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Supplementary issue paper
Searching for blood in Chinese lacquerware:
zhūxie
ˇhuī豬血灰
Silvia Miklin-Kniefacz1, Václav Pitthard2, Walther Parson3,4, Cordula Berger3,
Sabine Stanek2, Martina Griesser2,Šte
ˇpánka Hrdlic
ˇková Kuc
ˇková5
1
Studio for Conservation and Restoration of Metal Objects and East Asian Lacquer, Vienna, Austria,
2
Conservation Science Department, Kunsthistorisches Museum, Vienna, Austria,
3
Institute of Legal Medicine,
Medical University of Innsbruck, Austria,
4
Forensic Science Program, The Pennsylvania State University, USA,
5
Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech
Republic
The study gives an overview of the tests and analyses undertaken in the past 20 years to establish the
presence of blood in the foundation layers of Chinese lacquer artefacts and also shows the development
of analytical methods over that period. When undertaking the conservation of lacquer objects it is crucial
to know the type of binding medium as this influences the selection of any consolidants that may be
required in the treatment. Microchemical tests to identify blood using benzidine and luminol, various
chromatographic and mass spectrometric techniques and DNA analyses were assessed on selected
Chinese lacquer objects, and the results gained are summarized.
Keywords: Chinese lacquerware, Blood, Ground layer, Benzidine and luminol test, THM-Py-GC–MS, Nano-LC–MS/MS, DNA analyses
Introduction
For more than 2000 years blood from pigs or cattle was
used as a binding medium in the layers that were used to
prepare the surface of lacquerware in China. According
to Hsu (2014) early evidence for the use in China of a
ground layer containing pig’sbloodcanbeseenonlac-
querware from the Warring States period that dates
from approximately the fifth to third century BCE.
When mixed with lime and other filler materials such
as brick powder it made an appropriate binder when
preparing a ground layer to receive lacquer, due to its
adhesive properties and good water resistance.
The use of pig’s blood in the ground layers of
Chinese lacquerware is described in various historical
Chinese sources: ‘Before applying such varnish over
wood, the Chinese used —but not always —to
give it a bed or primer, as painters usually do before
painting, in the following way. Take the blood of a
pig […] and mix it with powdered quicklime, and
coat the wood with this mixture […]; once it is dry,
smooth it down with pumice stone or something
similar’(Bonanni, 2009, p. 23).
There are few detailed descriptions of the manufac-
ture of ground layers based on pig’s blood for lacquer,
which is known in Chinese as zhu
¯xie
ˇhuı
¯(豬血灰).
The same name is also used in Chinese architectural
decoration, which has been more thoroughly studied
(Chang, 2014, personal communication). There is a
long tradition of pig’s blood-lime mortars in Chinese
architecture, which dates back at least to Xianyang
Palace —the royal palace of the state of Qin (Qin
dynasty, 221–206 BCE)—in modern day Shaanxi pro-
vince (Zhao et al., 2014).
Blood, in the form of ox blood, can be found as a
binding medium in Europe. It is mentioned in old
recipes for waterproof putties to be applied to
wooden surfaces and buildings, sometimes referred
to as ‘Chinese blood-putty’(Lehner, 1877),
1
or in
Correspondence to: Silvia Miklin-Kniefacz, Bernardgasse 4/1, Vienna,
A-1070, Austria. Email: silvia.miklin@gmx.at
1
Recipes from Lehner (1877) (translated from German):
Page 51: Chinese blood putty: hydrated lime 100, whipped ox
blood 75, alum 2 (used in China to waterproof wooden containers
or the like);
Page 51: Blood ash putty for buildings: hydrated lime 100, sieved
ash from mineral coal 50, whipped ox blood 15 (for filling joints
between bricks and stones);
Page 49: Blood protein putty for buildings: hydrated lime 40, brick
powder 40, iron filings 10, ox blood 8, water 8 (for filling joints
between bricks). ‘When the oxblood comes from the body of the
slaughtered animal, it is whipped with a broom for ten minutes,
so that …the fibers segregate and the blood will not clot. Then
the blood is mixed with water and the powders are kneaded in.
This putty is especially useful to smear over the joints of bare
brickwork.’
© The International Institute for Conservation of Historic and Ar tistic Works 2016
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/Licenses/by-
nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received April 2015; revised paper accepted August 2016
DOI 10.1080/00393630.2016.1227039 Studies in Conservation 2016 VOL. 61 SUPPLEMENT 3S3-45
recipes for wall and wood paints.
2
There are no records
of the use of blood in Japan, except in lacquerware
from Okinawa Island, which is subject to a strong
Chinese influence as a result of its proximity (Hsu,
2014, p. 5).
In 1994 two Chinese lacquer artists, Wu Guofen 武
國芬und Wu Xi 吴晞were invited by the
Schönbrunn Palace in Vienna to demonstrate the
build-up of Chinese lacquerware with the aim of devel-
oping, together with conservators from the palace, a
conservation plan for the so-called Vieux-Laque
room. In the course of this workshop sample boards
were produced (Fig. 1) that gave important practical
insights into the preparation of the surface to receive
lacquer:
•the multiple uses to which blood was put, as sealing
medium for the wood, as glue for the paper layer
and as binding medium for the ground layer;
•the colour change of the blood from red to a greenish
shade as soon as it is mixed with lime; and
•the use of tung oil as an intermediate layer before the
lacquer layer is applied.
Wu Xi and Wu Guofen had brought a readymade
lime-blood mixture with them and during the three-
day journey from China this had developed a notice-
able smell, which points to the need to use freshly
made putty and not to keep it for long.
The normal procedure can be described as follows:
After collecting, the fresh blood is filtered as it soon
starts to clot; the filtration is carried out by kneading
the blood with the hands and forcing it through a
sieve. A simple mixture of the filtered blood with
lime is used to seal the surface (Hsu, 2014, personal
communication), but for the ground layers further
fillers are added to this mixture. In certain traditions
straw is added when filtering the blood and the
blood is warmed slightly, although never in excess of
70°C (Wu, 2014, personal communication); other
materials added to the mixture include tung oil
(Chang, 2014, personal communication).
The recipes for paints used in Europe suggest that
the serum and the blood cells were separated, but
this does not seem to be the case for putties, either in
Europe or China. As mentioned above, blood was
probably used as a binding medium because it
imparted good adhesive and waterproofing properties,
but was also a cheap alternative to other materials,
including the lacquer itself. For the conservation of
lacquer objects, it is important to know which type
of binding medium has been used in the preparatory
layers, as it influences the selection of any consolidants
that may be required in the treatment.
This contribution presents an overview of the tests
and analyses undertaken between 1995 and 2015 to
investigate the presence of blood in lacquer objects. It
sets out to address two questions —is blood present
and, if so, from which species does it originate —and
to give some information about the methods of analy-
sis have developed during this time. This development
in approaches to analysis is well exemplified by the
investigations conducted on the Chinese lacquer
panels of around 1720 that decorate the Vieux-Laque
room at the Schönbrunn Palace (1767–1770: Fig. 2)
(Miklin-Kniefacz et al., 2010).
The first attempts to look for blood in the ground
layers were undertaken in 1995 at the Institute of
Legal Medicine, University of Vienna, using benzidine
and luminol tests. The results were not convincing
because positive results were obtained from samples
of lacquer that lacked a ground layer, while other
samples comprising the ground layer alone gave nega-
tive results. Almost 10 years later, in 2006, Simone
Figure 1 Reference sample replicating the build-up of the ground layers for Chinese lacquer (produced by Wu Xi 吴晞in 1994 in
the workshop at the Schönbrunn Palace, Vienna). The layers are: (1) wood; (2) a mixture of pig’s blood whipped with lime; (3) pig’s
blood mixed with coarse brick powder; (4) pig’s blood and paper; (5), (6) and (7) pig’s blood with increasingly finely ground brick
powder; (8) and (9) tung oil.
2
Two recipes for oxblood paint are given by Kremer Pigmente. Oxblood
paint: 100 l of cow’s blood is left to stand three days yielding serum that
can skimmed off. To 30 l of this serum 250 ml of clove oil is added to
avoid spoiling, along with 26 kg slaked lime, 5 kg natural iron oxide, 3 l
of limewater and 100 ml of a defoaming agent. These are mixed together
and then 5 l linseed oil is added. The second recipe, attributed to
Wengerter, requires 4 Berliner quarts blood serum (ca. 4. 6 l), 8 pounds
of lime (ca. 3. 5 kg), 1 kg of red iron oxide pigment and 10% linseed oil;
see http:// kremer-pigmente.de/Texte/oxblood-paint.pdf (accessed 23
December 2015).
Miklin-Kniefacz et al. Searching for blood in Chinese lacquerware
Studies in Conservation 2016 VOL. 61 SUPPLEMENT 3S3-46
Mueller at the Institute of Veterinary Medicine,
University of Vienna, applied a newly developed
DNA test, finding both pig and cattle DNA by real-
time polymerase chain reaction (real-time PCR).
Advances made by Michael Schilling in characteriz-
ing Asian lacquers and European lacquers at the Getty
Conservation Institute by means of thermally assisted
hydrolysis and methylation pyrolysis-gas chromato-
graphy/mass spectrometry (THM-Py-GC/MS),
allowed markers for blood to be detected (Schilling,
2013).
Within the scope of a research project funded by a
grant from the Austrian Science Fund (2013–2016),
3
it was possible to test again the possibilities and
benefits of DNA analyses at the Austrian Central
DNA laboratory of the Institute of Legal Medicine
in Innsbruck, where special primers were developed
and pig DNA could be detected.
Finally, the most recent tests were undertaken at the
University of Chemistry and Technology in Prague in
2014, where pig’s blood and cattle blood could be
detected in a single measurement procedure using
nanoscale liquid chromatography coupled to tandem
mass spectrometry (nano-LC–MS–MS).
Different methods for blood identification in
detail
Benzidine and luminol
Ten samples of ground and lacquer layers from the
Vieux-Laque room (sample size c.5 ×3 mm) were
tested with hydrogen peroxide (H
2
O
2
) and a solution
of benzidine in glacial acetic acid. If blood is present
an enzymatic reaction causes the benzidine to change
colour to a dark blue (Odegaard et al., 2005).
In addition, a test using luminol was used, following
the procedure outlined by Weber: in a darkened space
samples were treated with a mixture of 5-amino-2,3-
dihydro-1,4-phthalazinedione (luminol) and hydrogen
peroxide. Three samples, which had previously given
positive results with the benzidine test, emitted blue
light when treated with the luminol reagent, indicating
the presence of blood. Seven other samples that gave a
negative result when tested with benzidine also gave a
negative result with luminol. The results were ambigu-
ous, as some of the samples from lacquer layers that
lacked a ground layer (and should therefore have con-
tained no blood) gave positive reactions while other
samples, which comprised solely the ground layer,
tested negative.
Further investigations were conducted to investigate
the selectivity of the test methods; the benzidine test
gave negative results when exposed to other proteins,
such as fish glue, casein or egg, but positive results
when applied to rabbit skin glue, which implies that
it is not entirely diagnostic of the presence of blood.
Benzidine and GC–MS
While it does not give incontrovertible proof of the
presence of blood, the benzidine test provides a good
initial screening method and can be applied in combi-
nation with other methods such as with gas chromato-
graphy–mass spectrometry analysis (GC–MS), which
can provide an amino acid profile of the sample that
can be matched to those of reference samples of
Figure 2 Ground layer on the rear of a sample from the Vieux-Laque room at the Schönbrunn Palace. The grey underside of the
black lacquer layer is seen at the top of the image, with a second ground layer beneath followed by a layer of paper and the first
ground layer, seen at the bottom of the image.
3
Austrian Science Fund FWF, Project No. P-25351 G2: Ostasiatische
Raumausstattungen in Schloss Schönbrunn/Asian Interior Decoration in
Schönbrunn Palace; project management Gabriela Krist, Institute of
Conservation, University of Applied Arts Vienna.
Miklin-Kniefacz et al. Searching for blood in Chinese lacquerware
Studies in Conservation 2016 VOL. 61 SUPPLEMENT 3S3-47
blood. In 2011, a sample from the ground layer of a
Chinese lacquer tea box (Canton, around 1820–1850,
private collection) was analysed in the Conservation
Science Department of the Kunsthistorisches
Museum Vienna (KHM) using this technique. The
sample was first treated with hydrogen peroxide and
benzidine, with the resulting blue colouration indica-
tive of the presence of blood. Subsequently, GC–MS
analysis was carried out to confirm the results
suggested by the chemical test. Comparison of the
chromatographic profile of the amino acids present
in the ground sample (after acid hydrolysis with 6 M
HCl at 105 °C for 24 hours) with that of a reference
sample of blood showed the ground to contain blood
(Fig. 3). In this case the GC–MS analyses were per-
formed using a 6890N gas chromatograph connected
to a quadrupole MS (model 5973N, Agilent
Technologies); the sample size was ∼1mm
2
and
yielded 0.5–1.0 mg.
DNA analysis
At the beginning of 2014 the question of whether pig
blood had been used as a key material in the Chinese
cabinets was addressed by the Institute of Legal
Medicine at the Medical University of Innsbruck.
The analysis of the nucleotide sequence of the mito-
chondrial cytochrome b gene is an established
method to determine from which animal species a
sample derives and has been used in a wide variety
of forensic genetic cases (Parson et al., 2000).
Although the method is sensitive and can be applied
to very small amounts of source material, it is also sen-
sitive to any external contamination that might be
present. When the sample is of putative animal
origin but has been handled by humans without
proper care taken to avoid contamination, then the
test results are expected to indicate a mixture of the
animal and the human sources. Such mixtures are gen-
erally difficult to interpret based on the nucleotide
sequence data.
The DNA was extracted from a set of samples from
the ground layers of different lacquer panels from the
Chinese cabinets from the Schönbrunn Palace using
the EZ1 Advanced instrument (Qiagen, Hilden,
Germany) and following the manufacturer’s protocols.
The cytochrome b gene was sequenced in the same
manner as in previous experiments (Parson et al.,
2000), and the resulting consensus sequences were
matched using GenBank (National Center for
Biotechnology Information, US National Library of
Medicine). In three samples of ground layers from
the reference sample board prepared by Wu Xi a con-
sensus sequence was found that matched GenBank
entries of Sus scrofa (wild pig). Two other panels
showed human cyt b sequences, most likely explained
by human contamination that masked the presence of
other species (Fig. 4).
In a second set of experiments new primers specifi-
cally designed for identification of DNA from the
Sus genus detected sequences corresponding to Sus
scrofa in these two panels, with some contamination
from the human cyt b gene sequence.
The authors believe that modern human contami-
nation in the laboratory can be excluded as a source
for the human sequences as the samples were taken
and handled with great care, explicitly following the
guidelines for forensic mtDNA testing (Carracedo
et al., 2000;Tully et al., 2001;Parson et al., 2014).
However, earlier human contamination cannot be
excluded and may derive from the steps taken to
prepare the ground layers, such as kneading the
blood with bare hands.
In a third set of experiments alternative cytochrome
b gen primers were employed (F15416-RMMd16) that
confirmed the results of the second analysis. The
sample size was about 1 cm
2
on average (Fig. 5).
The samples on which species identification was
attempted were very small for DNA analysis, and
spongy. Such samples are difficult to work with in
the lab and are likely to contain severely degraded
DNA because of their age. The material was used as
received in the laboratory; it was impossible to
dissect the different layers and the samples were
instead broken into smaller pieces to maximize DNA
Figure 3 Total ion chromatograms for (A) the ground layer;
and (B) a pig blood reference standard. The amino acids from
pig blood are: (Ala) alanine; (Gly) glycine; (Val) valine; (Leu)
leucine; (Ile) isoleucine; (Pro) proline; (Ser) serine; (Thr)
threonine; (Phe) phenylalanine; and (Asp) aspartic acid.
Miklin-Kniefacz et al. Searching for blood in Chinese lacquerware
Studies in Conservation 2016 VOL. 61 SUPPLEMENT 3S3-48
extraction efficacy. The small sample sizes meant that
other tests, such as chemical methods of detecting the
presence of blood, were not performed, as the risk of
losing the samples or introducing contamination
would have been too high.
THM-Py-GC–MS
Pyrolysis enables direct analysis of solid blood
samples without time-consuming pre-treatments.
The sample size can be very small, less than
0.5 mg, and the pyrolysis products are then sub-
sequently separated and identified by GC–MS. The
procedure was developed within the framework of a
research project on the characterization of Asian
and European lacquers by Michael Schilling and
Arlen Heginbotham and is described in detail else-
where in this publication.
The proteinaceous binder found in the ground layer
sample from one of the Chinese lacquer panels from
the Vieux-Laque room was examined by THM-Py-
GC–MS at the Getty Conservation Institute.
Markers characteristic for both animal glue and
blood were successfully detected (Schilling, 2013).
Nano-LC–MS–MS
Nanoscale liquid chromatography coupled to tandem
mass spectrometry (nano-LC–MS–MS) is used in the
field of biochemistry, called proteomics, as a funda-
mental tool for the identification and relative quantifi-
cation of proteins. The method does not analyse whole
proteins, but is based on the detection of shorter com-
ponents termed peptides. The peptides are created
from the proteins by the action of enzymes, which
cleave the peptide bonds between amino acids in pro-
teins. The mode of digestion is specific as particular
enzymes cause cleavage only at certain peptide
bonds. One of the most specific enzymes, which is
commonly used in proteomic laboratories, is trypsin,
which cleaves only the bonds behind arginine and
lysine. The resulting peptide mixture is analysed by
LC–MC–MS and the results compared to those in
publicly available databases.
Until recently, biochemists have worked only with
proteins in solution, but the use of trypsin has
allowed proteins that are embedded in solid phases,
such as those taken from works of art, to be analysed.
When trypsin cleaves whole or partial proteins on the
Figure 4 Sequence electropherograms of the cytochrome b gene, panels B and C.
Figure 5 Samples for DNA analyses, 20 ×5 mm (panel C,
Coromandel lacquer).
Miklin-Kniefacz et al. Searching for blood in Chinese lacquerware
Studies in Conservation 2016 VOL. 61 SUPPLEMENT 3S3-49
surface of a sample the resultant peptides are trans-
ferred into solution and subsequently analysed by
mass spectrometry. Nano-LC–MS–MS analysis deter-
mines the molecular weight and the sequence of amino
acids in the peptides, with identification of the
unknown proteins based on comparison of these
detected sequences with the sequences of proteins
stored in references libraries. The advantage of the
use of combined enzymatic digestion and mass spec-
trometric methods such as LC–MS–MS and
MALDI-TOF-MS in the field of cultural heritage is
to allow mass mapping of peptides that leads to unam-
biguous identification of the type of protein binder
from very small (microgram) fragments or even in
cross-sections made from samples from works of art
(Kuckova et al., 2013;Sandu et al., 2013). In many
cases the analyses could be considered as non-destruc-
tive, because after the enzymatic digestion it is fre-
quently possible to use the sample in a subsequent
analytical procedure. Moreover, the presence of more
than one type of protein binder in a single sample
does not impair the analyses as, although each
binder comprises a number of individual proteins,
LC–MS–MS is able to identify many hundreds of pep-
tides in a single analysis. The major drawback of mass
spectrometric methods —and of many other com-
monly used analytical procedures used for protein
identification —is that they are not able to determine
from which of the layers in the sample the proteins that
have been identified originate.
Initial tests of the method were carried out as part of
the investigation of particles in the ground layers of
different lacquer objects, such as a Chinese carved
lacquer screen from the Weltmuseum Wien (Pitthard
et al., 2016). During this investigation in Prague,
very tiny samples (<0.5 mg) were cleaved with a
trypsin solution to yield a peptide mixture that was
purified and then redissolved for analysis.
Measurements were carried out using a UHPLC
Dionex Ultimate3000 RSLC nano-LC connected to
an ESI-Q-TOF Maxis Impact mass spectrometer
(Bruker, Germany). The sample solution was loaded
on a trap column (Acclaim PepMap 100 C18) and
the peptides were eluted to an analytical column
(Acclaim PepMap RSLC C18) and then directly to
the ESI source (Captive spray, Bruker Daltonics,
Germany). The mass spectrometer was set up in the
positive ion mode with precursor selection in the
range of 400–2200 Da, and up to ten precursors
from each mass spectrum were selected for the frag-
mentation. The constituent proteins were identified
using the Uniprot database (version 2010–12). The
analyses confirmed the presence of pig’s blood in the
sample from the ground layers of the Chinese screen
at the Weltmuseum Wien (Fig. 6and Table 1).
Conclusion
Analysis using GC–MS and THM-Py-GC–MS are
commonly used and practicable methods to detect
blood as a binding medium in lacquer ground layers,
although it necessary to have good reference samples
available when GC–MS is employed.
DNA analysis can also be applied and has the
potential advantage of allowing species identification.
As samples from historic objects are particularly prone
to degradation, comparatively large samples are
needed in order to perform such analyses, which
Figure 6 Ground layers of the Chinese carved lacquer screen from the Weltmuseum Wien (No. 71.233).
Table 1 List of peptides and proteins detected by nano-
LC–MS–MS in the ground layers of the Chinese screen from
the Weltmuseum Wien
Accession Protein Number of peptides
HBB_PIG Hemoglobin subunit beta 5
HBA_PIG Hemoglobin subunit
alpha
4
TRYP_PIG Trypsin 2
HBB_TARBA Hemoglobin subunit beta 2
Miklin-Kniefacz et al. Searching for blood in Chinese lacquerware
Studies in Conservation 2016 VOL. 61 SUPPLEMENT 3S3-50
might be problematic when the samples must be
removed from a significant and valuable object.
The use of nano-LC–MS–MS allows both blood
and the species from which it derives to be determined
from very small samples, although the species identifi-
cation should not be regarded as definitive.
Acknowledgements
The authors wish to thank the Schönbrunn Kultur- und
Betriebsges. m.b.H., the Bundesmobilienverwaltung,
Vienna, this work was supported by FWF, the
Austrian Science Fund [Project No. P-25351 G21], the
University of Applied Arts, Vienna (Gabriela Krist)
and the Specific University Research (MSMT No. 20/
2015) from the Czech Republic for financial support.
They also thank their colleagues at the Institute of
Legal Medicine, Innsbruck (Daniel Entstrasser, Carola
Feßler, Anna König, Harald Niederstätter), the Getty
Conservation Institute, Los Angeles, especially
Michael Schilling and the RAdICAL (Recent
Advances in Characterizing Asian Lacquers) team
and Christiane Jordan, at the Weltmuseum Wien for
their invaluable support.
ORCiD
Martina Griesser http://orcid.org/0000-0001-8395-
1917
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