Bone Marrow Transplantation, (1997) 19, 829–834
 1997 Stockton Press All rights reserved 0268–3369/97 $12.00
Interphase FISH analysis of sex-mismatched BMT utilizing dual color
XY probes
V Najfeld1, W Burnett1 , A Vlachos2, E Scigliano1, L Isola1 and S Fruchtman1
1
Tumor Cytogenetics Laboratory and Bone Marrow Transplantation Service, Polly Annenberg Levee Division of Hematology,
Department of Medicine and 2Pediatric Hematology/Oncology, Department of Pediatrics, Mount Sinai Medical Center, New York,
NY, USA
Summary:
Interphase FISH analysis, utilizing dual color XY probes, was performed on 27 patients following allogeneic
sex-mismatched bone marrow transplantation and on
31 controls. Of the 123 167 examined interphase nuclei,
63 318 were from 19 of the 21 patients (54 specimens)
who engrafted, 31 827 from five of the six patients (29
specimens) who relapsed (four) or failed to engraft (one)
and 24 703 from the 31 control specimens. In patients
who engrafted, the mean percentage of host cells was
0.26% between day 29 and 5 years following BMT.
Microchimerism of 0.7% or less than 1–5 years following BMT was not predictive of relapse. Interphase FISH
analysis predicted relapse or failure of engraftment in
five of the six evaluable patients. In three of five patients
both conventional cytogenetics and interphase FISH of
bone marrow cells provided important information
regarding engraftment status and degree of chimerism.
Keywords: FISH; XY; interphase; cytogenetics; bone
marrow transplantation
powerful tool for monitoring engraftment, with a high sensitivity and low false positivity rate.6
The present study was undertaken to investigate the
efficiency, clinical validity and potential limitations prospectively of interphase FISH analysis after sex-mismatched BMT utilizing commercially available dual color
XY probes. Bone marrow and peripheral blood cells were
examined from 27 patients (86 specimens) and 31 normal
subject controls. These data were compared with conventional cytogenetic studies from the same patients. The
results obtained demonstrate that interphase FISH is a
highly reliable, efficient and sensitive technique which provides clinically important information. Routine specimens
can be processed within 4 h with analysis of over 1000
interphase nuclei. Both cytogenetics and interphase FISH
provide valuable information in patients who relapse or fail
to engraft.
Materials and methods
Patients
Following allogeneic bone marrow transplantation (BMT)
careful monitoring of engraftment is important because the
presence of residual host cells may precede the reappearance of the abnormal hematopoietic clone. Classic cytogenetics for detection of X and Y chromosomes in bone marrow cells from sex-mismatched transplant recipients is
labor-intensive, time-consuming and limited by the number
of cells that can be analyzed. To exclude mosaicism at the
level of 1%, 300 or more metaphase cells must be examined.1,2 Obtaining this many metaphase cells from bone
marrow following transplantation is nearly impossible in a
routine cytogenetic laboratory. Other methods of detecting
and monitoring chimerism are based on population studies
and have either poor sensitivity, a high rate of false positivity, or unproven biological or clinical significance.3–5 Fluorescence in situ hybridization (FISH) allows the rapid
screening of large number of cells and is becoming a
Correspondence: Dr V Najfeld, Tumor Cytogenetics Laboratory, Box
1079, Mount Sinai Medical Center, One Gustave L Levy Place, New York,
NY 10029-6574, USA
Received 12 August 1996; accepted 18 December 1996
Twenty-seven patients underwent allogeneic bone marrow
transplantation from sex-mismatched donors. Eleven had
CML, four AML, three myelodysplasia (MDS), three ALL,
and one patient each had thalassemia major, Waldenstrom’s
macroglobulinemia, familial hemophagocytic lymphohistiocytosis (FHL), severe combined immunodeficiency syndrome (SCID), severe aplastic anemia (SAA), and histiocytosis. Fifteen patients received marrow from their HLAidentical siblings, seven from unrelated donors, two from
cord blood, one received maternal haplo-identical and two
patients received maternal one antigen mismatched bone
marrow. Donor cells were not T cell depleted except for
marrow cells from haplo-identical maternal donor for
patient DM. Two patients (MP and SH) who relapsed were
treated with adoptive immunotherapy and received peripheral blood buffy coat infusions from their donors.
Three patients were not evaluable (DK and WT in
Table 2 and AF in Table 4) because they either expired
before day 28 following BMT, or follow-up study was not
performed. Criteria for engraftment and inclusion in
Table 2 were: presence of donor cells in the bone marrow
with recovering bone marrow function (WBC . 1000 for
3 days and untransfused platelet count .20 000 for 3 days).
Interphase FISH with XY in BMT
V Najfeld et al
830
Cytogenetics
Bone marrow cytogenetics, using standard procedures,
were performed in 23 of the 27 patients studied. ISCN
nomenclature was used to describe abnormal karyotypes.7
Interphase FISH
Interphase FISH testing was performed between day 8 and
day 1845 following BMT on 86 specimens obtained
between July 1994 and April 1996 and on 31 controls. An
aliquot of bone marrow following cytogenetic harvesting
preparation was used for FISH. Peripheral blood cells were
subjected to 0.8% of NH4Cl red cell lysis and incubated for
10–15 min at room temperature. A dual color X (spectrum
orange)/Y(sat.III spectrum green) probe (Vysis, Downers
Grove, IL, USA) mixed with hybridization buffer was
placed on the slide. The target and probe DNA were codenatured at 80°C for 1 min and 15 s. Hybridization was
carried out between 2 and 16 h at 37°C. Post-hybridization
washing was performed for 2 min in 0.4 × SSC pH 7.0, at
75°C. A centromere enumeration probe (CEP) for chromosome 8 (Vysis) and a locus-specific probe for chromosome
21 band region 21q22.13–22.2 (Vysis) were also used in
one patient who relapsed. The in situ hybridization method
was according to the manufacturer’s protocol. Interphase
nuclei were counterstained with DAPI. FITC, Texas Red
and DAPI hybridization signals were imaged sequentially
using a single-band pass filter. Images were merged using a
Cytovision (Applied Imaging, Pittsburgh, PA, USA) digital
imaging system. Scoring control cells was performed by
two observers using a triple-band pass filter. Scoring of
cells from all specimens was performed by the same
observer. Bone marrow cells from normal donors were used
as controls: 21 were male and 10 were female.
Results
Table 1 shows the results of FISH analysis performed on
bone marrow from 31 controls. The sensitivity of interphase
FISH to detect XY in males was 99% and XX in females
was 98.5%. False positive results, such as detection of XX
in male controls were found at the frequency of 0.011%.
No differences were noted in hybridization efficiency using
a co-denaturation protocol and decreased hybridization time
to 2 h (data not shown). All studies on specimens from
patients were performed using a co-denaturation protocol.
Bone marrow was investigated in 27 patients who
underwent allogeneic, sex-mismatched BMT. Post-trans-
Table 1
No. of
studies
31
plant results from 21 patients (56 specimens), day 8 to day
1845, are shown in Table 2. Two patients were not evaluable: patient DK expired on day 14 and patient WT was
not evaluated beyond day 8. Evaluation of 63 318
interphase nuclei demonstrated that the mean number of
host cells in 19 patients was 0.52% when evaluated during
the first 4 weeks following BMT and 0.26% when tested
between day 29 and day 1845 following BMT. A summary
of these data is presented in Table 3. Host cells were
not detected by bone marrow cytogenetics (8–100
metaphases/patient) in any of the 19 patients. Four patients
(Nos 2, 3, 10 and 19) who engrafted expired between day
35 and day 174 from a variety of causes (see legend of
Table 2).
The results in six patients (30 studies) who relapsed
(four) or failed to engraft (two) are shown in Table 4. Three
of the six patients had MDS, two had AML and one with
refractory ALL underwent a cord blood transplant but did
not engraft. Patient AF expired on day 29 from venoocclusive disease but was not evaluated beyond day 14 (and
thus excluded from this analysis). Patient EL expired on
day 615 from progressive disease. Evaluation of 31 827
interphase nuclei between day 23 and day 370 showed a
range in percentages of host cells of 0 to 100%. Host cells
in four patients were found in 6.7%, 29.2%, 71% and
99.7%, respectively. Subsequent relapse was confirmed by
hematological criteria. In patient AS, who failed to engraft,
6% of host cells were found on day 23 and by day 41 all
evaluated cells were of host origin.
Comparison of bone marrow cytogenetics and interphase
FISH in patients who relapsed or failed to engraft is shown
in Table 4. Patient MP, with myelodysplasia, relapsed on
day 287 when interphase FISH study of bone marrow cells
indicated 6.7% cells of host origin. At this time, three of
the eight (37%) evaluated metaphase cells had the host
abnormal 49,XX,+8,+8,+21 karyotype. This apparent difference in percentages between cytogenetics and interphase
FISH was most likely due to the small number of available
metaphases. By day 326, when repeat cytogenetic study
was adequate, the interphase FISH and conventional cytogenetics gave similar results (23% vs 22%). The treatment
for the patient’s post-transplant relapse was donor buffy
coat infusions. On day 370, following three donor buffy
coat infusions conventional cytogenetics of bone marrow
showed 82% host cells, while interphase FISH of bone marrow showed 28.4% of host cells.
To determine whether host (XX) cells contained an
abnormal copy number of chromosome 8, a co-hybridization in situ study was performed using dual color CEP
Interphase FISH: controls for dual color XY
Sex
(No.)
Males (21)
Females (10)
No. of cells with hybridization signals
Total
XX
XY
XO
YO
Other
2
7245
17 188
0
84
105
36
0
39
4
17 349
7354
XY in males = 99%; YO in males = 0.2%; XX in males = 0.000%; XX in females = 98.5%; XO in females = 1.4%; XY in females = 0%.
Interphase FISH with XY in BMT
V Najfeld et al
Table 2
Patient
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
831
Interphase FISH analysis of 21 patients who engrafted
CO
LPa
BMb
MM
AE
MG
AT
DKc
WJ
RS d
HL
SO
MG
HJ
JM
RMf
HR
SC
DMg
GC
WTi
Dx
Type of
transplant
Day after
BMT
No. of
tests
Tissue
No. of cells
evaluated
% of host
cells
CML
CML
CML
CML
CML
CML
CML
CML
CML
CML
CML
AML
AML
ALL
ALL
Thalassemia major
FHL
Waldenstrom’s macroglobulinemia
SCID
SAA
Histiocytosis
SIB
SIB
MUD
SIB
SIB
SIB
MUD
CORD
Maternal
SIB
SIB
SIB
MUD
SIB
Maternale
SIB
MUD
SIB
184–365
71
35–140
374
1845
1826
20–100
11
30
28–38
30
28–553
28–364
14–100
122–266
23–358
28–203
28–134
3
1
3
1
1
1
5
1
1
3
1
6
3
4
2
9
3
3
BM, PB
BM
BM
BM
PB
BM
BM, PB
PB
BM
BM, PB
BM
BM, PB
BM
BM, PB
PB
BM, PB, PB + PHA
BM, PB
BM, PB
1380
253
1799
1154
715
1031
5783
1745
1501
5632
1035
5264
3944
5231
3226
9277
3560
6387
0.23
0
0.4
0.7
0.4
0.1
1.0
0.1
0
0.06
0.9
0.05
0.03
0.9
0
0.34
0.66
0
Maternal h
MUD
MUD
16–28
29–44
8
2
2
1
PB
BM, PB
PB
3095
3051
1165
0
0
11.7
SIB = sibling; MUD = match unrelated donor; CORD = placental cord blood.
a
Expired on day 97 from multiple organ failure (MOF).
b
Expired on day 174 from MOF.
c
Expired on day 14 from MOF.
d
Expired on day 35 from GVHD.
e
One antigen mismatch.
f
Second BMT.
g
Expired on day 43 from liver failure.
h
T cell-depleted haplo-identical BMT.
i
Expired on day 57 from cerebral hemorrhage and sepsis.
Table 3
Percentages of host cells detected by interphase FISH in
patients who engrafted
Tissue
Days 14–28
No. of
samples
BM
PB
BM + PB
7
8
15
Days 29–1845
Host cells
% ± s.d.
Range
%
0.24 ± 0.6
0.77 ± 1
0.52 ± 0.7
0–0.6
0–2.9
0–2.9
No. of
samples
21
18
39
Host cells
% ± s.d.
Range
%
0.26 ± 0.4
0.26 ± 0.4
0.26 ± 0.4
0–1.5
0–1.5
0–1.5
probes for chromosomes X and 8 on day 326. The results
obtained showed that 22% of marrow cells and 6.5% of
peripheral blood cells had tetrasomy 8 in XX cells. To
determine whether abnormal cells with tetrasomy 8 carry
an abnormal copy number of chromosome 21, on day 352
dual color co-hybridization in situ was performed with CEP
probes for chromosome 8 and locus-specific probe for chromosome 21. Almost 14% of peripheral blood cells with
tetrasomy 8 also had four copies of band region 21q22.13–
22.2. These data suggest a proliferative advantage of the
abnormal host cells over normal donor cells.
Patient SH, with AML, who received an unrelated donor
transplant was cytogenetically normal at diagnosis. On day
92 following BMT interphase FISH analysis of peripheral
blood showed 0.1% host cells. By day 150, 29% of cells
in the blood were of host origin by interphase FISH. Cytogenetic results on day 168 indicated that over 53% of marrow host XX cells had an abnormal karyotype (Table 5).
On day 189, after discontinuation of immunosuppressive
therapy host cells were not detected among 2000 interphase
nuclei. Following relapse the patient was treated with donor
buffy coat infusions. Subsequent analyses showed the
absence of host cells among 9411 nuclei.
A comparison of FISH analysis of peripheral blood and
bone marrow specimens from the same day gave comparable results for patients who were in hematological
remission as shown in Table 5. The same comparison for
five specimens from two relapsed patients and one patient
who failed to engraft showed discrepancy in one specimen
from patient MP.
Discussion
This study was undertaken to determine whether interphase
FISH analysis, utilizing commerical dual color XY probes,
is a diagnostically reliable method to monitor engraftment
and detect chimerism after sex-mismatched BMT. Routine
cytogenetic study following BMT involves analysis of 30–
100 metaphases. Often, between day 7 and day 28 follow-
Interphase FISH with XY in BMT
V Najfeld et al
832
Table 4
Patient/Sex
1 EL/M
2 MC/M
3 MP/F
Comparison of interphase FISH with standard cytogenetics of marrow cells from patients who relapsed or did not engraft following BMT
Dx
MDS
MDS
MDS
Type
of
BMT
Sib
Sib
Sib
4 AS/M
ALL
Cord
5 SH/F
AML
MUD
6 AF/M
AML
Sib
Day
after
BMT
Tissue
No. of
cells
evaluated
% of
host
cells
119
365
25
287
326
326
352d
361
361
370f
370f
23
24
34
34
41–182
30
92
150
168
168
BM
BM
BM
BM
PB
BM
PB
BM
PB
BM
PB
PB
PB
BM
PB
BM
BM
PB
PB
PB
BM
2000
385
229
1077
480
533
1212
476
1051
1011
1056
534
345
515
159
3081
1025
1010
1035
1022
1001
71
99.7
1.4
6.7
6.5c
21.7c
13.9e
14.0
9.8
28.4
4.6
6.3
13
89.7
91.8
100
0
0.1
29.2
40.3
44.1
176
189–230h
14
PB
PB, BM
PB
3179
9411
409
5.4
0
52.3
Cytogenetics
Karyotype
No. of
studied
cells
% of host
cells
46.XX/45,XY,−7a
Non-diagnostic
46,XY
46,XY/49,XX,+8,+8,+21
50
—
47
8
85
—
0
37b
46,XY/50,XX,+8,+8,+21,+21
13
23
46.XY/50,XX,+8,+8,+21,+21
17
82
No mitosis
—
—
46,XY
20
0
46,XY/45,XX,?T(3:5),add(12),
der(20),−22 g
28
53.5
46,XY
Not done
22
—
0
—
a
The karyotype was 45,XY,−7/45,XY,t(3;15)(q28;q21),t(4;14)(q34;q12),−7,add(8).
3/8 evaluated metaphases had XX chromosomal complement. Due to the small number the % of host cells is not accurate.
Dual color X (red) and 8 (green) probes were used simultaneously to detect XX cells with trisomy or tetrasomy 8.
d
After the first buffy coat infusion from donor.
e
Dual color probes for chromosomes 8 and 21 were used simultaneously. The analysis was performed after the second buffy coat infusion.
f
Nine days after third buffy coat infusion.
g
The karyotype was 45,XX,?t(3;5)(q21;?q13),add(12)(p11),der(20)t(15;20)(q13;q21),−22.
h
Six studies were performed after three buffy coat infusions. Cytogenetics was performed on bone marrow cells on day 204.
b
c
Table 5
Comparison between bone marrow and peripheral blood in
detecting host cells by interphase FISH
Patient
Day after
BMT
Hematological remission
1 CO
365
7 AT
28
9 SO
553
11 HJ
27
12 RM
77
358
14 ST
28
Relapse or lack of engraftment
4 MP
326
361a
370b
5 AS
34
6 SH
168
a
% of host cells
BM
PB
0.1
0.6
0
0.2
1.5
0.5
0
0.2
0.9
0
0.9
0
0
0
21.7
14
28.4
89.7
44.1
6.5
9.8
4.6
91.8
40.3
After the second buffy coat infusion from the donor.
Nine days after the third buffy coat infusion from the donor.
b
ing BMT a minimum of 30 metaphases can be difficult to
obtain. Moreover, conventional cytogenetic analysis of
bone marrow cells is a time-consuming and labor-intensive
task and can take 8–10 h to complete. In this report, 117
specimens were studied by interphase FISH and 123 167
nuclei were examined from 27 patients and 31 controls.
The average time to complete the test, including 2 h of
hybridization and scoring of 1000 nuclei, was less than 4 h.
Therefore, the determination of XX/XY status in over 1000
cells by interphase FISH can be accomplished in less than
half the time needed for metaphase analysis of 30 cells.
Previously published reports on the use of FISH for
monitoring engraftment utilized either a Y-specific
probe6,8,9 or X-specific probe only, 10 or both probes with
the same color. One or both probes were non-commercial
probes without standard quality control.11 In these studies
the sensitivity of the test and the threshold for false positive
cells were not determined. The advantage of dual color
probes is that both X and Y hybridization signals can
be viewed simultaneously with a triple-band pass filter. In
this study, evaluation of 24 703 nuclei from 10 female and
21 male control bone marrow specimens showed XY-specific signals in 99% of male control cells and XX-specific
Interphase FISH with XY in BMT
V Najfeld et al
signals in 98.5% of female control cells. Therefore, in this
study the sensitivity of the interphase FISH test was 98.5%
but the threshold of detecting false positive cells containing
the opposite sex chromosomes by dual color interphase
FISH in normal controls was 0.011%. Reproducibility for
dual color XY probe for detection of XX and XY cells
was confirmed recently in a retrospective clinical validation
study.12 In this multi-center study, enumeration of both the
X chromosome and Y chromosome was performed on 120
slides prepared from two mixtures of hematologically
derived human cells with known percentages of XY/XX.12
The limit of detection for XY was determined to be 1%.
The mean percentage of residual host cells found in
patients who engrafted was 0.26 ± 0.4% in peripheral blood
using interphase FISH beyond 4 weeks following BMT.
However, a greater number (0.77 ± 1%) of host cells was
found within the first 4 weeks in the peripheral blood.
Blood specimens were analyzed earlier than marrow and
this may explain the difference. For example, in patient AT,
2.9% of residual host cells were detected on day 20 following BMT in the peripheral blood. In contrast, in a bone
marrow specimen, 8 days later, 0.6% of host cells were
found. Similarly, in patient HJ on day 14, 1.4% of the cells
in the blood were of host origin, whereas 0.2% were of
host origin in bone marrow on day 27. In most patients,
the percentage of residual host cells decreased as the time
from BMT increased irrespective of the tissue studied.
Although the results from patient DK, a 28-year-old male
with CML in blast crisis were not included in this analysis,
it is important to note that on day 11 following cord blood
BMT, peripheral blood analysis showed 99.9% donor cells.
This finding demonstrated the efficiency of the preparative
regimen in eradicating the leukemic clone and the potential
use of cord blood in adult BMT.
Between 0.1% and 0.7% host cells were detected by
interphase FISH in five patients (nine specimens) who were
between 1 and 5 years post-allogeneic BMT. These small
numbers of residual host cells may be explained by longlived T lymphocytes not being eradicated by the preparative
regimen. Host bone marrow metaphase cells were not
detected, indicating that these residual host cells were not
dividing, or were not detected among the 8–100 metaphases
studied in each patient. Further follow-up study may elucidate whether these residual host cells are predictive of
relapse. Microchimerism has been reported in a few cases
that showed no sign of relapse using highly sensitive PCR
technique for amplification of Y chromosome-specific
sequences.13 However, Elmeagacli et al14 reported that the
detection of mixed chimerism after BMT may represent
persistence of residual, apparently healthy, host-type cells.
In this study, chimerism was detected by PCR amplification
of Y chromosome-specific sequences with a sensitivity of
0.001%. These results correlated with the detection of minimal residual disease by BCR-ABL mRNA amplification in
28 male patients with CML who received BMT from the
opposite sex. In seven patients with mixed chimerism
longer than 24 months post-BMT the BCR-ABL transcript
was no longer detected. Our interphase FISH results of 0.1–
0.7% microchimerism, and those of Elmeagacli, provide
preliminary evidence that host cells present 1–5 years postBMT may not be associated with hematological relapse.
A comparison of FISH results obtained from peripheral
blood and bone marrow on the same day (Table 5) suggests
that routine studies of patients who are in hematological
remission could be performed using peripheral blood specimens alone. This suggestion is applicable only if relapse
or graft failure is not suspected.
Host cells were detected by interphase FISH in four
patients who relapsed and in one patient who did not
engraft. In two of the two patients metaphase analysis was
not available from bone marrow but interphase FISH provided evidence of host cells. In the other three patients
detection of chimerism was compared with standard cytogenetics and discordant results were obtained in two out of
four specimens from one patient.
Cytogenetics and interphase FISH studies were concordant in two of four studies performed on bone marrow cells
from patient MP. On day 287 an inadequate number of
mitoses prevented the accurate determination of the percentage of host cells. On day 370 a much higher proportion
(82%) of host cells was obtained by cytogenetics than by
interphase FISH (28.4%). These observations may be
explained by: (1) only 17 metaphases were available for
analysis; (2) abnormal host XX cells may have a proliferative advantage over XY normal donor cells in vivo; and (3)
on day 370 the patient had already received three buffy
coat infusions from the donor. Since the interphase FISH
constitutively enumerates all cells, irrespective of their proliferative state, it is likely that donor cells after the third
infusion were present but did not have a high proliferative
fraction. Therefore, both standard metaphase studies, which
requires proliferating cells, and interphase FISH, which
ascertains the XX/XY genotype of both dividing and nondividing cells, were instrumental in predicting relapse and
monitoring engraftment after buffy coat infusions in patient
MP. Similarly, in patients EL and SH, standard cytogenetics revealed a higher proportion of host cells than
interphase FISH, strongly suggesting that at relapse leukemic cells have a higher proliferative capacity than normal
donor cells. Moreover, cytogenetics detected further
karyotypic evolution in host cells of these three patients
(EL, MP and SH), providing evidence that cytogenetics is
instrumental in predicting and confirming relapse.
The results obtained in this study, along with previously
published reports using non-commercial probes,11 as well
as studies of X and Y chromosomes in preimplanation
embryos and uncultured amniocytes,15,16 provide strong
evidence that interphase FISH with X and Y probes is a
highly sensitive technique that may be useful for monitoring engraftment after BMT. In addition, quantifiable results,
the speed with which the analysis can be completed, and
the ability to analyze large number of cells make interphase
FISH a fundamental tool in molecular cytogenetic laboratories. Its high sensitivity, reproducibility and reliability
make interphase FISH an important diagnostic tool.
Furthermore, these results provide the basis for the use of
commercially available dual color probes to monitor
engraftment after sex-mismatched BMT by interphase
FISH.
Finally, standard cytogenetics and interphase FISH used
simultaneously may provide different, but important information on proliferating and nonproliferating cells regarding
833
Interphase FISH with XY in BMT
V Najfeld et al
834
engraftment, degree of chimerism and the outcome following adoptive immunotherapy.
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