Neurobiology of Disease 159 (2021) 105504

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Neurobiology of Disease
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Formalin RT-QuIC assay detects prion-seeding activity in formalin-fixed
brain samples from sporadic Creutzfeldt–Jakob disease patients
Thi-Thu-Trang Dong a, Akio Akagi b, Toshiaki Nonaka a, c, Takehiro Nakagaki c, Ban Mihara d,
Masaki Takao d, e, f, Yasushi Iwasaki b, Noriyuki Nishida c, Katsuya Satoh a, *a

Department of Health Sciences, Unit of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Japan
Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi 480-1195, Japan
Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japand
Department of Neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki 372-0006, Japane
Department of Neurology International Medical Center, Saitama Medical University, Saitama 350-1298, Japanf
Department of Clinical Laboratory National Center of Neurology and Psychiatry (NCNP), National Center Hospital, 4-1-1 Ogawa-higashi-cho, Kodaira, Tokyo 1878551, Japanbc

A R T I C L E I N F O

A B S T R A C T

Keywords:
RT-QUIC assay
Prion
Prion-seeding activity
Neuropathology

Background: The neuropathology of sporadic Creutzfeldt–Jakob disease (sCJD) is usually investigated using
formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interferewith immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other thanimmunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quan­titate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid.
Methods: We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients(14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed ac­
cording to Parchi’s classification. We assessed SD50 in brains after incubation in formalin solution for over 1
month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values
from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings.
Results: The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases
were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in
MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combinedformalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of
unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathologicalfindings in most brain regions examined.
Conclusion: RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously
been amenable to analysis.

1. Introduction(PRNP) or a relevant family history, including Gerstmann–Straussler–­
Scheinker disease, fatal familial insomnia (FFI), or genetic CJD (gCJD).
The acquired forms are dura-associated CJD (dCJD) and variant CJD
derived from bovine spongiform encephalopathy (Nozaki et al., 2010).
On the basis of the genotype at codon 129 of both PRNP alleles, thesize of protease-resistant PrPSc fragments, and disease phenotype, sCJD
is divided by Parchi’s classification into six subtypes: MM1/MV1, VV2,
MV2, MM2-cortical form (MM2C), VV1, and MM2-thalamic form
(MM2T) (Parchi et al., 1999). Japan Surveillance Meetings reported that

Human prion diseases, or transmissible spongiform encephalopa­
thies (TSEs), are fatal transmissible neurodegenerative diseases inhumans (Prusiner et al., 1998) and are classified into sporadic, genetic
and acquired forms; the most common being sporadic Creutzfeldt–Jakob
disease (sCJD). The annual incidence rate of sCJD is 1.4 per million in
Japan (Nozaki et al., 2010). Genetic prion disease is defined as a priondisease with causative mutations in the human Prion protein gene

* Corresponding author at: Department of Health Sciences, Unit of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1
Sakamoto, Nagasaki 852-8501, Japan.
E-mail addresses: ban.mihara@mihara-ibbv.jp (B. Mihara), iwasaki@sc4.so-net.ne.jp (Y. Iwasaki), satoh-prion@nagasaki-u.ac.jp (K. Satoh).
https://doi.org/10.1016/j.nbd.2021.105504
Received 3 July 2021; Received in revised form 1 September 2021; Accepted 2 September 2021
Available online 10 September 2021
0969-9961/© 2021 The Authors.
Published by Elsevier Inc.
This is an open(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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T.-T.-T. Dong et al.

Neurobiology of Disease 159 (2021) 105504

MM1 and MV1 patients constitute about 90% of the Japanese sCJD
population, and include cases previously classified as typical CJD of the
myoclonic type or as Heidenhain variant. Thus, the genotype distribu­
tion at codon 129 of PrP in sCJD disease in Japan reveals a higher
proportion of methionine homozygotes (96.8%) (Saborio et al., 2001).
The general Japanese population also presents with a higher frequency
of codon 129 homozygosity (methionine homozygotes: 0.92, heterozy­gotes: 0.08, valine homozygotes: 0) than populations in European
countries (methionine homozygotes: 0.37–0.49, heterozygotes:
0.42–0.49, valine homozygotes: 0.08–0.15).
The infectious agent, or prion, of TSEs appears to be composed of anabnormal prion protein (PrPSc). PrPSc is formed post-translationallyfrom the normal cellular prion protein (PrPc), which in purified form
can resemble amyloid fibrils. PrPSc induces the polymerization andconformational conversion of PrPc to infectious PrPSc or to PrPSc-likepartially protease-resistant PrP forms in a variety of in vitro reactions(Prusiner et al., 1998).
In vitro PrPSc amplification techniques, including protein misfolding
cyclic amplification (PMCA) (Atarashi et al., 2008), amyloid seeding
assay (ASA), and quaking-induced conversion (QuIC) (Atarashi et al.,2011) enable the highly sensitive detection of PrPSc. QuIC assays use
recombinant normal prion protein (rPrP-sen) as a substrate to amplify
very slight amounts of PrPSc with intermittent automated shaking,which can be performed more easily than sonication used in PMCA. We
recently developed a new in vitro amplification technology, designated“real-time quaking-induced conversion (RT-QuIC)”, for detecting prionseeding activity from many organs, including central nervous system(CNS) tissues and non-CNS tissues from sCJD patients (Takatsuki et al.,2015). Previously, we developed the RT-QuIC assay for detection of verysmall amounts of abnormal PrP in tissue and body fluids. These studies
demonstrated that RT-QuIC is more sensitive than bioassays. Impor­tantly, endpoint RT-QuIC can be used to evaluate human prion-seeding
activity in brains from patients with human prion disease (Akagi et al.,2018).
The neuropathology of sCJD is usually investigated using formalinfixed and formic acid-treated brain tissue. The neuropathology ofsCJD is characterized by the presence of spongiform changes, neuronalloss and the accumulation of PrPSc deposition. Pathologists treat tissue
with formic acid to reduce prion infectivity. Furthermore, to enhance
the immunoreactivity for PrPSc, many researchers use a combination of
formic acid and autoclaving pretreatment. In some cases, even if western
blotting detects similar levels of abnormal prion protein in the unfixed
brain tissue, the pretreatment methods for immunostaining can alter the
staining property of the tissues differently. This limitation has typicallyresulted in qualitative, rather than quantitative, pathological evalua­tions. Once brain tissue is fixed with formalin, its usefulness is limited
for uses other than neuropathological analysis. It is highly desirable tobe able to perform quantitative and biochemical analyses on formalinfixed brains. Therefore, in the present study, we used our previously
developed RT-QuIC method to quantitate the prion-seeding activity(SD50) in formalin-fixed brain tissue from sCJD patients.
2. Materials and methods
2.1. Human samples (Table 1)
We used endpoint RT-QuIC assays to analyze 19 brain samples ob­
tained from definite cases of 19 sCJD patients: 14 MM1 cases (10 cases
without formalin treatment and 4 cases fixed with formalin), 3 MM2T
cases, and 2 MM2C cases. Sporadic CJD was diagnosed according toParchi’s classification based on the genotype at codon 129 of PRNP
(methionine homozygous [MM], valine homozygous [VV], or hetero­
zygous [MV]), and the physicochemical properties of PrPSc. For
formalin-treated tissue, the entire cortex was treated with formalin for 1
month, and the samples that received combined treatment were subse­
quently exposed to 99% formic acid for 1 h.

Table 1
Summary of sporadic Creutzfeldt–Jakob disease patients.
Patient
number

Sex

Age
onset

sCJD subtype based on
Parchi’s classification

Log SD50/g tissue innative brain (mean ± SD)

Log SD50/g tissue in formalinfixed brains (mean ± SD)

Log SD50/g tissue in formalin-fixed brains
after formic acid treatment (mean ± SD)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

FemaleFemaleFemaleMale
FemaleMale
Male
FemaleMale
FemaleFemale
Male
Male
Male
FemaleMale
Female
Male
Male

69
80
50
68
81
86
64
84
79
72
74
73
75
64
68
40
69
82
67

MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM1
MM2T
MM2T
MM2T
MM2C
MM2C

10.38 ± 0.18
10.25 ± 0.35
10.00 ± 0.00
10.00 ± 0.35
10.13 ± 0.18
9.75 ± 0.00
9.88 ± 0.18
10.00 ± 0.35
10.13 ± 0.18
9.50 ± 0.00
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.

7.88 ± 0.53
7.88 ± 0.17
7.50 ± 0.35
7.75 ± 0.35
7.63 ± 0.53
7.63 ± 0.18
7.38 ± 0.17
7.75 ± 0.35
7.88 ± 0.53
7.13 ± 0.17
7.75 ± 0.71
8.38 ± 0.53
8.13 ± 0.89
8.13 ± 0.18
5.25 ± 0.00
5.88 ± 0.18
6.88 ± 0.53
7.50 ± 0.35
7.38 ± 0.17

4.75 ± 0.35
5.13 ± 0.18
4.75 ± 0.35
5.13 ± 0.18
5.00 ± 0.35
4.88 ± 0.18
4.63 ± 0.53
5.13 ± 0.18
4.50 ± 0.35
4.50 ± 0.35
5.00 ± 0.00
4.75 ± 0.35
5.00 ± 0.00
5.25 ± 0.00
3.50 ± 0.00
3.88 ± 0.18
4.50 ± 0.35
4.88 ± 0.17
4.62 ± 0.18

Clinical data and the SD50 concentrations in formalin-fixed brain homogenates from patients with prion disease. None of the patients have a PRNP mutation.
Based on the genotype at codon 129 on both PRNP alleles, the size of protease-resistant PrPSc fragments, and disease phenotype, we divided sporadic CJD into six
subtypes:sCJDMM1/sCJDMV1, sCJDVV2, sCJDMV2, sCJDMM2-cortical form, sCJDVV1, and MM2-thalamic form according to Parchi’s classification.
The MM1 patients: The pion seeding values of sCJD’s brain decreased 2.36 log after treating with formalin (SD50: 1010±0.29 → 107.64±0.36) which got further reduced
decreased 2.8 log (SD50: 107.64±0.36 → 104.84±0.34) after treating with formalin and formic acid of 10 cases MM1 (patients 1–10). the average of SD50 of 14 cases MM1(patients 1–14) decreased 2.89 log (SD50: 107.77±0.46 → 104.88±0.32) after combining formalin and formic acid treatment.
The MM2 patients: The SD50 values of sCJD’s brain of 2 cases MM2C (patients 18–19) decreased 2.69 log (SD50: 107.44±0.24 → 104.75±0.2), three cases MM2T (patients
15–17) reduced 2.04 log (SD50: 106.00±0.77 → 103.96±0.49) after formalin and formic acid treatment.
Data are presented as means ± standard deviation. MM2C: cortical form of MM2.
MM2T: thalamic form of MM2.
N.A.: not available.
2

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To examine prion distribution in different parts of the brain, we
analyzed prion-seeding activity by using endpoint RT-QuIC assay in six
different regions (frontal, temporal, parietal, occipital, thalamus, andcerebellum) of MM1 (n = 2), MM2T (n = 2), and MM2C (n = 1) samples.
We included 105 non-CJD brain specimens as negative controls.

2.5. Neuropathological examination
A postmortem study was performed within 22.5 h after death. The
brain and upper cervical spinal cord were fixed in 20% neutral-bufferedformalin for 4 weeks, and tissue blocks were immersed in 95% formic
acid for 1 h to inactivate prion infectivity. The specimens were thenembedded in paraffin and cut in 9-μm-thick sections. The sections weredeparaffinized in Lemosol, rehydrated through an ethanol gradient, andstained. For routine neuropathological examinations, sections were
subjected to hematoxylin and eosin (HE) and Klüver-Barrera (KB)
staining and modified Gallyas-Braak silver staining. We evaluated theseverity of the neuropathological changes as mild, moderate or severe,according to Parchi’s pathological staging for sCJD (Parchi et al., 1999).
Immunohistochemical analysis was performed with a mouse mono­clonal antibody against PrP (3F4, dilution 1:100; Dako, Glostrup,
Denmark) after hydrolytic autoclaving for antigen retrieval. PrP im­
munostaining was conducted as previously described (Tateishi et al.,1996). Peroxidase-conjugated streptavidin was visualized with 3, 3′ diaminobenzidine (DAB; Wako Pure Chemical Industries, Japan) as the
final chromogen. Immunostained sections were lightly counterstainedwith HE staining.

2.2. Preparation of tissue homogenate (non-fixed brains)
Brain tissue was subjected to RT-QuIC assay to evaluate SD50 (Akagi
et al., 2018). To prevent contamination from brain tissue into othersamples, we used single-use disposable tubes and beads, and all pro­cedures were performed on different days. Brain tissue samples were
homogenized in 10% (w/v) ice-cold phosphate-buffered saline supple­
mented with a protease inhibitor mixture (Roche, Mannheim, Germany)
using a multi-bead shocker (Yasui Kikai, Osaka, Japan). The samples
were clarified by centrifugation at 6000 rpm for 2 min and stored at− 80 ◦ C.
2.3. Preparation of tissue homogenate (formalin-fixed and formic acidtreated brains)
After treatment in formalin solution for over 1 month, each brain
sample was cut into small pieces, each weighing approximately 50 to
200 mg. The formalin-fixed brain samples were subjected to RT-QuIC
assay to evaluate SD50. To prevent contamination from brain tissue
into other samples, we used single-use disposable tubes and beads, andall procedures were performed on different days. Brain tissue samples
were homogenized in 10% (w/v) ice-cold phosphate-buffered saline
supplemented with a protease inhibitor mixture (Roche, Mannheim,
Germany) using a multi-bead shocker (Yasui Kikai, Osaka, Japan). The
samples were clarified by centrifugation at 10,000 ×g for 2 min andstored at − 80 ◦ C.
Alternatively, some formalin-fixed brain samples were subsequently
immersed in 20 ml 99% formic acid for 1 h and then washed three times
with 100 ml sterile distilled water. These samples were then processedusing the same procedures as for formalin-fixed brain samples.

2.6. Ethics approval and consent to participate
The protocol for investigation was approved by the Ethics Committeeof Nagasaki University Hospital (ID: 10042823), and the study was
registered with the University Hospital Medical Information Network(ID: UMIN000003301) and (ID: UMIN000038398). The protocol was
also granted ethical approval for the use of brain tissue by the Japan
Surveillance Unit for human prion diseases.
2.7. Statistical analysis
Statistical analysis was performed using IBM SPSS® Statistics
Version 22 (IBM Japan, Tokyo). We used the Mann–Whitney U test forcomparisons of SD50 between sCJD patients.
3. Results

2.4. Endpoint RT-QuIC (Fig. 1)
Analysis of the formalin RT-QuIC assays and endpoint formalin RTQuIC assay was similar to the previously described unfixed brain RTQuIC assays (Takatsuki et al., 2015). In brief, the RT-QuIC reaction
mix was composed of 50 mM PIPES (pH 7.0), 500 mM NaCl, 10 μμM
Thioflavin T (ThT), 0.1 mM ethylenediaminetetraacetic acid tetraso­
dium salt hydrate (EDTA), and recombinant human PrP (residues
23–231 with 129 M). Aliquots of the reaction mix (90 μl) were loaded
into each well of a black 96-well plate with a clear bottom (Nunc 96
well; Sigma-Aldrich, USA) and mixed with 10 μl of brain homogenate.
Four to eight replicates of each diluted sample were measured, and PrP
amyloid formation was monitored for 48 h. We calculated the 50%
seeding dose (SD50) in unfixed brains, formalin-fixed brains and formic
acid-treated brains using the Spearman–Kärber method.
We define a positive reaction by RT-QuIC assay when the following
three conditions are met: (1) the absorbance rises within 150 cycles, andafter reaching the maximum absorbance, the level of the absorbance isstable; (2) the maximum absorbance is more than six times higher than
the starting absorbance; and (3) only recombinant protein was used tomaintain the quality of the lot when the prepared recombinant protein
showed an SD50 ≥ 107 by RT-QuIC assay in the brain of MM1-type sCJD.
The assay was repeated at least twice, and Spearman–Kärber analysis
was used to estimate a seeding dose (SD50). The SD50 was calculatedusing the following formula: xp = 1 + 1/2d − dgp, where xp = 1 rep­resents the highest log dilution giving all positive responses, d is the log
dilution factor, p is the proportion positive at a given dose, and gp is thesum of values of p for xp = 1 and all higher dilutions (Wilham et al.,2010).

3.1. Analysis of formalin-fixed brain tissue in the frontal lobe from all
sCJD patients by endpoint RT-QuIC assay (Table 1 and Fig. 1,
Supplementary Fig. 1 [SF 1], and Supplementary Fig. 2 [SF 2])
We analyzed brain samples from all patients (unfixed, formalin-fixedand formic acid-treated tissue) by endpoint RT-QuIC assay. We first
analyzed patient 1 (sCJD MM1) alone to gain a better understanding of
the calculation method, which we showed in detail as a representative.
Brain homogenate was diluted from 1 × 10− 5 to 1 × 10− 10 and subjected
to endpoint RT-QuIC to quantitate seeding activity. The positive reac­tion percentage decreased in a sigmoidal curve within the dilution range
and the SD50 was calculated using the Spearman–Kärber method (Fig. 1
and SF 1).
We determined the optimal time for formalin fixation before pro­ceeding with the experiment. Generally, formalin fixation of brain tissue
is performed for 3–4 weeks. We measured the effect of time in formalin
on SD50 of the untreated brain. SD50 at 1 week, 2 weeks, 3 weeks, 4
weeks and 3 months of formalin fixation were 10.13, 10.08, 8.24, 7.88
and 7.85, respectively (SF2).
As shown in Fig. 1, unfixed brain homogenate from patient 1 had aconcentration of 1010.38±0.17 SD50 per gram of tissue, the formalin-fixedtissue contained 107.88±0.53 SD50 per gram of tissue equivalent, and theformalin-fixed and formic acid-treated tissue had 104.75±0.35 SD50 pergram of tissue equivalent. Our analysis showed that we successfully
determined prion-seeding activity in formalin-fixed and formic acidtreated sCJD brain tissue. It also showed that formalin treatment of3

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Neurobiology of Disease 159 (2021) 105504

the MM1 sCJD tissue for 1 month decreased the SD50 value by 2.5 log,
and formalin treatment with subsequent exposure to >99% formic acidfor 1 h decreased it by 5.63 (1010.38 in unfixed tissue vs. 107.88 after
formalin fixation vs. 104.75 after formic acid treatment) compared withthe unfixed tissue log SD50.
To confirm the above results, we determined the SD50 values forunfixed brain homogenate, formalin-fixed brain tissue and formalinfixed and formic acid-treated brain tissues from 10 MM1 cases (pa­tients 1–10). The prion seeding values of sCJD brains decreased by 2.36
log after formalin treatment (SD50: 1010±0.29 vs. 107.64±0.36), and further
decreased by 2.8 log (SD50: 107.64±0.36 vs. 104.84±0.34) after subsequent
formic acid treatment (Table 1, SF 1 and SF 3).
Using the same method, we analyzed 19 sCJD patients (Table 1),comprising 14 patients with MM1, 3 patients with MM2T and 2 patientswith MM2C. The averages of log10 SD50 in formalin-fixed brains ofMM1, MM2T and MM2C patients were 7.77, 6.00 and 7.44, respectively.
After the formic acid treatment, values for MM1, MM2T and MM2C
patients were 4.88, 3.96 and 4.75, respectively. MM1 and MM2C pa­tients exhibited a similar level of seeding activity (107.38–8.38/g), and theSD50 of MM2T patients was lower than those of MM1 and MM2C
patients.

SD50 per gram, but had also spread to the cerebral cortex and the cer­ebellum, with a range of 5.6–7.13 log SD50 per gram. As shown in
Fig. 2b, very mild spongy changes in the frontal lobe and the thalamus
were observed with HE staining. We also noticed very mild PrP depo­
sition in the thalamus and the frontal lobe by PrP immunostaining.
3.4. Analysis of white and gray matter in the frontal lobe of sCJD
formalin-fixed brains (Fig. 3)
We measured SD50 in formalin-fixed brain tissue from all sCJD pa­tients, and we analyzed prion-seeding activity of the white matter andgray matter from the frontal lobe of all sCJD patients. In all patients, theprion seeding values were higher in gray matter than in white matter.
(Fig. 2 and Supplementary Table 1).
After formalin fixation, the average log SD50 of 14 MM1 patients was
7.93 ± 0.38 and 7.88 ± 0.35 in gray matter and white matter, respec­tively. In 3 MM2C patients, the log SD50 was 7.63 ± 0.32 and 7.19 ±0.51 in gray matter and white matter, respectively. And in 3 MM2T
cases, the log SD50 was 6.67 ± 0.35 and 6.42 ± 0.47 in gray matter andwhite matter, respectively. Therefore, the difference between graymatter and white matter was the smallest in the MM1 cases, atapproximately 0.05, larger in the MM2T cases, at approximately 0.25,
and the largest in the MM2C case, at 0.44. After further treatment withformic acid, the difference between gray matter and white matter was
the smallest still in the MM1 cases (approximately 0.1), and in MM2T
and MM2C cases was 0.28 and 0.25, respectively.

3.2. Analysis of prion distribution in six brain areas by endpoint RT-QuIC
assay and the neurological findings of an MM2C patient (Fig. 2a)
To measure prion-seeding activity throughout the brain, we collected
six representative sites and measured prion-seeding activity in thoselocations (frontal lobe, temporal lobe, parietal lobe, occipital lobe,
thalamus and cerebellum). In an MM2C patient (patient 18), the spon­giform change typically consisted of large and coarse vacuoles. PrP
immunohistochemistry showed a coarse pattern of staining (Fig. 2a).
In the MM2C patient, the level of log SD50/g tissue in the six areas
ranged from 6.63 to 8.75; that of the temporal lobe was higher than thatof the frontal lobe, occipital lobe, parietal lobe and thalamus. Fig. 2
shows that the level of log SD50/g tissue was the highest in the temporal
lobe (108.75±0.35/g tissue), lower in the occipital lobe (108.5±0.35/g tis­sue) and parietal lobe (108.00±0.00/g tissue) and lowest in the frontal
lobe (107.5±0.35/g tissue), thalamus (107.5/g tissue) and cerebellum(106.63±0.53/g tissue).
In the neuropathological findings of patient 18, PrPSc accumulation
was highest in the temporal lobe, lower in the frontal lobe, occipital
lobe, and parietal lobe, and lowest in the thalamus and cerebellum.
Therefore, the neurological findings were consistent with the SD50
values in the MM2C patient.

4. Discussion
To conduct neuropathological analyses in patients with human priondisease, the brain tissue is routinely fixed with formalin and treated withformic acid to reduce the infectivity of abnormal prion proteins. Per­
forming biochemical analyses on formalin-fixed brain tissue is difficult
because proteins are rapidly denatured when the brain is immersed informalin solution. In this study, we successfully measured prion-seeding
activity in formalin-fixed brain samples and in formic acid-treatedformalin-fixed brain using RT-QuIC. To the best of our knowledge, noprevious studies have reported comparisons of infectivity when tissue isfixed with formalin and treated with formic acid, other than the infec­
tion experiment (Tateishi et al., 1996); the present study is the first toreport how infectivity changes in treated brain tissue of sCJD patients.
The level of SD50 decreased with the time that brains were immersed
in formalin solution (ranging from 1010.34 to 107.88; Table 1, Fig. 1 andSF 2b). The SD50 level also decreased with formic acid treatment after
formalin fixation (from 107.88 to 104.75). Formalin treatment of MM1
brain tissue for 1 month decreased the SD50 value by 2.36 log comparedwith unfixed tissue (SF3). Furthermore, the SD50 of unfixed tissue
decreased considerably 1 week from the start of formalin fixation (SF3b). The SD50 was stable after 3 weeks from the start of formalin fixation(SF 2). For neuropathological assessments, brain tissue is routinely fixedin formalin for at least 3 weeks, and our findings indicate that thisduration is adequate to reach stable SD50 values.
Subsequent >99% formic acid treatment after formalin fixation
decreased the value of log10 SD50 in MM1, MM2C and MM2T cases byapproximately 2.8, 2.69 and 2.04, respectively, compared with unfixedtissue. Therefore, the prion-seeding activity of MM1 cases with subse­
quent exposure to formic acid after formalin fixation was decreased by
5.16 logs compared with the MM1 unfixed brain tissue.
Bioassays for human prion disease can be performed in humanizedknock-in mice and sometimes can be used in 263 K mice or VM mice. In aprevious study, the infectivity titer “infectious dose (ID)50” of unfixed
brain from VM mice terminally affected with the 301 V strain of mousepassaged BSE agent was approximately 109–10 ID50/g; after formic acidtreatment of paraformaldehydelysine-periodate (PLP)-fixed brain,
infectivity was <102.5 ID50/g, and after formic acid treatment offormalin-fixed brain, infectivity was <103.0 ID50/g (Taylor et al., 1997).

3.3. Analysis of HE staining, PrP immunostaining and prion distribution,
endpoint RT-QuIC assay in an MM1 patient and an MM2T patient (Fig. 2b
and Table 2)
We measured SD50 in the frontal lobe and thalamus of formalin-fixedtissue from an MM1 patient and MM2T patient. We also examined theneuropathological findings of HE staining and PrP immunostaining in
the frontal lobe and thalamus.
In the HE stainings of the sCJD MM1 patient, the spongiform change
was characterized by small and typical vacuoles in the neuropil of thefrontal lobe. Synaptic-type PrP deposition in the frontal lobe and thal­amus was observed by PrP immunostaining in the MM1 patient, and PrP
deposition in the frontal lobe was higher than that in the thalamus.
Additionally, the log SD50 in the frontal lobe (8.13) was higher than thatin the thalamus (7.5). Therefore, we observed a positive relationshipbetween SD50 and neuropathological findings in the MM1 patient(Fig. 2b).
There was prominent atrophy of the thalamus and inferior olive with
only minor pathologic changes in the other areas of the MM2T brain.
Table 2 shows that the prion-seeding activity in the MM2T patient was
not only concentrated in the thalamus, with a range of 6.50–7.38 log
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Neurobiology of Disease 159 (2021) 105504

Fig. 1. Quantitation of SD50in patient brains by endpoint RT-QuIC.
(a). Quantitation of SD50 in the patient 1 brain.
(a1) Native brain homogenates were diluted (1 × 10− 5 to 1 × 10− 10) and subjected to the RT-QuIC reaction. ThT fluorescence was elevated at dilutions from 1 × 10− 5
to 1 × 10− 10.
(a2) Formalin-fixed brain homogenates were diluted (1 × 10− 3 to 1 × 10− 8) and subjected to the RT-QuIC reaction. ThT fluorescence was elevated at dilutions from1 × 10− 3 to 1 × 10− 8.
(a3) Formalin and formic acid-treated brain homogenates were diluted (1 × 10− 1 to 1 × 10− 6) and subjected to the RT-QuIC reaction. ThT fluorescence was elevatedat dilutions from 1 × 10− 1 to 1 × 10− 6.
(a4) Negative control reactions were seeded in quadruplicate with a 1 × 10− 6 dilution of brain tissue from a negative control brain.
(a5) Reduction of seeding activity by treatment with formalin and formic acid in patient 1 using endpoint RT-QuIC. Prion seeding values of an sCJD brain weredecreased by 2.5 log after formalin fixation and by approximately 5.63 log SD50 following combined formalin fixation and formic acid treatment.
(b). Quantitation of SD50 in six patient brains (2MM1, 2MM2T, and 2MM2C).
(b1) SD50 of a formalin-fixed brain determined by endpoint RT-QuIC.
(b2) Endpoint RT-QuIC to quantitate the SD50 in a formalin-fixed and formic acid-treated sample.
RT-QuIC assays were performed twice.

Although ID50 and SD50 correlate, the values do not represent the same
information. In addition, data obtained by qualitative assay after asimilar treatment of 301 V supports our results (Taylor, 1995). A pre­vious study reported that the infectivity titer in formalin- or PLP-fixedmouse brains infected with the 301 V BSE agent were reduced byaround 6 logs following treatment with formic acid compared with
unfixed brains. Similarly, formic acid-treated mouse brain infected with
the ME7 strain of scrapie agent showed a reduction of around 5 logs
compared with unfixed brain (Taylor et al., 1997). Another study informalin-fixed rodent brain infected with CJD agents showed that theinfectivity titers of scrapie were reduced by approximately 9 logs (from1010.2 to 101.3), and by approximately 6 logs (from 108.5 to 102.3) afteran exposure to >96% formic acid for 1 h (Brown et al., 1990). In thepresent study, the log10 SD50/g tissue values of brain samples from
MM1, MM2C and MM2T patients were 7.77, 7.44 and 6.00, respectively,
after formalin fixation for 1 month, and were approximately 4.88, 4.75
and 3.96, respectively, after combined treatment with formalin andformic acid. The changes reported in the infectivity titer (ID50) of PLPfixed mouse brain after formic acid treatment were consistent with thechanges in prion-seeding activity in human brain in the present study.
Infectivity decreased after formalin and formic acid treatment in pre­vious studies in mouse brains infected with the 301 V BSE agent or ME7
strain of scrapie agent by 5–6 logs. These findings show that the
reduction in infectivity after combined formalin and formic acid treat­ment is similar to the reductions in prion-seeding activity found in thepresent study using endpoint RT-QuIC assays.
We assumed that an SD50 titer of 7–7.5 is the limiting point oftransmission for humanized knock-in mice (Takatsuki et al., 2016).
Therefore, we conclude that in humanized knock-in mice, the formalinfixed brain would be infectious. In the tissue in the present study, whichwere treated with a combination of formalin and formic acid, the prionseeding activity in the treatment of formalin and formic acid was
100,000 times lower than that of the unfixed brain. Therefore, formic
acid treatment is a useful method to reduce the risk of exposure oflaboratory workers to highly infectious prions.
In the present study, we compared PrP deposition and prion-seeding
activity (SD50). The level of SD50 in formalin-fixed tissue was consistent
with the amount of PrPSc accumulation, detected by PrP immunostain­
ing (Fig. 2). PrP immunostaining of MM1 brains showed that the amountof PrPSc accumulation was similar in the cerebral cortex and cerebellum,
and was lower in the brainstem (substantia nigra, midbrain periven­
tricular gray, locus coeruleus and medulla) and hippocampus. Addi­
tionally, immunostaining showed slightly more PrPSc accumulation in
the occipital lobe (SD50: 8.75) than in the frontal lobe, temporal lobe andparietal lobe (SD50: 7.63–7.88) (Table 2).
The amount of the PrPSc accumulation detected by PrP immuno­
staining in the MM2C brains was similar in the cerebral cortex andcerebellum, and lower in the brainstem. Immunostaining also showedthat PrPSc accumulation was at the same level in all lobes of the cerebral
cortex in this MM2C patient. However, the SD50 assay in the MM2C

patient showed that SD50 in the frontal lobe (SD50: 7.5) was lower thanthat in the occipital lobe, temporal lobe and parietal lobe (SD50: 8–8.75).
These findings indicate that there was a consistent relationship between
SD50 and neuropathological findings throughout the cerebral cortex,
except for in the frontal lobe.
In a previous study, the levels of PrPSc accumulation detected by PrP
immunostaining in MM2T brains were similar throughout the cerebral
cortex and lower than those in MM1 brains (Tateishi et al., 1996).
Additionally, there was more PrPSc accumulation in the thalamus andcerebellum of MM2T brains than in the cerebral cortex, including thefrontal lobe, temporal lobe and parietal lobe. Our SD50 assay results in
the MM2T (patient 16) showed that the SD50 of the thalamus (SD50: 6.5)
and cerebellum (SD50: 6.63) were higher than those of the frontal lobe,
temporal lobe and parietal lobe (SD50: 5.6–5.88). Also, PrPSc accumu­
lation was higher in the thalamus than in other areas of this MM2T brain(Fig. 2). Thus, there was a consistent pattern between SD50 and theneuropathological findings throughout the cerebral cortex, except for in
the occipital lobe. These results suggest that the SD50 for formalin-fixed
brain tissue is compatible with the neuropathological findings in mul­
tiple sCJD subtypes.
The SD50 values in formalin-fixed sCJD MM1 brain were fairly
consistent between patients, ranging from 7 to 7.5. However, PrPSc
immunostaining was weaker in some MM1 patients than in others. Thisvariation is likely due to differences in storage conditions of formalinfixed brains, or differences in the degree of infiltration of formalinfrom brain to brain. Because of these limitations of PrP immunostaining,
SD50 calculation by RT-QuIC assay is a useful alternative to quantify
abnormal prion proteins. It is important to note that PrP immunostain­
ing allows for the quantitative analysis of an individual sCJD patient,whereas other neuropathological techniques to assess SD50 can only
provide semi-quantitative data of individual patients.
We evaluated the brains of three subtypes of sCJD patients, and theneuropathological findings indicated that the MM2T cases were the puretype of MM2T (Fig. 2b). In the pure type of MM2T, the amount of PrPSc
in the cerebral cortex is lower than that in the thalamus according to theneuropathological and SD50 analyses (Parchi et al., 1999). In a previous
study, the levels of PrPSc accumulation detected by PrP immunostaining
in MM2T brains were similar throughout the cerebral cortex and lower
than those in MM1 brains (Tateishi et al., 1996). In the present study, thelevel of SD50 in formalin-fixed brain was consistent with the amount of
PrPSc accumulation detected by PrP immunostaining (Fig. 2).
Finally, sCJD is primarily considered a disease of gray matter,although the extent of white matter involvement has not been welldescribed. In the present study, PrPSc accumulation levels detected by
PrP immunostaining in white matter were much lower than those ingray matter (Figs. 2a and 3). Many studies have reported that PrPSc is notseen or is very rarely seen in white matter (Parchi et al., 1996; Caverzasi
et al., 2014). Some previous studies have reported the occasional pres­ence of PrPSc in the white matter in sCJD (Muhleisen et al., 1995) and inmouse models, in which the PrPSc is not routinely detected in the white6

T.-T.-T. Dong et al.

Neurobiology of Disease 159 (2021) 105504

Fig. 2. Relationship between neuropathological findings and SD50 in sCJD patients.
(a) MM2-cortical form (patient 18): Immunostaining for PrP showed prominent perivacuolar-type and rough plaque-type (coarse-type) PrP deposits, whereas
neuropathological examination revealed large confluent vacuole-type spongiform changes in the cerebral cortex. In the cerebral cortex, there was a relationshipbetween the density of vacuoles and PrP deposition, as well as SD50, except for in the frontal lobe. In addition, mild neurodegeneration and PrP deposits wereobserved in the thalamus and cerebellum.
(b) MM1 (patient 14): HE staining in the frontal lobe with high gliosis and high neuronal loss and synaptic-type PrP deposition in the frontal lobe by PrP immu­
nostaining. Milder pathology was observed in the thalamus. MM2-thalamic form (patient 17): Mild spongy changes and very mild PrP deposition were observed in
the thalamus and the frontal lobe by PrP immunostaining. Moreover, the log10 SD50 had a low value, ranging from 6.88–7.38 in the different regions examined.

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T.-T.-T. Dong et al.

Neurobiology of Disease 159 (2021) 105504

Table 2Distribution of prion seeding in six locations in sCJD brains.
Patient
NumberMM1 (Patient 11)

MM1 (Patient 14)

MM2C (Patient 18)

MM2T (Patient 16)

MM2T (Patient 17)

end-point
RT-QuIC
assay(logSD50)

Formalinfixed brain
(mean
±SD)

Formic acidtreatedformalinfixed brain
(mean
±SD)

Formalinfixed brain
(mean
±SD)

Formic acidtreatedformalinfixed brain
(mean
±SD)

Formalinfixed brain
(mean
±SD)

Formic acidtreatedformalinfixed brain
(mean
±SD)

Formalinfixed brain
(mean
±SD)

Formic acidtreatedformalinfixed brain
(mean
±SD)

Formalinfixed brain
(mean
±SD)

Formic acidtreatedformalinfixed brain
(mean
±SD)

FrontalTemporalParietal
Occipital
ThalamusCerebellum

7.75 ± 0.71
7.88 ± 0.18
7.88 ± 0.18
8.75 ± 0.35
7.63 ± 0.53
8.88 ± 0.18

5.00 ± 0.00
4.62 ± 0.18
5.12 ± 0.18
5.75 ± 0.35
4.63 ± 0.18
6.12 ± 0.18

8.13 ± 0.18
N.A.
N.A.
8.00 ± 0.35
7.50 ± 0.00
8.38 ± 0.18

5.25 ± 0.00
N.A.
N.A.
4.88 ± 0.53
4.88 ± 0.18
5.5 ± 0.35

7.5 ± 0.35
8.75 ± 0.35
8.00 ± 0.00
8.5 ± 0.35
7.50 ± 0.00
6.63 ± 0.53

4.88 ± 0.17
5.88 ± 0.18
5.25 ± 0.00
5.25 ± 0.35
4.25 ± 0.35
3.88 ± 0.53

5.88 ± 0.18
5.6 ± 0.18
5.75 ± 0.35
5.88 ± 0.18
6.50 ± 0.35
6.63 ± 0.17

3.88 ± 0.18
3.63 ± 0.18
3.50 ± 0.00
3.75 ± 0.35
4.13 ± 0.18
4.25 ± 0.35

6.88 ± 0.53
N.A.
N.A.
7.13 ± 0.18
7.38 ± 0.18
7.00 ± 0.35

4.5 ± 0.35
N.A.
N.A.
4.75 ± 0.35
4.88 ± 0.18
4.5 ± 0.35

N.A.: not available. Data are presented as means ± standard deviation.

Fig. 3. Prion seeding in white and gray matter.
There was a slight difference between the SD50 in the white matter and gray matter in sCJD patients following formalin and formic acid treatment. The smallest
difference was in the sCJD MM1 case, with a difference of 0.05 after formalin treatment, and 0.1 after combined formalin and formic acid treatment. In the MM2T
group, the differences were 0.25 and 0.28, respectively. The largest differences were in the MM2C group, and were 0.44 and 0.25, respectively.

matter (DeArmond et al., 2002; Spilman et al., 2008), and suggest that it
might be transported along axons to distant gray matter regions (Spil­man et al., 2008). In the present study, we assessed the SD50 of whitematter and gray matter treated with formalin and formic acid in sCJD
patients and found a slight difference between the prion-seeding activity
in the white matter and gray matter. These findings indicate that PrPSc
accumulation was present in the gray matter, and that PrPSc accumu­
lation was present in the white matter, despite not being apparent from
pathological findings and PrP immunostaining.

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.nbd.2021.105504.
Consent for publication
We have obtained all authors’ consent for publication.
Availability of data and material
Data sharing is not applicable to this article, as no datasets weregenerated during the current study.

5. Conclusion
We successfully used endpoint RT-QuIC for analysis of formalin-fixed
brains of sCJD patients. We were able to detect SD50 in formalin-fixed
brains of sCJD patients, and pathological analyses and quantitative
biochemical methods can be performed after RT-QuIC. In the future,
endpoint RT-QuIC with formalin-fixed sCJD brain samples will be auseful tool in combination with neuropathological analysis to improveunderstanding of sCJD.

Author contributions
Conceived and designed the experiments: K.S. and N.N. Performed
the experiments: T.D. and T.Na. Analyzed the data: K.S., T.No., T.N., D.
I., and N.N. Contributed reagents/materials/analytical tools andanalyzed neuropathology: AA, B.M., M.T., and Y.I. Wrote the paper: K.S.
and N.N.
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Neurobiology of Disease 159 (2021) 105504

FundingAtarashi, R., Satoh, K., Sano, K., et al., 2011. Ultrasensitive human prion detection in
cerebrospinal fluid by real-time quaking-induced conversion. Nat. Med. 17 (2),
175–178.
Brown, P., Wolff, A., Gajdusek, D.C., 1990. A simple and effective method forinactivating virus infectivity in formalin-fixed tissue samples from patients withCreutzfeldt-Jakob disease. Neurology 40, 887–890.
Caverzasi, Eduardo, Mandelli, Maria Luisa, DeArmond, Stephen J., et al., 2014 Dec.
White matter involvement in sporadic Creutzfeldt-Jakob disease. Brain. 137 (12),3339–3354.
DeArmond, S.J., Kretzschmar, H.A., Prusiner, S.B., 2002. Prion diseases. In: Graham, D.I.,
Lantos, P.L. (Eds.), Greenfield’s Neuropathology, 7th edn. Arnold, New York,
pp. 273–323.
Muhleisen, H., Gehrmann, J., Meyermann, R., 1995. Reactive microglia in CreutzfeldtJakob disease. Neuropathol. Appl. Neurobiol. 21, 505–517.
Nozaki, I., Hamaguchi, T., Sanjo, N., et al., 2010. Prospective 10-year surveillance ofhuman prion diseases in Japan. Brain 133 (10), 3043–3057.
Parchi, A. Giese, Capellari, S., et al., 1999. Classification of sporadic Creutzfeldt-Jakob
disease based on molecular and phenotypic analysis of 300 subjects. Ann. Neurol. 46(2), 224–233.
Parchi, P., Castellani, R., Capellari, S., et al., 1996. Molecular basis of phenotypicvariability in sporadic Creutzfeldt-Jakob disease. Ann. Neurol. 39, 767–778.
Prusiner, S.B., Scott, M.R., DeArmond, S.J., et al., 1998. Prion protein biology. Cell. 93(3), 337–348.
Saborio, G.P., Permanne, B., Soto, C., 2001. Sensitive detection of pathological prion
protein by cyclic amplification of protein misfolding. Nature 411 (6839), 810–813.
Spilman, P., Lessard, P., Sattavat, M., et al., 2008 Jul 29. A gamma-secretase inhibitorand quinacrine reduce prions and prevent dendritic degeneration in murine brains.
Proc. Natl. Acad. Sci. U. S. A. 105 (30), 10595–10600.
Takatsuki, H., Satoh, K., Sano, K., et al., 2015. Rapid and quantitative assay of amyloidseeding activity in human brains affected with prion diseases. PLoS One 10 (6),e0126930.
Takatsuki, H., Fuse, T., Nakagaki, T., et al., 2016. Prion-seeding activity is widelydistributed in tissues of sporadic creutzfeldt-jakob disease patients. EBioMedicine.
12, 150–155.
Tateishi, J., Kitamoto, T., Kretzschmar, H., Mehraein, P., 1996. Immunhistologicalevaluation of Creutzfeldt-Jakob disease with reference to the type PrPres deposition.
Clin. Neuropathol. 15 (6), 358–360.
Taylor, D.M., 1995. Survival of mouse-passaged bovine spongiform encephalopathy
agent after exposure to paraformaldehyde-lysine-periodate and formic acid. Vet.
Microbiol. 44 i I 1–112.
Taylor, D.M., Brown, J.M., Fernie, K., McConnell, I., 1997. The effect of formic acid on
BSE and scrapie infectivity in fixed and unfixed brain-tissue. Vet. Microbiol. 58,
167–174.
Wilham, J.M., Orru, C.D., Bessen, R.A., et al., 2010. Rapid end-point quantitation ofprion seeding activity with sensitivity comparable to bioassays. PLoS Pathog. 6 (12),e1001217.

This study was financially supported by grants for scientific researchfrom the Ministry of Health, Labour and Welfare of Japan (KSat: No.
14507303), the Research Committee of Prion Disease and Slow Virus
Infection, Research on Policy Planning and Evaluation for Rare andIntractable Diseases, Health and Labour Sciences Research Grants, theResearch Committee of Surveillance and Infection Control of Prion
Disease, the Ministry of Health, Labour, and Welfare of Japan, and theJapan Agency for Medical Research and Development (AMED) (grant
number No. 18ek0109362h0001).
Credit author statement
Professor Katsuya Satoh, Corresponding author, is responsible for thecredit associated with this manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
We are grateful to the Japan Prion Disease Surveillance Committee.
We also thank Atsuko Matsuo for providing technical support during thedata analysis. Finally, we thank Lesley McCollum, PhD, and Angela
Morben, DVM, ELS, from Edanz (https://jp.edanz.com/ac) for editing a
draft of this manuscript.
References
Akagi, A., Iwasaki, Y., Mimuro, M., et al., 2018. Pathological progression of genetic
Creutzfeldt-Jakob disease with a PrP V180I mutation. Prion 12 (1), 54–62.
Atarashi, R., Wilham, J.M., Christensen, L., et al., 2008. Simplified ultrasensitive prion
detection by recombinant PrP conversion with shaking. Nat. Methods 5 (3),211–212.

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