Document ID: FDA-2009-P-0405-0005
Agency: fda
Document Type: Supporting & Related Material
Title: 
Posted Date: 2009-09-30T04:00Z

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  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"Table%201.%20The%20Prion%20Diseases"  Table 1. The Prion Diseases 

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  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"Table%202.%20Standard%20precautions*%20for%20autopsies%20of%20patients%
20with%20suspected%20prion%20disease"  Table 2. Standard precautions*
for autopsies of patients with suspected prion disease 

•

  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"Table%203.%20Autopsy%20suite%20decontamination%20procedures"  Table 3.
Autopsy suite decontamination procedures 

•

  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"Table%204.%20Brain%20cutting%20procedures"  Table 4. Brain cutting
procedures 

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  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"Table%205.%20Tissue%20preparation"  Table 5. Tissue preparation 

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  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"References"  References 

 	BMBL Section VII

Agent Summary Statements

Section VII-D: Prions

  

  

  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/biosfty.htm"  Biosafety
Documents 

  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4toc.htm"  BMBL
Table of Contents 

The Centers for Disease Control and Prevention and the National
Institutes of Health wish to express our appreciation to Jiri Safar,
M.D.; Darlene Groth, A.B.; Stephen J. DeArmond, M.D., Ph.D.; and Stanley
B. Prusiner, M.D., of the University of California San Francisco, San
Francisco, CA, for their contributions to this emerging field and the
preparation of this section. 

Prions are proteinaceous infectious particles that lack nucleic acids. 
HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_1_"  (1)  Prions are composed largely, if not entirely, of an
abnormal isoform of a normal cellular protein. In mammals, prions are
composed of an abnormal, pathogenic isoform of the prion protein (PrP),
designated PrPSc. The "Sc" superscript was initially derived from the
term scrapie because scrapie is the prototypic prion disease. Since all
of the known prion diseases (Table 6) of mammals involve aberrant
metabolism of PrP similar to that observed in scrapie, use of the "Sc"
superscript is suggested for all abnormal, pathogenic PrP isoforms. 
HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_2_"  (2)  In this context, the "Sc" superscript is used to designate
the scrapie-like isoform of PrP. 

A chromosomal gene encodes PrP and no PrP genes are found in purified
preparations of prions. PrPSc is derived from PrPC (the cellular isoform
of PRP) by a posttranslational process whereby PrPSc acquires a high
-sheet content.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_3_"  (3) 
Neither prion-specific nucleic acids nor virus-like particles have been
detected in purified, infectious preparations. In fungi, evidence for
three different prions has been accumulated.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_4_"  (4)  

The mammalian prions cause scrapie and other related neurodegenerative
diseases of humans and animals (Table 6). The prion diseases are also
referred to as the transmissible spongiform encephalopathies (TSEs). 
HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_5_"  (5)  

Table 1. The Prion Diseases 

Disease abbreviation)

Natural Host

Prion

Pathogenic PrP Isoform

Scrapie

sheep and goats

scrapie prion

OvPrPSc 

Transmissible mink encephalopathy (TME)

mink

TME prion

MkPrPSc

Chronic wasting disease (CWD)

mule deer and elk

CWD prion

MdePrPSc

Bovine spongiform encephalopathy (BSE)

cattle

BSE prion

BoPrPSc

Feline spongiform encephalopathy (FSE)

cats

FSE prion

FePrPSc

Exotic ungulate encephalopathy (EUE)

nyala and greater kudu

EUE prion

UngPrPSc

Kuru

humans

kuru prion

HuPrPSc

Creutzfeldt-Jakob disease (CJD)

humans

CJD prion

HuPrPSc

Gerstmann-Sträussler-Scheinker syndrome (GSS)

humans

GSS prion 

HuPrPSc

Gatal familial insomnia (FFI)

humans

FFI prion

HuPrPSc

 

Species-specificity of prions. Unlike many viruses, the properties of
prions change dramatically when they are passaged from one species to
another. The results of transgenic (Tg) mouse studies indicate that when
human prions are passaged into mice, their potential non-Tg
pathogenicity for humans probably declines drastically.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_6_"  (6) 
The prions that are propagated in the non-Tg mice are now mouse prions,
not human prions. The mouse prions contain mouse PrPSc, not human PrPSc.
This species-specific change in the PrPSc molecule is accompanied by an
alteration in the pathogenicity of the prion. In contrast to the human
prions, mouse prions are highly pathogenic for mice. Our understanding
of these species-specific changes in prion pathogenicity is derived
largely from studies of mice expressing a variety of PrP transgenes.
Because the PrPSc produced in the mouse is from mouse PrPC, it not
possible to determine the origin of the prion initially inoculated into
the mouse.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_7_"  (7)  

It is noteworthy that the susceptibility of a particular species to
prions from another species can be profoundly affected by different
prion strains.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_8_"  (8) 
The properties manifested by prion strains such as incubation times and
neuropathology profiles seem to be enciphered in the conformation of
PrPSc. 

Such considerations of the basic principles of prion biology help to
form the basis for the biosafety classification of different prions. 

Biosafety level classification. Human prions and those propagated in
apes and monkeys are manipulated at Biosafety Level 2 or 3, depending on
the studies being conducted. BSE prions are likewise manipulated at
Biosafety Level 2 or 3, due to the possibility that BSE prions have been
transmitted to humans in Great Britain and France.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_9_"  (9)  

All other animal prions are considered Biosafety Level 2 pathogens.
Thus, based on our current understanding of prion biology described
above, once human prions are passaged in mice and mouse PrPSc is
produced, these prions should be considered Biosafety Level 2 prions,
even though the human prions are Biosafety Level 3 under most
experimental conditions. An exception to this statement is in the case
of mice expressing human or chimeric human/mouse transgenes. These
transgenic mice produce human prions when infected with human prions and
should be treated as Biosafety Level 2 or 3 in accord with the
guidelines described above. The mechanism of prion spread among sheep
and goats developing natural scrapie is unknown.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_10_"  (10) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_11_"  (11)  CWD, TME, BSE, FSE, and EUE are all thought to occur
after the consumption of prion-infected foods.   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_12_"  (12) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_13_"  (13)   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_14_"  (14) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_15_"  (15)  

Human prion diseases. In the care of patients dying of human prion
disease, the precautions used for patients with AIDS or hepatitis are
certainly adequate. In contrast to these viral illnesses, the human
prion diseases are not communicable or contagious.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_16_"  (16) 
There is no evidence of contact or aerosol transmission of prions from
one human to another. However, they are infectious under some
circumstances, such as ritualistic cannibalism in New Guinea causing
kuru, the administration of prion-contaminated growth hormone causing
iatrogenic CJD, and the transplantation of prion-contaminated dura mater
grafts.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_17_"  (17) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_18_"  (18)   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_19_"  (19) 
Familial CJD, GSS, and FFI are all dominantly inherited prion diseases;
five different mutations of the PrP gene have been shown to be
genetically linked to the development of inherited prion disease. Prions
from many cases of inherited prion disease have been transmitted to
apes, monkeys, and mice carrying human PrP transgenes.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_20_"  (20) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_21_"  (21)   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_22_"  (22) 

Surgical procedures. Surgical procedures on patients diagnosed with
prion disease should be minimized. It is thought that CJD has been
spread from a CJD patient to two other patients who underwent
neurosurgical procedures in the same operating theater shortly
thereafter.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_23_"  (23) 
Although there is no documentation of the transmission of prions to
humans through droplets of blood or cerebrospinal fluid, or by exposure
to intact skin, or gastric and mucous membranes, the risk of such
occurrences is a possibility. Sterilization of the instruments and
decontamination of the operating room should be performed in accordance
with recommendations described below. 

Because it is important to establish a 'definitive' diagnosis of a human
prion disease and to distinguish between sporadic and familial cases and
those acquired by infection as a result of medical procedures or from
prion-contaminated food products, unfixed brain tissue should be
obtained. For all cases of suspected human prion disease, a minimum of
one cubic centimeter of unfixed cerebral cortex should be part of any
biopsy. This specimen should be bisected from the cortical surface
through to the underlying white matter with one half of the specimen
formalin-fixed and the other half frozen. 

Autopsies. Routine autopsies an-d the processing of small amounts of
formalin-fixed tissues containing human prions require Biosafety Level 2
precautions.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_24_"  (24) 
At autopsy, the entire brain should be collected and cut into coronal
sections about 1.5 inches (~4 cm) thick; small blocks of tissue can
easily be removed from each coronal section and placed in fixative for
subsequent histopathologic analyses. Each coronal section is immediately
heat sealed in a heavy-duty plastic bag. The outside of this bag is
assumed to be contaminated with prions and other pathogens. With fresh
gloves or with the help of an assistant with uncontaminated gloves, the
bag containing the specimen is placed into another plastic bag which
does not have a contaminated outer surface. The samples should then be
frozen on dry ice or placed directly in a -70C freezer for storage. At
the very minimum, a coronal section of cerebral hemisphere containing
the thalamus and one of the cerebellar hemisphere and brainstem should
be taken and frozen. 

The absence of any known effective treatment for prions disease demands
caution. The highest concentrations of prions are in the central nervous
system and its coverings. Based on animal studies, it is likely that
high concentrations of prions are also found in spleen, thymus, lymph
nodes, and lung. The main precaution to be taken when working with
prion-infected or contaminated material is to avoid puncture of the
skin.  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm"
\l "N_25_"  (25)  The prosector should wear cut-resistant gloves if
possible. If accidental contamination of skin occurs, the area is
swabbed with 1N sodium hydroxide for 5 minutes and then washed with
copious amounts of water. Tables 2-5 provide guidelines to reduce the
chance of skin punctures, aerosols, and contamination of operating room,
morgue surfaces and instruments. Unfixed samples of brain, spinal cord,
and other tissues containing human prions should be processed with
extreme care at Biosafety Level 3. 

Bovine spongiform encephalopathy. The risk of infection for humans by
BSE prions is unclear. Perhaps the most prudent approach is to study BSE
prions in a Biosafety Level 2 or 3 facility depending on the samples to
be studied, as noted above for human prions (i.e., brain, spinal cord). 

Experimental rodent prion diseases. Mice and hamsters are the
experimental animals of choice for all studies of prion disease. With
the development of transgenic mice that are highly susceptible to human
prions, the use of apes and monkeys is rarely needed. The highest titers
of prions (~109.5 ID50/g) are found in the brain and spinal cord of
laboratory rodents infected with adapted strains of prions;  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_26_"  (26) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_27_"  (27)  lower titers (~106 ID50/g) are present in the spleen and
lymphoreticular system.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_28_"  (28) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_29_"  (29)  

Physical properties of prions. The smallest infectious prion particle is
probably a dimer of PrPSc; this estimate is consistent with an ionizing
radiation target size of 55 ±9 kDa.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_30_"  (30) 
Therefore, prions may not be retained by most of the filters that
efficiently eliminate bacteria and viruses. Additionally, prions
aggregate into particles of non-uniform size and cannot be solubilized
by detergents, except under denaturing conditions where infectivity is
lost.  HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm"
\l "N_31_"  (31)   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_32_"  (32) 
Prions resist inactivation by nucleases,  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_33_"  (33) 
UV-irradiation at 254 nm,  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_34_"  (34) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_35_"  (35)  and treatment with psoralens,  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_36_"  (36) 
divalent cations, metal ion chelators, acids (between pH 3 and 7),
hydroxylamine, formalin, boiling, or proteases.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_37_"  (37) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_38_"  (38)  

Inactivation of prions. Prions are characterized by extreme resistance
to conventional inactivation procedures including irradiation, boiling,
dry heat, and chemicals (formalin, betapropiolactone, alcohols). While
prion infectivity in purified samples is diminished by prolonged
digestion with proteases,  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_39_"  (39) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_40_"  (40) results from boiling in sodium dodecyl sulfate and urea
are variable. Sterilization of rodent brain extracts with high titers of
prions requires autoclaving at 132C for 4.5 hours (h). Denaturing
organic solvents such as phenol or chaotropic reagents such as guanidine
isothiocyanate or alkali such as NaOH can also be used for
sterilization.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_41_"  (41) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_42_"  (42)   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_43_"  (43) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_44_"  (44)   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_45_"  (45) 
Prions are inactivated by 1N NaOH, 4.0 M guanidinium hydrochloride or
isocyanate, sodium hypochlorite (2% free chlorine concentration), and
steam autoclaving at 132C for 4.5 h.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_46_"  (46) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_47_"  (47)   HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_48_"  (48) 
 HYPERLINK "http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l
"N_49_"  (49)  It is recommended that dry waste be autoclaved at 132C
for 4.5 h or incinerated. Large volumes of infectious liquid waste
containing high titers of prions can be completely sterilized by
treatment with 1N NaOH (final concentration) or autoclaving at 132C for
4.5 h. Disposable plasticware, which can be discarded as a dry waste, is
highly recommended. Because the paraformaldehyde vaporization procedure
does not diminish prion titers, the biosafety cabinets must be
decontaminated with 1N NaOH, followed by 1N HCl, and rinsed with water.
HEPA filters should be autoclaved and incinerated. 

Although there is no evidence to suggest that aerosol transmission
occurs in the natural disease, it is prudent to avoid the generation of
aerosols or droplets during the manipulation of tissues or fluids and
during the necropsy of experimental animals. It is further strongly
recommended that gloves be worn for activities that provide the
opportunity for skin contact with infectious tissues and fluids.
Formaldehyde-fixed and paraffin-embedded tissues, especially of the
brain, remain infectious. Some investigators recommend that
formalin-fixed tissues from suspected cases of prion disease be immersed
for 30 min in 96% formic acid or phenol before histopathologic
processing,  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_50_"  (50) 
but such treatment may severely distort the microscopic neuropathology. 

Handling and processing of tissues from patients with suspected prion
disease. The special characteristics of work with prions require
particular attention to the facilities, equipment, policies, and
procedures involved. The related considerations outlined in the
following tables should be incorporated into the laboratory's risk
management for this work. 

Table 2. Standard precautions* for autopsies of patients with suspected
prion disease 

*Not to be confused with "Standard Universal Precautions" 

1. Attendance should be limited to at least one experienced pathologist
and minimal staff. One of the staff avoids direct contact with the
deceased but assists with handling of instruments and specimen
containers.

2. Standard autopsy attire is mandatory. 

a. A disposable, waterproof gown is worn in place of a cloth gown. 

b. Cut-resistant gloves are worn underneath two pairs of surgical gloves
or chain mail gloves are worn between two pairs of surgical gloves. 

c. Aerosols are mainly created during opening of the skull with a
Stryker saw. Appropriate respiratory protection should be worn (i.e.,
PAPR). 

3. To reduce contamination of the autopsy suite: 

a. The autopsy table is covered with an absorbent sheet that has a
waterproof backing. 

b. Contaminated instruments are placed on an absorbent pad. 

c. The brain is removed while the head is in a plastic bag to reduce
aerosolization and splatter. 

d. The brain can be placed into a container with a plastic bag liner for
weighing. 

e. The brain is placed onto a cutting board and appropriate samples are
dissected for snap freezing (see Table 4). 

f. The brain or organs to be fixed are immediately placed into a
container with 10% neutral buffered formalin. 

g. In most cases of suspected prion disease, the autopsy can be limited
to examination of the brain only. In cases requiring a full autopsy,
consideration should be given to examining and sampling of thoracic and
abdominal organs in situ. 

Table 3. Autopsy suite decontamination procedures 

1. Instruments (open box locks and jaws) and saw blades are placed into
a large stainless steel dish, soaked for 1 h in 2N sodium hydroxide or 2
h in 1N sodium hydroxide, and then rinsed well in water before
autoclaving at 134C (gravity displacement steam autoclaving for 1 h;
porous load steam autoclaving for one 18-minute cycle at 30 lbs psi or
six 3-minute cycles at 30 lbs psi). 

2. The Stryker saw is cleaned by repeated wetting with 2N sodium
hydroxide solution over a 1 h period. Appropriate washing to remove
residual NaOH is required. 

3. The absorbent table cover and instrument pads, disposable clothing,
etc. are double bagged in appropriate infectious waste bags for
incineration. 

4. Any suspected areas of contamination of the autopsy table or room are
decontaminated by repeated wetting over 1 h with 2N sodium hydroxide

Table 4. Brain cutting procedures 

1. After adequate formaldehyde fixation (at least 10-14 days), the brain
is examined and cut on a table covered with an absorbent pad with an
impermeable backing. 

2. Samples for histology are placed in cassettes labeled with "CJD
precautions." For laboratories that do not have embedding and staining
equipment or microtome dedicated to infectious diseases including CJD,
blocks of formalin-fixed tissue can be placed in 96% absolute formic
acid for 30 minutes, followed by fresh 10% neutral buffered formalin
solution for at least 48 h.  HYPERLINK
"http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s7d.htm" \l "N_51_"  (51) 
The tissue block is then embedded in paraffin as usual. Standard
neurohistological or immunohistochemical techniques are not obviously
affected by formic acid treatment; however, in our experience, tissue
sections are brittle and crack during sectioning. 

3. All instruments and surfaces coming in contact with the tissue are
decontaminated as described in Table 3. 

4. Tissue remnants, cutting debris, and contaminated formaldehyde
solution should be discarded within a plastic container as infectious
hospital waste for eventual incineration. 

Table 5. Tissue preparation 

1. Histology technicians wear gloves, apron, laboratory coat, and face
protection. 

2. Adequate fixation of small tissue samples (e.g. biopsies) from a
patient with suspected prion disease is followed by post-fixation in 96%
absolute formic acid for 30 minutes, followed by 48 hours in fresh 10%
formalin. 

3. Liquid waste is collected in a 4L waste bottle containing 600 ml 6N
sodium hydroxide. 

4. Gloves, embedding molds, and all handling materials are disposed of
as biohazardous waste. 

5. Tissue cassettes are processed manually to prevent contamination of
tissue processors. 

6. Tissues are embedded in a disposable embedding mold. If used, forceps
are decontaminated. 

7. In preparing sections, gloves are worn, section waste is collected
and disposed of in a biohazard waste receptacle. The knife stage is
wiped with 1-2N NaOH, and the knife used is discarded immediately in a
"biohazard sharps" receptacle. Slides are labeled with "CJD
Precautions." The sectioned bloc is sealed with paraffin. 

8. Routine staining: 

a. Slides are processed by hand. 

b. Reagents are prepared in 100 ml disposable specimen cups. 

c. After placing the coverslip on, slides are decontaminated by soaking
them for 1 h in 2N NaOH. 

d. Slides are labeled as "Infectious-CJD." 

9. Other suggestions: 

a. Disposable specimen cups or slide mailers may be used for reagents. 

b. Slides for immunocytochemistry may be processed in disposable petri
dishes. 

c. Equipment is decontaminated as described above. 

References

1. Prusiner S.B. 1997. Prion diseases and the BSE crisis. Science
278:245-251. 

2. Prusiner S.B., Baron H., Carlson G., Cohen F.E., DeArmond S.J.,
Gabizon R., Gambetti P., Hope J., Kitamoto T., Kretzschmar H.A.,
Laplanche J.-L., Tateishi J., Telling G., Weissmann C., Will R., In
press. "Prions." In: Virus Taxonomy. 7th Report of the International
Committee on Taxonomy of Viruses. Academic Press. 

3. Pan K.-M., Baldwin M., Nguyen J., Gasset M., Serban A., Groth D.,
Mehlhorn I., Huang Z., Fletterick R.J., Cohen F.E., Prusiner S.B. 1993.
Conversion of -helices into -sheets features in the formation of the
scrapie prion proteins. Proc Natl Acad Sci USA 90:10962-10966. 

4. Wickner R.B. 1997. A new prion controls fungal cell fusion
incompatibility [Commentary]. Proc Natl Acad Sci USA 94:10012-10014. 

5. Gajdusek, D.C. 1977. Unconventional viruses and the origin and
disappearance of kuru. Science 197:943-960. 

6. Telling G.C., Scott M., Mastrianni J., Gabizon R., Torchia M., Cohen
F.E., DeArmond S.J., Prusiner S.B. 1995. Prion propagation in mice
expressing human and chimeric PrP transgenes implicates the interaction
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