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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2002-01-0295.
IMMUNOBIOLOGY
From the Fred Hutchinson Cancer Research Center; the
University of Washington Medical Center; and the Virginia Mason Medical
Center, Seattle, WA; and the Department of Chemical Pathology, The
Chinese University of Hong Kong, Hong Kong Special Administrative
Region of the People's Republic of China.
Male DNA, of presumed fetal origin, can be detected in the maternal
circulation decades after delivery and is referred to as fetal
microchimerism (FM). We previously found quantitatively greater FM in
the circulation of women with the autoimmune disease scleroderma (SSc)
than of healthy women. However, it is unknown whether this difference
is due to intact circulating cells or free DNA released from breakdown
in disease-affected tissues. To distinguish the origin of FM, we
developed a real-time quantitative polymerase chain reaction
(PCR) assay for the Y-chromosome-specific sequence
DYS14, and tested 114 women in peripheral blood mononuclear cells (PBMCs) and/or plasma. Fifty-seven controls and 57 SSc
patients were studied, 48 and 43 of whom, respectively, had given birth to at least one son. Circulating FM was quantitatively greater in
PBMCs from SSc patients (n = 39; range,
0.0-12.5 male genome-equivalent cells per million maternal
cells), compared with healthy women (n = 39;
range, 0.0-4.4; P = .03). In contrast, there was no
difference between patients (n = 25) and controls (n = 22) in
plasma, and no evidence of free DNA. FM was enriched among T
lymphocytes compared with PBMCs (P = .01) in controls
(n = 14), but not in SSc patients (n = 14); the latter finding
was most likely due to immunosuppressive medications. In
conclusion, this real-time quantitative assay showed that
quantitative differences in the circulation of women with SSc are due
to cells and not to free DNA. As FM was not uncommon in healthy women,
including among T cells, and because graft-versus-host disease has
similarities to SSc, these results also suggest that FM merits
investigation in pheresis products used for stem cell transplantation.
(Blood. 2002;100:2845-2851) During normal human pregnancy, fetal DNA and cells
pass into the maternal circulation.1 The term
microchimerism refers to the presence of a small amount of DNA or cells
from one individual in another individual. Persistent microchimerism of
presumptive fetal origin (fetal microchimerism [FM]) has
recently been described in the circulation of women with systemic
sclerosis (SSc), also called scleroderma, and in healthy
women.2,3 SSc is an autoimmune disease characterized by an
excessive deposition of collagen in the skin and internal organs and by
vascular and immunologic abnormalities. Studies of FM in SSc were
initiated on the basis of a constellation of
observations,4 including the clinical similarities of SSc to chronic graft-versus-host-disease (cGvHD),5 the
increased incidence of SSc observed in women during the
postreproductive years,6 and the long-term persistence of
FM after pregnancy.2 In prior studies, we tested this
hypothesis using an assay that had been developed for use in prenatal
diagnosis.7 The assay tested multiple aliquots of
whole blood for a Y-chromosome-specific sequence by means of a direct
quantitative polymerase chain reaction (PCR) test. Using this
assay to test whole peripheral blood from women who had given birth to
at least one son, we found quantitatively greater levels of FM in women
with SSc compared with healthy controls.3 Recently, Lo et
al8 reported that during pregnancy, plasma affords
a better source for measuring FM than does the cellular fraction of
maternal blood. Plasma fetal DNA is rapidly cleared following
delivery.9 Thus, the increase of FM observed in whole blood samples from women with SSc compared with controls could be
derived from circulating cells or from fetal DNA released into the plasma from disease-affected tissues. The latter possibility would
be analogous to increased levels of FM seen in the serum of patients
with pre-eclampsia thought to reflect DNA liberated from fetal
nucleated cells.10 We therefore developed a sensitive real-time quantitative PCR assay and employed it to study FM in peripheral blood mononuclear cells (PBMCs), plasma, and also T lymphocytes of parous healthy women and women with SSc.
Study subjects
Procurement of blood specimens and preparation of PBMCs
and plasma
DNA extraction To prepare samples for the quantitative PCR assay, DNA was extracted from PBMCs by means of an Isoquick Nucleic Acid Extraction Kit (ORCA Research, Bothell, WA) according to the manufacturer's instructions and resuspended in 10 mM Tris-HCl Tris (tris(hydroxymethyl)aminomethane-Hcl), (pH 9.0) or was extracted from plasma by means of a High Pure PCR template Preparation Kit (Roche, Basel, Switzerland), following the isolation procedures recommended by the manufacturer. For DNA extraction, 500 µL plasma sample was used. An elution volume of 100 µL was used.Real-time quantitative PCR The theoretical and practical aspects of real-time quantitative PCR have previously been described.12 The amplification and product-reporting system used was based on the 5' nuclease assay.13 In this system, in addition to the 2 amplification primers used in conventional PCR, a dual-labeled fluorogenic probe is included. Two fluorescent dyes, a reporter and a quencher, are attached to the probe. With both dyes attached to the probe, reporter dye emission is quenched. During each extension cycle, the 5'-3' nuclease activity of the Taq DNA polymerase cleaves the reporter dye from the probe. Once separated from the quencher, the reporter dye emits its characteristic fluorescence. Amplification data were collected by a Perkin-Elmer Applied Biosystems 7700 sequence detector, stored in a Macintosh computer and analyzed by means of Sequence Detection System software (PE Applied Biosystems, Foster City, CA).The DYS14 sequence (GenBank sequence accession number, X06325) was selected as the Y-chromosome-specific sequence for use in the quantitative PCR (QPCR) assay. The reason for this choice was that, while single-copy genes, such as the SRY, can be reliably used in developing a QPCR assay, the use of a single-copy gene results in less sensitivity than a multiple-copy gene. On the other hand, some multiple-copy genes, for example, DYZ1, have been postulated to exhibit autosomal cross-reactivity.14,15 The DYS14, on the other hand, is believed to be strictly Y specific.16 QPCR amplification primers were designed and used with the following
sequences: DYS14 forward primer, 5'-CATCCAGAGCGTCCCTGG-3'; and DYS14
reverse primer, 5'-TTCCCCTTTGTTCCCCAAA-3'. The dual-labeled fluorescent
DYS14-probe sequence was 5' (FAM)-CGAAGCCGAGCTGCCCATCA (TAMRA)-3'.
Simultaneously, the same amount of target DNA was tested for the
Real-time quantitative PCR reactions were set up in a reaction volume
of 50 µL. The target consisted of either 66 ng DNA (in 5 µL) from PBMCs or 5 µL resuspended DNA after
extraction from plasma. A conversion factor of 6.6 pg DNA per cell was
used for expressing the results as male genome-equivalent
cells per milliliter of maternal plasma or per million maternal
cells, with 66 ng DNA corresponding to 10 000 male genome-equivalent
cells.17 Each PCR reaction consisted of 5 µL 10 × platinum buffer (Gibco BRL, Burlington, ON,
Canada), 300 nM each amplification primer, 100 nM dual-labeled
probe, 200 nM each deoxynucleoside triphosphate (dNTP) (Gibco
BRL), 3.5 mM MgCl2 (Gibco BRL), 1.5 U platinum
Taq (Gibco BRL), and 60 nM the standard reference dye ROX
(Synthegen, Houston, TX). The amplification conditions
consisted of an initial incubation at 50°C for 2 minutes, followed by
incubation at 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 56°C for 1 minute. For quantitative measurement of fetal
DNA, a calibration curve was constructed with the use of DNA from a
healthy male individual diluted in DNA from a healthy female
individual. Fourteen aliquots of 10 000 male genome-equivalent
cells (66 ng) were tested for each patient and control,
including a duplicate for T-lymphocyte isolation by fluorescence-activated cell sorting (FACS) PBMCs were filtered on nylon wool (Du Pont Biotechnologies, Boston, MA) to avoid cell aggregation and were resuspended in phosphate-buffered saline (PBS)/1% fetal calf serum. Staining was performed as previously described18 on 10 to 20 × 106 cells with anti-CD3-cychrome and/or anti-CD4-phycoerythrin (PE) and/or anti-CD8-fluorescein isothiocyanate (FITC) (Becton Dickinson, Mountain View, CA) at 3 µL per antibody per million cells. After staining incubation, DNase (Boehringer-Mannheim, Indianapolis, IN, or GIBCO BRL) was added (30 U per million cells) after the last wash to avoid cell aggregation during the FACS process. Cells were sorted by a single laser on a FACS (Becton Dickinson), on the basis of the markers described above. An aliquot of the sorted cells was run, and the percentage of sorted and correctly gated cells were calculated to assess the purity. Purity consistently exceeded 99%. Cells were collected in RPMI. Cell sorting was immediately followed by centrifugation and 2 washes to remove all remaining DNase. Cell aliquots were stored at 80°C in cell lysis buffer until DNA
extractions were performed.
Statistical analysis We investigated whether the number of male genome-equivalent cells per million maternal cells in PBMCs, or per milliliter in maternal plasma, was higher in women with SSc compared with healthy women. The analysis considered women who had given birth to at least one son. P was calculated by means of a regression model; robust variances were estimated by means of the method of generalized estimating equations. For multiple measurements based on blood drawn on the same day, the DNA-equivalent fetal cells per million maternal cells values were averaged. Multiple measurements based on repeated blood draws per woman were entered into the analysis as repeated measures, with adjustment for possible correlation between values within a subject.
Quantitative assessment of male DNA in plasma of healthy women and women with SSc Plasma samples from 66 women were investigated for quantitative assessment of male DNA. Study subjects included 29 healthy women and 37 women with SSc. Twenty-two healthy women and 25 women with SSc had given birth to at least one son. The majority of women who had given birth to a son (healthy women as well as women with SSc) had no detectable male DNA in the plasma, 73% and 64%, respectively (Figure 1). The range of male genome-equivalent cells was 0 to 10 per million maternal cells among controls and 0 to 8.2 per million maternal cells among patients, both with median values of 0 (Table 1). There was no significant difference between healthy women and women with SSc. Of the 6 healthy women with positive results (6 women, 8 observations; Figure 1), all had given birth to a son. Among the 11 SSc patients with positive results, 9 had given birth to a son and 1 had a history of blood transfusion. For one SSc patient with a low positive result, the source of male DNA was unclear as she had daughters but had not received a blood transfusion. In addition, she was not a twin and did not have a history of known miscarriage. All women with SSc had given birth to sons 10 or more years previously whereas 6 healthy women had given birth to sons fewer than 10 years from the time of testing. However, results did not differ if women with more recent births were excluded from the analysis.
A positive result in plasma could represent free DNA or could be the
result of apoptotic fetal cells in plasma, as has been described by
others.19 Our method of centrifugation was gentle, using
400g, as compared with other methods that have been
described as using consecutive rounds of centrifugation or
centrifugation followed by filtration.20 To explore the
explanation that positive results derived from apoptotic cells that
were not removed with the gentle centrifugation method, we conducted
additional experiments in women with positive results. Samples from 5 women (4 healthy controls and 1 woman with SSc) with high levels of
plasma male DNA were studied according to 2 protocols: a 10-minute
centrifugation at 400g, as initially done, and a 10-minute
centrifugation followed by filtration using 0.45-µm filters to remove
apoptotic cells but to leave any floating DNA in the plasma. All women
had a negative result after filtration (Figure
2), indicating that the positive results observed in the plasma samples were due to apoptotic cells and
not to free circulating DNA.
Quantitatively greater levels of male DNA in PBMCs in women with SSc compared with healthy women PBMCs were quantitatively assessed for male DNA for 93 women, in 46 healthy women, and 47 women with SSc. In each group, healthy women and women with SSc, 39 women had given birth to at least one son. The frequency of any detectable male DNA in PBMCs was somewhat higher in SSc patients (51%) compared with controls (31%) but was not significant (P = .07). A significant difference in FM levels between patients and controls was observed (P = .03). The range of male DNA among controls was 0 to 4.4 and among patients 0 to 12.5. Table 2 summarizes a comparison between healthy women and women with SSc who had given birth to at least one son. Two patients had outlying values. When we excluded these values to see if they were influencing the estimates, the results remained significant (P = .047). Six SSc patients gave a history of blood transfusion; if these patients were excluded, the difference was not statistically significant. All statistical analyses were adjusted for some subjects who were tested on more than one occasion. Three women in the control group and 2 SSc patients were tested at 2 time points: 1 control and 1 SSc patient at 3 time points, and 1 control at 4 time points.
SSc is a disease that is most often diagnosed in women in the late 40s
or even early 50s. Most of the SSc patients we studied had sons who
were older than 10 years at the time of the patient's blood draw (95%
of cases), whereas controls not infrequently had given birth fewer than
10 years previously. We therefore conducted a second analysis comparing
only women for whom the son was born more than 10 years previously
(Table 3; Figure
3). The difference between
patients and controls was significant both overall
(P = .021) and after adjustment for time from birth of the
most recent son (P = .006). Results were also significant
when large outlying values were excluded (P = .018). The
difference between patients and controls was also significant when
patients who had received a blood transfusion were excluded from the
analysis (P = .035). Statistical analysis was adjusted for
2 control women and 6 women with SSc who were tested on 2 different
dates, and 1 SSc patient who was tested at 3 points.
Some patients and controls who were parous but had no history of a male birth or who were nulligravid had positive results for male DNA in PBMCs. Among the 8 patients tested, 5 had positive results, all but 1 at a very low level (0.07 to 0.6 male genome-equivalent fetal cells per million maternal cells); 1 with a high result (26 male genome-equivalent fetal cells) had given birth only to a daughter but reported 3 pregnancies. Among the 4 patients who had a low level of FM, 1 reported an incomplete pregnancy, but the 3 other patients with low levels did not report any pregnancies. Among healthy controls who did not give birth to a son, 2 of 7 also had a very low-level positive result (equivalent to 0.08 and 0.2 male fetal cells per million maternal cells). One had given birth to a daughter, but the pregnancy history, in terms of the possibility of miscarriage, was unknown. The second was a nulliparous woman. Although not specifically investigated, FM is presumed to occur after an incomplete pregnancy, and it is known that large amounts of FM are found in the maternal circulation after an induced abortion.21 Thus, potential explanations include an early unrecognized miscarriage, an unreported induced abortion, an unrecognized loss of a male twin in utero, or possibly cells from sexual intercourse or transfer, via the maternal circulation, from an older male sibling. Male DNA among T lymphocytes of healthy women and women with SSc As FM was cellular in origin, and because T lymphocytes are implicated in a number of autoimmune diseases including SSc, we also employed the QPCR assay to test T lymphocytes isolated to greater than 99% purity by flow cytometry. Forty-one women, including 22 healthy controls and 19 SSc patients, were studied after isolation of cells with antibodies to CD3 (T lymphocytes) and/or CD3/CD4 (T-helper cells) and/or CD3/CD8 markers (T-cytotoxic cells).Among the healthy women, 67% (12 of 18) had FM in the T-cell
compartment (CD3 marker only) (Figure 4).
The range of positive results extended from 0.3 to 68.7 male
genome-equivalent fetal cells per million maternal T cells.
The levels of FM were greater in T lymphocytes (only CD3 markers) than
in PBMCs for 71% of the healthy women for whom quantitative testing
was done from the same draw date (n = 14). This result indicated FM
was significantly enriched in T lymphocytes compared with PBMCs
(P = .01 by Wilcoxon signed rank test). T-helper (CD3/CD4)
and T-cytotoxic (CD3/CD8) cells were studied from some healthy controls
(n = 13 and n = 11, respectively). Positive results were found in
both populations. Thirty-one percent (4 of 13) healthy women had
positive results for CD4 T cells, ranging from 0.2 to 50.3 male
genome-equivalent cells per million maternal T cells.
Sixty-four percent (7 of 11) had positive results in CD8 T cells,
ranging from 0.6 to 16.3 male genome-equivalent cells per million
maternal T cells.
Among women with SSc, 26% (5 of 19) had a positive result for FM in
the T cells isolated by antibodies to CD3 only, with a range from 1.6 to 44.8 male genome-equivalent cells per million maternal T
cells (Figure 5). Thirty percent
(3 of 10) had FM in T cells isolated by antibodies to CD3 and CD4 (T
helper cells), with a range from 0.5 to 13.7 male genome-equivalent
cells per million maternal T cells. Thirty-eight percent (3 of
8) had FM in T cells isolated by antibodies to CD3 and CD8 (T-cytotoxic cells), with a range from 1.1 to 24.2 male genome-equivalent cells per
million maternal T cells. These results suggest a lower
frequency and generally lower levels of FM among T cells in SSc
patients than in healthy women. Additionally, no significant difference was observed in the levels of FM among T cells compared with PBMCs from
the same draw date in women with SSc (n = 14). However,
interpretation of results from SSc patients is confounded by the fact
that the majority of patients studied were taking immunosuppressive
medications. Consistent with modulation of results from
immunosuppressive medication use, the total lymphocyte cell count was
within the normal range (15% to 45%) for 4 SSc patients who had high
levels of FM in T cells (patients no. 3, 6, 8, and 10), in
contrast to the majority of patients who had low total lymphocyte
counts (4% to 10%) and were negative for FM. One patient (no. 6, Figure 5) was tested on 2 draw dates with high levels in FM in T cells
while taking low-dose methotrexate and undetectable FM in T cells while
taking cyclophosphamide.
Recent studies indicate that fetal DNA and cells routinely pass into the maternal circulation during normal pregnancy and that persistent FM can be found in the maternal circulation for decades after pregnancy completion.1,2 Persistent FM, when considered in concert with other observations, including the female predilection to autoimmunity and clinical similarities of cGvHD and some autoimmune diseases, led to the hypothesis that microchimerism is involved in autoimmune diseases such as SSc.4 In a prior study, we found that women with SSc had quantitatively greater levels of FM in peripheral blood than healthy women.3 The assay used in this study was developed for use in prenatal diagnosis and was employed in testing of multiple aliquots of whole blood targeting the Y-chromosome-specific sequence 49a, a moderately repetitive Y segment with no autosomal homology.22 This study was unable to determine the origin of fetal DNA in the maternal circulation. In women who are currently pregnant, fetal DNA has been found in almost 1000-fold higher amounts in plasma than in the cellular compartment of maternal peripheral blood,8 with levels rapidly dropping off within hours of delivery.9 These observations raise the question as to whether the increased FM that we previously found among women with SSc compared with controls is due to circulating fetal cells or to free fetal DNA released into the circulation from cellular breakdown in disease-affected tissues. To elucidate the origin of long-term persistent FM from pregnancy in patients with SSc and healthy women, we developed a quantitative assay using real-time PCR targeting DYS14, a pseudogene of a multigene family,16,23,24 and employed the QPCR assay to test plasma and PBMC compartments of peripheral blood. In a large study of more than 100 women, we found that the majority of healthy women and women with SSc had no detectable male circulating DNA in plasma, and no difference was observed between women with SSc and healthy women. Studies from other investigators have shown that not all fetal DNA in plasma is soluble and cell-free, with some deriving from "plasmatic cells" in the process of apoptosis.19 Therefore additional studies were conducted for some women who had positive results in the plasma samples. All plasma samples became negative after being subjected to filtration, a process that removes cells, but keeps free circulating DNA. Thus, it is likely that the few positive results derived not from free DNA but from DNA associated with cells in the process of apoptosis. In contrast to results for plasma, FM was quantitatively greater in the cellular component, in PBMCs, in women with SSc compared with healthy women. The difference between SSc patients and controls was statistically significant before and after adjusting for time since the last birth of a son. Results were also statistically significant when restricted to the study of subjects for whom sons had been born more than 10 years previously (examined because sons of some healthy women were born more recently than sons of women with SSc). Similarly, results were significant when women with SSc who had received a blood transfusion25 were excluded from the analysis. To our knowledge, this is the first study to employ a quantitative assay to test the origin of long-term, persistent FM in maternal peripheral blood. Following our initial description of quantitatively greater levels of FM in whole blood samples,3 other studies were reported that tested for FM in peripheral blood from women with SSc and controls.26-30 The results of these studies have been variable. Most studies have used qualitative rather than quantitative methods and asked whether the frequency of any detectable FM differs in SSc patients compared with controls. Some studies have used a nested PCR test (nonquantitative) and described an increased frequency of detectable FM in women with SSc compared with controls,26 while others have used the same method and reported no significant difference.27 Variability of results may also arise owing to the particularY-chromosome-specific sequence used as a measure of FM, as these studies used a repetitive DNA family, DYZ1, that has homology with autosomal sequences, raising concern with respect to false-positive results.14,15 Other studies described a semiquantitative PCR method for the Y-chromosome-specific sequence 49a with no significant difference in SSc patients compared with controls.28 We found a marginal difference in the frequency with which FM could be detected in SSc patients compared with controls using a nonquantitative technique targeting the DYS14 sequence.29 In the current study, using the same Y-chromosome sequence, but using a real-time assay to quantify FM, we similarly observed an increased frequency of FM in women with SSc compared with controls (although prior results were just above the point of statistical significance, whereas current results were just short of significance). However, the quantitative assay clearly demonstrated a statistically significant difference in FM levels in PBMCs between patients and controls. Another source of variability in studies of peripheral blood for FM is the compartment of blood that is investigated. Most studies have used DNA extracted from PBMCs, although some have tested DNA from whole blood, and others have tested DNA white blood cells. Thus, both differences in the techniques and differences in the source of sample from peripheral blood are likely, at least in part, to explain variability in reports. Differences in technique include whether an assay was qualitative or quantitative, the level of sensitivity of the assay, and the specific Y-chromosome sequence targeted. Further variability may derive from the fact that most studies use male DNA as the measure of FM, but some studies summarize results for women with sons while others have summarized results without providing pregnancy history for the subjects tested. The use of male DNA as a measure of FM can be confounded by other potential sources of microchimerism. Cells are known to engraft and persist between twins, so that a woman with a fraternal male twin may have positive result.31 Twinning is also not uncommon, so that a woman could even potentially have microchimerism from an unrecognized twin that was lost early in gestation. Blood transfusion is another source of persistent microchimerism, as has been described in the circulation of trauma patients who have received multiple transfusions.25 Although FM has not yet been formally studied, it is presumed to also occur after a miscarriage or an induced abortion. Consistent with this likelihood, FM has been detected as early as 5 weeks of gestation,32 and large amounts of fetal DNA have been reported in the blood of women undergoing elective pregnancy termination.21 Similar to other reports, our study found that some women who did not have a history of a male birth had a positive test for male DNA. Some of the women with positive results had either had a previous blood transfusion or reported a previous miscarriage. The origin of male DNA in others is not known, although the most likely source may be an unrecognized miscarriage. It is at least theoretically possible that male cells could be transferred from an older male sibling via the maternal circulation, or possibly even from sexual intercourse. Results are not likely to be due to contamination, since the QPCR assay used is a homogeneous assay, which did not require the opening of the amplification wells following PCR. We also found that FM was enriched among T lymphocytes as compared with PBMCs among healthy women. Few SSc patients had FM among T lymphocytes; however, most were taking immunosuppressive drugs, thus limiting the ability to draw conclusions. The finding of FM among T cells in healthy women, in any case, indicates that the presence of FM among T lymphocytes per se is not likely to be detrimental to the host. Factors that could be important in determining whether FM has a neutral or a detrimental (or possibly even a beneficial) effect on the host include the particular human leukocyte antigen (HLA) genotype of the host, the HLA genotype of the microchimeric cell population, and the HLA-relationship between host and nonhost cells. In prior studies, we found that HLA compatibility for DRB1 of a previously born child was associated with almost a 9-fold increased risk of SSc in the mother.3 Additionally, although donor CD4 and CD8 T cells are implicated in cGvHD,33 which has similarities to SSc, the mechanism by which FM putatively contributes to SSc would not necessarily expected to be similar. There are clinical and pathological differences in the 2 disorders, and most importantly, quantitative levels of chimerism differ dramatically. In cGvHD, donor cells essentially replace circulating host cells, whereas, in SSc, circulating fetal cells at most are estimated at less than 1 in a million host cells.34 Other ways by which FM could contribute to SSc include through the disruption of host-to-host interactions, by providing, for example, a source of peptides that are presented by host cells to other host cells, referred to as the "indirect" pathway of allorecognition.35 The real-time QPCR assay described in the current studies provides a tool for future studies in which other specific cellular subsets can be quantitatively assessed, thereby helping to clarify the direction for functional studies directed at elucidating the role of FM in the healthy host and in patients with SSc. Careful quantitative assessment of FM will be useful in resolving variability among initial reports investigating FM in SSc and other autoimmune diseases, including primary biliary cirrhosis and Sjögren syndrome.36 Additionally, because FM was also found among healthy women, including among T lymphocytes, and because SSc resembles graft-versus-host disease for which a parous donor increases risk,37,38 results of the current study also indicate that FM merits investigation in pheresis products used for stem cell transplantation.
We would like to thank the Scleroderma Registry, Jennifer Brackensick, and Gretchen Agee for their contributions to this study. We gratefully acknowledge Jennifer Pang and Heidi Hermes for technical support and Wendy Leisenring for statistical advice.
Submitted January 31, 2002; accepted March 25, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2002-01-0295.
Supported by the Scleroderma Foundation (Grant no. 007/01) and National Institutes of Health grants AI41721 and AR48084.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nathalie Lambert, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, D2-100, Seattle, WA 98109-1024; e-mail: nlambert{at}fhcrc.org.
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© 2002 by The American Society of Hematology.
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  T. H. Brix, P. S. Hansen, K. O. Kyvik, and L. Hegedus Aggregation of Thyroid Autoantibodies in Twins from Opposite-Sex Pairs Suggests that Microchimerism May Play a Role in the Early Stages of Thyroid Autoimmunity J. Clin. Endocrinol. Metab., November 1, 2009; 94(11): 4439 - 4443. [Abstract] [Full Text] [PDF]
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 Y. Fujiki, K. L. Johnson, I. Peter, H. Tighiouart, and D. W. Bianchi Fetal Cells in the Pregnant Mouse Are Diverse and Express a Variety of Progenitor and Differentiated Cell Markers Biol Reprod, July 1, 2009; 81(1): 26 - 32. [Abstract] [Full Text] [PDF]
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 J. M. Rak, P. P. Pagni, K. Tiev, Y. Allanore, D. Farge, J.-R. Harle, D. Launay, E. Hachulla, R. Didelot, J. Cabane, et al. Male microchimerism and HLA compatibility in French women with sclerodema: a different profile in limited and diffuse subset Rheumatology, April 1, 2009; 48(4): 363 - 366. [Abstract] [Full Text] [PDF]
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 J. A. Sawicki Fetal Microchimerism and Cancer Cancer Res., December 1, 2008; 68(23): 9567 - 9569. [Abstract] [Full Text] [PDF]
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Y. Fujiki, K. L. Johnson, H. Tighiouart, I. Peter, and D. W. Bianchi Fetomaternal Trafficking in the Mouse Increases as Delivery Approaches and Is Highest in the Maternal Lung Biol Reprod, November 1, 2008; 79(5): 841 - 848. [Abstract] [Full Text] [PDF]
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V. Cirello, M. P. Recalcati, M. Muzza, S. Rossi, M. Perrino, L. Vicentini, P. Beck-Peccoz, P. Finelli, and L. Fugazzola Fetal Cell Microchimerism in Papillary Thyroid Cancer: A Possible Role in Tumor Damage and Tissue Repair Cancer Res., October 15, 2008; 68(20): 8482 - 8488. [Abstract] [Full Text] [PDF]
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 Y. Uemura, M. Suzuki, T.-Y. Liu, Y. Narita, S. Hirata, H. Ohyama, O. Ishihara, and S. Matsushita Role of human non-invariant NKT lymphocytes in the maintenance of type 2 T helper environment during pregnancy Int. Immunol., March 1, 2008; 20(3): 405 - 412. [Abstract] [Full Text] [PDF]
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 K. Khosrotehrani, M. Leduc, V. Bachy, S. N. Huu, M. Oster, A. Abbas, S. Uzan, and S. Aractingi Pregnancy Allows the Transfer and Differentiation of Fetal Lymphoid Progenitors into Functional T and B Cells in Mothers J. Immunol., January 15, 2008; 180(2): 889 - 897. [Abstract] [Full Text] [PDF]
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 I. C L Kremer Hovinga, M. Koopmans, H. J Baelde, E. de Heer, J. A Bruijn, and I. M Bajema Tissue chimerism in systemic lupus erythematosus is related to injury Ann Rheum Dis, December 1, 2007; 66(12): 1568 - 1573. [Abstract] [Full Text] [PDF]
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V. K. Gadi and J. L. Nelson Fetal Microchimerism in Women with Breast Cancer Cancer Res., October 1, 2007; 67(19): 9035 - 9038. [Abstract] [Full Text] [PDF]
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 K. Khosrotehrani, R.R. Reyes, K.L. Johnson, R.B. Freeman, R.N. Salomon, I. Peter, H. Stroh, S. Guegan, and D.W. Bianchi Fetal cells participate over time in the response to specific types of murine maternal hepatic injury Hum. Reprod., March 1, 2007; 22(3): 654 - 661. [Abstract] [Full Text] [PDF]
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 S. Nguyen Huu, M. Oster, S. Uzan, F. Chareyre, S. Aractingi, and K. Khosrotehrani Maternal neoangiogenesis during pregnancy partly derives from fetal endothelial progenitor cells PNAS, February 6, 2007; 104(6): 1871 - 1876. [Abstract] [Full Text] [PDF]
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I. C. L. Kremer Hovinga, M. Koopmans, E. de Heer, J. A. Bruijn, and I. M. Bajema Chimerism in systemic lupus erythematosus--three hypotheses Rheumatology, February 1, 2007; 46(2): 200 - 208. [Abstract] [Full Text] [PDF]
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 M. Klintschar, U.-D. Immel, A. Kehlen, P. Schwaiger, T. Mustafa, S. Mannweiler, S. Regauer, M. Kleiber, and C. Hoang-Vu Fetal microchimerism in Hashimoto's thyroiditis: a quantitative approach Eur. J. Endocrinol., February 1, 2006; 154(2): 237 - 241. [Abstract] [Full Text] [PDF]
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N C Lambert, J M Pang, Z Yan, T D Erickson, A M Stevens, D E Furst, and J L Nelson Male microchimerism in women with systemic sclerosis and healthy women who have never given birth to a son Ann Rheum Dis, June 1, 2005; 64(6): 845 - 848. [Abstract] [Full Text] [PDF]
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 K. Khosrotehrani and D. W. Bianchi Multi-lineage potential of fetal cells in maternal tissue: a legacy in reverse J. Cell Sci., April 15, 2005; 118(8): 1559 - 1563. [Abstract] [Full Text] [PDF]
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A. M. Stevens, H. M. Hermes, N. C. Lambert, J. L. Nelson, P. L. Meroni, and R. Cimaz Maternal and sibling microchimerism in twins and triplets discordant for neonatal lupus syndrome-congenital heart block Rheumatology, February 1, 2005; 44(2): 187 - 191. [Abstract] [Full Text] [PDF]
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 K. L. Johnson and D. W. Bianchi Fetal cells in maternal tissue following pregnancy: what are the consequences? Hum. Reprod. Update, November 1, 2004; 10(6): 497 - 502. [Abstract] [Full Text] [PDF]
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 R Bulla, F Fischetti, F Bossi, and F Tedesco Feto-maternal immune interaction at the placental level Lupus, September 1, 2004; 13(9): 625 - 629. [Abstract] [PDF]
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A E Turco and L M Bambara Pregnancy, microchimerism and autoimmunity: an update Lupus, September 1, 2004; 13(9): 659 - 660. [Abstract] [PDF]
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 K. Khosrotehrani, K. L. Johnson, D. H. Cha, R. N. Salomon, and D. W. Bianchi Transfer of Fetal Cells With Multilineage Potential to Maternal Tissue JAMA, July 7, 2004; 292(1): 75 - 80. [Abstract] [Full Text] [PDF]
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 R. J.P. Rijnders, G. C.M.L. Christiaens, A. A. Soussan, C. E. van der Schoot, P. Invernizzi, G. Simoni, and M. Podda Cell-Free Fetal DNA Is Not Present in Plasma of Nonpregnant Mothers * Authors of the article cited above respond: Clin. Chem., March 1, 2004; 50(3): 679 - 681. [Full Text] [PDF]
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 K. M. Adams, N. C. Lambert, S. Heimfeld, T. S. Tylee, J. M. Pang, T. D. Erickson, and J. L. Nelson Male DNA in female donor apheresis and CD34-enriched products Blood, November 15, 2003; 102(10): 3845 - 3847. [Abstract] [Full Text] [PDF]
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J.-M. Costa, A. Benachi, M. Olivi, Y. Dumez, M. Vidaud, and E. Gautier Fetal Expressed Gene Analysis in Maternal Blood: A New Tool for Noninvasive Study of the Fetus Clin. Chem., June 1, 2003; 49(6): 981 - 983. [Full Text] [PDF]
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T.-W. Lau, T. N. Leung, L. Y.S. Chan, T. K. Lau, K.C. A. Chan, W. H. Tam, and Y.M. D. Lo Fetal DNA Clearance from Maternal Plasma Is Impaired in Preeclampsia Clin. Chem., December 1, 2002; 48(12): 2141 - 2146. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-globin gene to confirm the quality (ability to amplify)
of the extracted DNA. The quantitative results were used to calculate
the fractional concentration of FM (by DYS14 PCR) among
total DNA (by 
