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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 683-689
Detection of Microchimerism After Allogeneic Blood
Transfusion Using Nested Polymerase Chain Reaction Amplification With
Sequence-Specific Primers (PCR-SSP): A Cautionary Tale
By
Anthony S. Carter,
Mike Bunce,
Lucia Cerundolo,
Ken I. Welsh,
Peter J. Morris, and
Susan V. Fuggle
From the Nuffield Department of Surgery, University of Oxford, John
Radcliffe Hospital, Headington, Oxford, UK.
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ABSTRACT |
In bone marrow transplantation, the detection of chimerism is an
important adjunct to the repertoire of tests available for determining
acceptance of the graft. In solid organ transplantation, there is
currently intense interest in the role that chimerism plays in both
short- and long-term host reactivity to the graft. Allogeneic blood
transfusion has been associated with a subtle immunosuppressive effect
in renal transplantation and chimerism is implicated as a possible
mechanism for this effect. To assess the survival of allogeneic cells
after blood transfusion or transplantation, we have developed a
technique based on molecular typing for HLA class II alleles, which
enables the detection of donor-derived cells in patients receiving
blood transfusions. While developing this technology, we investigated
why we and others observe false amplification. Sequencing of false
products has shown that they arise from amplification of both
pseudogenes and non-pseudogenes present in the DNA under test.
Elucidation of this phenomenon allows the amplification of these false
products to be predicted in any given combination and hence avoided by
the judicious selection of primers. Validation has been achieved by
following donor alleles after transfusion of blood containing defined
numbers of leukocytes expressing selected mismatched antigens.
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INTRODUCTION |
THE DEVELOPMENT OF chimerism after
allogeneic bone marrow transplantation is paramount to its success.
However, the connection between the development of chimerism after
solid organ transplantation and graft outcome is tenuous. In solid
organ transplantation, microchimerism is commonly used to define the
presence of donor cells/DNA outside of the graft environment. It has
been hypothesized that spontaneous hematopoietic microchimerism may be
essential for the development and maintenance of immunologic
unresponsiveness to organ allografts.1,2 Hematopoietic
microchimerism has been demonstrated after transplantation of
livers,3-5 kidneys,6,7 hearts,8 and
lungs9,10 using methods based on polymerase chain reaction
(PCR) amplification of the Y-chromosome3 and the
HLA-DR region of the major histocompatibility complex
(MHC).6-8,10 An accurate assessment of the level of
donor-derived cells surviving within recipients may lead to an improved
understanding of the relative importance of microchimerism after
either blood transfusion or solid organ transplantation.
The transfusion of allogeneic blood is in many ways analogous to a bone
marrow transplant without the accompanying immunosuppression and for
this reason is often termed mononuclear cell
transplantation.11 It has been associated with an
improvement in subsequent renal allograft survival,12
increased susceptibility to viral or bacterial infection,13,14 and to the possible increased risk of
cancer recurrence.15,16 Experimental evidence suggests that
the transfusion effect depends on recipient exposure to viable
allogeneic donor cells.17,18 Although several studies have
documented the survival of donor cells within recipients after blood
transfusion in animal models,19-21 there has been limited
investigation into the development of such microchimerism after
allogeneic blood transfusion in humans. Human studies of donor
leukocyte survival after HLA-unrelated transfusions have shown little
evidence for long-term microchimerism, showing the presence of donor
cells only in the first few days after transfusion.22-26
These assays were restricted, as they were based on the detection of
male donor cells in female recipients. In addition, the techniques may
not have been sensitive enough to detect the low levels of donor
leukocytes that may be present after allogeneic transfusions.
There are many factors, both immunologic and nonimmunologic, that may
influence the survival of donor cells after allogeneic blood
transfusion including the length and method of storage of the blood,
HLA matching, and natural killer cell activity. To be able to assess
the relative influence of such factors, a reproducible and accurate
assay system for the identification of donor cells in the recipient is
required. We have now developed a technique based on published
methods,27-29 which enables the detection of donor-derived
cells in patients receiving blood transfusions. While investigating the
commonly used techniques for the detection of microchimerism after
solid organ transplantation, we frequently observed the presence of
what are euphemistically termed "false positives". In this
report, we describe the conditions under which these false positives
occur and how to compensate for their presence.
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MATERIALS AND METHODS |
DNA Isolation
Genomic DNA was isolated from leukocytes obtained from anticoagulated
blood using the salting out procedure,30 precipitated with
ethanol, and resuspended in sterile water at a concentration of 200 ng/µL.
HLA DR Typing
PCR-SSP typing (PCR amplification with sequence-specific primers).
Allele specific primers (0.5 µmol/L), designed on the basis of
published sequences,29,31 were used in multiple
amplification reactions (Table 1)
consisting of 200 ng genomic DNA, 67 mmol/L Tris pH 8.8, 16.6 mmol/L
NH4SO4 (ammonium sulphate), 200 µmol/L of
each deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine
triphosphate (dTTP), 2.0 mmol/L MgCl2
(magnesium chloride), and 0.25 U BioTaq polymerase (Bioline, London,
UK). PCR amplifications were performed in a PTC200-96v thermal cycler
(Genetic Research Instrumentation, Essex, UK) according to the method
of Bunce et al.29 All products were analyzed by agarose gel
electrophoresis.
Nested PCR-SSP typing.
Forward primers DRFR1 CCCCACAgCACgTTTCTTg and DRFR2 CCCCACAgCACgTTTCCTg
and reverse primer DRFR3 CCgCTgCACTgTGAAgCTCT amplify a 280-bp region
of exon 2 of DRB1-9.32 The combination of primers is
necessary to amplify all of the currently known HLA-DR
alleles.
Amplification.
A total of 200 ng DNA was initially amplified in a buffer containing 67 mmol/L Tris pH 8.8, 16.6 mmol/L NH4SO4 , 200 µmol/L of each dATP, dCTP, dGTP, dTTP, 1.0 mmol/L MgCl2
and 0.25 U BioTaq polymerase for 35 cycles (95°C for 30 seconds,
65°C for 45 seconds, and 72°C for 45 seconds) in a PTC200-96v
thermal cycler. The resultant PCR product was diluted 1:200 in water
before PCR-SSP typing.
Determination of Sensitivity and Specificity of Typing
Methods
Sensitivity of PCR-SSP typing.
Decreasing amounts of DNA (200 ng/µL) from heterozygous individuals
were mixed into DNA of known HLA type (200 ng/µL) to give final
concentrations of 5%, 1%, 0.5%, and 0.1% (vol/vol). The resulting
DNA mixtures were subsequently typed using equimolar HLA-DR
sequence-specific primers and analyzed by agarose gel electrophoresis.
Sensitivity of nested PCR-SSP typing.
As for PCR-SSP typing, the sensitivity of nested PCR-SSP typing was
analyzed by mixing DNA from healthy volunteers (200 ng/µL) with DNA
of known type (200 ng/µL) at the following relative concentrations: 1%, 0.1%, 0.01%, 0.001%. The mixtures were then subjected to nested PCR-SSP typing and analyzed by agarose gel electrophoresis.
Specificity.
Specificity of nested PCR-SSP was determined by sequencing seven PCR
products resulting from the amplification by primer mix 118 (Table 1).
Amplicons were isolated from a low melting temperature agarose gel
using the Wizard PCR Preps Purification System (Promega, Hampshire,
UK). 33P cycle sequencing was performed with the Thermo
Sequenase cycle sequencing kit (Amersham Life Science, Buckinghamshire,
UK) using either [ -33P]-dATP or
[-33P]-dCTP and the specific forward or reverse
primer. The products were run on a 7.5% polyacrylamide gel at 65 W for
2.5 hours. The gel was dried and autoradiography performed.
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RESULTS |
Determination of the Sensitivity of PCR-SSP Typing
The results summarized in Table 2 show that
there is immense variation in the efficiency of amplification of HLA-DR
alleles when using this system. While certain primer mixes (115 and
119) were capable of detecting DNA to a level of 0.1%, other primer mixes (120, 125, 126, and 127) only detected DNA to a level of between
1% and 5%. These results demonstrated that a greater sensitivity was
required to detect donor-derived DNA after blood transfusion.
Nested PCR-SSP Typing
Sensitivity.
The results summarized in Table 2 demonstrate that the
sensitivity of this nested PCR-SSP typing system is approximately 100-fold higher than that of PCR-SSP typing, and that 11 of 14 primer
mixes tested could detect DNA to a level of 0.001%. However, primer
mixes 114, 126, and 122 could only detect DNA to a level of between
0.01% and 0.001%. Figure 1 shows a
typical set of results where all HLA-DR alleles from one donor
(DRB1*0102, *1101/4, DRB3*0202/3) can be detected to a level of 0.001%
in a recipient of HLA type DRB1*1501, *0701, DRB5*01, DRB4*0101. There
is also an extra band that appears on the gel, in each case, which is
neither donor nor recipient in origin (band detected with primer mix
118).
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| Fig 1.
Sensitivity and specificity of nested PCR-SSP typing. DNA
from one individual (HLA-DRB1*0102, *1101/4; DRB3*0202/3) was mixed with DNA of known type (HLA-DRB1*1501, *0701; DRB4*0101; DRB5*01) at
relative concentrations of 1%, 0.1%, 0.01%, and 0.001%. DNA mixtures were typed using nested PCR-SSP and products analyzed by
agarose gel electrophoresis. All HLA alleles from the donor can be
detected to a level of 0.001% (donor and recipient lanes are in bold
type, with donor lanes also shaded. The lane yielding the false
amplification is outlined).
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Specificity.
When nested PCR-SSP typing was used, extra bands appeared on the gel,
one of which appeared every time, regardless of donor/recipient HLA
type. Primer mix 118 yielded a band almost every time nested PCR-SSP
typing was used (Fig 1). The only instance when this primer mix failed
to yield a band was when DNA homozygous for DRB1*1001 was used (data
not shown). To investigate the nature of the products amplified by this
primer mix, DNA samples of known HLA type were amplified using this
primer mix in nested PCR-SSP typing and sequenced by 33P
Cycle sequencing. In conventional PCR-SSP typing systems, primer mix
118 amplifies the alleles DRB1*0301-8, with the exception of DRB1*0302
and DRB1*0305. Seven individuals, two of whom were DRB1*0301
(Table 3) and five who were not DRB1*0301-8
and, hence, should not be amplified by this primer mix, were sequenced
and the results are summarized in Fig 2. As
predicted, the two DNA samples typed as DRB1*0301 (A and B) had a
sequence that corresponded exactly to that of the published sequence
for DRB1*0301.31 When the remaining samples were compared
with known HLA class II sequences, five of six corresponded to one or
other of the HLA-DRB alleles present in the reaction. Interestingly,
one of the samples (F) showed a number of differences to the sequence
of DRB1*0301, which were only present in the sequence of HLA-DRB7*0101
(previously HLA-DRB ), a pseudogene associated with the DR4, 7 and 9 haplotypes.33 The sequencing results show that the extra
products seen when nested PCR-SSP typing is used are the result of
mispriming events leading to the nonspecific amplification of HLA
alleles or pseudogenes present in the PCR reaction.
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| Fig 2.
Investigation into products detected when performing
nested PCR-SSP typing with primer mix 118. Seven DNA samples (A to G) were subject to nested PCR-SSP typing and the resultant amplicons were
purified and sequenced using 33P cycle sequencing. The
resultant amplicon sequences were aligned to known HLA-DRB
sequences31 and compared with HLA DRB1*0301 (redundancies
are identified using the International Union of Biochemistry Group Codes where K = G/T, M = A/C, R
= A/G, S = C/G, V = A/C/G, W = A/T, Y = C/T; * indicates base
unknown).
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DISCUSSION |
The use of PCR-SSP typing alone for detection of HLA alleles was found
not to be applicable to the detection of microchimerism as the level of
sensitivity is poor for many alleles. The sensitivity experiments
performed using deliberate mixing of DNA showed significant differences
in the efficiency of amplification of the individual HLA-DR primer
mixes (Table 2). These experiments suggested that a more
sensitive method was required to enable donor-derived HLA alleles to be
detected after allogeneic transfusion. A number of investigators
attempting to assess the levels of microchimerism after solid organ
transplantation have used a nested PCR approach,8,10,34,35 primarily amplifying exon 2 of HLA DRB1, followed by a second PCR based
on standard HLA typing procedures.27,28 We have used a
similar technique and have found, in accordance with other groups, an
increased sensitivity in the detection of donor-derived alleles in our
mixing experiments (Table 2). We were able to detect
HLA-DR alleles tested to a level of 0.001%, equivalent to a
sensitivity of 1:100,000. However, we have shown that this nested PCR
technique results in the presence of extra bands on the gel, bands that can easily be misinterpreted to indicate the presence of donor alleles.
On sequencing a number of these nonspecific products, using primers
that amplify DRB1*0301 in PCR-SSP typing, we have determined that one
product is solely the result of a mispriming event which leads to the
amplification of the HLA class II-associated pseudogene, HLA-DRB7,
present on the DR4, 7 and 9 haplotypes. There are an additional four
known pseudogenes associated with the class II region of the MHC;
(Fig 3). The primers used in the first
round amplification will amplify exon 2 of HLA-DRB1-9, with the
exception of DRB2 and DRB8, as these pseudogenes do not contain exon
2.36,37 Although all alleles of HLA-DRB6, and HLA-DRB9 will
be amplified by the first round primers, the forward and reverse
primers from mix 118 are sufficiently mismatched to prevent any
amplification of these alleles in the second round of amplification. On
analysis of the other nested PCR-SSP amplicons, we have found that most of the nonspecific products are the result of mispriming events leading
to the amplification of one or both of the HLA alleles present in the
reaction.
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| Fig 3.
Organization of the known HLA DRB loci within the class
II region of the human MHC (known pseudogenes are shaded).
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The specificity of PCR-SSP typing relies on a mismatch at the 3
residue of one of the primers being sufficient to prevent misamplification of alleles under carefully established
conditions.38 However, we have shown that the addition of
the preliminary amplification, so necessary for increased sensitivity,
alters the stringency required for the subsequent PCR-SSP reactions. In
many cases, the single base pair mismatch at the 3 end of either
primer is not then sufficient to prevent mispriming events, this in
turn resulting in nonspecific amplification of many alleles. We have analyzed the sequences of the HLA alleles (including all pseudogenes) used in determining the specificity of nested PCR-SSP typing for their
ability to bind to the individual primers in mix 118 (Table 4). The allele DRB1*0101, present in
sample C, has a single base pair mismatch with the forward primer and
two with the reverse, all being located at the 3 end of the
primers. Sequencing showed that this allele was amplified, hence the
3 mismatches are not sufficient in this case to prevent
amplification. All of the alleles present in sample D are also
amplified with this primer mix, as shown by the sequencing result (Fig
2). We believe this is because there is complete homology between the
sequences of DRB1*1201 and DRB3*0201/4 and the reverse primer with only
a single residue mismatched with the forward primer in both cases.
There is obviously a hierarchy in terms of which alleles are amplified.
This is illustrated by samples E, F, and G where only those alleles
with one mismatch with the forward or reverse primers are amplified.
The remaining alleles are not detected by sequencing and one can assume
that they are not amplified by this primer set in these samples.
Interestingly, the pseudogene DRB7*0101 was amplified in samples F and
G. This is due to the fact that the forward primer will bind, possibly with complete homology to the DRB7*0101 sequence (there are still two
bases unknown) and the reverse primer merely has one residue mismatched
with DRB7*0101, the mismatch being located internally. This analysis
shows that we can now predict which HLA alleles will be amplified in
any given donor-recipient combination and we can control for their
presence by the judicious selection of primers.
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Table 4.
Degree of Mismatching of Forward and Reverse Primers
Included in Primer Mix 118 With the HLA Alleles Used for
Specificity Determination
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Our data suggests that before using these techniques for the detection
of microchimerism, adequate controls are mandatory. In addition to a
negative or water control, a specificity control (pretransfusion/pretransplant recipient DNA) must be used to validate the technique for each recipient/donor combination, otherwise a band
that is present after transfusion/transplant may be wrongly assumed to
be indicative of the presence of a donor allele. Indeed, this has been
demonstrated recently in a study evaluating microchimerism after solid
organ transplantation using nested PCR for HLA genes, where 17% of
patients would have yielded false positive results had a recipient
pretransplant DNA sample not been included in the
analysis.35
To validate our nested PCR-SSP technique, we have followed a number of
different donor MHC class II alleles after transfusion of blood
containing defined numbers of leukocytes bearing selected mismatches.
Preliminary results indicate that donor-derived alleles are present in
the early period after transfusion, and we are now in the process of
analyzing further samples to give a clearer understanding of the
patterns of survival of donor cells after HLA matched or mismatched
transfusions.
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FOOTNOTES |
Submitted January 28, 1998;
accepted March 10, 1998.
Supported by a grant from the National Kidney Research Fund
(Huntingdon, Cambs, UK).
Address reprint requests to Anthony Carter, Nuffield Department of
Surgery, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK;
e-mail: anthony.carter{at}nds.ox.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract