Coagulation and Transfusion Medicine / RFLP AND PCR
Restriction Fragment Length Polymorphism
and Polymerase Chain Reaction
HLA-DQA1 and Polymarker Analysis of Blood Samples
From Transfusion Recipients
Rajiv I. Giroti, MSc,1 Rajesh Biswas, PhD,1 and Kanchan Mukherjee, MS2
Key Words: RFLP; Restriction fragment length polymorphism; PCR; Polymerase chain reaction; Blood transfusion; DNA profiling
Abstract
The effect of blood transfusion on DNA profiles of
an individual is a significant issue in the forensic
context. In the present study, the effects of blood
transfusion in 5 recipients were studied by performing
restriction fragment length polymorphism and
polymerase chain reaction HLA-DQA1 and Polymarker
(LDLR, GYPA, HBGG, D7S8, and GC) assays (Roche
Molecular Systems, Branchburg, NJ) on serial
posttransfusion blood samples. Pretransfusion and
posttransfusion DNA profiles of all 5 recipients were
consistent with no evidence of the donor genetic
material. Currently used DNA profiling techniques in
forensic science are reliable and informative for
paternity and identity purposes in situations involving
transfusion of 1 or 2 U of blood up to 24 hours
posttransfusion.
382
Am J Clin Pathol 2002;118:382-387
It is known that the blood of each person carries in it the
secrets of individuality. A battery of potentially individualizing genetic markers in blood has been recognized and used
as a valuable tool in human identification.1-3 Unfortunately,
transfusion is known to dilute the recipient’s identity by the
introduction of donor material and the dilution of the recipient’s own cells and proteins.4-6 There have been some recent
problems in serologic typing and interpretation of autologous blood group phenotypes and allozymes after multiple
transfusions.7,8
With the increasing use of blood transfusion, occasional
cases with a history of massive transfusions may be encountered in a forensic laboratory for the establishment of identity
or paternity. From the standpoint of forensic science, it is
notable that the conventional approach of RBC antigen
typing by agglutination or isozyme typing may be misleading
in such cases.8,9
During the past few years, DNA analysis has become
routine in laboratories conducting identity and paternity
testing, while serologic methods are falling into disuse.
Therefore, the special situation arising owing to blood transfusion has led to a growing interest in typing the DNA from
posttransfusion blood samples. In forensic literature, with
currently available DNA analysis techniques it has been
suggested that reliable typing of blood recipients is possible
even after massive transfusion.10,11 On the other hand, there
are a few reports of successful detection of blood
microchimerism after transfusion.12,13 Blood transfusion
already has been important in a few cases concerned with
forensic and paternity identification.7,8 As the use of DNA
evidence in courts of law has increased and the admissibility
of this evidence is under strict legal scrutiny,14 it has become
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
necessary to systematically address the issue of detection of
blood microchimerism after transfusion.
This study, which also is a part of the internal validation
and accreditation program of our laboratory, was undertaken
to determine whether currently practiced DNA methods of
forensic and paternity identity testing are suitable and informative for providing unambiguous identification after blood
transfusion.
Materials and Methods
Recipients Studied
Five men scheduled for a neurosurgical procedure
requiring blood transfusion were selected for the study. All
5 underwent surgery and received at least 1 U of whole
blood (>72 hours from donation). The serial blood samples
following transfusion were obtained as summarized in
❚Table 1❚.
The pretransfusion and serial posttransfusion blood
samples from the recipients were stored along with the reference blood samples from the respective donors at –70°C
immediately after collection. The pretransfusion sample and
the serial posttransfusion samples from each recipient were
batched along with the respective donor samples and
processed together 2 days after the collection of the last
serial posttransfusion sample for that recipient.
DNA Isolation
DNA was extracted from 200 µL of whole blood using
the QIAamp protocol (QIAGEN, Hilden, Germany). We
incubated 200 µL of liquid blood for 10 minutes at 56°C in
200 µL of QIAGEN lysis buffer and 20 µL of Proteinase K.
After addition of ethanol, the lysate was applied to a
QIAamp spin column and microcentrifuged for 1 minute.
Subsequently, the spin column was loaded once each with
the 2 ethanol-containing high salt buffers (AW1 and AW2)
provided in the kit. The spin column was centrifuged at
8,000 rpm for 1 minute after the AW1 wash, and a 3-minute
centrifugation at 14,000 rpm followed the AW2 wash.
Finally, the sample column was loaded with 200 µL of
QIAGEN elution buffer, and, after incubation at room
temperature for 5 minutes, DNA was eluted by microcentrifuging for 1 minute.
Restriction Fragment Length Polymorphism Analysis
Restriction fragment length polymorphism (RFLP)
analysis was performed according to the protocol developed
by the US Federal Bureau of Investigation.15,16 High-molecular-weight DNA obtained from the aforementioned samples
was restricted with HaeIII as described in Federal Bureau of
Investigation procedures.17 The restriction mixture was incubated at 37°C overnight. Samples were reprecipitated and
suspended in 16 µL of TE–4 buffer. A test gel then was run to
assess the restriction process.
Restricted samples were electrophoresed on a 1%
agarose analytic gel overnight and transferred to BIODYNE
A membrane (Pall Life Sciences, Ann Arbor, MI) by
Southern blot analysis. DNA fragments were linked covalently to the nylon membrane by baking for 1 hour at 80°C
in a vacuum oven and then cross-linking in a UV-cross linker
at 200 J. Membrane then was hybridized with GIBCO BRL
ACES DNA probe PH 30 (Life Technologies, Gaithersburg,
MD) that is specific for the D4S139 locus. The chemiluminescent substance Lumi-Phos Plus (Life Technologies) was
applied to the membranes, which then were sealed in a
PhotoGene development folder (Life Technologies). The
hybridized membranes then were placed into an x-ray film
cassette adjacent to a sheet of Kodak BioMax film (Eastman
Kodak, Rochester, NY) at room temperature. After development, the resulting lumigrams were analyzed.
Polymerase Chain Reaction Analysis
About 10 ng of DNA were used for polymerase chain
reaction (PCR). HLA-DQA1, LDLR, GYPA, HBGG, D7S8,
and GC loci were amplified simultaneously by using the
AmpliType Polymarker and DQA1 PCR amplification and
typing kit (Roche Molecular Systems, Branchburg, NJ).
Amplification was carried out in a thermal cycler (Gene
Amp PCR system 2400, Perkin Elmer Applied Biosystems,
Foster City, CA). Typing of the 6 loci was performed by
❚Table 1❚
Transfusion Summary*
Recipient No.
1
2
3
4
5
*
†
No. of Transfusions
1
1
2
1
1
Units of Whole Blood Transfused
1
1
2
1
1
Sampling Period After Transfusion†
Immediately; 1 h; 24 h
Immediately; 1 h
Immediately after first; immediately after second
Immediately; 1 h; 24 h
Immediately; 1 h; 24 h
All units of blood were more than 72 hours old.
Samples were obtained from all recipients midtransfusion in addition to the times indicated.
© American Society for Clinical Pathology
Am J Clin Pathol 2002;118:382-387
383
Giroti et al / RFLP AND PCR
reverse dot blot with allele specific oligonucleotide probes,
using the AmpliType Polymarker and DQA1 PCR amplification and typing kit.
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Results
RFLP Analysis
The pretransfusion and serial posttransfusion samples
from the 5 recipients and the samples from the 6 transfusion
donors yielded high-molecular-weight DNA. We obtained
100 ng of human DNA from each sample for complete
RFLP analysis. Examinations of the resulting lumigrams
showed that the banding patterns produced from the serial
blood samples obtained from all recipients were indistinguishable from their respective pretransfusion blood
samples. The RFLP profile of pretransfusion blood samples
from 2 representative recipients, the corresponding transfusion donors, and the serial posttransfusion blood samples are
shown in ❚Image 1❚.
PCR HLA-DQA1 and Polymarker Analysis
Pretransfusion samples from all recipients and their
corresponding donors and the serial blood samples obtained
after transfusion were amplified with specific primer pairs to
HLA-DQA1 and Polymarker loci to identify donor alleles.
All samples were amplified and typed successfully. The
pretransfusion and posttransfusion HLA-DQA1 and Polymarker profiles of the 2 representative recipients at different
time intervals are shown in ❚Table 2❚, along with the profiles
of their corresponding donors. The pattern of alleles generated by the PCR typing of the pretransfusion samples of the
recipient remained consistent in the serial posttransfusion
blood samples. ❚Image 2❚ shows HLA-DQA1 results with no
apparent differences in allelic patterns between pretransfusion and posttransfusion profiles of the recipient.
Discussion
We performed the RFLP and PCR-HLA-DQA1 and
Polymarker analyses on serial blood samples that were
obtained from transfusion recipients at various intervals to
study the effect of the blood transfusion on the DNA typing
results. Pretransfusion blood samples from the recipients and
all corresponding donors were included in the study to
obtain reference DNA profiles for comparison.
Transfusion of 1 to 2 U of whole blood and blood
sampling up to 24 hours posttransfusion had no effect on the
posttransfusion DNA profiles of all 5 recipients studied. All
donor samples were typed successfully by PCR and the
384
Am J Clin Pathol 2002;118:382-387
❚Image 1❚ Lumigram of D4S139 profiles from pretransfusion
and serial posttransfusion samples obtained from recipients
1 and 2 and their respective donors. Lanes 1,7, and 12
contain 0.5- to 22-kilobase markers (Life Technologies,
Gaithersburg, MD); lane 2, 100 ng of HaeIII-digested K562
DNA; lanes 3 and 4, 100 ng each of DNA extracted from
pretransfusion samples from recipient 1 and his donor,
respectively; lanes 5, 6, 8, and 9, 100 ng each of DNA
extracted from serial posttransfusion samples from recipient
1; lanes 10 and 11, 100 ng each of DNA extracted from a
pretransfusion sample from recipient 2 and his donor,
respectively; lanes 13, 14, and 15, 100 ng each of DNA
extracted from serial posttransfusion samples from
recipient 2. Exposure time was 30 minutes after an
overnight ramp on Kodak BioMax film (Eastman Kodak,
Rochester, NY).
RFLP technique, indicating detectable levels of DNA in the
unit of blood stored for transfusion. However, no donor
alleles were observed in any of the posttransfusion samples.
This observation supports the findings of several other
workers who have reported that the DNA patterns are not
influenced by blood transfusion.6,9-11
Previous studies have illustrated that less abundant DNA
in artificially generated paired mixtures was detectable by
PCR-based short tandem repeat assays, even when it represented only 10% of the total DNA within the mixture.18,19
Another study reported detection of 0.0001% of male DNA
against a background of female DNA using nested primers
for the Y chromosome microsatellite marker, and, similarly,
the D1S80 minisatellite locus has been reported as a useful
marker for detecting microchimerism with a sensitivity of
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
❚Table 2❚
Pretransfusion and Posttransfusion HLA-DQA1 and Polymarker Results for Transfusion Recipients 1 and 3 and Their
Respective Donors
Polymarker Loci*
Sample
Donor for recipient 1
Recipient 1
Midtransfusion
Time after transfusion
Immediately
1h
24 h
Donor 1 for recipient 3
Donor 2 for recipient 3
Recipient 3
Midtransfusion
Time after transfusion
Immediately (transfusion 1)
Immediately (transfusion 2)
*
LDLR
GYPA
HBGG
D7S8
GC
HLA-DQA1
AB
AA
AA
AB
BB
BB
BB
BB
BB
AA
AB
AB
AC
AA
AA
1.3,3
1.1,1.1
1.1,1.1
AA
AA
AA
AB
BB
AA
AA
BB
BB
BB
AB
AB
AA
AA
BB
BB
BB
AB
AB
AA
AA
AB
AB
AB
AA
AB
AB
AB
AA
AA
AA
AC
AC
AB
AB
1.1,1.1
1.1,1.1
1.1,1.1
4.1,4.1
1.3,4.1
1.1,3
1.1,3
AA
AA
AA
AA
AA
AA
AB
AB
AB
AB
1.1,3
1.1,3
Roche Molecular Systems, Branchburg, NJ.
0.1%.13 There is a recent report of the detection of natural
DNA mixtures in fraternal twins using short tandem repeat
analysis.20 While, to our knowledge, no study has been done
on the sensitivity of the RFLP technique for detecting mixed
samples, it is reported that it requires at least 20 to 50 ng of
high-molecular-weight DNA for successful analysis.21 The
reported sensitivity of HLA-DQA1 is about 400 pg,22,23
which is comparable to that of short tandem repeats.24,25 The
HLA-DQA1 and Polymarker systems have been evaluated
for their ability to detect mixed samples. The HLA-DQA1
could detect a 1:100 mixture,22,23,26,27 while the Polymarker
system could detect a 1:16 mixture in 4 ng of total DNA.28
There is reported evidence of the detection of Y-chromosome specific DNA following nested PCR in female
trauma patients who received blood transfusions from male
donors.29 Furthermore, various PCR amplification strategies
have been proposed and applied successfully for the detection of microchimerism in blood.30 The absence of transfusion effects in our results can be understood in terms of the
survival kinetics of donor leukocytes in the recipient’s circulation and the sensitivity of the assay system.
An average unit of blood might have about 1 to 3 × 108
leukocytes.31 As soon as a donor’s blood enters the recipient’s
circulation, the transfused leukocytes begin to disappear from
❚Image 2❚ Effects of blood transfusion on polymerase chain reaction DNA typing at the locus HLA-DQA1. D1 and D2, profiles of
the blood donors; S, pretransfusion profile of the recipient. Posttransfusion profiles of the recipient are ET1, end of transfusion
1; ET2, end of transfusion 2, and MT1, midtransfusion 1. The pretransfusion HLA-DQA1 types (1.1,3) of the recipient are
consistent with his posttransfusion profiles.
© American Society for Clinical Pathology
Am J Clin Pathol 2002;118:382-387
385
Giroti et al / RFLP AND PCR
the recipient’s circulation and move into the tissue spaces.
White32 showed that donor leukocytes persisted in the circulation for only 30 to 90 minutes after transfusion. Rosse and
Gurney33 found a longer leukocyte survival of 30% to 50% at
6 hours and 5% to 10% at 24 hours. It is inevitable, therefore,
that blood samples obtained from transfusion recipients will
contain few leukocytes. This presents a problem, since the
RFLP technique requires a relatively larger amount of intact
DNA as a starting point. However, it is intriguing that a highly
sensitive PCR-initiated analysis, which was most likely to
detect 2 different sources of DNA, did not reveal any alleles
corresponding to the transfusion donors in the posttransfusion
profiles of the recipients. The techniques we used for our set
of DNA samples indeed did not permit us to detect minor-type
nuclear fragments of the donor in the posttransfusion samples
of the recipients after transfusion of 1 to 2 U of blood up to 24
hours posttransfusion. However, further study is necessary to
determine the suitability of these techniques for obtaining
DNA profiles of recipients after massive transfusion. Furthermore, DNA profiling studies on the recipient’s blood up to 3 to
5 days posttransfusion will be of considerable importance, as a
study on the kinetics of clearance of donor leukocytes in transfusion recipients showed a transient recirculation of donor
lymphocytes on days 3 to 5 after transfusion.34,35 Additional
studies on the effect of the age (<72 hours and >3-4 weeks) of
the transfused blood on the recipient’s DNA profile and
enumeration of donor leukocytes in the recipient’s circulation
by quantitative PCR amplification assay also are warranted.
In forensic science, a suitable individualizing method is
the one that is reliable and sensitive enough to retain specificity. The present study documents that the RFLP and PCRHLA-DQA1 and Polymarker techniques are finely tuned to
reveal the secrets of individuality, even after the transfusion
of 1 to 2 U of blood.
From the 1Genetic Profiling Laboratory, Central Forensic Science
Laboratory, and the 2Department of Neurosurgery, Post Graduate
Institute of Medical Education and Research, Chandigarh, India.
Address reprint requests to Mr Giroti: Genetic Profiling
Laboratory, Central Forensic Science Laboratory, Plot No. 2,
Dakshin Marg, Sector 36-A, Chandigarh-160 036, India.
Acknowledgment: We thank Ranjeet Singh Verma, PhD,
Director, Central Forensic Science Laboratory, Chandigarh,
India, for encouragement.
References
1. Sensabaugh GF. Uses of polymorphic red cell enzymes in
forensic science. Clin Haematol. 1981;10:185-207.
2. Henke L, Fimmers R, Josephi E, et al. Usefulness of
conventional blood groups, DNA-minisatellites and short
tandem repeat polymorphisms in paternity testing: a
comparison. Forensic Sci Int. 1999;103:133-142.
386
Am J Clin Pathol 2002;118:382-387
3. Lincoln PJ. From ABO to DNA… [editorial]. Med Sci Law.
2000;40:3-7.
4. Culliford BJ. The Examination and Typing of Blood Stains in the
Crime Laboratory. Washington, DC: US Department of
Justice, Law Enforcement Assistance Administration, and
Superintendent of Documents; 1971:38-40.
5. Legler TJ, Eber SW, Lakomek M, et al. Application of RHD
and RHCE genotyping for correct blood group determination
in chronically transfused patients. Transfusion. 1999;39:852-855.
6. Wenk RE, Chiafari PA. DNA typing of recipient blood after
massive transfusion. Transfusion. 1997;37:1108-1110.
7. Brauner P. DNA typing and blood transfusion. J Forensic Sci.
1996;41:895-897.
8. Davidson AK, Lee LD. Unusual results due to transfused
blood. Sci Justice. 1999;39:179-180.
9. Huckenbeck W, Rand S. Serological findings and efficiency of
DNA profiling in transfused patients and their significance for
identity and paternity tests. Int J Legal Med. 1994;106:178-182.
10. Brauner P, Shpitzen M, Freund M, et al. The effects of blood
transfusions on PCR DNA typing at the CSF1P0, TPOX,
TH01, D1S80, HLA-DQA1, LDLR, GYPA, HBGG, D7S8
and GC loci. J Forensic Sci. 1997;42:1154-1156.
11. Reid ME, Rios M, Powell VI, et al. DNA from blood samples
can be used to genotype patients who have recently received a
transfusion. Transfusion. 2000;40:48-53.
12. Carter AS, Bunce M, Cerundolo L, et al. Detection of
microchimerism after allogeneic blood transfusion using
nested polymerase chain reaction amplification with sequence
specific primers (PCR-SSP): a cautionary tale. Blood.
1998;92:683-689.
13. Sahota A, Yang M, McDaniel HB, et al. Evaluation of seven
PCR-based assays for the analysis of microchimerism. Clin
Biochem. 1998;31:641-645.
14. Pitluck HM. DNA evidence will be admissible if the proper
foundation is laid: advice for a forensic medicine expert. Croat
Med J. 2001;42:221-224.
15. Budowle B, Baechtel FS. Modifications to improve the
effectiveness of restriction fragment length polymorphism
typing. Appl Theor Electrophor. 1990;1:181-187.
16. Alan M, Giusti BS, Budowle B. A chemiluminescence-based
detection system for human DNA quantitation and restriction
fragment length polymorphism (RFLP) analysis. Appl Theor
Electrophor. 1995;5:89-98.
17. Budowle B, Waye J, Shutler G, et al. HaeIII: a suitable
restriction endonuclease for restriction fragment length
polymorphism analysis of biological evidence samples.
J Forensic Sci. 1990;35:530-536.
18. Hammond H, Caskey CT. Personal identification via short
tandem repeats. Proceedings of the Third International
Symposium on Human Identification. Madison, WI: Promega;
1992:163-175.
19. van Oorschot RAH, Gutowski SJ, Robinson SL, et al.
HUMTH01 validation studies: effect of substrate,
environment and mixtures. J Forensic Sci. 1996;41:142-145.
20. Rubocki RJ, McCue BJ, Duffy KJ, et al. Natural DNA
mixtures generated in fraternal twins in utero. J Forensic Sci.
2001;46:120-125.
21. Kobilinsky L. Recovery and stability of DNA in samples of
forensic science significance. Forensic Sci Rev. 1992;4:67-87.
22. Comey CT, Budowle B. Validation studies on the analysis of
the HLA DQalpha locus using the polymerase chain reaction.
J Forensic Sci. 1991;36:1633-1648.
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
23. Wilson RB, Ferrara JL, Baum HJ, et al. Guidelines for internal
validation of the HLA-DQalpha DNA typing system. Forensic
Sci Int. 1994;66:9-22.
24. Budowle B, Lindsey JA, DeCou JA, et al. Validation and
population studies of the loci LDLR, GYPA, HBGG, D7S8
and GC (PM loci), and HLA-DQalpha using multiplex
amplification and typing procedure. J Forensic Sci.
1996;40:45-54.
25. Wallin JM, Buoncristiani MR, Lazaruk KD, et al. TWGDAM
validation of the AmpF/STR Blue PCR amplification kit for
forensic casework analysis. J Forensic Sci. 1998;34:117-133.
26. Blake E, Mihalovich J, Higuchi R, et al. Polymerase chain
reaction (PCR) amplification and human leukocyte antigen
(HLA)-DQalpha oligo-nucleotide typing on biological
evidence samples: casework experience. J Forensic Sci.
1992;37:700-726.
27. Schneider PM, Rittner C. Experience with the PCR-based
HLA-DQalpha DNA typing system in routine forensic
casework. Int J Legal Med. 1993;105:295-299.
28. Dimo-Simonin N, Brandt-Casadevall C. Evaluation and
usefulness of reverse dot blot DNA–PolyMarker typing in
forensic casework. Forensic Sci Int. 1996;81:61-72.
© American Society for Clinical Pathology
29. Viëtor HE, Hallensleben E, van Bree SPMJ, et al. Survival of
donor cells 25 years after intrauterine transfusion. Blood.
2000;95:2709-2714.
30. Reed WF, Lee TL, Trachtenberg E, et al. Detection of
microchimerism by PCR is a function of amplification
strategy. Transfusion. 2001;41:39-44.
31. Koerner K, Sahlmen P, Zimmermann B, et al. Preparation of
leukocyte-poor red cell concentrates: comparison of five
different filters. Vox Sang. 1991;60:61-62.
32. White LP. The intravascular life span of transfused leucocytes
tagged with Atabrine. Blood. 1954;9:73.
33. Rosse WF, Gurney CW. The Pelger-Huët anomaly in three
families and its use in determining the disappearance of
transfused neutrophils from peripheral blood. Blood.
1959;14:170.
34. Lee T-H, Donegan EA, Slichter S, et al. Transient increase in
circulating donor leucocytes after allogeneic transfusions in
immunocompetent recipients compatible with donor cell
proliferation. Blood. 1995;85:1207-1214.
35. Goodarzi MO, Lee TH, Pallavicini MG, et al. Unusual
kinetics of white cell clearance in transfused mice.
Transfusion. 1995;35:145-149.
Am J Clin Pathol 2002;118:382-387
387