Prion biology in transfusion medicine: Implications for lab testing

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From: TSS ()
Subject: Prion biology in transfusion medicine: Implications for lab testing
Date: November 7, 2005 at 7:34 am PST

10 September 2005 ¦ MLO www.mlo-online.com

C O N T I N U I N G

E D U C A T I O N

To o earn CEUs, see test on page 24.

LEARNING OBJECTIVES

Upon completion of this article, the

reader will be able to:

1. Define and discuss the relevance

of prion biology in transfusion

medicine.

2. Discuss laboratory testing for

prion disease.

3. Discuss symptoms of various TSE

clinical syndromes.

Prion biology in

transfusion medicine:

Implications for

lab testing

By Girolamo A. Ortolano, PhD;

Samuel O. Sowemimo-Coker, PhD;

Jeffrey Schaffer, DVM;

and Joseph S. Cervia, MD, FACP, FAAP

he word prion — suggested by Nobel Laureate Stanley

Prusiner — is a contrived abbreviation for an infectious protein

believed to be the causative agent of a family of progressive

neurodegenerative diseases known as transmissible

spongiform encephalopathies (TSE). The most notorious TSE is

bovine spongiform encephalopathy (BSE), which is responsible for

"mad cow" disease.

Concern over BSE has heightened since it was identified that ingestion

of beef can cause TSE in humans. The United Kingdom

(U.K.) has experienced most of the reported cases of BSE, and citizens

of the U.K. have contracted the majority of cattle-derived TSE

cases referred to as variant Creutzfeldt-Jakob Disease (vCJD). At this

time, references to BSE and vCJD are found regularly in the news.

vCJD has captured considerable attention among politicians, government

groups, and academic researchers alike with a disproportionately

large rate of growth shown in publications in the medical

literature (see Figure 1).

This review provides a basic understanding of the pathogenesis

of prion diseases and highlights the magnitude of the current concern

over BSE and vCJD with an emphasis upon transmissibility

and the implications for transfusion and blood testing in both humans

and animals.

Prion history

The first TSE was identified in sheep in 1759. Since afflicted animals

would appear to scrape their sides along the fences of their pens,

the condition was commonly referred to as scrapie. Although the experimental

transmissibility was not reported until 1936, postmortem

examination revealed that the brains of infected animals were spongelike,

resulting in the rapid acceptance of the term "transmissible

spongiform encephalopathy" as a common and recurring characteristic

of the disease in sheep and other animals.

In the 1920s, two separate reports appeared describing a progressive

neurodegenerative disease in humans presenting with central

C O V E R S T O R Y

Continues on page 12

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"Raskolnikov" wood engraving by Harry Brockway. See Table of Contents for details.

12 September 2005 ¦ MLO www.mlo-online.com

C O V E R S T O R Y

nervous system dysfunctions reminiscent of those seen in

scrapie. The first was reported by Hans Creutzfeldt (1920)

and the second by Anton Jakob (1921). Thus, the constellation

of symptoms they described became known as Creutzfeldt-

Jakob disease (CJD).

In 1966, T. Alper and colleagues provided the first clue to

demonstrate the unique pathogenesis associated with TSE.

They showed that chemical manipulations resulting in the destruction

of nucleic acids, known as the infective component

of viruses, did not alter prion infectivity. It was Prusiner, however,

who is credited with providing overwhelming evidence

suggestive of the radical idea that a protein was the infective

agent — an effort he published in 1982 and for which he was

subsequently awarded a Nobel Prize.

In 1956 and 1957, another form of human TSE referred to

as kuru reached epidemic proportions through the practice of

cannibalism among the Fore people of Papua New Guinea.

As the practice subsided, the incidence of kuru among these

people decreased. The cultural practice reflected a devotion

to their ancestors through the ingestion of brain matter of the

deceased— predominantly by the women and children — and

the consumption of their muscle by the men. Women often

succumbed to kuru, but men did not. This further implicated

neural tissue in the pathogenesis of TSE.

In the mid-1980s, the U.K. experienced an outbreak of

TSE among cattle. The brains were affected in all afflicted

cattle, and the symptoms included awkward movements and

abnormal gait with exaggerated behaviors characterized by

some as madness — hence, the term "mad cow disease" was

coined for BSE.

In 1987, it was thought that scrapie was passed to cattle

because they were fed sheep-derived meat and bone meal. In

1989, selected parts of slaughtered cattle (brain, spinal cord,

intestine, thymus, tonsils, and spleen — collectively referred

to as specified bovine offal, or SBO) were banned from becoming

byproducts to be fed to cattle, thus preventing prions

from entering the human food chain.

Recently, there has been some concern over the rising incidence

of prion disease in deer and elk (called chronic wasting

disease, or CWD). It is also appreciated that many animals

are capable of acquiring TSEs, including cats (feline

spongiform encephalopathy, or FSE), mink (transmissible mink

encephalopathy, or TME), and a number of exotic zoo animals

as well.

Prion biology

Since the advance of molecular biological techniques, we have

gained considerable knowledge of basic prion biology beginning

with the molecular structure. The molecular structure of

the prion protein (PrP) is dictated by the prion gene, the human

form of which is abbreviated as PRNP. PRNP encodes

for a protein of 254 amino acids in length. PRNP undergoes

post-translational modifications in two important ways —

cleavage and glycosylation. First, the leading 22 amino acids

and trailing 23 amino acids are cleaved to leave a protein with

210 amino acids. Since the average molecular weight of an

amino acid is 118 daltons, multiplication by 210 produces an

unglycosylated protein of about 25 kilodaltons (kd).

The protein undergoes glycosylation at three sites. The first

site is a small C-terminal glycophospha-tidylinositol (GPI)

moiety, which links the prion to cell membranes; this tells us

that the prion protein has a cell-associated form. The other

two sites are more extensive glycosylations at the asparagine

amino acids in positions 181 and 197. Sulfide-containing amino

acids at positions 178 and 213 form a disulfide bridge that constrains

the motion of the molecule. In fact, the molecule is

quite restricted in its movement for all except the first

(N-terminus) 90 amino acids.

Molecular biology techniques have been extended to functional

analyses of prion proteins. Mice have been raised in

whom Prnp (the designation for the prion protein gene in mice)

has been eliminated or knocked out of their genome. Such

knock-out mice are apparently capable of surviving and reproducing

normally, suggesting that prion protein does not

appear to be important for these functions. Interestingly, pathogenic

prion infection does not lead to disease in Prnp knockout

mice. Therefore, pathogenic prion must recruit normal

prion to convert to an isoform that is pathogenic. With advancing

age, some Prnp knock-out mice present with symptoms

of neurological abnormalities, suggesting that prions may

play a role in normal nerve-impulse transmission and the production

of an insulative coating around the nerve fiber that

aids in electrical conduction within the length of the nerve

cells. Some knock-out mice also show disturbances in sleep

patterns (oddly enough, the major symptom of a human TSE

called familial fatal insomnia (fFI) is sleep disturbance, specifically

insomnia as the name indicates).

Prion diseases can develop spontaneously through a single

nucleotide polymorphism (SNP) or replacement of one nucleotide

for another in the sequence of nucleotides that comprise

genetic information within DNA. Every three nucleotides encode

for a particular amino acid and are referred to as a codon.

If one nucleotide is replaced for another, the replaced nucleotide

can result in a codon that translates into a different amino

acid in the programmed sequence comprising the prion pro-

Continues on page 14

Note: Data for the number of publications cited in the last five years from

Medline has been omitted because of the artificial inflation that would

appear since the National Library of Medicine expanded the scope of entries

into its database to include published works from 1949 to 1965 – a period

when no citations for prion or TSE appear in the database.

Figure 1. Exponential growth of prion research activity compared with

the linear rate of growth of medical research

14 September 2005 ¦ MLO www.mlo-online.com

C O V E R S T O R Y

tein. Such SNPs can occur naturally; when they result in disease

in humans, the disease is called sporadic CJD (sCJD).

Prion diseases are, of course, transmissible and have been

shown to develop in response to biological products (human

growth hormone, chorionic gonadotropin administration),

through the use of grafts (cornea or dura mater), or via contaminated

surgical instruments used in neurosurgery. Such

prion diseases are commonly referred to as iatrogenic CJD

(iCJD).

Finally, transmission can occur through ingestion within

the gut, and recent data, discussed further below, show that it

is possible for infected subjects to be free of symptoms for

quite some time. Infected persons who become blood donors

may be a source of infection in recipients of transfused blood

products. Ingestion of beef from asymptomatic BSE-infected

cattle has led to over 150 cases of vCJD, predominantly in the

U.K. Among these, there are two suspected cases of transfusion-

transmitted vCJD.

Normal prion protein is widely distributed throughout the

body but has its highest concentrations in neural tissue.

It is found within the central nervous system and in secondary

lymph organs including lymph nodes, the spleen, and

Peyer’s patches in the gut.

Prion protein has been

found in the placenta and

muscle. Another interesting

observation is that

noninfectious prion protein

has been detected in

the urine of TSE-affected

animals and humans. We

have already learned that

through the presence of

GPI linkages, prion protein

has a cell-associated

form. Now, with the observation

that prion protein

can be found in urine,

which is largely free of

cells, we know that prion

can exist in a soluble form

as well. The fact that

prion can have both cellassociated

and soluble

forms may have implications

for transmissibility,

particularly as it relates to

transfusion.

The W Western estern blot assay

Before moving on to

transmissibility, however,

it is important to understand

how prion is detected. Here, too, techniques applied

within the discipline of molecular biology are important, with

the Western blot being the most informative method of detection

to date. The Western blot can help differentiate

pathogenic from normal prion protein. The Western blot

assay involves subjecting a liquid sample or tissue homogenate

to the proteolytic action of the enzyme Proteinase K

(PK). There are rare exceptions to the rule that PK degrades

normal prion protein whereas it leaves the pathogenic form

of prion largely unaffected and therefore resistant. The nonpathogenic

form of prion is often referred to as PrPC because

it is found normally in cells or PrPsen because it is sensitive

to PK. In contrast, the pathogenic form of prion is more

commonly referred to as PrPres because it is resistant to PK.

PK-digested sample, when placed onto a polyacrylamide

gel and subjected to electrophoreses, results in the separation

of the sample into three bands of proteins in most species.

In the electroblotting step, the proteins are transferred

with the aid of an electric current to nitrocellulose or another

suitable membrane. The membrane containing the

transferred protein is subjected to immersion into a solution

of antibody directed against prion protein. After the membrane

is washed, a secondary antibody conjugated to an enzyme

is employed. Then the prion protein can be visualized

by exposing the membrane to substrates for the linked enzyme,

which in turn catalyzes a chemiluminescent reaction

wherein the light that is given off can be captured on film.

The developed images appear much like the one shown in

Figure 2A.

In 1956 and 1957, another form of human TSE referred

to as kuru reached epidemic proportions through

the practice of cannibalism among the Fore people

of Papua New Guinea.

Continues on page 16

For Approved R&D For Approved R&D

sale sale

Table able 1. Summary of BSE diagnostics tests on the market or in development

**Approved for use by the European Food Safety Authority as of February 16, 2005

Company Technology echnology Postmortem Antemortem

Immunoassays

Abbott/Enfer/ ELISA, Automated X

Protherics Sample Prep** X X X (ELISA)

Bio-Rad ELISA X X X

(ELISA)

Prionics/Roche Western, estern, ELISA, X

Lateral Flow Immunoassay** X X X (ELISA)

InPro/Beckman CDI X X X

CediT CediTect** ect** ELISA X X

Institut Pourquier ELISA X X

Speed’it BSE**

Roboscreen Beta ELISA X X

Prion BSE**

Roche Applied ELISA X X

Science PrionScreen**

Synthetic ligand technologies

Idexx/Microsens** Spherion polymer ligand X X X

Adlyfe Synthetic peptide X X

16 September 2005 ¦ MLO www.mlo-online.com

C O V E R S T O R Y

The foregoing discussion left us with an understanding of

the size of the prion protein estimated at 25 kd in the

unglycosylated form, and the image shows it — along with

two other bands — representing the mono- and

diglycosylated forms of the prion protein. In contrast, PrPC

(or PrPsen) will not appear on the Western blot because the

PK-digested fragments would be so small as to cause them

to migrate off the gel.

PK sensitivity or resistance reflects just one of a number of

differences in the chemical properties of the normal versus

the pathogenic form of the prion protein. PrPC is soluble in

aqueous solution, but PrPres is not. Consequently, it has been

difficult to study the three-dimensional structure of PrPres since

many of the methodological approaches for such studies require

the target compound to be present in solution. PrPres

may have the same amino acid sequence as PrPC, along with

the same post-translational glycosylations and disulfide bond.

PrPsen, however, shows a three-dimensional structure, which

contains an alpha-helical configuration along about 40% of

its length, whereas only 3% is in what is known as a beta-sheet

configuration. In contrast, PrPres appears to possess less (about

20%) alpha-helical conformation, and a major portion (over

50%) is in the beta-sheet form. Therefore, pathogenic prion

would be expected to be more apparent in tissue than the

soluble, innocuous counterpart, and it is not surprising that

the brains of afflicted individuals and laboratory animals appear

riddled with holes that look like fat globules.

The Western blot has revealed differences in the intensity

and migration patterns of the three bands representing the

un-, mono- and diglycosylated forms of prion. The relative

intensity of the bands in the Western blot reflect different

amounts of glycosylated prion and may be used to differentiate

one subtype of prion disease from another. Moreover, the

migration of the unglycosylated form may present in one of

two positions on the gel, which co-migrate with a protein

marker of either 19 kd or 21 kd — the larger and more slowly

migrating band is referred to as type 1 and the faster 19 kd

band as type 2. Some of the clinical syndromes can be described

in terms of their homozygous or heterozygous property at

codon 129 combined with a type 1 or 2 electrophoretic mobility

pattern in the Western blot.

Clinical syndromes

Although symptoms characterizing different clinical syndromes

vary, the brains of afflicted individuals show remarkable

similarity upon gross inspection. Human prion diseases

are referred to as TSEs because they may be passed from animal

to animal of the same species; and the brain degenerates

so visibly as to appear, upon gross inspection and histopathologic

examination, like a sponge with holes. Damage is confined

largely to the gray matter. This is often accompanied, as

in vCJD, with abundant amyloid plaque formation. Amyloid

plaque is a translucent proteinaceous substance with a waxy

consistency. It is made of protein in combination with sugars

(polysaccharides) and may be associated with Alzheimer’s disease

and other disorders.

Furthermore, it has been noted that SNPs in codon 129 of

the human prion coding region [i.e., methionine (MET) homozygosity,

versus valine (VAL) homozygosity, versus methionine/

valine (MET/VAL) heterozygosity] along with the

types of bands present on Western blot [i.e., type 1 (21 kd)

versus type 2 (19 kd)] may be used in a system to characterize

the clinical and histopathological manifestations of various subtypes

of spontaneously occurring CJD or sCJD.

Occurring in roughly one in 1 million persons, sCJD is

generally characterized by varying degrees of awareness or

cognitive impairment and psychosis, along with ataxia (lack

of coordination), visual field defects, and other neurological

manifestations, with onset most common in the seventh decade

of life. The most common subtypes are characterized

by the SNPs and migration patterns on the Western blot as

seen in Figure 2B. They include MET homozygosity and

type 1 (19 kd) migration, referred to as MM1. Another subtype

is characterized by VAL homozygosity and type 2 (21

kd) migration, denoted as VV2. Finally, heterozygous codon

129 with type 1 migration, referred to as MV1, is the third

most frequent subtype. These subtypes run their courses to

a fatal outcome in a matter of four to six months, while other

subtypes may proceed more gradually over 15 to 17 months

on average.

Some prion diseases appear to occur on a familial basis.

Familial CJD (fCJD) is very similar to sCJD in terms of its

clinical and histopathological manifestations. Familial fatal

Continues on page 21

diglycosylated

unglycosylated

monoglycosylated

1 2 3 4 markers

Serial dilutions

30 kd diglycosylated

27 kd unglycosylated

1 2 3 4 5 6

21 kDa type 1

19 kDa type 2

sFI-MM2 MM1 VV2

MV2

MM2

VV1

Panel B. Adapted from Gambetti P, Kong Q, Zou W, Parchi P, Chen SG.

Sporadic and familial CJD: classification and characterisation. Br Med

Bull. 2003;66:213-239 and Collinge J, Sidle KC, Meads J, Ironside J, Hill

AF. Molecular analysis of prion strain variation and the aetiology of ‘new

variant’ CJD. Nature. 1996;383(6602):685-690.

Panel A. Adapted from Thackray AM, Madec JY, Wong E, et al. Detection

of bovine spongiform encephalopathy, ovine scrapie prion-related protein

(PrPSc) and normal PrPC by monoclonal antibodies raised to copper-refolded

prion protein. Biochem J. 2003;370(Pt 1):81-90.

Figure 2. Western estern blot of various dilutions of Proteinase-K-treated tissue

from a prion-infected animal.

www.mlo-online.com MLO ¦ September 2005 21

P R I O N B I O L O G Y

insomnia is another such disorder characterized by intractable

insomnia, myoclonus (muscle twitches), and autonomic

dysfunction with a mean age at onset of 49 years and a duration

of 11 to 23 months. Finally, a familial prion disease due

to a PRNP open reading frame mutation, or SNP, is known

as Gerstmann-Straussler-Scheinker syndrome (GSS). It is

characterized by a gradual progression of cerebellar ataxia,

dementia, spastic paraparesis (weakness of the legs), and extrapyramidal

signs over five to six years beginning in the fifth

decade. Extrapyramidal signs include involuntary movements

of the mouth, lips, and tongue as well as tremors, restlessness,

or rigidity, among other things.

Most importantly, a number of prion diseases have a recognized

mode of transmission. Kuru, as discussed earlier, was

one of the first to be uncovered. The Fore people, indigenous

to Papua New Guinea, gave this disorder its name, which

means "to shiver." Characterized by progressive and ultimately

fatal cerebellar ataxia over six to nine months, onset usually

occurred between the second and fourth decades.

It is possible to contract prion disease from contaminated

surgical instruments as well as biological preparations, and the

resulting disease is referred to as iatrogenic CJD (iCJD). Depending

upon the source of infection, the onset varies among

those contracting iCJD. Infection sources include neurosurgical

instrumentation (with onset in 12 to 28 months), human

growth hormone (50 to 450 months), corneal transplantation

(16 to 320 months), dural patches (18 to 216 months), and

human gonadotropin (144 to 192 months).

Of greatest concern, however, is vCJD, which occurs years

after the ingestion of meat products containing traces of neural

tissue from cows infected with BSE. Despite this long latency

period, there has been a mean age at onset of 28 years.

vCJD is manifested by psychiatric and sensory disturbances

and dementia that progresses to its inexorably fatal outcome

in an average of just over one year. It is believed that

neuroinvasion of pathogenic prion is facilitated by white blood

cells, particularly B-lymphocytes, that are present in intestinal

Peyer’s patches. Recently, two cases of vCJD transmitted

by blood transfusion have been reported. This raises concerns

about protecting the blood supply from prion diseases. Screening

methods for prion detection in blood have been elusive.

Adding to the level of concern is the fact that the incidence

of progressive neurological diseases, such as

Alzheimer’s disease, has increased dramatically in recent

years, even when adjusted for the increasing population of

elderly individuals. These progressive disorders are diagnosed

almost exclusively based upon the clinical observation of signs

and symptoms that overlap with those of CJD. Recently, histopathological

examination of the brains of patients thought

to have died from Alzheimer’s disease revealed that 8% of

patients in one group and 26% of patients in another group

actually died of prion disease. Therefore, TSEs may be under-

reported illnesses. If substantiated, such diagnostic uncertainty

will make laboratory testing of pathogenic prion in

man and animals an important addition to the testing armamentarium

of laboratorians. But just how prevalent TSEs may

become is related to the resistance to infection across species

— the so-called species barrier.

Species pecies barriers

There are many examples of the transmission of prion disease

within a given species. There is, however, a barrier to disease

transmission when the source pathogenic prion derives from

a different species. The essence of the protein-only hypothesis

of prion propagation is that PrPSc replicates itself by recruiting

PrPc molecules and inducing a conformational change,

which results in the accumulation of more PrPSc. This PrPSc

may, in turn, convert more of the cellular isoform to the pathogenic

form. Based upon this hypothesis, it might be expected

that prion diseases are transmissible between different species.

Indeed, experimental transmission across mammalian species

has been well established.

Interspecies transmission, as measured by the appearance

of clinical signs in the host, is frequently limited by a species

barrier. This barrier has been characterized by (1) longer

latency periods between infection and symptom onset, which

may even exceed the animal’s typical lifespan, (2) atypical signs

of disease in the recipient animal, and (3) a reduced rate of

recipient animals succumbing to disease relative to the rate

noted in the species of the source animal. Following the transmission

of PrPSc across a species barrier, serial passage of

pathogenic prion within that same recipient species is characterized

by shorter latency periods and more uniform signs

of disease. The number of passages required for this to occur

is one way to quantify the magnitude of the particular

species barrier. Additional evidence for the species barrier

has been demonstrated in transgenic mice that overexpress

Syrian hamster PrP transgenes. These mice, in contrast with

their wild-type littermates, show no species barrier when infected

with hamster prions.

Several factors appear to contribute to the species barrier.

One is the degree of variability in PrP gene sequences between

that of the source animal and that of the recipient. Species

barriers can also be affected by PrPSc conformation, a feature

that also characterizes prion strains. Different strains

appear to have different transmission profiles. The form and

degree of PrPSc glycosylation are thought to be additional variables

determining transmissibility. Finally, an as yet unknown

factor termed "Protein X" has been hypothesized to have an

effect on the species-barrier phenomenon. Protein X, if it exists,

has not been characterized and may indeed be PrP itself.

The implications of the species barrier are that disease may

be more prevalent than is recognized. This is compounded by

the protracted latency from infection to manifestation of symptoms.

It is thought that the central nervous system tissue of

scrapie-infected sheep entered the food supply and was fed to

cows, which resulted in BSE. Thus, scrapie transmission to

humans represents an example of a species barrier that is

bridged by cows. There are other examples of TSEs from one

species affecting a second, and that species in turn becoming

infectious for yet another species. Therefore, the extent to

Continues on page 22

Recently, there has been some concern over the rising

incidence of prion disease in deer and elk (called

chronic wasting disease, or CWD).

22 September 2005 ¦ MLO www.mlo-online.com

veillance of vCJD. In the absence of an effective antemortem

test that can be used to detect the presence of infectious prion

in potential blood donors who may be carrying the causative

agent of vCJD, an alternative strategy must be considered to

protect the safety of transfusion recipients. One such approach

is to filter the blood to reduce or remove pathogenic prions.

Results from experimental models of TSE suggest that approximately

half of infectious prions are found within leukocytes

and the balance is found in plasma. Therefore, removal

of leukocytes from blood is a prudent and necessary first step

in minimizing the risk of transmission of vCJD. A recent report

suggests, however, that the current generation of leukocyte-

reduction filters was effective in removing only 42% of

the total TSE infectivity in endogenously infected blood.

A new filtration technology that reduces both leukocytes

and prions from the most often transfused blood product,

packed red blood cells, is nearing commercialization. This filter

reduces leukocytes and their associated prions by more than

three orders of magnitude. Concerning the plasma-associated

infectivity, in a preliminary study, a prototype of the filter was

shown to reduce prion infectivity by approximately four orders

of magnitude. Preliminary results of an endogenous infectivity

study show the filter also prevents the transmission

of prion disease in hamsters. Although the risk of transfusiontransmitted

vCJD is not quantifiable at this time, the use of

prion-reducing filters will provide a necessary measure of protection

until such time as the risk is better understood either

through antemortem blood testing or clinical surveillance.

Summary

Although eerily silent for many years after the recognition of

scrapie in 1759, TSEs remained present within the genome of

some mammals. Not since the mid-1950s when Dr. Carleton

Gadjusek visited the Fore Indians of New Guinea to study

kuru, however, has there been a more frenetic interest by governmental

investigators. Certainly, the U.K. experience has

heralded a renewed interest in TSEs due to the notoriety associated

with younger subjects succumbing to a variant CJD

traced to the ingestion of beef. Human TSEs and the potential

for their transmission among and across species of mam-

C O V E R S T O R Y

which other species may be potential sources of human TSE

is not well understood. The best information we have comes

from the National CJD Surveillance effort in the U.K. as shown

in Figure 3. The peak of exposure in the human food chain to

BSE-infected cattle occurred in 1989, and the peak in vCJD

appears to have occurred in 2001, suggesting a latency of 12

years from infection to symptom onset.

It appears that some of those asymptomatic citizens of the

U.K. became blood donors and, as noted previously, there are

two cases of vCJD victims who received blood product from

confirmed vCJD donors. There may be other species incubating

TSEs with longer latencies for which humans may be

susceptible; thus, the sense of urgency for developing tests for

TSEs is warranted.

BSE testing

Aside from the Western blot assay, the only tests on the market

are enzyme-linked immunosorbent assays (ELISA) or other

immunoassays based upon the use of antibodies that do not

possess specificity for pathogenic prion as opposed to normal

prion protein. Like the Western blot, sample pretreatment with

Proteinase K is a requirement. Table 1 provides an overview

of the assays currently available and some of those known to

be in development. There is a lack of antemortem assays, which

would be most widely used to screen cattle for their status as

satisfactory subjects for introduction into the food chain. The

only available antemortem test requires a veterinarian to access

the lymphoid tissue in the nictitating membrane (so-called

third eyelid) of cattle. The sampling technique shows about a

60% success rate, and the assay has a sensitivity of 99% and

specificity of only 70%, leaving quite a margin of opportunity

for the development of better assays.

Protecting the safety of the blood supply is being approached

in yet another way. There are ongoing efforts to

manufacture products that can either reduce or remove prions,

including pathogenic prions, that are either cell-associated or

free in plasma or both.

Filtration of blood for prion removal

The recent occurrences of probable cases of transfusion-transmitted

vCJD raise concerns about the safety of the blood supply.

Because there is a long latency from infection to symptom

onset and there is no antemortem test to screen for infectivity,

aymptomatic blood donors may be compromising the safety

of our nation’s blood supply. Although the U.K. experience of

susceptibility to vCJD suggests that codon 129 MET homozygotes

may predispose individuals to disease, one of the two

reported cases of transfusion-transmitted vCJD was detected

in a patient who was a heterozygote MET/VAL at codon 129.

The distribution of codon 129 is such that M/M and M/V

together comprise about 88% of the Caucasian population of

the developed nations. According to some thought leaders,

these findings have major implications for results from sur-

The peak of exposure in the human food chain to BSEinfected

cattle occurred in 1989, and the peak in vCJD

appears to have occurred in 2001, suggesting a latency

of 12 years from infection to symptom onset.

Figure 3. TSE Mortality for the U.K., 1990- (Data from 2003 are incomplete).

www.mlo-online.com MLO ¦ September 2005 23

P R I O N B I O L O G Y

mals has also captured the attention of many. Yet, to date, there

is no reliable antemortem test available to screen for infected

animals or humans. Antibody-based assays are difficult to develop

because most of them do not have specificity for the

pathogenic form of prion protein.

Whether or not prion testing efforts will change dramatically

depends upon the incidence of disease. Some speculate a

reduction in testing, because BSE incidence is waning since

the adoption of remedial steps in the U.K. in 1989. Others

remind us, however, of the long latency of prion diseases and

of the recent observations of two patients who succumbed to

vCJD after having received blood products from donors who

subsequently died of vCJD. The growing incidence of CWD,

combined with the emerging observation that as many as 26%

of Alzheimer’s patients may have been misdiagnosed — having

died instead of prion disease — maintains pressure for legislators

to adhere to the precautionary principle and support

blood-donor exclusionary criteria, antemortem-test development,

and pathogen removal from donated blood. The laboratorian

can expect to see new tests for prion disease work

their way into clinical-testing practice in the near future. In

addition, the adoption of newer filtration technologies holds

the promise of improved protection from transfusion-transmitted

prion disease.

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Girolamo A. (Jerry) Ortolano, PhD, graduated from Columbia University (BS)

and the University of Rhode Island (MS, PhD - Pharmacology), completed a postdoctoral

fellowship at the University of Michigan Hospital, and continued research

there before joining Pall Corporation. He has authored over 60 scientific articles

and abstracts and co-authored five book chapters. Samuel O. Sowemimo-Coker,

PhD, is principal scientist and Jeffrey Schaffer, DVM, is New Initiatives staff scientist

at Pall Medical in Port Washington, NY. Joseph S. Cervia, MD, FACP, FAAP,

is medical director and senior vice president, Biomedical Division at Pall Medical

in East Hills, NY, as well as professor of Clinical Medicine and Pediatrics at Albert

Einstein College of Medicine in the Bronx, NY.