, London, UK) and for B cells
was CD22 (Leul4; Becton Dickinson, Mountain View, CA). The
myeloid-associatedantibody for granulocytes/monocytes was CD1 IC
(3.9; a gift from Dr N. Hogg, London, UK), for immature granulocytes/monocytes was CD13 (MY7; Coulter Electronics Ltd, Luton,
UK), for monocytes was CD14 (MY4; a gift from Dr J. Griffen,
Boston, MA), and for megakaryocytes was CD41 (J15; a gift from
Dr A. McMichael, Oxford, UK). The antibody for hematopoietic
progenitors was CD34 (QBENDlO Quantum Biosystems, Cambridge, UK). The antibody for erythrocytes was antiglycophorin A
(AGA)(R10; a gift from Dr P. Edwards, Sutton, UK).
FZSH. Cytocentrifuged cells from both colony cells and MNC
(following the APAAP technique) and standard cytogenetic slide
preparations were treated using the same in situ hybridization
protocol. In preliminary experiments, we found that chromosomespecific centromeric repeat probes (for chromosomes 1, 4, 8, and
X) all gave strong signals in interphase nuclei from cytospun MNC
(data not shown). We concluded that, for efficient hybridization of
these probes, pretreatment, with the consequent loss of morphologic integrity, was not necessary. Cytogenetic slides were stored
for up to 2 weeks at room temperature before hybridization.
APAAP preparations were kept at room temperature for up to 2
months before hybridization without any obvious deterioration in
the hybridization signal. Shortly before hybridization the cover
slips were removed in hot water and the slides allowed to air dry.
Cell preparations were heat denatured in 2 x SSC/70% formamide
at 70°C for 2 minutes, dehydrated through 70%, 90%, and 100%
alcohol, and air dried at room temperature. Hybridization buffer
(50% formamide/lO% dextran sulphate in 2 x SSC, pH 7.0)
containing 2 ng/kL of biotin-labeled probe (chromosome 8 specific
a-satellite probe D8Z1; Oncor Science, Gaithersburg, MD) was
denatured at 70°C for 7 minutes and cooled on ice at 4°C.
Hybridization solution (15 pL) was added to each slide under a
sealed coverslip and incubated overnight in a moist chamber at
37°C. The coverslips were removed in 2 x SSC and the slides
washed in 50% formamide/2~SSC (pH 7.0) at 45°C for 40
minutes, twice in 2 x SSC (pH 7.0) for 5 minutes at room
temperature, and once in 1x SSC (pH 7.0) for 5 minutes at room
temperature. Slides were stored at room temperature in phosphate
buffer (0.1 mol/L NaHzP04,O.l mol/L NazHP04, pH 8.0).
Detection. The slides were blocked in 3% BSA in 4X SSC at
37°C for 10 minutes. Fluoresceinated avidin (200 pL) (Vector
Laboratories, Burlingame, CA), at a concentration of 5 kg/mL in
4 x SSC containing 5% lowfat milk and 0.05% Triton X, was placed
on the slide under a coverslip and incubated for 40 minutes at 37°C.
The signal was amplified using biotinylated anti-avidin (Vector) at
a concentration of 5 p,g/mL and a further layer of fluoresceinated
avidin. Between detection steps, the slides were washed twice for 5
minutes in 4 x SSC/O.O5% Triton X, pH 7.0. After a final wash in
phosphate buffer, the cells were mounted in fluorescence antifade
(Citifluor; Nottingham University, Nottingham, UK) or antifade
containing the counterstains propidium iodide (3 ng/kL) or
diamidino-2-phenyl-indole dihydrochloride (DAPI; 200 ng/mL)
(both Sigma, Poole, UK). Double-labeled fluorescent cells were
analyzed using either a Zeiss photomicroscope 111 equipped with
epifluorescence and a dual band-pass filter (Omega, Brattleboro,
VT) or a Bio-Rad MRC-600 laser scanning confocal microscope
equipped with a kryptodargon ion laser (488/568 nm line excitation with dual channel, 522 nm and 585 nm, emission filters). Cells
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
L
Fig 3. A confocal image of FISH/APAAP-stained lymphocytesfrom
patient 1 viewed under dual fluorescence. (A) CD3+ T call with two
chromosome 8-specific signals. (B) CD22+ B cell with two chromosome 8-specific signals.
. ..
Fig 1.
I
31L
L
c
I
-
. - :.**
Fig 2. A n interphase cell from
patient 2. (A) Viewed with a
phase contrast light microscope
t o show APAAP staining for antiglycophorin A R10 antibody (Fast
Red). (B) A confocal image of the
same cell after in situ hybridization with the chromosome 8 - s ~ e cific probe viewed under dual
fluorescence (APAAP R10+: red
fluorescence; FlTC chromosome
8 probe: green fluorescence)
A
B
Fig 4. Confocal images of FISHiAPAAP-stained C D l l c + cells from
patient 1 viewed under dual fluorescence. (A) Cell w i t h t w o chromosome 8-specific signals. (E) Cell w i t h three chromosome 8-specific
signals. (C) Two cells w i t h four chromosome &specific signals. The
fourth signal in the cell t o the right (arrow) is in a slightly different
confocal plane t o remaining three signals.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PRICE ET AL
1036
viewed on the Zeiss photomicroscope were photographed on
Kodak Ektar 400 color film (Eastman Kodak, Rochester, NY).
Confocal images were printed on Sony Mavigraph video print film
(Sony Corporation, Tokyo, Japan).
Table 2. Frequency of In Situ HybridizationSignals of the
Chromosome 8-Specific Probe in BFU-E and CFU-GM Colonies
RESULTS
In situ hybridization. In situ hybridization with the
chromosome 8-specificprobe confirmed the presence of the
trisomy in cytogenetic preparations from the peripheral
blood cells of both patients (Fig 1).
The false negative rate for the probe was assessed by
scoring 200 cells from APAAP control slides (Ig subclass
specific) from the normal control (Table 1). As with other
centromeric repeat probes, the false negative rate (1 signal
per cell) for the chromosome 8 probe is higher (2% to 5%)
than the false positive rate (3 signals per cell) at less than
1% in the normal contr01.'~ Scorable cells with no signal
contributed to less than 0.5% of the total cells observed.
Cells with three signals in APAAP control slides from
patients 1 and 2 were observed in 21% and 71%, respectively (Table 1). In addition, patient 1 had a small percentage (3%) of cells with four signals.
Colony analysis. Colonies from patients 1 and 2 showed
the presence of the trisomy in both myeloid (1 of 2 and 13 of
13) and erythroid (6 of 6 and 3 of 3) hematopoietic
progenitors (Table 2). The number of cells analyzed per
colony and the percentage of cells showing 1, 2, or 3
chromosome 8-specific signals are given in Table 2. The
hybridization efficiency in colony cytospin cells is reduced
so that the number of informative cells is lower in comparison with normal MNC cytospins. Consequently, the false
negative rate for centromeric probes in this type of preparation is higher at 5% to 17%, whereas the false positive rate
remains similar to that observed in normal MNC cytospins
(2% to 2.5%) (Price, unpublished observation). This problem, however, is offset by the clonal nature of the preparations, whereby only a few cells in the colony need be scored
to confirm the genotype.
Immunophenotype analysis. Results of the immunophenotyping of the MNC for both patients are given in Table 3.
Patient 1 was positive for the following antibodies: CD3
(9%), CD14 (7.5%), C D l l c (87%), CD22 (2.5%), and
CD34 (2.5%). No megakaryocytes (J15) or nucleated red
blood cells (R10) were observed. Patient 2 was positive for
the erythroid antibody R10 (48%), although faint staining
was seen on several cells using the myeloid (MY7) and
T-lymphocyte (T28) antibodies. Morphologic staining of
the latter showed that the cytoplasm was damaged, which
would account for the diffuse cytoplasmic staining. Frozen
Table 1. In Situ HybridizationSignal for the Chromosome 8-Specific
Probe in MNC Cytospin Slides
No. of Signals
(%from 200 cells)
Control
Patient 1
Patient 2
1
2
3
4
2
5
3
97.5
71
26
0.5
21
71
-
Slides were treated with APAAP control reagents (see text).
Abbreviation: -, no cells with four signals observed.
No. of Signals
No. of Cells
Patient 1
Colony
Colony
Type
No.
BFU-E
CFU-GM
Patient 2
BFU-E
CFU-GM
(Oh
Scored
cells)
percolony
1
2
3
1
2
3
4
5
6
1
2
55
32
23
94
18
39
154
160
2
15
5
4
5
2
3
6
7
13
0
7
6
0
5
92
91
72
87
89
89
90
92
2
1
2
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
13
39
17
11
39
6
10
46
17
4
3
57
5
18
24
11
39
6
0
0
12
9
8
17
0
2
0
25
0
0
0
11
0
0
8
17
0
2
23
18
13
0
0
15
6
0
0
8
0
0
21
9
13
0
100
98
65
73
79
83
100
83
94
75
100
92
100
89
79
91
79
83
MNC from other patient samples (stored for up to 8 years
and not detailed in this report) produced suitable intact
cells for APAAP/FISH, suggesting that patient sample
variation rather than storage method was responsible for
the cell damage.
In situ and immunophenotype analysis. In situ results
with the chromosome 8 probe on the immunophenotyped
cells are given in Table 4. Erythroid (R10)-positive cells
only could be scored for patient 2 and of these the majority
(90%) had three signals confirming the involvement of the
erythroid lineage in the neoplastic clone. An R10+ cell
photographed before (Fig 2A) and after FISH (Fig 2B)
illustrates the dual-fluorescence signal obtained in antibodypositive cells. B and T lymphocytes from patient 1showed a
normal two-signal distribution of chromosome 8 (Fig 3A
and B), whereas monocytes and granulocytes showed two
separate populations: 63% to 76% with two signals (Fig 4A)
and 21% to 35% with three signals (Fig 4B). A small
proportion of CDllc+ cells (1%)had four signals (Fig 4C).
These cells either represent a third clone with two additional copies of chromosome 8 or are polyploid cells
resulting from aberrant mitosis. A mixed population of
normal (46%) and trisomic cells (64%) was also observed in
the CD34+ cells from patient 1.
3
-
DISCUSSION
In this report, we present a method in which it is possible
to analyze both phenotype and genotype simultaneously in
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
SIMULTANEOUS IMMUNOPHENOTYPING AND FISH
1037
Table 3. Frequency of APAAP-Positive Cells in MNC Cytospins
Antibodv
Myeloid
Associated
Lymphoid
Associated
Erythroid
Precursor
CDllc
(3.9)
CD13
(MY71
CD14
WY4)
CD41
AGA
(T28)
CD22
(Leul4)
(J15)
(R10)
CD34
(QBEND10)
9
1,789
2.5
500
87
2,819
ND
7.25
400
-
-
2.5
*
*
*
CD3
Patient 1
% Positive
No. cells scored
Patient 2
% Positive
No. cells scored
Abbreviations: -, no positive cells observed; ND, not done; AGA, anti-glycophorinA;
the same interphase cell. Two major advantages of this
method are (1) that it allows a direct cell by cell analysis of
lineage and chromosomal markers and negates the need for
sequential photography followed by relocation of specific
cells, and (2) that the number of analyzable cells is high due
to the absence of harsh pretreatment steps that damage
cytoplasmic and cell surface features. This technique has
been used successfully to show discordant lineage involvement of the neoplastic clone in the myeloproliferative
disease PV.
In both patients studied the clonal marker (trisomy 8)
was present in early myeloid (CFU-GM) and erythroid
(BFU-E) cells. In patient 1, trisomy 8 was present in a
subset of mature myeloid cells (CDllc+, CD13+, CD14+),
but was absent in all B and T lymphocytes scored. The
CD34+ cells from this patient included both normal and
trisomic populations and because CD34+ cells include both
lymphoid and myeloid progenitors,14J5we assume that the
trisomy is most likely to be myeloid restricted.
Previous studies of hematopoiesis in patients with PV
using glucose-6-phosphate dehydrogenase (G-6-PD), phosphoglycerate kinase (PGK), or hypoxanthine phosphoribosyl transferase (HPRT) X-linked polymorphisms have shown
clonality of erythrocyte, granulocyte, and platelet lineages,16-18whole blood,Is and blood leukocyte^.^^^^^ In a
recent study of PGK polymorphism using polymerase chain
reaction (PCR) amplified DNA from different cell populations,21 two patients with PV were shown to have clonal
granulocytes and BFU-E, whereas in a third patient the
granulocytes were polyclonal but most of the BFU-E
Table 4. Frequency of In Situ HybridizationSignals of the
Chromosome 8-Specific Probe in lmmunophenotyped Cells
No. of
Signals (%)
Total No.
Cells
Scored
1
Patient 1
Lymphoid associated CD3 (T28)
CD22 (Leul4)
(3.9)
Myeloid associated
CD1IC
CD14 (My4)
Precursor
CD34(QBEND10)
100
155
200
200
100
2
4
2
2
0
Patient 2
Erythroid
100
8
Antibody
AGA (R10)
2
3
4
9 8 0 0
9 6
0 0
76 21 1
63 35 0
46 64 0
2
9 0 0
ND
*
48
680
*
*, damaged cytoplasm (see text).
expressed the same PGK allele, implying a clonal derivation. These studies provide convincing evidence of clonality
of leukocytes and myeloid/erythroid cells in most, but not
all, cases of PV. In addition, a study of clonality in PV using
PGK polymorphisms has shown that whole separated blood
granulocytes were clonal in four cases assessed, but T
lymphocytes from the same patients were p o l y ~ l o n a lThis
.~~
result could indicate that the “target” cell for clonal
selection in PV is not the lymphoid/myeloid stem cell but a
myeloid lineage-committed cell. Alternatively, the longevity
of T cells and the slow rate of new T cell production in
adults could obscure a clonal contribution to the lineage.
Rare cases of lymphoblastic transformation of PV,22,uas in
T cell blastic crisis of chronic myeloid leukemia (CML),24-26
indirectly implicate the lymphoid/myeloid stem cell. A
detailed study of Epstein-Barr virus-transformed B-lymphoblastoid lines from a patient with PVhas provided evidence
of clonality of the B-lymphoid lineage, thus indicating this
disease can involve a stem cell pluripotent for both the
lymphoid and myeloid series.”
In our study, trisomy 8 could not be found in either B or T
lymphocytes. This observation may be a true reflection of
the lineage restriction of the disease in patient 1or could be
a consequence of the trisomy failing to fully define the
myeloproliferative clone. Similar arguments apply to those
myeloid cells in patient 1 with only two copies of chromosome 8. The incidence of clonal chromosomal aberrations
in PV at diagnosis or preceding cytotoxic therapy is relatively low (13% to 17%)1°; it is possible, therefore, that
trisomy 8, although a nonrandom finding in PV, occurs as a
secondary event in an already established neoplastic clone.
Studies to determine clonality of specific cell lineages using
HPRT/PGK polymorphism in combination with the dualimmunophenotype and chromosomal marker analysis presented in this report should provide additional information
on the lineage relationships of cells involved in the initiation and progression of PV, other myeloproliferative disorders, and leukemia.
ACKNOWLEDGMENT
The authors are grateful to Prof T. Pearson and Dr T.Kumaran
for allowing the study of patients under their care, and to Dr I.
Dokal for his assistance. W e also thank Dr H.Walker and B.
Czepulkowski for the original cytogenetic analysis and Dr H.
Paterson for his assistance with the confocal imaging system.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PRICE ET AL
1038
REFERENCES
1. Poddighe PJ, Moesker 0, Smeets D, Awwad BH, Ramaekers
FCS, Hopman A H N Interphase cytogenetics of hematological
cancer: Comparison of classical karyotyping and in-situ hybridization using a panel of eleven chromosome specific DNA probes.
Cancer Res 51:1959,1991
2. Tkachuk DC, Westbrook CA, Andreeff M, Donlon TA,
Cleary ML, Suryanarayan K, Monge M, Redner A, Gray J, Pinkel
D: Detection of bcr-ab1fusion in chronic myelogenous leukemia by
in-situ hybridization. Science 250559, 1990
3. Craig JM, Hawkins JM, Yamada T, Ganeshaguru K, Mehta
AB, Secker-Walker LM: First intron and M-bcr breakpoints are
restricted to the lymphoid lineage in Philadelphia positive acute
lymphoblastic leukemia. Leukemia 4:678, 1990
4. Anastasi J, Thangavelu M, Vardiman JW, Hooberman AL,
Bian LM, Larson RA, Le Beau MM: Interphase cytogenetic
analysis detects minimal residual disease in a case of acute
lymphoblastic leukemia and resolves the question of origin of
relapse after allogeneic bone marrow transplantation. Blood 77:
1087,1991
5. Losada AP,Wessman M, Tiainen M, Hopman AHN, Willard
HF, Sole F, Caballin MR, Woessner S, Knuutila S: Trisomy 12 in
chronic lymphocytic leukemia: An interphase cytogenetic study.
Blood 78:775,1991
6. Knuutila S, Teerenhovi L Immunophenotyping of aneuploid
cells. Cancer Genet Cytogenet 41:1,1989
7. Wessman M, Knuutila S: A method for the determination of
cell morphology, immunologic phenotype and numerical chromosomal abnormalities on the same mitotic or interphase cell. Genet
(Life Sci Adv) 7:127,1988
8. Tiainen M, Poop S, Partier V, Emmerich P, Bellama MJ,
Ruutu T, Cremer T, Knuutila S: Chromosomal in-situ suppression
hybridization of immunologically classified mitotic cells in hematological malignancies. Genes Chromosomes Cancer 4:135,1992
9. Murdoch A, Jenkinson ET,Johnson GD, Owen J J T Alkaline
Phosphatase-Fast Red, a new fluorescent label: Application in
double labelling for cell surface antigen and cell cycle analysis. J
Immunol Methods 13245,1990
10. Mertens F, Johansson B, Heim S, Kristofersson U, Mitelman F Karyotypic patterns in chronic myeloproliferative disorders: Report on 74 cases and review of the literature. Leukemia
5:214,1991
11. Wasserman LR: The management of polycythaemia vera. Br
J Haematol21:371, 1971
12. Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z,
MacDonald S, Pulford KAF, Stein H, Mason D Y Immunoenzymatic labelling of monoclonal antibodies using immune complexes
of alkaline phosphatase and monoclonal anti-alkaline phosphatase
(APAAPcomplexes). J Histochem Cytochem 32:219,1984
13. Kiechle-Schwarz M, Decker HJH, Berger CS, Fiebig HH,
Sandberg AA: Detection of monosomy in interphase nuclei and
identification of marker chromosomes using biotinylated alpha
satellite DNA probes. Cancer Genet Cytogenet 51:23,1991
14. Gore SD, Kastan MB, Civin CI: Normal human bone
marrow precursors that express terminal deoxynucleotidyl transferase include T-cell precursors and possible lymphoid stem cells.
Blood 77:1681,1991
15. Peault B, Weissman IL, Baum C, McCune JM, Tsukamoto
A Lymphoid reconstitution of the human fetal thymus in SCID
mice with CD34+ precursor cells. J Exp Med 174:1283,1991
16. Adamson JW, Fialkow PJ, Murphy S, Prchal JF, Steinmann
L Polycythemia vera: Stem cell and probable clonal origin of the
disease. N Engl J Med 295:913,1976
17. Adamson JW, Singer JW, Catalan0 P, Murphy S, Lin N,
Steinmann L, Ernst C, Fialkow PJ: Polycythemia vera: Further in
vitro studies of hematopoietic regulation. J Clin Invest 66:1363,
1980
18. Lucas GS, Padua RA, Masters GS, Oscier DG, Jacobs A
The application of X-chromosome gene probes to the diagnosis of
myeloproliferative disease. Br J Haematol72:530,1989
19. Anger B, Janssen JWG, Schrezenmeler, Hehlmann R,
Heimpel H, Bartram CR: Clonal analysis of chronic myeloproliferative disorders using X-linked DNA polymorphisms. Leukemia
4:258,1990
20. Taylor KM, Shetta M, Talpaz M, Kantarjian HM, Hardikar
S, Chinault AC, McCredie KB, Spitzer G: Myeloproliferative
disorders: Usefulness of X-linked probes in diagnosis. Leukemia
3:419,1989
21. Gilliland DG, Blanchard KL, Levy J, Perrin S, Bunn H F
Clonality in myeloproliferative disorders: Analysis by means of the
polymerase chain reaction. Proc Natl Acad Sci USA 88:6848, 1991
22. Hoffman R, Estren S, Marks SM: Lymphoblastic-like leukemic transformation of polycythemia vera. Ann Intern Med 8971,
1978
23. Aitchison R, Black AJ, Greaves M F Polycythaemia rubra
vera transforming to acute lymphoblastic leukaemia. Clin Lab
Haematol9:201,1987
24. Griffin JD, Tantravahi R, Canellos GP, W i s h JS, Reinherz
EL, Sherwood G, Beveridge RP, Daley JF, Lane H, Schlossman
S F T cell surface antigens in a patient with blast crisis of chronic
myeloid leukaemia. Blood 61:640, 1983
25. Jacobs P, Greaves MF: Phl-positive T lymphoblastic transformation. Leuk Res 8:737,1984
26. Chan LC, Furley AJ,Ford AM, Yardumian DA, Greaves
M F Clonal rearrangement and expression of the T cell receptor p
gene and involvement of the breakpoint cluster region in blast
crisis of CGL. Blood 67:533, 1986
27. Raskind WH, Jacobson R, Murphy S, Adamson JW, Fialkow
PJ: Evidence for the involvement of B lymphoid cells in polycythemia vera and essential thrombocythemia. J Clin Invest 75:1388,
1985
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1992 80: 1033-1038
Simultaneous genotypic and immunophenotypic analysis of
interphase cells using dual-color fluorescence: a demonstration of
lineage involvement in polycythemia vera
CM Price, EJ Kanfer, SM Colman, N Westwood, AJ Barrett and MF Greaves
Updated information and services can be found at:
http://www.bloodjournal.org/content/80/4/1033.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.