Mollison’s Blood Transfusion in Clinical Medicine - part 9 pdf

Self-exclusion ‘self-deferral’ of donors Retrospective analysis indicates that the risk of con-tracting AIDS from the transfusion of blood and blood components other than products of pla

The first reported case of transfusion-associated

AIDS (TAA) turned out to be an 18-month-old infant

with severe combined immunodeficiency who had

been transfused repeatedly at birth and had received a

unit of platelets from a donor who subsequently

devel-oped AIDS (Amman et al 1983) By 1984, 38 cases

of AIDS had been reported in patients with no risk

factors other than a history of transfusion Nineteen

of the patients were adults who, during the previous

5 years, had received blood components derived from

unpooled donations In those cases in which all the

donors could be followed up, an individual in a

‘high-risk’ group could always be identified (Curran et al.

1984) An expanded study of 194 patients showed that

in most cases the high-risk donor was anti-HIV

posit-ive, and in those few cases in which the high-risk

donor lacked anti-HIV, another donor tested positive

(Peterman et al 1985) A total of 157 525 cases of

AIDS was reported in the USA by the end of 1990, and

5371 (3.4%) of these were attributed to the

trans-fusion of blood or blood components The median

incubation period, estimated as the time from

expos-ure by transfusion to diagnosis of AIDS, has been

estimated to be 34 months in adults and 22 months in

children (Peterman 1987), although later estimates

indicated a longer period with a median of 7–8 years

for adults and 3 –5 years for children (Rogers 1992)

In total, 50% of untreated subjects infected with HIV

by transfusion will develop AIDS within 7 years

com-pared with 33% of subjects infected by other routes

(Ward et al 1989).

Following the first reports, the number of cases of

TAA in the USA increased rapidly By the end of 1991,

out of a total of 206 392 cases of AIDS, there were

6060 adults and 472 infants or children who had

acquired the disease by transfusion of blood or blood

products, representing 3% and 13.6% of the total

cases in adults and children respectively (CDC 1992)

The risk of transmission of HIV by blood transfusion

is now trivial in countries without major heterosexual

spread and in which donor education, encouragement

of self-exclusion and screening for HIV antibodies

have been established since 1985 In these countries,

the risk of transmission of HIV is almost solely

attributable to donations given during the window

period (see below)

In western Europe, HIV prevalence among blood

donors has declined progressively since the onset of

systematic testing and, according to the European

Centre for Epidemiologic Monitoring of AIDS,approximated 1.3 positives per 100 000 donations

in 2002 (www.eurohiv.org) Since 1995, the ence has remained relatively constant in Belgium,Scandinavia, Ireland, the Netherlands and the UK, hasdecreased regularly in France and Spain, but hasremained above 2 per 100 000 in Italy, Greece andPortugal (Hamers and Downs 2004) In EasternEurope, prevalence has increased markedly since

preval-1995, now exceeding 30 per 100 000 in 2002 Thehighest levels are reported in the Ukraine (93), Estonia(54) followed by Azerbaijan, Georgia and the RussianFederation

Because HIV is both cell associated and present inplasma, all blood components are potentially infec-tious (Curran 1985) The viral load has been estimated

at between 1.5 × 104tissue culture infective doses in a250-ml unit of blood from an asymptomatic donor,

to 1.75 × 106from a unit drawn from a symptomatic

person (Ho et al 1989) The relative importance of the

viral strain, concurrent infection by other blood-borneagents, cellular receptors for HIV and other geneticand acquired host factors, both for infection and forclinical course of HIV in transfusion recipients, hasreceived a great deal of attention, but is still an area

of intense research (Vicenzi et al 1997; Keoshkerian

et al 2003; Zaunders et al 2004).

Follow-up studies have shown that 90–95% ofpatients receiving blood or blood components fromanti-HIV-positive donors have become infected (Ward

et al 1987; Donegan et al 1990b) The virus is well

preserved in refrigerated and frozen blood; however,components that are washed, leucoreduced or coldstored for several weeks, procedures that diminish the number of viable leucocytes or the amount of virus, reduce the likelihood of transfusion transmis-

sion (Donegan et al 1990a) Neither donor status nor

recipient characteristics affect the likelihood of HIV

transmission (Busch et al 1990a) However, when

AIDS developed in the donor shortly after donation,the period of asymptomatic infection in the recipient

was also shortened (Ward et al 1989) Albumin

pre-parations, immunoglobulins, antithrombin III andhepatitis B vaccine have not been associated with HIVinfection (Desmyter 1986; Melbye 1986; Morgenthaler1989; Cuthbertson 1991) Furthermore, when HIV isadded to plasma and the plasma is then fractionated bythe cold-ethanol process, HIV does not appear in the Igfraction (Morgenthaler 1989)

TAA in infants, and AIDS in infants and children in

general, has a shorter incubation period than in adults

and many of the clinical manifestations of the disease

are different (Rogers 1992) Infants born to

anti-HIV-positive mothers who become infected perinatally and

infants transfused during the first years of life have the

shortest median incubation period (less than 5 years),

and usually develop AIDS in the first year of life The

increased susceptibility of infants to AIDS may be

related to their immature immune system and to the

larger viral load relative to body size Children likely to

develop TAA are those who are likely to be transfused:

premature infants and children with haemophilia,

thalassaemia and sickle cell anaemia In the USA, of

2734 children with AIDS, 250 (9.1%) were

transfu-sion associated and 138 (5.0% of the total) occurred in

children with coagulation factor deficiencies (CDC

1991)

In much of Asia and Africa, the transmission of HIV

by blood transfusion is still an important source of

infection Reasons for an alarmingly high rate of

trans-mission, reported to be up to 10% of all cases, include:

(1) the high demands for blood for inpatients with

severe anaemia and haemorrhage, mainly in obstetrics,

gynaecology and paediatrics; (2) the prevalence of

HIV infection amongst the donor population, which

can be as high as 20%; (3) the fact that HIV infection is

not confined to a minority of the population who can

be requested to refrain from blood donation; and

(4) the inability of many laboratories to test for HIV or

to perform and control the tests properly The patient

groups at greatest risk of acquiring HIV-1 or -2 by

blood transfusion in tropical Africa are children with

malaria and anaemia, patients with sickle cell anaemia

(120 000 infants with sickle cell disease are born each

year in Africa), anaemic antenatal patients, women

with severe obstetric haemorrhage and trauma victims

(Fleming 1990)

Transfusion-associated AIDS and haemophilia

To December 1996, 4674 cases of AIDS were reported

in patients with haemophilia, accounting for less

than 1% of the 581 429 AIDS cases reported in

adults and children Of the 7629 cases of AIDS

reported in children under the age of 13 years, 373

(5%) were recipients of blood or tissue and 231 (3%)

were haemophiliacs (Centers for Disease Control and

Prevention 1997) All but 39 of these infections

occurred prior to testing blood and plasma donors for HIV

Studies of patient cohorts and specimens in serumrepositories revealed that more than 90% of severehaemophiliacs (subjects with less than 1% factor VIIIactivity) treated with factor VIII concentrate had been

infected prior to 1984 (Evatt et al 1985) In a study of

a 16-centre cohort of haemophiliacs in the USA andEurope, infections first appeared in 1978, peaked

in October 1982 and declined to an estimated fourinfections per 100 person-years by July 1984 (Kroner

et al 1994) For patients who were high-dose

recipi-ents, peak risk appeared even earlier, indicating thatthe majority of patients with haemophilia wereinfected before the disease was widely recognized andlong before it was attributed to transfusion The riskwas related to each patient’s mean annual dose of clot-ting factor concentrate As clotting factor concentratesare prescribed on the basis of patient weight or plasmavolume, older patients with severe disease generallyreceived more concentrate, had more ‘donor expos-ures’ and seroconverted sooner than did children andpatients with milder disease When corrected for doseand severity of disease, the association between ageand early seroconversion disappears The cumulativeincidence of infection was 96% for high-dose recipi-ents, 92% for moderate-dose recipients and 56% forlow-dose recipients Subjects who received only single-donor products (plasma and/or cryoprecipitate) hadthe lowest cumulative incidence of infection, 16%

(Kroner et al 1994) This experience is consistent with other reports (Andes et al 1988; Gjerset et al 1991).

These startling numbers underscore the potential public health risks of using transfusion products manufactured from pools of plasma drawn from

20 000 donors or more

Although AIDS was first reported in threehaemophiliacs in 1982, studies of serum samplesstored from as far back as 1968 have shown that the first cases of the development of anti-HIV inhaemophiliacs occurred in 1978 in the USA and in

1981 in the UK (Evatt et al 1983; Machin et al 1985; Ragni et al 1986) Plasma for preparing the implicated

clotting factor concentrates may have been collected ayear prior to that The prevalence of HIV seropositiv-ity and of AIDS varies from one haemophilia centre toanother depending on the source, volume and type ofconcentrate used In a study of 13 haemophilia centres

in western Europe, Canada and Australia involving

2370 patients with haemophilia A and 434 patients

with haemophilia B, the overall incidence of

anti-HIV was 53.6% (CDC 1987a) The percentage of

haemophiliacs infected with HIV in different countries

has varied from 4% to more than 60%, with the higher

rates in those countries using mainly concentrates

from plasma imported from the USA Some batches of

factor VIII concentrate from European plasma have

also transmitted HIV (Madhok and Forbes 1990) A

clear correlation was also found between the severity

of haemophilia and HIV seropositivity (Melbye 1986;

UK Haemophilia Centre 1986) Haemophiliacs treated

before 1985 with cryoprecipitate alone have shown

a low risk of HIV infection (Ragni et al 1986; UK

Haemophilia Centre 1986)

Patients with haemophilia B fared somewhat better

(Evatt et al 1984; Mannucci et al 1985; Ragni et al.

1986; UK Haemophilia Centre 1986) The difference

may be due partly to the uneven partitioning of HIV in

infected plasma during fractionation, with HIV

sepa-rating preferentially in the cryoprecipitate fraction

(Aronson 1979; Morgenthaler 1989; Madhok and

Forbes 1990) The source of plasma used for

fractiona-tion is also partly responsible for this difference In

many countries all the factor IX is prepared locally,

whereas at least part of the factor VIII is imported

from the USA Approximately 70% of patients in the

USA with haemophilia A and 35% with haemophilia

B developed HIV antibodies before the introduction

of methods for viral inactivation in blood products

(CDC 1987b) In a multicentre study of haemophiliacs

treated in the UK between 1980 and 1984, 896 (44%)

of 2025 patients with haemophilia A were positive for

anti-HIV; 20 (6%) of 324 patients with haemophilia B

and 11 (5%) of 215 patients with von Willebrand’s

disease were seropositive

Although a large number of severe haemophiliacs in

the UK were treated up to 1983 with unheated factor

VIII concentrate imported from the USA, the incidence

of anti-HIV in haemophiliacs in the UK is much lower

than in countries such as Germany, Spain and the USA,

where the frequency of anti-HIV ranges between 68%

and 94% (Kitchen et al 1984; 1985) On the other

hand, in Groningen, in the Netherlands, only 1 out

of 18 severe haemophiliacs treated with commercial

factor VIII concentrates between 1978 and 1983

developed HIV antibodies (van der Meer et al 1986).

There has been no transmission of HBV, HCV

or HIV by US-licensed plasma derivatives since the

introduction of effective virus inactivation procedures(Tabor 1999)

Prevention of transfusion-transmitted HIVinfection

Reducing the residual risk

The reduction in risk of transfusion-transmitted HIV over the past 15 years has been dramatic and reassuring Nevertheless, enormous public concernpersists As in almost no other area, blood safety, and specifically the possibility of HIV transmission,provokes emotions and measures to further reducerisks that defy the conventional cost–benefit calculus.Actions to reduced residual risk fall into three generalcategories: (1) measures that can be introduced byblood collection facilities, such as improved donorscreening, testing, education and exclusion techniques;(2) enlightened transfusion practice, such as judicioususe of allogeneic blood components, appropriate use

of autologous blood, and alternatives to transfusion(see Chapter 17); and (3) measures that depend on thedevelopment of new technologies, such as viral inac-tivation of cellular components and safe substitutes for blood

Donor demographics have proved effective at fying and excluding donors at high risk for infection

identi-and transmission of HIV (Busch et al 1991a) Donors

with high-risk profiles include men who have had sexual contact with other men since 1977, intraven-ous drug users, residents of high prevalence regions,prisoners, prostitutes, haemophiliacs who have received

‘unsafe’ clotting factor concentrates and the sexualpartners of people in all of these groups The year 1977was chosen as the point of reference, because the firstclinical cases of AIDS in the USA were diagnosed retro-

spectively as far back as 1978 (Jaffe et al 1985) The

rate of seropositivity has been higher in paid plasmadonors than in volunteers Seven HIV positives werefound out of 35 000 plasma donors attending centres

located outside high prevalence areas (Stramer et al.

donor education are not infallible (Leitman et al.

1989) Between 1% and 2% of donors do not report

risks that would disqualify them from blood donation,

and donation incentives such as complimentary

labor-atory testing increases this rate (Glynn et al 2001)

In a study of blood donors found positive for hepatitis

C antibody, 42% admitted to intravenous drug use on

subsequent questioning, despite denying such use on

predonation screening (Conry-Cantilena et al 1996).

In an anonymous mail survey of 50 162 volunteer

allo-geneic blood donors, 1.9% of the 34 726 respondents

reported one or more risk factors that should have led

to their deferral at the time of their last donation

(Williams et al 1997) Refinements in blood donor

screening techniques, such as the use of illustrated

risk-related activities, expansion of the screening

question-naire and interactive computer-based screening, have

been proposed, but data supporting the effectiveness

of such measures are lacking (Mayo et al 1991)

How-ever, the reasons why some volunteer blood donors

appear to disregard certain screening criteria are

un-known and may have more to do with donor

psycho-logy than with inadequate screening and education

Donors who are confirmed positive for HIV should

be counselled and referred to specialized centres for

follow-up Counselling should be performed by

spe-cially trained staff Appropriate interventions will not

only help the donor to obtain long-term supportive care

and to prevent further spread, but will also aid

transfu-sion services to understand which groups of the

popu-lation are HIV seropositive and still come forward

to donate (Lefrere et al 1992) Donor education and

selection methods can then be modified accordingly

Self-exclusion (‘self-deferral’) of donors

Retrospective analysis indicates that the risk of

con-tracting AIDS from the transfusion of blood and blood

components (other than products of plasma

fractiona-tion) prior to focused screening and testing was far

higher than the one infection per million units

trans-fused that had been estimated during the 1983–1985

interval The risk of HIV transmission from

trans-fusion in San Francisco has been calculated from its

first appearance in 1978 to rise exponentially to a peak

risk of approximately 1.1% per unit transfused in

1983 (Busch et al 1991a) A retrospective study of

heavily transfused patients with leukaemia in New

York City revealed an overall risk between 0.02% and

0.11% per component transfused (Minamoto et al.

1988) The major blood collectors published a Joint

Statement of Recommendations in January 1983

(American Association of Blood Banks et al 1983),

and the Public Health Service published tions in March 1983 (CDC 1983) that proposed suchmeasures as public education, self-deferral for donorsengaging in high-risk activity and confidential unit

recommenda-exclusion procedures (Pindyck et al 1984, 1985) These

measures proved unusually effective An estimated90% of men in high-risk cat-egories self deferred By

1984, the risk in San Francisco had dropped to lessthan 0.2% per unit Screening with anti-HIV-1 in 1985

reduced the risk to about 1 in 40 000 units (Busch et al.

1991a)

Routine screening tests for HIV in blood donors

In most countries, screening tests for anti-HIV by the enzyme immunoassay (EIA) format are now com-pulsory for all blood donations If reactive (‘repeatreactive’), additional tests for HIV using independentmethods are used to confirm the diagnosis of HIV

infection (Stramer et al 2004) The current algorithm

in the USA requires that anti-HIV-1/2 repeat reactivespecimens be further tested with the HIV-1 Westernblot (see below) and a specific anti-HIV-2 EIA EarlyELISA assays using disrupted purified virus wereplagued by false-positive reactions Current genera-tion screening assays, using recombinant and syntheticantigens, have reduced false reactives dramatically whileincreasing test sensitivity for both the predominantand the variant viral strains (Busch and Alter 1995).Less than 10% of reactive assays confirm positiveusing this strategy Western blot is notoriously subjec-tive and complicated by non-viral bands (Kleinman

et al 1998) Alternative strategies use a second EIA or

a NAT assay of HIV RNA

Approximately 25.6 million donations were screened

by the American National Red Cross from September

1999 to 30 June 2003, resulting in 17 090 HIV repeat

reactive blood donations (Stramer et al 2004) Only

4.8% of these donors (818) were Western blot positiveand approximately 90% (759) of those also tested positive by NAT Follow-up testing of the remaining10% demonstrated that almost all of these donors represent Western blot false-positive results

Although some antibodies to core and other

anti-gens of HIV-1 and HIV-2 crossreact (Sazama et al.

1992), currently available tests are designed to detectboth anti-HIV-1 and anti-HIV-2 Such combined

assays are available as antiglobulin ELISAs and as

sandwich ELISAs and are routinely used in several

countries Seroconversion in infection with HIV-1 is

detected earlier in these combined assays than by

anti-HIV-1 assays (Gallarda et al 1994; Fiebig et al.

2003) Most of the combined assays have been found

to be less sensitive for the detection of anti-HIV-2

than most anti-HIV-2 specific tests (Christiansen et al.

1996)

Some samples with anti-HIV are repeatedly positive

in some, but negative in other screening assays If only

one screening test is used, such samples may give

false-negative results (Hancock et al 1993) One cause of

such false negatives is that antibodies against subtype

O, a variant found predominantly in West Africa, are

not recognized in all screening assays (Loussert-Ajaka

et al 1994; Schable et al 1994) At present group O

prevalence is low in the USA and in western Europe

(Sullivan et al 2000) False-negative reactions have

also been found to be due to contamination with glove

powder, inhibition by serum proteins, haemoglobin

and certain anticoagulants (Sazama 1995)

Currently available ELISAs for HIV-1 antibodies

detect HIV-1 subtype group 0 Soon after the

dis-covery of an assay for HIV-Ab, transmission of HIV by

blood from seronegative donors had been recognized

(Esteban et al 1985; Raevsky et al 1986; Ward et al.

1988; Cohen and Munoz 1989) Studies reported

detection of HIV-1 p24 antigen in analyses of stored

blood specimens from plasma donors as early as 1986,

and confirmed cases of HIV-Ag-positive, Ab-negative

blood in primary HIV infection were reported by 1988

(Allain et al 1986; Clark et al 1991; Irani et al 1991).

The utility of this test as a screening assay was not

so obvious Antigen tests are positive for only part of

the initial antibody-negative viraemic phase In some

subjects antigen can be detected as early as 2 weeks

after infection, persisting for between 3 weeks and

3 months, and is no longer detectable when anti-p24

appears in the serum, although it may reappear

inter-mittently during the asymptomatic phase (Allain et al.

1986; Fiebig et al 2003) Later, antigen may reappear

with a loss of anti-p24 A prospective study of 515 494

units donated at 13 blood centres in the USA failed

to detect a single instance of Ag-positive/Ab-negative

donated blood A retrospective analysis of 200 000

repository specimens and prospective studies of blood

donors in Europe confirmed these findings (Backer

et al 1987; Busch et al 1990a) However, after three

anti-HIV seroconversions followed transfusion of p24antigen-positive units, testing of donated blood for p24antigen was mandated in the USA in 1996 (Busch andAlter 1995) Mathematical models predicted that uni-versal antigen screening would detect eight additionalpotentially infectious units per year In fact it took

5 years before eight antigen-positive/antibody-negative

units were interdicted (Kleinman et al 1997; Kleinman

and Busch 2000) Because of this limited usefulnessand troubling frequency of false positives, HIV p24antigen screening was not adopted widely outside ofthe USA With the adoption of universal NAT for HIV

in the USA in 1999 and its licensure in 2002, HIV-Ag

screening was rendered unnecessary (Busch et al 2000).

Confirmatory tests: they do not always confirm

The Western blot is the most widely used additional

or ‘confirmatory’ test for HIV The criteria for theinterpretation of Western blot results have been re-evaluated several times because of greater sensitivityand specificity of screening assays and Western blotreagents, better insight into the serological patterns ofHIV infection, experience with patterns of non-specificreactivity in low- and high-risk populations andknowledge of the serological basis of non-specificity

(Sayre et al 1996) Samples are now considered to

be WB positive demonstrate reaction with the gp41

and gp120/160 env bands or with either of these bands and the p24 gag band The earlier requirement for a reaction with a third gene product (e.g p31 or p66 pol

bands) has been abandoned and reactivity with more

than one env antigen alone is enough for confirmation (O’Gorman et al 1991) If there is reactivity with only

one band, the result of the Western blot is considered

to be indeterminate The absence of reactivity in theWestern blot indicates that the donor has not devel-oped anti-HIV (Dodd 1991)

In the original Western blot assay, viral lysate wasused as a source of antigen In the newer assays, recom-binant or synthetic viral antigens are applied Theseassays have been found to be both more sensitive and

specific than the original WB (Soriano et al 1994).

Nevertheless, the assay is still relatively subjective and beset by indeterminate and false-positive resultswhen compared with NAT as the ‘gold standard’ orwhen investigated with sequential sampling follow-up

(Kleinman et al 1998; Mahe et al 2002; Stramer

2004)

Detection of HIV DNA and HIV RNA

Donations during the window period constitute the

predominant risk for HIV transmission through

transfusion (Busch et al 2000) A more sensitive and

specific alternative to testing for p24 antigen is NAT

for HIV DNA in PBMC (Ou et al 1988) or HIV-1

RNA in plasma by reverse transcription PCR or a

similar amplification assay (Henrard et al 1992) All

blood in the USA, Japan and most European

coun-tries is tested by NAT in pools of 16 –90 specimens

Ultrasensitive assays can detect fewer than 10 genomic

copies/ml (Busch et al 2000) However, tests have

been optimized for HIV subtype B and may lack

sensi-tivity when applied to non-B subtypes (Triques et al.

1999) Single-donor assays are inevitable, but await the

development of fully automated combination (HIV/

HCV/HBV) assays

Current risk of transmission of HIV by blood

transfusion

Although the various measures outlined above have

dramatically reduced the risk of TAA-AIDS, a small

residual risk remains (Delwart et al 2004; Phelps et al.

2004) Most of this risk results from ‘window period’

donations Before the introduction of HIV-Ag and

NAT, prospective studies estimated this risk at about

one infection in 60 000 units (Busch et al 1991b;

Nelson et al 1992) Subsequent estimates rely on

models based on calculations of HIV incidence and

window period In the USA, residual risk has been

calculated as 1 per 2 135 000 repeat donors (Dodd

et al 2002) The incidence rate is approximately

two times greater among first-time donors In most

European countries where the prevalence of HIV in

blood donors is lower than in the USA, the residual

risk is probably lower still However, in countries with

a high percentage of infected subjects and where HIV

is spread mainly by heterosexual intercourse, the risk

of transmission of HIV by blood transfusion is still

considerable

Human T-cell leukaemia viruses types I

and II

The human T-cell leukaemia virus type I (HTLV-I), the

first human retrovirus to be described, was isolated

from cultured cells from a patient with an aggressive

variant of Mycosis fungoides and from a patient with Sézary syndrome (Poiesz et al 1980; Gallo et al 1981).

The virus has subsequently been shown to be identical

to the adult T-cell leukaemia virus (Yoshida et al 1982; Watanabe et al 1984) HTLV-I is the causative

agent of adult T-cell leukaemia (ATL) and is ciated with a chronic demyelinating neurological disease called tropical spastic paraperesis (TSP), known

asso-in Japan as HTLV-associated myelopathy (HAM)

(Vernant et al 1987; Roman and Osame 1988)

HTLV-seropositive individuals appear to have a 0.25% time risk of developing TSP, compared with a 2–5%

life-risk of developing ATL (Kaplan et al 1990) The

virus is also associated with lung infections, cancer ofother organs, monoclonal gammopathy, renal failure,

infection with Strongyloides stercoralis, intractable

non-specific dermatomycosis, lymphadenitis anduveitis These effects may be due to the immunodefici-ency induced by HTLV-I infection (Takatsuki 1996)

The association of HTLV-I with Mycosis fungoides is

controversial, as no HTLV-related DNA sequencescould be detected in patients with this disease

(Bazarbachi et al 1993; Vallejo et al 1995) In Japan,

only 2.5% of HTLV-I carriers develop ATL (Takatsuki1996)

HTLV-I and -II belong to the oncovirus subtype ofthe retrovirus family and are able to induce polyclonal

proliferation of T lymphocytes in vitro and in vivo.

Like the lentiviruses HIV-1 and -2, these viruses arelymphotropic and neurotropic, and have the essential

structural genes gag (group antigen), pol (reverse scriptase) and env (envelope) in addition to regulatory genes In HTLV-I, the gag gene codes for the structural proteins p55/24/19; pol codes for a protein of approx- imately 100 kDa, and env codes for glycoproteins

tran-gp61/46/21 In HTLV-II the structural proteins aresimilar to those in HTLV-I with a high degree of cross-

reactivity; gag encodes the polypeptides p53/24/19; pol a protein of approximately 100 kDa; and env

codes for the glycoproteins gp61/46/21

Areas endemic for HTLV have been found, larly in south-west Japan, with prevalence as high as

particu-15% (Maeda et al 1984), in the Caribbean with a 1–8% prevalence (Clark et al 1985), in regions of

Central and South America and in parts of

sub-Saharan Africa (Gessain et al 1986; Vrielink and

Reesink 2004) Populations in these areas show ent prevalence rates for anti-HTLV-I, as do emigrants

differ-from these regions (Sandler et al 1991; Vrielink and

Reesink 2004) It has been estimated that more than

1 million Japanese people are healthy carriers of

HTLV-I Carriers of HTLV have also been found

in the USA, especially in Florida and states on the

Pacific, and in France, UK, the Netherlands and many

other countries The prevalence in blood donors has

been reported to be one in 6250 in the USA (CDC

1990), about one in 30 000 in France (Pillonel et al.

1996), one in 45 000 in the Netherlands (Zaaijer

et al 1994) and one in 20 000 in London (Brennan

1992)

HTLV-II, the second human retrovirus to be

dis-covered, has a 65% nucleotide sequence identity with

HTLV-I and a significant serological crossreactivity

(Hjelle 1991) However, HTLV-II antibodies are not

detected by all HTLV-I assays The distinction

between HTLV-I and -II can be made by DNA PCR

(Reesink et al 1994), and in the recently developed

WB assays in which specific HTLV-I and -II

recombin-ant recombin-antigens are used The relative prevalence of the

two viruses in blood donors in the USA was found to

be about equal (Glynn et al 2000), but in many other

countries HTLV-I predominates in blood donors

There is a high prevalence of HTLV-II among i.v drug

users and their sexual contacts in the USA and other

countries (Vrielink and Reesink 2004) A large

pro-portion of HTLV-II-positive subjects in the USA were

Hispanics and American Indians (Hjelle et al 1990a;

Sandler et al 1991) HTLV-II has been found to be

associated with a HAM-like neurological disease

(Hjelle et al 1992; Murphy et al 1997) Although

the virus was first found in a patient with hairy

cell leukaemia (Kalyanaraman et al 1982) and

sub-sequently in other T-cell malignancies, no viral RNA

could be detected in the malignant cells (Manns and

Blattner 1991)

Transmission of human T-cell leukaemia virus

HTLV is mainly transmitted by sexual contact, by the

sharing of infected needles and from mother to child,

particularly by breast-feeding (Kajiyama et al 1986).

If infected mothers refrain from breast-feeding,

trans-mission of HTLV to their infants is prevented in 80%

of cases (Hino et al 1996) Infection of infants is also

prevented when the milk is freeze-thawed or heated at

56°C for 30 min (Ando et al 1986; Hino et al 1987).

Transmission from mother to fetus has been

demon-strated by culture studies of cord blood lymphocytes in

2 out of 40 cord blood samples from HTLV-I-positive

mothers (Satow et al 1991).

Human T-cell leukaemia virus and bloodtransfusion

HTLV-I has been transmitted by cellular components,but not by cell-free plasma or plasma derivatives(Okochi 1985) However, HTLV-RNA is detectable inplasma from infected subjects The lack of infectivity

of HTLV-positive plasma may be explained by thepresence of neutralizing antibodies, the fact that it isintegrated in viral DNA and the requirement of cell–

cell interactions for infectivity (Rios et al 1996).

Antibodies are usually first detectable 14 –30 daysafter transfusion, although the interval may be as long

as 98 days (Inaba et al 1989; Gout et al 1990) Of 85

recipients of anti-HTLV-I-positive cell concentrates inJapan, 53 (62%) developed antibodies 3–6 weeks aftertransfusion: IgM antibodies were present only in theearly stages, whereas IgG antibodies persisted at hightitre throughout the period of follow-up (Sato andOkochi 1986) Storage of blood appears to decreasethe risk of transmission of HTLV This may explainthe lower rate of transmission reported in USA trans-fusion recipients, whose blood may have been storedfor a longer period than the units in Japan and the

Caribbean (Donegan et al 1994) Antibodies became

detectable in 79.2% of recipients of blood stored for1–5 days, but in only 55% of recipients of blood stored11–16 days (Okochi 1989)

Recipients of HTLV-I-infected concentrates may

develop HAM (Gout et al 1990; Araujo and Hall

2004) It has been estimated that 2–8% of subjectsinfected with HTLV-I by blood transfusion will even-

tually develop HAM (Murphy et al 1997; Araujo and

Hall 2004) ATL developed in two immunosuppressedpatients who had received multiple transfusions 6 and

11 years earlier (Chen et al 1989) HTLV-II has also been transmitted by blood transfusion (Hjelle et al.

between HTLV-I and -II, the sensitivity of

anti-HTLV-I assays for detecting anti-HTLV-anti-HTLV-Ianti-HTLV-I was found to be

only 55–91% (Wiktor et al 1991; Cossen et al 1992).

As infection with HTLV-II is probably associated with

HAM and as many HTLV-positive donors (more than

50% in the USA) are HTLV-II positive, tests designed

to detect both antibodies are now used (US Food and

Drug Administration 1998) In these ELISAs,

recom-binant proteins, including HTLV-I and -II-specific

ones, have been added to lysate or are used exclusively

(Hartley et al 1991) The sensitivity of such assays has

been claimed to be 100% (Vrielink et al 1996a).

In Japan, an agglutination test in which gelatin

particles are coated with HTLV-I-antigens has been

developed and used extensively The sensitivity of

the commercially available agglutination test Serodia

HTLV-I has been claimed to be 100% for detecting

anti-HTLV-I; all of 12 anti-HTLV-II-containing sera

gave positive reactions (Vrielink et al 1996a) An

ELISA for the combined detection of anti-HIV-1 and

-2 and anti-HTLV-I and -II in which synthetic peptides

of all four viruses are used, has been developed

(McAlpine et al 1992) In a study 242 samples

from anti-HIV-1/-2 or I/-II panels, two

HTLV-II-positive samples and two very weak

anti-HIV-1-positive samples were negative The specificity of the

test was slightly less than that of specific assays

(Flanagan et al 1995).

Despite the improved specificity of anti-HTLV-I/-II

screening tests, many repeatedly positive reactions that

cannot be confirmed are still found and all repeatedly

reactive samples must therefore be tested in

confirm-atory assays

Human T-cell leukaemia virus confirmatory

assays

Confirmatory testing for HTLV-I/-II continues to be

challenging, primarily because of a dearth of licensed

reagents Western blot and radioimmunoprecipitation

assays (RIPAs) are used (Anderson et al 1989; Hartley

et al 1990) For a positive reaction, antibody

reactiv-ity with both a gag (p19 and /or p24) and an env

pro-tein (gp46 and /or gp68) are required (WHO 1990)

All other reaction patterns were considered to be

indeterminate As the sensitivity of the Western blot

in detecting antibodies against env proteins was low,

many indeterminate results had to be checked in RIPA,

a much more elaborate assay (Lillehoj et al 1990; Lal

et al 1992) A report on the transmission of HTLV-I

by blood from a donor with an indeterminate pattern

in the WB and RIPA (p19 and gp68 reactivity) to fourout of six recipients, confirmed by PCR, demonstratedthe insufficient sensitivity of these original confirma-

tion assays (Donegan et al 1992) A modified WB has

been developed in which both shared (r21e) and

speci-fic (rgp46Iand rgp46II) HTLV-I and -II recombinant

env proteins are used (Lillehoj et al 1990; Lal et al.

1992) For a positive reaction in this modified Western

blot, a reaction with at least one gag protein (p19 and /or p24) and with env r21e as well as rgp46Iorrgp46II is required All other patterns are indeter-minate This Western blot, in which a reaction withHTLV-I or -II can be distinguished, has been found

to be more sensitive and specific than the originalWestern blot Instead of 66, only two positive samplesrequired confirmation by RIPA and none of 158 inde-terminate samples (original Western blot) reacted

(Brodine et al 1993) A recombinant immunoblot

assay (RIBA) in which the same antigens are applied

gave similar results (Vrielink et al 1996b) NAT has

not been licensed for confirmatory testing

Cytomegalovirus

Characteristics of the virus and ofcytomegalovirus infectionCytomegalovirus (CMV) is a large, enveloped, double-stranded DNA, beta herpes virus that is cell associated,but may also be found free in plasma and other body

fluids (Drew et al 2003) CMV has a direct cytopathic

effect on infected cells The result may lead to tropenia, some depression of cellular immunity andinversion of T-cell subset ratios, with a consequentincrease in susceptibility to bacterial, fungal and protozoa infections in immunosuppressed patients

neu-(Grumet 1984; Landolfo et al 2003) CMV infection

causes parenchymal damage, such as retinitis, monitis, gastroenteritis and encephalitis, and canresult in substantial morbidity and mortality

pneu-CMV can cause primary acute clinical and ical infections Chronic subclinical infections may occur

subclin-in which the virus is shed subclin-in saliva and ursubclin-ine CMVremains latent in a large proportion of infected sub-jects The presence of anti-CMV does not guaranteeimmunity As with HIV and HCV infection, specificantibody is a marker of potential infectivity although

only in the case of CMV, a relatively small proportion

of seropositive subjects seem to be infectious (Drew

et al 2003) CMV antibody-positive subjects may

infect others through sexual contact, breast-feeding,

transplacental transmission or transfusion (Tegtmeier

1986) In subjects with antibody, CMV infection may

be reactivated, or the subject may become re-infected

with exogenous strains of CMV

Primary CMV infection is generally more severe

than is re-infection (co-infection with a different strain)

or reactivation In view of the difficulty of

distinguish-ing between reactivation and re-infection, the term

recurrent infection has been coined to embrace both.

However, when necessary to distinguish between the

two, donor viral DNA can be distinguished from

recipi-ent viral DNA by restriction enzyme analysis (Glazer

et al 1979; Chou 1990) In practice, the diagnosis of

recurrent infection is limited to demonstrating a

four-fold increase in antibody titre or the presence of IgM

anti-CMV In immunosuppressed patients, serological

tests cannot be relied upon for a diagnosis of CMV

partly because the patients may not make antibody

and partly because any anti-CMV detected may have

been derived from transfused blood Viral culture is

impractical because the virus grows slowly in vitro

On the other hand, immunofluorescence techniques

to detect viral antigen using monoclonal antibodies on

biopsies or bronchial washings provide results within

hours (Griffiths 1984)

Prevalence of anti-CMV

The frequency of subjects with anti-CMV varies

widely in different populations Seroprevalence is lower

(30 –80%) in developed than in developing countries,

where the figure may reach 100% (Krech 1973;

Preiksaitis 1991) The prevalence of anti-CMV

corre-lates with age and socioeconomic status (Lamberson

1985; Tegtmeier 1986) The frequency of

anti-CMV-positive donors may vary widely within a given

coun-try, for example 25% in southern California and

70% in Nashville, Tennessee (Grumet 1984; Tegtmeier

1986)

Transmission of cytomegalovirus by transfusion

The transmission of CMV by blood transfusion was

first reported in the 1960s (Kaariainen et al 1966;

Paloheimo et al 1968; Klemola et al 1969) CMV is

now known as one of the infectious agents most quently transmitted by transfusion The pathogenesis

fre-of transfusion-transmitted CMV infection is notclearly understood In most cases CMV appears to

be transmitted in a latent, particulate state only by cellular blood components, and the virus reactivatesfrom donor leucocytes after transfusion Host as well

as donor factors are involved in CMV infection(Tegtmeier 1989; Preiksaitis 1991) CMV has beenisolated from the mononuclear and polymorpho-nuclear cells of patients with acute infections Thespecific cell type responsible for carrying the virus has not been identified, although mononuclear cellsare the favourite candidates as hosts of CMV in latentinfection Fresh blood appears more likely than storedblood to transmit CMV infection, although no con-trolled studies document this impression (Tegtmeier1986)

Some 3 –12% of units have the potential to mit CMV (Adler 1984), although most authors havereported a carrier rate of 1% or less (Tegtmeier 1986;

trans-Drew et al 2003) The discrepancy may be related to

the frequency of donor testing and the sensitivity andspecificity of the assay used Primary infection ratesdepend on the number of transfusions, age of blood,time of year and immunocompetence of the recipient

(Tegtmeier 1989; Preiksaitis et al 1988; Preiksaitis

2000) Donors with IgM anti-CMV appear to be more likely than others to transmit CMV (Lamberson

et al 1984) One large study found that only 0.5% of

antibody-positive donors have detectable CMV DNA

in their leucocytes (Roback et al 2003).

At present, no rapid, easy way to identify infectioussubjects exists Viral excretion in urine is a good index

of infectivity, but blood donors would probably rebel

at this screening strategy Virus can also be culturedfrom saliva, and PCR-based assays are available for the detection of viral genome in peripheral blood.Detection of pp65 antigen in leucocytes (pp65 anti-genaemia) is considered the ‘gold standard’ amongdiagnostic tools for diagnosing CMV infection and initiating antiviral therapy Both CMV DNA andimmediate early-messenger RNA detection have beencompared with pp65 antigenaemia, but none of themshowed advantages in terms of earlier diagnosis andbetter prognosis PCR’s major advantage is its semi-automation compared with the immunofluorescenceemployed for pp65 antigenaemia Isolation of CMV

by culture is reliable for the diagnosis of active

infection, but is less sensitive and requires more time

for viral detection

Transfusion-transmitted cytomegalovirus

infection in immunocompetent subjects

Before the era of universal (or near universal)

leuco-reduction, some 30% of anti-CMV-negative recipients

undergoing cardiac surgery involving transfusion

developed infection, as confirmed by virus isolation or

the development of CMV In addition, some

anti-CMV-positive patients developed recurrent infection

In almost all cases, the infection is asymptomatic

Of patients who develop a primary or recurrent

CMV infection following transfusion, fewer than 10%

develop a mononucleosis-like syndrome This

syn-drome, originally termed the post-perfusion synsyn-drome,

but now referred to as the post-transfusion syndrome,

appears 3 – 6 weeks after transfusion Common

fea-tures include fever, exanthema, hepatosplenomegaly,

enlargement of lymph nodes and the presence in

peripheral blood of atypical lymphocytes resembling

those found in infectious mononucleosis (Foster 1966)

Recovery is usually complete The development of

atypical lymphocytes due to post-transfusion CMV

infection should be distinguished from the

develop-ment of atypical lymphocytes 1 week after transfusion

as a response to allogeneic lymphocytes

Consequences of transfusion in patients with

impaired immunity

During the past decade, major advances have been

achieved regarding the management of CMV infection

through the development of new diagnostic techniques

for the detection of the virus and through the

perform-ance of prospective clinical trials of antiviral agents

(Meijer et al 2003) Nevertheless, in

immunosup-pressed patients, or in fetuses and premature infants

with an immature immune system, CMV infections,

particularly primary infections, still cause severe

dis-ease that can be fatal

1 The fetus in utero Following maternal primary

CMV infection, the fetus becomes infected in 30 – 40%

of cases Approximately 5–10% of infected infants

develop sequelae such as mental retardation, hearing

loss or chorioretinitis (Stagno et al 1986).

2 Premature infants The risk of serious CMV

infection is high when the infant’s birthweight is less

than about 1300 g and when the mother is anti-CMVnegative In two large prospective studies, 25–30% ofinfants with these risk factors, transfused with a total

of 50 ml or more of blood, some of which was CMV positive, acquired CMV infection, and 25% ofthese infants died Infants transfused with anti-CMV-negative blood did not develop CMV infection (0 out

anti-of 90) (Yeager et al 1981; Adler et al 1983) A lower

incidence of CMV infection (7–9%) has been reportedfrom two other centres: in infants weighing less than

1500 g born to anti-CMV-negative mothers and fused with blood, some of which was anti-CMV posit-

trans-ive (Smith et al 1984; Tegtmeier 1984) All reports

agree that clinically significant CMV infection in newborn infants develops only when the infant is premature and of low birthweight, when the motherlacks anti-CMV and when anti-CMV-positive blood

is transfused (Tegtmeier 1986)

3 Bone marrow transplant recipients frequently

develop primary or recurrent CMV infections thatmay prove fatal (Tegtmeier 1986) Blood transfusionrepresents the main risk factor for CMV acquisition inCMV-negative patients receiving bone marrow from

a CMV-negative donor In a prospective randomizedtrial of 97 anti-CMV-negative patients, 57 receivedanti-CMV-negative marrow: 32 out of the 57 receivedanti-CMV-negative blood components and only one

of these developed a CMV infection; of the 25 whoreceived blood components unscreened for anti-CMV,eight developed CMV infection Among the 40 recipi-ents of anti-CMV-positive bone marrow, the rate ofCMV infection was no lower in those who receivedonly anti-CMV-negative blood components (Bowden

et al 1986) Granulocyte concentrates, which contain

large numbers of leucocytes that can harbour CMV,reportedly carry the greatest risk of transmitting CMV

infection (Winston et al 1980; Hersman et al 1982).

However, when only two prophylactic transfusionswere given, the risk appeared to be no higher in thosewho received granulocytes than in those who did not

(Vij et al 2003).

4 Renal transplant recipients are at high risk of

prim-ary or recurrent CMV infection; the main source ofinfection lies in the transplanted kidney (Tegtmeier1986) In anti-CMV-negative recipients of a kidneyfrom an anti-CMV-negative donor, blood transfusionplays a significant role in CMV transmission (Rubin

et al 1985).

5 Heart and heart–lung transplant recipients may

develop severe primary CMV infection, which can

lead to opportunistic infections with fungi or bacteria

In anti-CMV-negative recipients the main sources of

infection are anti-CMV-positive transplanted organs

or organ blood donor units (Preiksaitis et al 1983)

If both donor and recipient are anti-CMV negative,

blood transfusion is a major source of CMV disease;

infection can be minimized by the use of

anti-CMV-negative red cells and platelets (Freeman et al 1990)

If a heart or heart–lung from a donor with CMV

anti-bodies is transplanted to an anti-CMV-negative

recipi-ent, prophylactic administration of specific CMV

hyperimmune IVIG seems to lessen the severity of

CMV disease (Freeman et al 1990), although data in

support of this practice are unimpressive

6 Following splenectomy due to trauma, patients

receiving massive transfusion may develop serious

CMV infections (Baumgartner et al 1982; Drew and

Miner 1982)

7 Subjects infected with HIV and especially those

with AIDS, if anti-CMV negative, may acquire

prim-ary CMV infection by transfusion (Jackson et al 1988;

Sayers et al 1992) In these patients CMV may cause

sight-threatening infection that may lead to blindness

in up to 25% of patients not receiving antiviral

therapy Because the rate of reactivation of CMV in

already infected patients is high, it is difficult to

deter-mine the contribution of transfusion-transmitted virus

(Bowden 1995)

8 Liver transplant recipients should also be

con-sidered at risk, especially children or pregnant women

Now that smaller amounts of blood and blood

com-ponents are needed in liver transplantation, it has been

possible to give only anti-CMV-negative blood and

platelets to negative recipients of

anti-CMV-negative grafts

Prevention of transfusion-transmitted

cytomegalovirus infection

Subjects who are at highest risk of severe primary

CMV infection are anti-CMV-negative patients with

impaired immunity For such patients, preventive

measures are available to reduce transmission of CMV

by transfusion The selection of CMV-seronegative

donors has proven to be effective, but not infallible

(Tegtmeier 1989; Miller et al 1991) Seropositivity,

especially the presence of IgM, is a marker of previous

infection and latent, but potentially infectious virus

However, antibody assays vary in sensitivity and asmall risk of transmission even from seronegative units

remains (Kraat et al 1993; Bowden et al 1995).

Window period infections are the most likely source ofantibody screening failures Although the windowperiods for HIV-1, HCV and HBV have been reason-ably well defined, the length of the CMV-seronegativewindow, estimated at 6–8 weeks, is less well character-ized Several recent studies using PCR technology documented CMV DNA in both plasma and cellularblood components from several weeks before sero-conversion to several months after seroconversion,although culture positivity was found for a muchshorter period in white blood cells (WBCs) and not

observed in plasma (Zanghellini et al 1999).

When only red cells are required, frozen lysed red cells can be used and have not been shown

deglycero-not to transmit CMV (Brady et al 1984; Taylor et al 1986; Sayers et al 1992).

As CMV is a white cell-associated virus, an ative approach to preventing CMV transmissioninvolves filtration removal of leucocytes from red cell

altern-and platelet concentrates (Graan-Hentzen et al 1989).

Leucoreduction by filtration may fail to prevent CMVtransmission, as 105to 106WBCs may still be trans-fused and an estimated 1 in 1000 to 1 in 10 000 WBCs

are infected by CMV during latency (Drew et al 2003).

In 10 patients whose WBCs were CMV antigen andculture positive before filtration and culture negativeafterwards, 2 out of the 10 did, however, have CMVDNA detected in leucocytes after filtration (Lipson

et al 2001) Plasma viraemia, if present, would not be

diminished by leucoreduction and might also accountfor CMV transmission following leucoreduced com-ponents Although the exact number of residual leuco-cytes that is sufficiently small to pose no risk of CMVtransmission is unknown and may not exist, a largeprospective study has shown that leucocyte removal

is as safe as selection of anti-CMV-negative donors

In this study, 502 patients were randomized prior

to bone marrow transplantation to receive filtered,three-log leucocyte-depleted cellular components orcomponents from anti-CMV-negative donors Therewas no significant difference in the probability ofCMV infection between the recipients of anti-CMV-negative or filtered concentrates, although more CMV-associated disease was observed in the filtration group (Bowden 1995) The same investigators havesubsequently questioned their finding that filtered red

cells are equivalent to CMV-seronegative cells (Nichols

et al 2003) When granulocyte transfusions are needed,

selection of anti-CMV-negative donors is the only

solution

The success of leucocyte removal in the prevention

of CMV transmission has raised the question of the

importance of infection with a second strain of CMV

in anti-CMV-positive recipients and the value of

trans-fusing such recipients with leucocyte-depleted

concen-trates Although co-infection with a second strain of

CMV does occur (Boppana et al 2001), the clinical

consequences of such infection resulting from

trans-fusion are less clear

Tests for anti-CMV

Several tests for anti-CMV are available Complement

fixation used to be the diagnostic reference test, but it

proved too complicated for routine donor screening

Indirect immunofluorescence, solid-phase fluorescence,

ELISA and particle agglutination assays are also

avail-able Competitive ELISAs seem to be the most reliable

of the currently available screening tests (Bowden et al.

1987) CMV PCR technology has been used in clinical

diagnostics since the 1980s (Bowen et al 1997; Lipson

et al 1998) Conventional PCR requires detection and

confirmatory testing of the amplicon by gel

elec-trophoresis, incorporating an isotopically labelled or

non-radioactive hybridization assay or a nested PCR

to detect targets present in very low copy numbers

(Lipson et al 1995) A simplified, more rapid

PCR-solid-phase enzyme immunoassay (EIA) plate

tech-nology system has been developed (Davoli et al 1999).

The quantitative real-time CMV PCR assay using

TaqMan chemistry and an automated sample

prepara-tion system has also been applied to CMV detecprepara-tion

(Piiparinen et al 2004).

An inexpensive, uncomplicated CMV-Ag (pp65

antigen) assay is available and well suited for most

diagnostic microbiology laboratories (Lipson et al.

1998) Assay for the early antigen pp65 is considered

the ‘gold standard’ for the initiation of antiviral therapy

Other viruses

Epstein–Barr virus

Infection with Epstein–Barr virus (EBV), a herpes

virus-like CMV, is endemic throughout the world

EBV can cause primary symptomatic infection tious mononucleosis), but most commonly causesasymptomatic infection followed by latent infection(Henle 1985) In most countries more than 90% ofblood donors have neutralizing anti-EBV, which coex-ists with latent virus in B lymphocytes of peripheralblood and lymph nodes At least one in 107circulatinglymphocytes of carriers harbour EBV genomes, butpost-transfusion EBV infection is a rare occurrence,

(infec-and symptomatic infection is even rarer (Rocchi et al.

1977) The virus is found in three of every 104eral lymphocytes during acute infection The majority

periph-of susceptible recipients are young children Even inchildren, the chance of acquiring EBV infection fol-lowing the transfusion of anti-EBV-positive blood isminimal, because the donor’s neutralizing antibodiespersist in the recipient’s circulation long after the EBV-infected lymphocytes disappear In a study of 25 EBVantibody-negative patients aged 3 months to 15 yearsand transfused with 1–11 units of blood stored for notmore than 4 days, only one developed EBV antibodiesand this patient had no symptoms (Henle 1985)

Of five patients transfused during cardiac bypass,four who were initially anti-EBV negative producedantibody that persisted at high titre for many monthspostoperatively Two out of the four patients had concomitant CMV infection and no heterophile anti-bodies; one developed transient fever, and the otherhepatitis Only one of the four patients developed aninfectious mononucleosis-like syndrome with hetero-

phile antibodies (Gerber et al 1969).

Although most cases of ‘post-transfusion syndrome’are caused by CMV, two adult patients who were anti-EBV negative before transfusion developed post-

transfusion syndrome due to EBV (McMonigal et al.

1983) In a survey of some 800 patients, fewer than8% lacked EBV antibody before transfusion, and only5% of these developed antibodies following trans-fusion These patients suffered no clinical illness or disturbance of liver function (MRC 1974) Possibly,the discrepancy between these observations and those

of others may have been related to the use of fresh

blood in the last-named series (Gerber et al 1969).

Post-transfusion infectious mononucleosis is seenonly rarely in anti-EBV-negative immunocompetentpatients and usually occurs when only a single unit ofblood or blood component, obtained from the donorduring the incubation phase is given within 4 days ofcollection When more than 1 unit is transfused, one

of the units is almost certain to contain anti-EBV

In the reported cases, the donors developed symptoms

of mononucleosis 2–17 days after blood donation

The incubation period in recipients has been 21–

30 days (Solem and Jorgensen 1969; Turner et al.

1972) Transfusion-transmitted EBV infection with

symptomatic mononucleosis has occasionally been

reported in patients transfused for splenectomy (Henle

1985)

Post-transfusion EBV infections may contribute to

the development of lymphomas in severely

immuno-suppressed patients such as haematopoietic graft

recipients T-lymphocyte suppression allows

EBV-infected B lymphocytes to outlive the passively

trans-fused antibodies and to proliferate (Marker et al.

1979; Hanto et al 1983).

Other herpes viruses

Herpes simplex and Herpes varicella zoster have never

been shown to be transmitted by blood transfusion;

viraemia occurs only during primary infections, which

usually occur in childhood (Henle 1985)

HHV-6 is a recently characterized human herpes

virus, originally named HBLV (human B lymphotropic

virus) for its ability to infect freshly isolated B cells

The virus was found in patients with various

lympho-proliferative disorders HHV-6 can infect monocytes,

macrophages, T cells and megakaryocytes (Ablashi

1987) The virus is cytopathic for selected T-cell lines

Infection is acquired usually within the first year of life

and the virus was found to be ubiquitous in blood

donors when tested in London and the USA (Briggs

et al 1988; Saxinger et al 1988) No

transfusion-associated disease has been reported

HHV-8, Kaposi’s sarcoma-herpes virus is white cell

associated and may be present in up to 30% of normal

donors Despite a relatively high HHV-8

seropre-valence in a Texas blood donor cohort (23%), HHV-8

DNA was not detected in any sample of donor whole

blood using a highly sensitive PCR assay (Hudnall

et al 2003) Blood components from HHV 8-infected

donors apparently carry little transfusion risk (Engels

et al 1999).

Human parvovirus B19

HPV B19 infection has long been known to cause

erythema infectiosum (Fifth disease), a common febrile

exanthem of childhood (Anderson et al 1983) HPV is

also associated with polyarthritis and rash in adults as

a result of antigen–antibody immune complex

deposi-tion in skin and joints (Reid et al 1985; White et al.

1985) Because of its specific cytotoxic effect on erythroblasts, HPV B19 can precipitate aplastic crises

in children who have haematological disorders withshortened red cell survival, such as those with sicklecell anaemia and other chronic haemolytic anaemias,

particularly hereditary spherocytosis (Pattison et al.

1981; Young and Mortimer 1984) The virus may alsocause thrombocytopenic purpura (Pattison 1987).Intrauterine infection may cause hydrops fetalis and

spontaneous abortion in early pregnancy (Brown et al 1984; Anand et al 1987).

HPV B19 was discovered by an Australian virologistwho noted viral particles in an antigen–antibody line

of detection (plate B, well 19) in an assay for hepatitis

B (Cossart et al 1975) HPV B19 is a small

single-stranded, non-enveloped, thermostable DNA member

of the Parvoviridae family (Shade et al 1986; Young

and Brown 2004) The parvoviruses are dependent

on help from host cells or other viruses to replicate.Parvovirus B19 is the type member of the erythrovirusgenus, which propagates best in erythroid progenitorcells The red cell P antigen, a globoside present on avariety of cells in addition to erythrocytes, has beendocumented as the specific receptor for HPV (Brown

et al 1994) This may account for reports of

poly-arthritis nephropathy, myocarditis and pathy (Young and Brown 2004) Persistent infectionwith anaemia occurs in immunosuppressed subjects

cardiomyo-(Kurtzman et al 1987) PCR assays have revealed the

presence of viral DNA along with the simultaneouspresence of specific IgG in 0.55–1.3% of normal blood

donors (Candotti et al 2004), but the long-term

persistence, infectivity and clinical importance of thevirus in these subjects has not been well studied.Current methods of viral inactivation may not be able

to eliminate HPV B19 completely (Williams et al.

1990a)

Infection with the virus normally occurs via atory droplets HPV B19 has been transmitted byplasma fractionation products derived from largepools of plasma, particularly by factor VIII and factor

respir-IX concentrates (Santagostino et al 1997; Blumel et al.

2002) One episode of fulminant hepatitis has beenattributed to the intravenous route of infection

(Hayakawa et al 2002) Use of recombinant products

should eliminate this risk (Soucie et al 2004).

Transmission of the virus by single-donor components

is unusual, but a red cell unit has been associated with

HPV B19 transmission and possible cardiac

involve-ment in a 22-year-old woman with thalassaemia major

(Zanella et al 1995).

The classic diagnosis of infection with HPV B19

is based on the detection of IgM or IgG antibodies

with an EIA (Cohen et al 1983) Alternatively viral

DNA can be detected by PCR (Salimans et al 1989;

McOmish et al 1993) HPV B19 has been detected by

PCR in solvent–detergent-treated clotting factor

con-centrates (Lefrere et al 1994), in heat-treated factor

VIII and IX concentrates and in IVIG (McOmish et al.

1993; Santagostino et al 1994) and in clotting factor

concentrates prepared by different purification and

inactivation procedures (Zakrzewska et al 1992;

Blumel et al 2002) The virus has also been detected in

plasma pools designed for fractionation (in 64 out of

75 pools), in 3 out of 12 albumin preparations, in 3 out

of 15 IVIG and in three out of four IMIG preparations,

as well as in seven out of seven factor VIII

prepara-tions There is some indication that treatment of

IVIG preparations at low pH may result in removal

of detectable HPV B19 DNA (Saldanha and Minor

1996) Since 2002, major plasma fractionators have

screened plasma units with quantitative measurements

of B19 DNA to reduce the risk of transmission The

prevalence of HPV B19 in blood donors has been

estimated at 0.03% in the UK (McOmish et al 1993).

A similar prevalence was found in Japan (1 in 35 000)

but, during an epidemic of erythema infectiosum, the

prevalence was much higher (1 in 4000) (Tsujimura

et al 1995) B19 prevalence varies according to season

and from year to year (Young and Brown 2004)

A rapid test for HPV B19, suitable for large-scale

screening of donors has been developed (Sato et al.

1996) The test is based on agglutination of blood

group P-positive gluteraldehyde-treated red cells by

the virus for which the P antigen is the receptor (see

Chapter 4) Although only intact viruses can bind to P,

the test has been found to be sensitive and it could be

used to select donors for patients at risk Whether

rou-tine screening of donors is necessary is an unanswered

question Although 81.6% of 136 haemophiliacs

studied had anti-HPV B19, B19 DNA was detectable

in none and there were no signs of lasting clinical or

haematological sequelae (Ragni et al 1996).

Commercial immune globulins are a good source of

antibodies against parvovirus; a persistent B19 tion responds to a 5- or 10-day course of immuno-globulin at a dose of 0.4 g per kilogram of bodyweight, with a prompt decline in serum viral DNA, asmeasured by hybridization methods, accompanied

infec-by reticulocytosis and increased haemoglobin levels

(Kurtzman et al 1987; Frickhofen et al 1990).

West Nile VirusSince its importation into the USA in 1999, West Nilevirus (WNV) has become a significant transfusion-transmitted infection with a calculated mean risk oftransfusion transmission of 3.02 per 10 000 donations

in high-risk metropolitan areas during epidemic tions (Biggerstaff and Petersen 2003) Transfusiontransmission was first documented when four organsharvested from a common multi-transfused cadaverdonor transmitted virus to all four recipients; thedonor’s pretransfusion sample tested negative forWNV, while one of 63 blood donors tested positivewith a nucleic acid assay and developed IgM antibody

condi-to WNV over the subsequent 2 months (Iwamocondi-to et al.

2003) Using strict case definition criteria, logists document 23 transmissions and another 19inconclusive investigations of 61 case investigations

epidemio-during a 12-month period (Pealer et al 2003) The true

number of transmissions was probably higher by atleast an order of magnitude

As was the case with HIV and the hepatitis viruses,blood transfusion of WNV represents a small, buthighly visible portion of a large epidemic WNV is amosquito-borne flavivirus transmitted primarily tobirds and some small mammals Humans serve as anincidental host Approximately 80% of human infec-tions are asymptomatic; 20% result in a febrile illnessknown as West Nile fever About 1 in 150 patientsdevelop meningoencephalitis and residual neurolo-gical deficits have been reported Although there is noparticular susceptibility to mosquito-borne infection,elderly and immunosuppressed subjects appear par-ticularly vulnerable to severe, progressive disease The virus is present for a week or more during initialinfection Most symptomatic subjects describe fever,headache and malaise, although these are not suffi-ciently specific for effective donor screening Viralshedding may persist for 7–8 weeks after infection,usually in the presence of specific antibody The period

of infectivity has not been well defined

Of the 23 well-studied patients with

transfusion-transmitted infection, 14 were identified because West

Nile virus-associated illness came to the clinician’s

attention following transfusion Overall, 15 patients

had recognized illness (13 meningoencephalitis, two

fever) The illness began between 2 and 21 days after

the implicated transfusion The highly

immunosup-pressed transplant recipients appeared to have the

longest incubation periods (median 13.5 days) Red

cells, platelets and fresh-frozen plasma (FFP) have

all been implicated in transmissions Sixteen blood

donors were implicated in transmission, with no

pre-dilection for age or gender Nine out of the sixteen

recalled symptoms compatible with a viral illness

around the time of donation, although all donors

passed the donor screening procedures Three donors

developed symptoms prior to donation, one on the day

of donation and five post donation Fever, headache

and weakness were the most common symptoms

Samples obtained at the time of donation had virus

lev-els less than 80 pfu per millilitre and all were negative

for West Nile virus IgM antibody

Since screening of blood was undertaken in the USA,

approximately 6 million units were tested during the

June–December 2003 interval, resulting in the removal

of at least 818 viraemic blood donations from the

blood supply Nevertheless, six cases of WNV

trans-mitted by transfusion occurred because of transfusion

of components containing low levels of virus not

detected by the testing of pooled specimens (Macedo

et al 2004) All blood donations in the USA are

cur-rently screened by rtPCR, and it is likely that testing of

individual donations will begin in areas of epidemic

transmission and eventually be practised universally

(Custer et al 2004).

Other flaviviruses such as St Louis encephalitis

virus, Japanese encephalitis virus and dengue virus

are likely to be blood transmissible as well, although

documentation is lacking

Simian foamy virus

Simian foamy virus (SFV) (spumaretrovirus) is a highly

prevalent primate retrovirus that has been shown to

infect human cells, replicate and produce cell-free

infectious virus Tropism is broad and includes B and

T lymphocytes, macrophages, fibroblasts, endothelial

cells and kidney cells Simian retroviruses are spread

in areas of the world such as Central Africa, where

non-human primates are hunted for food and amongpeople who handle non-human primates as pets or laboratory animals In total, 1% of people living insouthern Cameroon were found to harbour antibodies

to SFV; all had reported exposure to fresh primate

blood or body fluids (Wolfe et al 2004).

Studies from the USA CDC report a prevalence ofinfection of 2–5% with simian foamy viruses amonglaboratory and zoo workers occupationally exposed

to non-human primates (Heneine et al 1998; Switzer

et al 2004) Persistent viraemia has been detected

in peripheral blood lymphocytes (PBLs) and 11 infected workers are known to have donated blood.Six donors were confirmed positive at the time ofdonation SFV transmission through transfusion wasnot identified among four recipients of cellular bloodcomponents (two RBC, one filtered RBC, one plateletconcentrate) from one SFV-infected donor (Boneva

SFV-et al 2002) Derivatives containing plasma from that

donor tested negative for SFV

Evidence of SFV infection included seropositivity,proviral DNA detection and isolation of foamy virus.There is no evidence as yet that SFV causes human dis-ease but, recombination within the host, especially inimmunocompromised hosts that may allow persistentinfection, remains a concern

Prion proteins

A fundamental feature of prion diseases involves anormal protein constituent of human tissue, prion

protein (PrP) (Bendheim et al 1992) PrP can exist

in either a natural cellular form (PrPC) or as variantpathological forms known collectively as ‘scrapie isoforms’ or PrPSc Different prion strains result in distinct clinical and pathological diseases (Prusiner1998) Both forms of prion protein have an identicalamino acid sequence, but differ in the secondary struc-ture, beta-pleated structures in the variant formsinstead of the alpha-helical structure of the native pro-

tein (Pan et al 1993) The structural difference has two

major consequences First, the beta-pleated tion allows PrPSc molecules to form protease-resistantaggregates Second, the abnormal protein seems to becapable of suborning normal protein to the abnormalform The molecules with beta-pleated regions causekey alpha-helical regions of the native protein toassume the pleated conformation, thus converting normal protein to abnormal aggregates that cause the

conforma-spongiform degeneration of the brain that is

character-istic of these diseases

Prion proteins are linked to the cell plasma

mem-brane by the lipid glycophosphatidyl inositol (GPI)

GPI-linked proteins are released from cells by several

different mechanisms and may be taken up by other

cells, thus facilitating dissemination (Devetten et al.

1997) Prion proteins are taken up selectively by

motile follicular dendritic cells, suggesting that

infec-tive proteins may be present in circulating blood (Klein

et al 2001).

Creutzfeldt–Jakob disease

Creutzfeldt–Jakob disease (CJD) is a rare, but

invari-ably fatal neurodegenerative disease, with an

incid-ence of about one per million of the UK and USA

population (Collinge and Rossor 1996) CJD may be

‘sporadic,’ arising from a random change in a normal

individual, ‘familial,’ arising from a point mutation

in the DNA coding for the protein, which leads to an

increased susceptibility to production of the abnormal

form, or ‘acquired,’ by the transfer of infectious

mater-ial, as in individuals injected with pituitary-derived

hormones or treated with dura mater transplants

(Brown et al 1992) A single case of transmission

from a corneal transplant has also been reported The

proportion of recipients acquiring CJD from growth

hormone varies from 0.3% to 4.4% in different

coun-tries, and acquisition from dura mater varies between

0.02% and 0.05% in Japan, where most cases occurred

(Brown et al 2000) Patient follow-up after point

exposures to contaminated materials indicate that

clinical latency for iatrogenic CJD may exceed 30 years

(Fradkin et al 1991; Brown et al 2000).

Classic CJD typically affects older subjects with

a median age of 60 years Memory loss is an early

manifestation, but the disease progresses rapidly after

the first symptoms through confusion, motor and

cere-bellar symptoms and dementia Death often occurs

within a year of the first symptom

Because of the possibility of transfer of infectious

prions by transfusion, donors have been questioned

to discover (1) whether there is any family history of

CJD; (2) whether they have received pituitary-derived

hormones; or (3) in some countries, whether they have

received corneal grafts or grafts of dura mater

How-ever, a systematic review of five case–control studies

from the UK, Europe, Japan and Australia, involving

2479 patients, failed to find an association betweenCJD and blood recipients No evidence of transmis-sion of CJD by blood transfusion exists, despite theidentification of individuals who were exposed toblood donated by people who later developed this dis-

ease (Wilson et al 2000) A study of preserved brain

samples of 25 haemophilic patients who have highexposure to blood transfusions and potentially higherexposure to blood infected with the agent responsiblefor Creutzfeldt–Jakob disease found no evidence of

the disease (Evatt et al 1998) ‘Look-back’ studies

have not identified any cases of Creutzfeldt–Jakobdisease developing in recipients who received bloodfrom a donor in whom the disease was later diagnosed.The risk of CJD transmission through transfusion isnow considered negligible, and plasma pools are nolonger discarded if a contributing donor developsCJD Deferrals designed to eliminate ‘high-risk’ donorcategories, such as growth hormone and dura materrecipients, remain in force

Variant Creutzfeldt–Jakob disease

A neurodegenerative disease affecting cattle was firstidentified in England in 1984 and subsequently branded

‘mad cow disease’ Bovine spongiform encephalopathy(BSE) is now recognized as a prion-related disorderwith more than 179 000 confirmed clinical cases ofBSE, driven by the recycling of infection through theinclusion of bovine protein in cattle feed A ban onfeeding tissues from one ruminant animal to otherruminant animals, introduced in 1988, brought theBritish BSE epidemic under control However, because

of the long delay between infection and the onset

of clinical signs of disease (5 years on average), theannual incidence of clinical cases did not peak until

1992 BSE has been found in several other Europeancountries and in Canada More rapid and accuratedescription of the epidemiology of BSE has been hampered by the absence of a diagnostic test that can be applied to live animals to detect those that areincubating the infection

The human disease equivalent, new variantCreutzfeldt–Jakob disease (vCJD), was not identifieduntil 1996, 7 years after the ban was introduced A disease-related isoform of prion resembling that ofBSE is consistent with the conclusion that BSE hascrossed the species barrier, probably from ingestion of

infected beef (Collinge and Rossor 1996; Bruce et al.

1997) Unlike classic CJD, vCJD usually affects

younger subjects (age 18–53 years) and is

charac-terized by early psychiatric and sensory symptoms

The disease progresses over 8 months to 3 years, with

progressive dementia, ataxia and myoclonus Brain

biopsy has a characteristic pattern and Western blot

analysis shows a diagnostic migration pattern of

pro-teinase-treated PrPSc Pathological PrPSc can be found

in tonsils, spleen lymph nodes and appendix (Collinge

et al 1996) Prion protein gene analysis has shown

that all cases were homozygous for methionine at

codon 129 (Ironside and Head 2004) More than 150

cases have been found in the UK and cases have been

described in France and Italy Although the number

of vCJD ‘carriers’ remains unknown, early estimates

of as many as 100 000 cases have in recent years

dwindled to a maximum of just a few hundred cases,

assuming an estimate of 15 to 20 years as the average

incubation period (Valleron et al 2001) One model

indicates that the primary epidemic in the susceptible

genotype (methionine-methionine (MM)-homozygous

at codon 129 of the prion protein gene) has now

peaked, with an estimate of 40 future deaths (Ghani

et al 2003) However, the prevalence of vCJD

infec-tion in the UK populainfec-tion, estimated to be as high as

1 per 4000 based on a study of routinely acquired

tonsils and appendices, raises the possibility of a

second and third ‘wave’ of clinical cases in subjects

heterozygous (methionine-valine) and homozygous

for valine at codon 129

Prion protein has been identified on a wide range

of circulating cells including platelets, myeloid cells,

lymphocytes and red cells (Dodelet and Cashman

1998; Holada and Vostal 2000; Bessos et al 2001;

Li et al 2001) Findings in experimental models show

that blood not only contains infective agents of prion

diseases, but that no barrier to transmission exists with

intraspecies transmission, and that the intravenous

route of exposure to prions is fairly efficient (Casaccia

et al 1989; Cervenakova et al 2003) The seminal

experiments in sheep have shown transmission of BSE

and scrapie by blood transfusion, and blood for

trans-fusion in these experiments was obtained from sheep

midway through the incubation period (Houston et al.

2000; Hunter et al 2002) Infectivity has also been

noted in the incubation period and symptomatic phase

in a rodent model of vCJD (Cervenakova et al 2003).

In 1997, the UK set up a surveillance system

between the national CJD surveillance unit and the UK

national blood services In 2004, the first probabletransfusion-associated case of vCJD was described

(Llewelyn et al 2004):

A 62-year-old man received 5 units of red cells for a surgical procedure Six and a half years later, the patient developed irritability and depression, followed by a shuffling gait, blurred vision, motor dysfunction and cognitive impairment Thirteen months after the onset of symptoms, he died of autopsy-confirmed vCJD One of the red cell units had been donated by a 24-year-old subject who developed symptoms

of vCJD 3 years and 4 months later, and subsequently died of vCJD confirmed at autopsy Red cells from a second donation were traced to a patient who died of cancer 5 months later Platelets from the donation could not be traced to a recipient The clinical presentation of the transfusion recipient was typical of vCJD, and diagnosis was confirmed by neuropatho- logical examination Transfusion took place before universal leucoreduction Of particular note in this case is the age of the recipient, the second oldest patient with vCJD, making it even less likely that this case was caused by ingestion of tainted beef.

Statistical analysis, taking account reported vCJDmortality to date and details of the recipients of vCJDdonations indicate that the probability of recording acase of vCJD in this population in the absence of trans-fusion-transmitted infection ranges between about 1 in

15 000 and 1 in 30 000 (Llewelyn et al 2004).

A case of preclinical vCJD has been reported in apatient who died from a non-neurological disorder

5 years after receiving a blood transfusion from adonor who subsequently developed vCJD:

In 1999, an elderly patient received a unit of non-leucodepleted red blood cells from a donor who developed symptoms of vCJD 18 months after donation The donor died in 2001 and vCJD was confirmed at autopsy The recipient, who had no evidence of a neurological disorder, died 5 years after receiv- ing the transfusion Western blot analysis of splenic tissue showed the presence of PrP res with the mobility and glyco- form ratio of the signals similar to those seen in spleen from patients with clinical vCJD and distinct from those of sporadic CJD cases.

Protease-resistant prion protein (PrPres) was detected

by Western blot, paraffin-embedded tissue blot andimmunohistochemistry in splenic tissue, but not in the brain However, animal studies suggest that migra-tion is inevitable given sufficient time Prion proteinwas also present in a cervical lymph node Perhaps the most disturbing observation is that this patient was

a heterozygote at codon 129 of prion protein gene

(PRNP), suggesting that susceptibility to vCJD

infec-tion is not confined to the methionine homozygous

genotype This finding suggests that a second, later

wave of vCJD cases in heterozygotes and even a third

may appear long after the initial peak of the epidemic

(Peden et al 2004) The chance of observing vCJD

transmission in the absence of a transfusion infection

in this second recipient of blood from a donor with

vCJD is far less than the 1 in 15 000 to 1 in 30 000

chance for the first reported case

Transmissable spongiform encephalopathy (TSE)

agents are resistant to the range of physical and

chem-ical means that have been used to inactivate viruses in

plasma products However, a range of studies using

animal TSE models have demonstrated that the

pro-cesses used to purify proteins, including factor

concen-trates, over the course of plasma fractionation can also

contribute significantly to removing both abnormal

prions and infectivity (Lee et al 2001) Extensive

washing of cellular components may also deplete the

prion content; however, the risk of infection has not

been determined A prototype prion removal filter has

been designed to remove about four logs of PrP, below

the level of detection in the Western blot assay, and

has prevented infection in a hamster scrapie model

(SO Sowemino-Coker, personal communication)

Bacteria

Transmission of bacteria represents the most frequent

infectious complication of blood transfusion in the

developed world and a major cause of

transfusion-associated mortality (Andreu et al 2002) The reported

frequency of contamination varies depending upon

the nature of the blood component and the method

of study technique

Treponema pallidum

Treponema pallidum is a motile spirochaete that

spreads by sexual contact, transfusion, percutaneous

exposure and transmission from mother to infant The

incubation period from transfusion to clinical

pres-entation varies from 4 weeks to 4.5 months, averaging

9 –10 weeks, and the infected recipient usually exhibits

a typical secondary eruption Donors at any stage of

disease, including late, latent syphilis, can transmit the

infection (Hartmann and Schone 1942)

Transfusion-transmitted syphilis was once ered a serious problem Kilduffe and DeBakey (1942)identified more than 100 cases that had been pub-lished after 1915, all from direct transfusion Some

consid-138 cases had been reported by 1941 (De Schryver and Meheus 1990) Since then, few cases have beenreported in the developed world The last case pub-lished in the USA was reported from the ClinicalCenter at the National Institutes of Health (Chambers

et al 1969).

In 1966, a patient was admitted to the Clinical Center with a diagnosis of lymphoma His serological test for syphilis (STS) was negative on admission He received 5 units of RBCs and

25 units of fresh platelets, none of which reacted in the VDRL test Two months later, he developed a maculopapular rash consistent with secondary syphilis Serological tests and con- firmatory assays for syphilis turned positive The patient was treated with penicillin and the rash cleared.

Of the 30 donors, 27 tested negative repeatedly, but two could not be traced and one refused to be retested The investigators presumed that one out of those three donors, a platelet donor, was infectious for syphilis.

The chief reasons for the decline of transmitted syphilis seem to be the almost universalpractice of storing blood at 4°C before transfusion,universal donor testing and the decline in the preval-ence of syphilis in many countries since the advent

transfusion-of penicillin Other factors that have probably tributed to the decline include the administration ofantibiotics to a large proportion of patients requiringtransfusion and the exclusion of donors with high-risksexual practices

con-Spirochaetes are unlikely to survive in citrated bloodstored for more than 72 h at 4 – 6°C (Bloch 1941;Turner and Diseker 1941) However, organisms can

be detected for as long as 6 days (Selbie 1943) There

is a close relationship between the number of ponemes added to donor blood and the survival times

tre-of T pallidum determined by a sensitive assay in

rab-bits; in blood heavily contaminated with spirochaetes(1.3 × 106 and 2.5 × 107/ml blood), surviving tre-

ponema were found at 120 h (Garretta et al 1977; van der Sluis et al 1985).

Several tests are available for screening blood tions for syphilis, including the automated reagin test(ART), the rapid plasma reagin test (PRP) and theVenereal Disease Research Laboratory (VDRL) slidetechnique in which non-specific reagins (antibodies)

dona-are detected and the T pallidum haemagglutination

assay (TPHA), the fluorescent treponemal antibodies

absorbed with Reiter’s treponeme assay (FTA-Abs)

and ELISAs for the detection of specific antibodies

Non-specific reagin tests detect antibody to lipoidal

antigen and are often considered to be insufficiently

sensitive as screening tests (Barbara et al 1982; Young

et al 1992) Positive screening tests should be tested

for FTA-Abs or some other specific assay in a reference

laboratory A lack of demonstrable T pallidum DNA

or RNA suggests that blood donors with confirmed

positive results in STS are unlikely to have circulating

T pallidum in their blood, and that that their blood

is unlikely to be infectious for syphilis (Orton et al.

2002)

Serological tests cannot prevent all cases of

trans-fusion syphilis because most remain negative in early

primary syphilis, when spirochaetaemia is most

prom-inent (Spangler et al 1964).

The rationale for continued syphilis testing relies

upon the increasing demands for fresh blood

compon-ents, especially platelets and fresh blood for exchange

transfusion in newborn infants, the very occasional

reported case (Chambers et al 1969; Soendjojo et al.

1982; Risseeuw-Appel and Kothe 1983) and its

ques-tionable value as a surrogate test to exclude donors

who are in high-risk groups for HIV and HBV

infec-tion A positive test in a transfusion recipient may

result from antibody acquired passively from the

donor (Rossi et al 2002) However, passively acquired

antibody rarely remains detectable for more than a few

months after transfusion (Ravitch et al 1949; Rossi

et al 2002).

In countries with a high incidence of syphilis, some

consultants recommend that recipients of fresh blood

receive 2 megaunits of penicillin G or its equivalent

(Bruce-Chwatt 1985) In endemic areas, subjects who

have had non-venereal trepanomatosis, such as yaws

or pima caused by T pallidum pertenue or T pallidum

carateum, may also have a positive screening test for

syphilis

Brucella abortus

This organism can survive for months in stored blood

and there are several reports of blood

transfusion-transmitted symptomatic infection, mainly in children

and splenectomized patients (Wood 1955; Tabor 1982)

Antibody-positive donors are common in Mexico,

Greece, Spain and in some rural areas of the USA,although transfusion-transmitted brucellosis has notbeen reported in the USA Infected donor blood hasvery low concentrations of brucella and poses littlerisk except for immunosuppressed patients (Fernandez

et al 1981).

After an incubation period ranging from 6 days to

4 months, recipients of infected blood may developundulant fever, headache, chills, excessive sweating,muscle pains and fatigue Hepatosplenomegaly, lym-phadenopathy, leucopenia and arthritis occur and, veryrarely, complications such as purpura, encephalitis orendocarditis develop (Tabor 1982)

In view of the chronic nature of the disease, personswith a history of brucellosis should not be used asdonors However, 80% of infections are asymp-tomatic Even in endemic areas screening tests are not practical; most subjects with high-titre brucellaantibodies do not transmit infection by transfusion

(Fernandez et al 1981).

Miscellaneous

Lyme disease The organism responsible for this disease is Borrelia burgdorferi, a tick-borne spirochaete whose primary

reservoir is the white-footed mouse Humans areinfected in the nymphal stage of the cycle and white-tailed deer are infected during the second year of life

of the tick Most cases of Lyme disease have beenreported in the USA, but thousands of cases have alsobeen reported in Europe

The disease has three stages: in the acute stage oferythema migrans, a skin rash starting at the site of the tick bite spreads locally and is often accompanied

by ‘flu-like symptoms’ If this stage is not treatedpromptly with antibiotics, the second stage progresses

to disseminated infection with cardiovascular and neurological manifestations In the third stage, arthritis develops

Although no cases of transmission of B burgdorferi

by blood transfusion have been reported so far, mission is theoretically possible Subjects in the primarystage of infection pose the most risk However, as thesepatients are generally ill and as spirochaetaemia is oflow intensity and of short duration, infectious donorsprobably do not present often for blood donation(Popovsky 1990; Westphal 1991a) The spirochaete

trans-has been isolated and cultured from blood as old as

14 days

Four transfusion recipients of blood components

from a donor who gave blood between the

disappear-ance of erythema migrans and the second stage of

the disease have been studied; none of the recipients

showed signs of disease or developed antibodies against

B burgdorferi (Halkier et al 1990).

Mycobacterium leprae

Mycobacterium leprae is known to have been

trans-fused inadvertently from a donor incubating leprosy to

the two recipients without apparent harm M leprae

has also been injected intravenously into human

volunteers without the recipients becoming infected,

although the period of follow-up is not known (Tabor

1982)

Tick-borne rickettsial disease

Tick-borne rickettsial diseases are caused by two

groups of intracellular bacteria belonging to the order

Rickettsiales: (1) bacteria belonging to the spotted

fever group of the genus Rickettsia within the family

Rickettsiaceae; and (2) bacteria within the family

Anaplasmataceae, including several genera, such as

Anaplasma and Ehrlichia Rickettsiosis (Rocky

Mountain spotted fever) has been reported to have

been transmitted by blood from a donor incubating

the disease who subsequently died (Wells et al 1978).

The rarity of the transmission of rickettsiosis by blood

transfusion probably reflects the clinical illness

suf-fered during most of the period of rickettsial infection

that makes blood donation unlikely (Tabor 1982)

In addition to the spotted fever group rickettsioses,

human anaplasmosis (formerly human granulocytic

ehrlichiosis) has also emerged in Europe (Parola

2004) This disease was first described in the USA

in 1994 and presents most commonly as a febrile

illness occurring in summer or spring The causative

organism is Anaplasma phagocytophilum, formerly

known as Ehrlichia equi, Ehrlichia phagocytophila

and human granulocytic ehrlichia The vector in

Europe is I ricinus, which is also the vector of

Lyme borreliosis Anaplasma are well preserved in

refrigerated blood components (McKechnie et al.

2000) Transfusion-transmitted ehrlichiosis has been

reported (Eastlund et al 1999).

Exogenous and various endogenous bacteria andbacterial products contaminating stored blood orblood components

Bacteria may affect blood or blood components in one

of the following ways:

1 Bacteria may contaminate solutions or equipment

that are to be used for transfusions but which have notyet been sterilized After sterilization the solutions orequipment may remain contaminated with heat-stablebacterial products (pyrogens) capable of producingfebrile reactions when they are introduced into the cir-culation (see Chapter 15) In contaminated equipment

or solutions such as hydroxyethyl starch used in pheresis, bacteria may survive ‘sterilization’ or maycontaminate solutions that have previously been steril-ized, for example when a glass container is cracked

leuca-during shipment (Wang et al 2000) This kind of

con-tamination has become rare

2 Bacteria originating from skin flora, such as

Staphylococcus epidermidis, Micrococcus species, Sarcina species and diphtheroids, may gain entrance to

the blood pack during venesection, especially if the site

of venepuncture is scarred It is virtually impossible todisinfect the deeper layers of the skin and a skin plugoften enters the blood pack upon collection (Anderson

et al 1986; Puckett 1986a).

3 Bacteria in the environment (Pseudomonas species,

Flavobacterium species, Bacillus species) may gain

entrance to blood components through minute lesions

in the packs, during collection or processing in open

systems (Szewzyk et al 1993).

4 The ports of packs of cryoprecipitate or FFP may

become contaminated, if not protected by a secondaryplastic bag, during thawing in waterbaths contami-

nated with pseudomonas (e.g Pseudomonas cepacia,

P aeruginosa) (Casewell et al 1981).

5 Bacteria circulating in the blood of an apparently

healthy donor suffering from asymptomatic bacteraemiamay proliferate in red cell components stored at 4°C or

in platelet concentrates stored at room temperature.Bacteraemia in donors may be chronic and low grade,

as in the case of the incubation or convalescence periods

of salmonella, Yersinia enterocolitica or Campylobacter jejuni infections, or acute and transient as occurs

within the first few hours after dental extractions when

the organisms involved are usually Streptococcus dans, Bacteroides species and, less often, Staphylococcus aureus A particularly notorious case reported from the

viri-Clinical Center at NIH involved a repeat platelet donor

with asymptomatic Salmonella cholerasuis

osteomyeli-tis, whose donations resulted in sepsis in seven patients

and death in one of these before platelets were

identi-fied as the source of infection (Rhame et al 1973).

Frequency of bacterial contamination of blood

components

Bacteria are usually prevented from growing by the

antibacterial properties of blood Complement, even

in the absence of specific antibodies may kill bacteria,

which may then be phagocytosed by leucocytes

(Högman et al 1991; Gong et al 1994) Nevertheless

positive cultures in samples from blood or blood

com-ponents are found fairly frequently In an extensive

survey in which approximately 2500 units of red cells

and an equal number of platelet concentrates were

sampled annually, over a period of 10 years the mean

rates of positive cultures were 0.3% and 0.4%

respect-ively (Goldman and Blajchman 1991) Bacterial

con-tamination of whole blood cultured after 2–20 h of

storage at room temperature ranges from 0.34% to

2.2% (Bruneau et al 2001; de Korte et al 2001).

Survival of most organisms is reduced by storage at

refrigerated temperatures However, platelet

con-centrates are stored at room temperature so that

bacterial contamination and continued growth present

an urgent clinical problem In one early study, 6 out

of 3141 (0.19%) pooled concentrates from random

donors were found to contain bacteria just prior

to transfusion (Yomtovian et al 1993) In another

prospective study, 7 out of 15 838 (0.04%)

concen-trates were confirmed positive (Blajchman et al 1994).

A higher percentage (0.79%) was found in samples

taken from freshly prepared concentrates: of 11

posit-ive cultures only one was confirmed on repeated culture

from the same concentrate (Högman and Engstrand

1996) Only 5 out of 17 928 (0.03%) single-donor

concentrates obtained by apheresis contained bacteria

(Barrett et al 1993) A 6-year experience using a

semi-automated system for routine platelet cultures in

Denmark reported an initial positive reaction in 84

samples (0.38%) from 22 057 platelet units Growth

was confirmed in 70 of these (Munksgaard et al.

2004) The risk of collecting a contaminated unit of

platelets is estimated at 1 in 2000 (Chiu et al 1994).

A prospective study using a conservative case

definition found the rate of transfusion-transmitted

bacteraemia (in events/million units) to be 9.98 for single-donor platelets, 10.64 for pooled platelets and0.21 for RBC units (1/500 000); for fatal reactions, therates were 1.94, 2.22 and 0.13 respectively (Kuehnert

et al 2001) From a 12-year retrospective analysis

of transfusion data from a single institution, tomatic sepsis from transfused platelets was estimated

symp-to occur at between 1 in 5000 (pooled derived platelets) and one in 15 000 single-donor

whole-blood-apheresis platelet transfusions (Ness et al 2001).

The incidence of clinical reactions due to ated blood is much lower than that expected from thereported rates of positive bacterial cultures Althoughsome contaminated components, especially those containing large numbers of organisms or endotoxin,result in immediate, catastrophic reactions includingshock and death, others may be quite mild with little

contamin-more than low-grade fever or chills (Yomtovian et al.

1993) As many patients who receive platelet fusions already have an infection, febrile reactions orsymptoms of septicaemia may not be ascribed to the

trans-transfusion (Chiu et al 1994) Furthermore many of

these patients are being treated with antibiotics thatblunt detection and diagnosis of septic reactions

Autologous blood

As might be anticipated, autologous donations arealso susceptible to bacterial contamination At leastfive such cases have been reported The majority of

the infected units contained Y enterocolitica, and the

donors recalled in retrospect gastrointestinal episodes

in the weeks prior to donation (Haditsch et al 1994).

Some properties of organisms that grow in stored blood

Organisms isolated from refrigerated blood are ally Gram-negative rods capable of metabolizing citrate (Pittman 1953) Many strains of organisms isolated from contaminated stored blood cause clotformation by consuming citrate, yet another reason toexamine all units carefully at the time of issue (Braude

usu-et al 1952; Pittman 1953) Blood that is heavily

con-taminated with organisms is not necessarily lysed Among bacteria isolated from stored blood, 25%

haemo-produce no haemolysis (Braude et al 1952) Many

organisms that grow in stored blood are psychrophilic– organisms that grow preferentially in the cold

Unless blood is heavily contaminated with

organ-isms, microscopic examination is an unreliable method

of detecting infection After serial dilutions of an

inoculum of a coliform organism to blood, 24 × 105

organisms/ml could be detected readily but 24 × 104/ml

(one organism in every 100 fields) could be detected

only with difficulty (Walter et al 1957) On the other

hand, when an aliquot of 0.3 ml was cultured for 24 h,

contamination could be detected when the number of

organisms added was as low as 24/ml

The effect of antibiotics on bacteria in stored blood

has been investigated Chlortetracycline,

oxytetracy-cline and polymyxin B in concentrations as low as

10 mg in 500 ml of blood (i.e 20 µg/ml) would all

pre-vent the inocula of either cold- or warm-growing

bac-teria from multiplying in human blood either in the

cold or at room temperature Nevertheless, routine

addition of antibiotics to stored blood cannot be

recommended First, antibiotics cannot be autoclaved

and would therefore have to be added later, and this

addition might itself contaminate the collection

Second, no antibiotics are effective against all

organ-isms Third, there is the risk of immunizing patients to

particular antibiotics or of inducing a hypersensitivity

reaction in a patient already immunized

Importance of maintaining refrigeration

After the first 24 h or so of storage, strict maintenance

of refrigeration at a temperature of 4°C becomes

essential When blood has to be transported for any

considerable distance the use of refrigerated containers

can maintain blood below 10°C for as long as 48 h

Some transfusion centres routinely keep blood at

approximately 20°C for up to 24 h after collection

before it is processed The storage at room

temper-ature does not seem to affect the yields of factor VIII in

plasma subsequently frozen and fractionated or the

shelf-life of platelets or red cells (Pietersz et al 1989).

Leucocytes in fresh blood have a bactericidal effect for

a few hours after collection and before processing into

blood components with removal of the buffy coats

From experiments in which defined numbers of

colony-forming units of different bacteria were added to fresh

blood, leucocytes were shown to effect clearing of

bac-teria, the efficiency varying according to the bacterial

species: Staphylococcus epidermidis and Escherichia

coli were cleared more readily than S aureus, which

was engulfed by the leucocytes and then released after

causing cell death (Högman et al 1991) The clearing

effect of leucocytes sometimes requires as long as 24 h.Some Gram-negative bacteria are killed by plasma fac-tors, presumably antibodies and complement Certain

anaerobic bacteria such as Propionibacterium species

do not grow in stored blood, regardless of the presence

or absence of white cells Yersinia enterocolitica was

cleared temporarily, but reappeared within a few hours;

if leucocytes were removed from the blood after 5 h,the units remained sterile Although some bacteria arekilled intracellularly, others kill the cells, which then

disintegrate releasing the bacteria (Högman et al 1991).

Bacteria contaminating different blood components

The type of bacterial contamination largely depends

on the type of blood component:

1 Red cells involved in cases of bacterial infection

have been contaminated mainly with Pseudomonas fluorescens, P putida and Y enterocolitica Yersinia is

normally sensitive to complement and is phagocytosed

by leucocytes in fresh blood However, it is capable ofproducing a virulence plasmid that expresses a surfaceprotein rendering the organism resistant to comple-ment and intracellular killing after phagocytosis (Lian

et al 1987) Yersinia grows well at 4°C, uses citrate

as a source of energy and, owing to its lack ofsiderophores, requires iron for optimum growth Redcell concentrates provide an ideal culture medium Theorganism produces a potent endotoxin during storage.Transfusion of contaminated red cells can cause fatalsepticaemia In almost all serious reactions due to

Y eneterocolitica, the red cell concentrates had been

stored for more than 3 weeks Phagocytes containinglive bacteria may persist after a donor has otherwisecontained a yersinia infection During storage of redcell concentrates the leucocytes may disintegrate,releasing live bacteria, which can then multiply

(Högman et al 1992) Leucocyte-depleted red cell concentrates, previously inoculated with Y eneteroco- litica remain sterile, whereas growth develops after

2–3 weeks in leucocyte-containing concentrates (Gong

et al 1994) Between April 1987 and the end of

February 1991, eight fatalities associated with thetransfusion of red cells contaminated with bacteriawere reported to the FDA; seven of these were due to

Y enterocolitica During the same period, 10 cases

of Y enterocolitica caused by contaminated red cell

transfusions were reported in the USA, three cases

in France, one in Belgium and one in Australia The

patients developed fever and hypotension within 50 min

of the start of transfusion In the 10 cases that occurred

in the USA, the blood had been stored in the cold for a

mean of 33 days (range 25– 42 days) Although reports

of septicaemia due to yersinia are rare, the numbers

have increased in the past 10 years If patients are

tak-ing antibiotics, infection by blood contaminated with

yersinia can be asymptomatic (Jacobs et al 1989).

2 Platelet concentrates stored at 20–24°C have caused

fatal bacterial sepsis when contaminated with any of

several Gram-negative or -positive organisms such as

staphylococci, streptococci, Serratia species,

flavo-bacteria and salmonellae (Goldman and Blajchman

1991) One plateletpheresis donor with subclinical

Salmonella cholerae suis osteomyelitis caused sepsis

in seven platelet recipients (Rhame et al 1973).

3 Cryoprecipitates and FFP can become contaminated

with P cepacia and P aeruginosa during thawing in

con-taminated waterbaths (Goldman and Blajchman 1991)

4 Any of the above (1–3) can become infected if

the exterior of blood packs is massively contaminated

during manufacture by bacteria such as Serratia

marcescens Septicaemia has been reported in

recipi-ents of such contaminated units (Heltberg et al 1992).

Effect of transfusing bacterially

contaminated blood

Shock following the injection of bacteria is

pre-sumably due to bacterial toxins, although an immune

reaction between naturally occurring antibodies and

bacteria may also produce ill effects The transfusion

of contaminated blood may produce immediate

col-lapse, followed by profound shock and hyperpyrexia;

haemorrhagic phenomena due to disseminated

intra-vascular coagulation are common; in a series of

25 cases, mortality was 58% (Habibi et al 1973).

A review of more than 30 reports of post-transfusion

sepsis due to contaminated red cells, platelets or a

few other blood components showed that septic shock

developed in many cases and that the overall mortality

rate was 26% Common signs and symptoms reported

were fever, chills, vomiting, tachycardia and

hypo-tension, often during the transfusion, but sometimes

developing a few hours later In the case of

Pseudomonas cepacia, septicaemia or wound infection

developed several days or weeks after transfusion

(Goldman and Blajchman 1991) However, atypicalpresentations have been well documented in prospect-ive studies and suggest that clinical syndromes are

under-reported (Yomtovian et al 1993).

Investigations following the suspected transfusion of injected blood

Any blood remaining in the container should be cultured at various temperatures, including 4°C and20°C, in appropriate culture media A negative cultureexcludes the possibility that the blood was heavilyinfected at the time of transfusion A positive culturedoes not enable one to say with certainty whether theblood was contaminated before the transfusion orbecame contaminated during or after transfusion, orupon sampling Techniques to exclude contaminationupon sampling have been described (Puckett 1986b)

It is still useful to culture implicated containers thathave been left at room temperature for 24 h Most sterile units remain sterile or at worst lightly con-taminated In fatal cases, blood for culture should becollected from the body (e.g by cardiac puncture) assoon as possible after death Blood contaminated with

a coliaerogenes bacterium cultured positive, althoughwhen pseudomonads were involved, blood collected atautopsy was sterile (Pittman 1953)

Specific identification of the contaminating ism can be important for determining the extent ofdonor follow-up and the donor counselling message(Haimowitz 2005)

organ-Inspection of red cells: the dark side of transfusion

Colour change in stored red cell concentrates mayindicate that the unit has been contaminated with bac-teria In 13 units of red cells that supported the growth

of Y enterocolitica, a darkening related to haemolysis and a decrease in PO2was observed in the primary

container (Kim et al 1992) The attached sample

seg-ments, which were sealed from the main unit, remainedsterile and did not darken The colour change wasapparent in all the contaminated units by day 35, or1.5 to 2 weeks after the bacteria were first detected incultures of the blood A keen observer can identify

units grossly contaminated with Y enterocolitica by

comparison of the colour of the segment tubing

with that of the unit (Kim et al 1992) Review of

photographs on file at the Centers for Disease Control

revealed this colour change in 2 units of blood that

caused transfusion-transmitted sepsis (Enterobacter

agglomerans and an unidentified Gram-negative

bacil-lus), which demonstrates that the colour change is not

limited to contamination by Y enterocolitica.

Screening for bacterial contamination

Avoidance of bacterial contamination is preferable

to detecting organisms downstream Examination

of the donor’s arms and rigorous skin preparation

reduce quantitative bacterial growth by as much as

50% (Goldman et al 1997) (see Chapter 1) Diversion

of an aliquot of the initial blood collection removes

some of the organisms that populate the phlebotomy

site (de Korte et al 2002) Screening collected blood for

bacterial contamination is complicated by the small

initial inoculum (sampling error), differential growth

rates of various species, and the potential for

introduc-tion of organisms at the time of the screening procedure

Despite the absence of convenient and inexpensive

detection methods, routine surveillance of platelet

preparations for bacterial contamination has been

instituted in several countries The two most

com-monly used methods are culture based A sensitive and

specific automated, continuous monitoring culture

system measures carbon dioxide production as a

marker of bacterial growth (Brecher et al 2001,

2004) Both aerobic and anaerobic cultures are

pos-sible The procedure increases the storage interval

before platelets can be released and cultures may turn

positive after platelets have been issued or transfused

A second system monitors consumption of oxygen

after 24 h at room temperature in aliquots of platelet

concentrates as a marker of microbial growth (Rock

et al 2004) The disadvantages include inability to

detect anaerobic organisms, reliance on analysis at a

single time point and limited ability to detect

slow-growing bacteria However only one example of a

fatality associated with an anaerobic organism has

been reported (McDonald et al 1998) Both methods

have interdicted contaminated transfusions

Malaria

Transmission of malaria by blood transfusion

Malaria can be transmitted by the transfusion of any

blood component likely to contain even small numbers

of red cells; platelet and granulocyte concentrates,fresh plasma and cryoprecipitate have all been incrim-inated An inoculum containing as few as 10 parasites

can cause Plasmodium vivax malaria (Bruce-Chwatt

1972)

Despite intensive eradication programmes, theprevalence of malaria in tropical and subtropicalcountries is extremely high, with about 300 millioncases reported each year, and more than 1 millionannual fatalities attributed to malaria in tropicalAfrica (Wyler 1983) The increasing resistance of themosquitoes to insecticides and the increasing appear-

ance of drug-resistant strains, particularly of P parum and the recurrence of malaria in areas where

falci-the disease had previously been eradicated, have greatlyincreased the incidence of malaria in recent years.Malaria parasites of all species can remain viable instored blood for at least 1 week (Hutton and Shute

1939) Cases of P falciparum malaria have been mitted by blood stored for 14 days (Grant et al 1960) and for 19 days (De Silva et al 1988) Malaria para-

trans-sites may survive longer in blood stored in containing solutions (WHO 1986) In a review ofnearly 2000 cases of transfusion malaria due to

adenine-P malariae, in the great majority of cases the blood

had been stored for less than 5 days and cases ing the transfusion of blood stored for 2 weeks wererare (Bruce-Chwatt 1974) The parasites survive well

follow-in frozen blood (Kark 1982) Plasma that has beenfrozen or fractionated has never been known to trans-mit malaria

In non-malarious countries, mainly because of delay

in diagnosis, post-transfusion malaria has a relativelyhigh fatality rate Malaria is particularly serious inpregnant women and in splenectomized or immuno-suppressed patients (Kark 1982; Bruce-Chwatt 1985).Transfusion-transmitted malaria usually responds

to conventional drug therapy In severe cases in whichthe diagnosis has been delayed and in fulminant cases,the patient may benefit from exchange transfusion

in the period before antimalarial drugs have had time

to become effective (Yarrish et al 1982; Kramer et al 1983; Files et al 1984) However, no controlled trials

have been performed and antimalarial medical therapy

is effective in most cases (Mordmuller and Kremsner1998)

The red cells in a renal allograft and in a bone

mar-row graft have been known to transmit P falciparum

malaria (Kark 1982; Dharmasena and Gordon-Smith

1986) The plasmodia can also be transmitted by

transplacental passage

Frequency of post-transfusion malaria

Estimates of the frequency of post-transfusion malaria

vary from less than 0.2 cases per million units of blood

transfused in non-endemic countries to 50 or more

cases per million in some endemic countries

(Bruce-Chwatt 1985) In countries where malaria is

non-endemic the frequency of reported transfusion malaria

is relatively low The UK has reported eight cases in

50 years, the USA 26 cases in 10 years from 1972 to

1981, and France 110 cases in 20 years (Bruce-Chwatt

1985), although between 1980 and 1986 only 14 cases

were observed On the other hand, nine cases of

transfusion-transmitted malaria were reported to the

Centers for Disease Control (CDC) in the USA in

1982, although only 11 cases in the following 4 years

(Westphal 1991b) In Canada, only three cases of

transfusion-transmitted malaria were reported between

1994 and1999, a rate of 0.67 cases per million

dona-tions (Slinger et al 2001) The rate of

transfusion-transmitted malaria in the USA since 1992 has

fluctuated from 0.18 to 0.6 per million units

trans-fused (Mungai et al 2001) In many malarious areas

where reporting is inefficient, the frequency of

post-transfusion malaria is unknown

Frequency with which the different parasites

are involved

In the period 1950 –72, P malariae was responsible

for about 50% of cases of transfusion malaria and

P vivax for 20% (Bruce-Chwatt 1974) In the period

1973–80 a global survey showed a large relative

increase of cases due to P vivax, which, with a few

cases of P ovale, accounted for 42% of all cases;

cases due to P malariae fell to 38% whereas those due

to P falciparum increased four-fold (Bruce-Chwatt

1982) In more than one-half of the cases reported,

the species involved was not identified The relative

increase in cases due to two of the parasites has been

attributed to the failure of donors from highly endemic

areas to comply with rules laid down by transfusion

services (Guerrero et al 1983; Shulman et al 1984).

The simultaneous transmission of two different strains

of malaria (P falciparum and P malariae) by

trans-fusion has been described (Aymard et al 1980) Of

93 cases of transfusion-transmitted malaria reported

to the CDC from 1963 to 1999, 33 (35%) were due to

P falciparum, 25 (27%) were due to P vivax, 25 (27%) were due to P malariae, 5 (5%) were due to

P ovale, 3 (3%) were mixed infections and 2 (%) were

due to unidentified species; 10 out of the 93 patients(11%) died

Incubation period

The incubation period of transfusion malaria depends

on the numbers and strain of plasmodia transfused, onthe host and on the use of antimalarial prophylaxis;

with P falciparum and P vivax it is between 1 week and 1 month, but with P malariae it may be many

months (Bruce-Chwatt 1974)

Period for which donors remain infectious

P falciparum infections, prevalent in West Africa, are

usually eliminated within 1 year but have been known

to persist for as long as 5 years after the last exposure

to infected mosquitoes (Guerrero et al 1983) P parum transmitted from asymptomatic blood donors

falci-can cause fulminant infection and death in an alreadyill, non-immune recipient in whom the diagnosis,

being unexpected, is usually delayed P vivax

infec-tions, prevalent on the Indian subcontinent, mayrelapse for up to 2.5 years after infection but only

rarely after more than 3 years With P ovale a clinical

attack may occur up to 4 years after returning from anendemic area In immune carriers who have lived in

endemic areas for most of their lives, P falciparum or

P vivax may reappear long after the limits given above.

P malariae infections persist for longer than those of

any of the other malaria parasites Transmission ofmalaria has been known to occur up to 46 years afterthe last exposure to infection (Miller 1975)

Rules to prevent transmission of malaria in non-endemic areas

In non-endemic areas, eligibility of potential blooddonors who have visited endemic areas is restricted.Regulations and standards are not harmonized andnot always consistent Two different approaches havebeen adopted: the USA relies solely on the deferral ofsubjects who have been in endemic areas, whereas in

Europe deferral and testing for malaria parasites can

be combined

All donor clinics should have an up-to-date WHO

map showing the areas endemic for malaria together

with an alphabetical list of the countries

In the USA, those who normally live in a

non-endemic area but have travelled in an area considered

to be endemic for malaria may be accepted as regular

blood donors 1 year after returning to a non-malarious

area, provided that they have had no symptoms

Pro-spective donors who have had malaria are deferred for

3 years after the cessation of therapy or after leaving

the malarial area if they have been asymptomatic

meanwhile In the USA, of 64 implicated donors

whose country of origin was reported, 38 (59%) were

foreign born Among those for whom complete

informa-tion was available, 37 out of 60 donors (6%) would

have been excluded from donating according to

guide-lines in place since 1994, and 30 out of 48 donors

(62%) should have been excluded under the guidelines

in place at the time of donation (Mungai et al 2001).

The Council of Europe in 1995 adopted the

recom-mendation that individuals born or brought up in

endemic areas may be accepted as donors of whole

blood 6 months after their arrival in a non-endemic

area, provided that an approved immunological test

has given a negative result (Council of Europe 2003)

Others from non-endemic areas, for example

trav-ellers, can be accepted as blood donors 6 months after

their return if they have been afebrile and have not

taken antimalarial prophylaxis; those who have had

febrile illnesses may be accepted if an antibody test

is negative at 6 months Although a proportion of

antibody-positive subjects are non-infectious and are

thus unnecessarily rejected, application of these

proced-ures makes it possible to use 75 –90% of units of blood

from donors returning from malarious areas to France,

the UK and Belgium (Soulier 1984; Wells and Ala 1985)

The foregoing rules will not prevent the occasional

transmission of P malariae and, especially, of P vivax.

Donations to be used for the preparation of plasma

for fractionation may be accepted from donors who do

not meet the above criteria

Tests to detect carriers

When blood films are examined by simple microscopy,

a density of less than 100 parasites per microlitre of

blood cannot be detected, although a unit from a

donor whose blood contained even one parasite permicrolitre would contain about half a million parasites(Bruce-Chwatt 1985)

The indirect fluorescent antibody test or ELISAoffers a good chance of detecting latent malarial infec-

tion (Deroff et al 1982; Voller and Draper 1982).

ELISA is more suitable for large-scale screening in the blood transfusion service and its specificity isimproving with the use of chromogenic substrates Acommercial ELISA for malarial antibodies has under-gone a successful trial in the UK Ideally, potential car-riers who volunteer as blood donors in non-malariousareas should be screened for malaria antibodies using

a test covering three homologous antigens However,

only P falciparum is readily available for in vitro tests:

although there is some degree of crossreactivity betweenthe different strains of malaria parasites it is not known

how valuable a test based on the use of P falciparum

would be in a country such as Mexico, where none ofthe 44 cases of transfusion malaria reported in 5 years

was due to this strain (Olivares-Lopez et al 1985).

Sensitive tests, using monoclonal antibodies, arebeing developed for use in endemic areas for detection

of parasites in red cells, and soluble antigens and bodies in plasma (Soulier 1984; Prou 1985) In a study

anti-in blood donors anti-in Chandigarh, India, a comparison

of different screening techniques for malaria carriersshowed that a test for malaria antigen, using mono-clonal antibodies, had good sensitivity and specificity.The test was considerably more sensitive than the

direct examination of blood films (Choudhury et al.

1991) Serological tests for malarial antibodies and testsfor malarial antigens are useful in the identification ofdonors implicated in cases of transfusion-transmittedmalaria in non-endemic countries when blood filmsare negative PCR has been used to determine theprevalence of malaria in potential blood donorsidentified as being at high risk of having malaria Only4% of blood donors tested positive for malaria byPCR Furthermore, 42% of blood donors who werenegative on PCR testing returned to donate, and

no cases of transfusion-transmissible malaria were

reported (Shehata et al 2004).

Prevention of transfusion malaria in malarious countries

In countries such as Nigeria, Zambia and Papua NewGuinea, where up to 10% of donors have plasmodia

detectable by simple microscopy, the contribution

of blood transfusion to the overall problem of the

transmission of malaria is negligible In such countries,

the transmission of malaria to the recipient can be

prevented by giving antimalarial treatment to donor

or recipient Officer (1945) injected blood containing

some 300 million malaria parasites into each of five

healthy volunteers All volunteers were given

anti-malarial treatment after transfusion and none

devel-oped malaria

Trypanosoma cruzi Chagas’ disease –

American trypanosomiasis

In Latin America 24 million people are infected with

Trypanosoma cruzi Infection is most commonly

transmitted to humans, usually in childhood, by

triatomid bugs Until recently, Chagas’ disease was

limited to rural areas with mud huts and thatched

roofs but, due to migration of the rural population,

infected people are also found in urban areas (Wendel

1995) The next most common mode of transmission

is by blood transfusion

Treatment of acute infection cures 50 –90% of

patients, but most acute infections are subclinical and

untreated individuals remain infected throughout life

Depending on the country, between 5% and 40% of

untreated patients may develop serious chronic

com-plications such as cardiomyopathy, megaoesophagus

and megacolon, 10 or more years after infection

(Marsden 1984; Schmunis 1991)

In chronic infection, antibodies can be detected by

complement fixation, direct or indirect

haemagglu-tination, immunofluorescence or ELISA (Schmunis

1991) ELISA appears to be more sensitive than

indirect immunofluorescence and haemagglutination

(Magnaval et al 1985) Although tests for antibody

are positive in about 95% of chronic cases and in 50%

of acute cases, they are often negative during the first

few months of infection (Wolfe 1975) At least 50% of

those infected have parasitaemia and are thus likely

to be able to transmit infection by blood transfusion

(Cerisola et al 1972) Parasitaemia is diagnosed by

allowing triatomid bugs to feed on the patient’s

blood and then examining the insects for infestation

(‘xenodiagnosis’) Most Latin American countries test

part, but not all, of their blood donations for T cruzi

antibodies; the frequency of a positive test amongst

donors varies from about 0.3% to 28% and is as high

as 62% in parts of Bolivia (Schmunis 1991) Owing tomigration to urban areas and to the existence of paiddonors, antibody-positive donors are often found incities that lack the triatomid vector When blood from

an antibody-positive donor is transfused, the incidence

of infection in recipients is between 12% and 18% andcan be as high as 50% (Schmunis 1991) The parasites

of Chagas’ disease survive well in stored blood, andblood stored for more than 10 days can still be infec-

tious (Cerisola et al 1972) T cruzi can be transmitted

by plasma as well as by whole blood: the parasite vives in plasma which has been frozen at –20°C for

sur-24 h but it does not resist lyophilization (Wendel andGonzaga 1992) The addition of 125 mg of crystal(gentian) violet to a unit of stored blood kills the para-sites after storage at 4°C for 24 h without damagingthe red cells Blood treated with gentian violet causes

no obvious toxic reaction in the recipient, but cancause minor side-effects and rouleaux formation of the red cells Artificial light and sodium ascorbateaccelerate the effect and reduce the dose of gentian violet required to kill the parasites Gentian violet

has been reported to have mutagenic effects in vitro

(Wendel and Gonzaga 1992)

Latin Americans who migrate do so mainly to NorthAmerica where more than 5 million now live Morethan 100 000 may be infected with Chagas’ disease(Wendel 1993) Chagas’ disease acquired by bloodtransfusion has been reported in the USA, Canada and

Spain (Villalba et al 1992) Six cases of transmitted T cruzi have been reported in the USA and

transfusion-Canada (four and two cases respectively), with theimplicated donor in at least five of these cases from a

T cruzi endemic country (Leiby et al 2002a) Platelets

may be the blood component that is most often implicated in transfusion-transmitted Chagas’ disease,possibly because of their relatively short storage period

at temperatures favouring parasite survival (22–24°C)

or because platelet recipients are more likely to beimmunocompromised and therefore are more likely

to manifest clinical infection than recipients of other

components (Leiby et al 1997).

Other protozoaToxoplasma gondii

Toxoplasma gondii is an obligate intracellular

proto-zoan that replicates in the intestine of members of the

cat family, its definitive host Humans may be infected

by eating undercooked lamb or pork or by exposure to

the faeces of infected cats Seroprevalence of T gondii

rises with age and the incidence of infection varies with

the population group and geography In the USA,

almost one-quarter of the population has been infected

(Jones et al 2001) T gondii has been isolated from

donors’ blood up to 4 years after the onset of infection;

entry of parasites into the blood occurs for only a

few weeks, but as T gondii is an obligate intracellular

parasite it persists in the white cells and can survive

storage at 4°C for 4–7 weeks (Tabor 1982) Congenital

transmission occurs, particularly in women infected

shortly before or during pregnancy

Most infections with toxoplasma are asymptomatic

or result in mild fever and lymphadenopathy However,

infection in immunocompromised patients is

poten-tially life threatening Transmission of T gondii from

seropositive solid organ donor to seronegative recipient

is well documented, but for recipients of haematopoietic

stem cell transplants, reactivation of latent infection

appears to be more important Leucocyte transfusions

from donors with high levels of toxoplasma

anti-bodies have resulted in severe acute toxoplasmosis

in immunosuppressed patients (Siegel et al 1971;

Tabor 1982) As the risk of severe

transfusion-acquired toxoplasmosis is confined almost entirely to

immunosuppressed patients transfused with leucocyte

concentrates, some centres have elected to maintain a

panel of donors negative for toxoplasma antibodies

for leucapheresis and for the provision of granulocyte

concentrates for premature babies (M Contreras,

per-sonal communication)

Babesia

In North America, the parasite causing babesiosis in

humans is Babesia microti, which occurs most often

in the north-east and in Wisconsin and Minnesota

However, the geographic range of B microti is

expanding Other babesia species have been

implic-ated in transfusion transmission in the western USA,

and the mobility of blood donors and blood

com-ponents may result in the appearance of

transfusion-transmitted babesiosis in areas less familiar with these

parasites (Cable and Leiby 2003) In Europe, the

responsible parasite is Babesia bovis

Transfusion-transmitted cases have been reported only in the USA

(Popovsky 1990; Mintz et al 1991) Babesia microti,

like Anaplasma phagocytophila and B burgdorferi,

is transmitted to humans from animal reservoirs (primarily deer and mice) by black-legged ticks of the

genus Ixodes (Cable and Leiby 2003) For donor

screening purposes, self-reported tick bites are neithersensitive nor specific indicators of serological status

(Leiby et al 2002b).

B microti reproduce only in red cells, and on blood smear the organisms appear similar to Plasmodium falciparum The clinical symptoms also resemble those

of malaria The diagnosis must be confirmed by testing

for Babesia antibodies and by inoculating hamsters

with the patient’s blood Parasites can survive for up to

35 days in blood stored at 4°C, and persist in frozenred cells for years

Although normally mild and frequently tomatic, the disease can be fulminant and rapidly fatalfor immunosuppressed and splenectomized patients.Since 1980, more than 40 USA cases of post-transfusionbabesiosis, including at least two cases of WA-1, thewestern US variant, have been reported, most of them

asymp-in immunocompromised or splenectomized patients

In two splenectomized patients infected by transfusion(one by a platelet concentrate), exchange transfusionusing whole blood helped bring about control of the

disease (Jacoby et al 1980; Cahill et al 1981) As in the

case of malaria, the role of exchange transfusion is notwell documented, but the combination of clindamycinand quinine seems to eradicate the parasite from most

immunosuppressed patients (Smith et al 1986).

Microfilariae

Microfilariae of five common species have been shown

to persist in the circulation of infected subjects for

years and to survive in stored blood Wuchereria bancrofti can be detected in refrigerated blood for 3

weeks Microfilariae can be transmitted to transfusionrecipients Three varieties of filariae can cause disease

in humans: Brugia malayi in South-East Asia, Loa loa

in Africa and W bancrofti in southern Asia, tropical

Africa and some tropical areas of Latin America.However, blood transfusion-transmitted microfilariaenever reach the adult stage in non-endemic areasbecause of the absence of the vector necessary for thesecond passage to humans Recipients of infectedblood usually have no symptoms, although they mayoccasionally experience acute inflammation of thespleen, lymph nodes or lungs, sometimes with tropical

pulmonary eosinophilia Recipients may develop

fever, headache and rash as a hypersensitivity response

to the dead microfilariae (Choudhury et al 2003).

Signs and symptoms are self-limited and so is the

sur-vival of microfilariae in transfusion recipients (Tabor

1982; Westphal 1991a)

Leishmaniasis

In 1903, Leishman and Donovan independently

described the protozoan now called Leishmania

dono-vani in patients from India with the life-threatening

disease now called visceral leishmaniasis or kala-azar.

Leishmaniasis and its major syndromes (visceral,

cutan-eous and mucosal) have changed little over the past

century, but information regarding the endemic areas,

the vectors and the transfusion risks have evolved

considerably (Herwaldt 1999) More than 20 different

species of Leishmania have been recognized, some

anthroponotic but most zoonotic The organism is

transmitted by the female phlebotomine sand fly,

of which some 30 species are vectors Visceral

leish-maniasis or kala-azar caused primarily by Leishmania

donovani is found most often in China, India and the

eastern Mediterranean L major, L tropica and

L infantum are found in the Middle East However, the

organisms are found in jungles, deserts, rural areas and

cities of some 88 countries Leishmaniasis is endemic

in much of Latin America between northern Argentina

(L braziliensis, L panamensis) and southern-central

Texas (L mexicana), and in south-western Europe

(France, Spain, Portugal, Italy, Greece) and, because

the sand fly has a limited range, microfoci of activity

are found within countries where the disease is

con-sidered endemic The cutaneous form of the disease

goes by many names, including Baghdad Aleppo boil,

Oriental sore and bush yaws These characteristics

make screening questions difficult to formulate There

is no screening test and no licensed drug therapy

No vaccine has been developed, although paediatric

inoculation using preparations from active sores is

widely practised in endemic areas

Leishmania multiply in mononuclear phagocytes

and clinical disease transmitted by blood

trans-fusion has been reported in transplant recipients, in

haemodialysis patients and in newborn infants When

present in blood, the parasite has been found in

large mononuclear cells and granulocytes (Shulman

1990) The incubation period can vary from 10 days

to 2 years, but is generally 2– 6 months and results inchronic infection Leishmania species are clearly bloodtransmitted, although fewer than 15 transfusion caseshave been reported, one of which was fatal Canine

transmission is well recognized (Owens et al 2001).

Studies of asymptomatic blood donors in southern

France indicated that L infantum circulates

intermit-tently at low levels and mononuclear cells from these

donors can infect Syrian hamsters (le Fichoux et al.

1999) Although the conventional teaching holds thatvisceral leishmaniasis poses the greatest risk of blood

transmission, ‘dermatotropic’ organisms such as L tropica, L braziliensis and L panamensis have been

found in blood decades after the patient’s exposure.Human transfusion transmission results in viscerocu-taneous disease

L tropica has been demonstrated to survive in

stored blood under blood bank conditions for 25 days

(Grogl et al 1993) A 1-year deferral was instituted by

the USA Department of Defense in 1991 and again in

2003 for service personnel stationed in endemic areas

in Iraq Deferral for diagnosed infection is indefinite.Cases of possible transmission of kala-azar by transfu-sion in India, China, Brazil and four European coun-

tries have been reported (Chung et al 1948; Cummins

et al 1995; Singh et al 1996) Subjects with a history

of visceral leishmaniasis must not be accepted asdonors (Council of Europe 1992)

African trypanosomiasis (sleeping sickness) has

rarely been transmitted by transfusion (Wolfe 1975;Tabor 1982)

Methods of inactivating pathogens in blood and blood products

Despite careful donor selection and sensitive atory testing, a small risk of transmission of infectiousagents by transfusion remains, particularly with prod-ucts obtained from large pools of plasma The frac-tions obtained at an early stage of the cold-ethanolfractionation, for example fibrinogen, factor VIII, factor IX and prothrombin complex concentrate, con-tain intact virus Plasma fractions obtained later in thefractionation process, such as the immunoglobulinsand albumin, are safer, although HCV can be detected

labor-in the fraction from which IVIG is prepared (Yei and

Yu 1992) HCV has been transmitted by IVIG

pre-parations (Weiland et al 1986; Bjoro et al 1994; Meisel

et al 1995; Power et al 1995).

Inactivation of viruses in blood products or their

removal, or both, should further increase the safety

of transfusion and might protect against unknown

pathogens or those for which no screening tests are

available The ideal inactivation technology should

reduce the risk of infection from the blood component,

result in minimal damage to the component cell or

protein content and function, and pose minimal

toxicological risk to the recipient, particularly to

sus-ceptible patients such as gravid women and neonates

The risk–benefit estimates have clearly favoured

inactivation technologies for protein concentrates

derived from large plasma pools The calculus differs

for cellular components derived from single donors

Plasma and plasma products

Heat treatment

Pasteurization and vapour treatment of liquid clotting

factor concentrates and heat treatment of lyophilized

concentrates (‘dry heat’) have been used for decades

(Kingdon and Lundblad 2002) The efficacy of these

methods differs Hepatitis viruses have been

trans-mitted by lyophilized concentrates heated between

60°C and 68°C for periods of between 24 and 72 h

(Colombo et al 1985; Blanchette et al 1991) Even the

more heat-labile HIV has been transmitted by such

concentrates (Mariani et al 1987; Williams et al.

1990b) Dry heating at 80° for 72 h, however,

effec-tively inactivates HIV and hepatitis viruses No

evid-ence of infection with these viruses was detected in 38

previously untreated haemophiliacs treated with

dry-heated concentrates (Skidmore et al 1990; Williams

et al 1990b) Pasteurized concentrates are also free

from the risk of transmitting HIV (Schimpf et al 1987)

and in prospective studies have been found not to

transmit hepatitis viruses (Mannucci et al 1990; Azzi

et al 1992; Kreuz et al 1995) On the other hand,

sev-eral cases of hepatitis B or C have been ascribed to the

use of pasteurized concentrates in patients not enrolled

in controlled studies (Kernoff et al 1987; Brackmann

and Egli 1988; Shulman et al 1992).

A major disadvantage of the heat treatment

methods is that they may not inactivate non-enveloped

viruses such as HAV and parvovirus B19 Some

recipi-ents have developed signs of parvovirus B19 infection

(Azzi et al 1992) However, dry heat treatment at

100°C for 1 h more effectively inactivates both

lipid-enveloped and non-lipid-enveloped viruses and, whenapplied to IVIG preparations, leads to only a very

small loss of IgG (Rubinstein et al 1994).

Solvent–detergent treatment

The combination of a solvent and a detergent disruptslipid-enveloped viruses such as HIV and hepatitis

viruses A non-volatile solvent, tri-(n-butyl) phosphate

(TNBP) is used in combination with a detergent such

as sodium cholate (Prince et al 1986) The solvent–

detergent (S–D) mixture is removed at the end of theprocedure by precipitation of the product or by chro-matography The viral safety of S–D treatment hasbeen shown by clinical trials in over 100 haemophiliacsand in hundreds of thousands of transfusions (Mannucci

1993; Klein et al 1998).

The S–D method has been adapted for whole plasmaand involves incubation of pooled thawed plasma withthe organic solvent TNBP and the detergent Triton

X-100 for 4 h at 30°C (Horowitz et al 1992; Klein

et al 1998) A drawback of the S–D method is that

non-enveloped viruses are not inactivated Severalreports of transmission of HAV and parvovirus B-19

by S–D-treated concentrates have been documented

(Mannucci 1992; Temperley et al 1992; Peerlinck and

Vermylen 1993) Factor VIII or IX concentrates tain diminished levels of anti-HAV which in otherfractions, such as IVIg, neutralizes the virus

con-S–D treatment has a few other flaws The reducedlevel of alpha(2)-antiplasmin in S–D plasma has causedconcern when the product is used in patients with liver disease; however, controlled studies have not

confirmed a clinical problem (Williamson et al 1999).

S–D plasma also has reduced levels of protein S, aplasma factor with anticoagulant properties Reducedprotein S may contribute to reports of venous throm-bosis during massive plasma infusion In 68 con-secutive patients with thrombotic thrombocytopenicpurpura (TTP) (25 men, 43 women), eight documentedthromboembolic events (six deep venous thromboses,three pulmonary emboli) were identified in sevenpatients during therapeutic plasma exchange All sixwere associated with central venous lines at the site

of thrombosis (Yarranton et al 2003).

One little appreciated benefit of S–D plasma is theabsence of association with TRALI, almost certainly

as a result of the pooling of 12 000 units per batch.However, for a variety of reasons, including safety and

commercial failure, S–D plasma is no longer available

in the USA

Heating in supension with n-heptane

Heating with n-heptane at 60°C for 20 h has been used

for factor VIII and IX concentrates but, as hepatitis

viruses have been transmitted by concentrates treated

in this way, S–D treatment is preferred

Methylene blue treatment

Methylene blue was first used clinically by Paul Ehrlich

in the nineteenth century and has been used as a

viruci-dal agent for more than half a century As with other

photoactive chemicals, methylene blue and similar

phenothiazine dyes have a high affinity for both

nucleic acids and the surface structures of viruses On

exposure to red (visible) light at 620–670 nm,

excita-tion of the dye causes chemical modificaexcita-tion of

adja-cent molecules, a process which involves oxygen

radicals (Lambrecht et al 1991) Methylene blue

treat-ment is currently the only method that can be applied

to individual units of plasma Residual dye is minimal

(1 µM) and can be further reduced 10-fold from the

final product by filtration

Methylene blue treatment does not inactivate

intra-cellular viruses efficiently but, as the plasma is frozen

and thawed, leucocytes are disrupted with release

of viral particles Neither does it effectively reduce

transmission of bacteria or protozoa For reasons that

remain unexplained, non-enveloped viruses, especially

HAV, are relatively resistant to methylene blue (Wagner

2002) An unfortunate consequence of methylene blue

treatment of plasma is the loss of 10% or more of some

clotting factors, particularly factor VIII and

fibrino-gen (Cardigan et al 2002; Williamson et al 2003).

Cryoprecipitate prepared from this plasma has not

been extensively studied and is not in current use

Commercial manufacturing systems have been

de-veloped and millions of single units of plasma treated

with methylene blue have now been transfused in

Europe without unexpected side-effects (Williamson

et al 2003) Concern remains in some quarters about

the potential mutagenic effects of methylene blue and

its derivatives, even in low concentration To date, no

immediate problems have been encountered in

trans-fusion recipients, although the number of neonates,

children and parous women treated remains small

Other methods of inactivation: at the bench but not yet at the bedside

Other methods include the use of the virucidal agent

hypericin (Lavie et al 1995) and photochemical

treat-ment with virucidal short-wavelength ultraviolet light

with the addition of rutin or psoralen (Chin et al 1995; Hambleton et al 2002).

Cellular componentsSome viruses such as CMV and HTLV are transmittedexclusively by leucocytes, whereas others, such asHIV, HBV and HCV are transmitted both by cellularcomponents and by plasma HIV can be internalized

by platelets, which may contain large amounts of the

virus (Zucker-Franklin et al 1990; Lee et al 1993).

HIV associates with red cells, probably as antibody–complement adherent complexes, but it is not clearwhether this ‘pool’ of virus is associated with transmis-

sion (Hess et al 2002) HPV B19 is known to adhere to

red cells To be effective, inactivation methods shoulddeal with free virus, virus attached to cells and intracel-lular virus

Physical methods

As mentioned above, the removal of leucocytes fromred cell and platelet concentrates reduces transmis-sion of CMV and HTLV-I and -II (Kobayashi 1993).Extensive washing and the process of freezing and deglycerolization are also effective in reducing trans-

mission (Brady et al 1984; Taylor et al 1986; Sayers

et al 1992).

Chemical and photochemical techniques

Several chemical and photochemical processes targetnucleic acid to inactivate residual contaminatingviruses, bacteria and protozoa in blood components(Klein 2005) As an added benefit, nucleic acid-targetedprocesses have the potential to inactivate residual lymphocytes and prevent transfusion-associated graft-

versus-host disease (Fast et al 2002, 2003, 2004).

Whole blood and red cells: the hurdles remain

For red cell concentrates, agents must be selected thatare photoactivated with a wavelength above that of

haemoglobin that would otherwise absorb the agent.

Optimal properties of sensitizing dyes for use in red

cell suspensions include selection of dyes that traverse

cell and viral membranes, bind to nucleic acids, absorb

light in the red region of the spectrum, inactivate a

wide range of pathogens, produce little red cell

photo-damage from dye not bound to nucleic acid and do not

haemolyse red cells in the dark Some agents inactivate

both free and cell-associated virus, but subtle changes

in the red cell membrane may affect storage, survival or

immunogenicity (Sieber et al 1992; Wagner et al 1993).

A red cell additive, S-303 or frangible anchor linker

effector, is an alkylating agent that inactivates

nuc-leated pathogens by crosslinking DNA and RNA in a

rapid, light-independent reaction (Klein 2005) The

initial enthusiasm for this agent has been tempered

by the report of red cell antibody formation in two

subjects enrolled in clinical trials (L Corash, personal

communication) Antibody formation has also been

reported during a trial of chronic transfusion of

pati-ents with sickle cell disease using red cells treated

with PEN110 (J Chapman, personal communication)

PEN110, a binary ethyleneimine that binds covalently

to nucleic acid is also light independent and interferes

with polymerase-mediated replication (Lazo et al.

2002; Ohagen et al 2002)

A 50-µM riboflavin solution added to diluted red

cells that are exposed to visible light of 450 nm, a

wavelength with minimal absorbance by haemoglobin,

effects a two- to four-log reduction of a number of

pathogens (Klein 2005) Clinical data for riboflavin

treatment of red cells are not yet available No method

has been licensed for inactivating pathogens in whole

blood or red cells

Methods for treating platelets

PEN110 treatment, which involves covalent binding

of drug to nucleic acids (see above), requires an

extens-ive washing procedure to remove residual additextens-ive, so

the process has not been applied to plasma or platelet

components Riboflavin, an essential nutrient (vitamin

B2) that absorbs both visible and UVA light, has been

investigated with both components (Li et al 2004).

Riboflavin’s three-ringed planar structure intercalates

between bases of DNA and RNA, and upon light

exposure, oxidizes guanosine through electron transfer

reactions, resulting in single-strand breaks of nucleic

acids and formation of covalent adducts (Dardare and

Platz 2002) The process has proved effective against

a wide range of human and animal pathogens, ing bacteria, intracellular HIV-1, West Nile virus andporcine parvovirus in preclinical studies of platelets

includ-and plasma (Goodrich 2000; Ruane et al 2004).

Initial toxicology assessment of the photoproducts ofriboflavin generated under proposed treatment condi-tions have been encouraging

By far the greatest experience with pathogen tion of platelet components involves the use of pso-ralen additives and UVA light activation Psoralenspass through the cell membrane and the capsids ofviruses and intercalate reversibly into helical regions

reduc-of nucleic acids Upon illumination with UVA (300–

400 nm) covalent crosslinks to pyrimidines in RNA andDNA form and block the replication and transcription

of nucleic acids (Hanson 1992) 8-Methoxypsoralen(8-MOP) inactivates free virus and even HIV incorp-

orated in the genome (Corash et al 1992) This very

high-energy dose of UV damages platelets, but whenaminomethyl-trimethyl psoralen (amotosalen, AMT,S-59) is used, lower doses of UV can be applied forshorter periods without loss of virucidal activity

(Corash et al 1992; Benade et al 1994) When

pso-ralens are used, the platelets must be suspended inplasma-reduced medium, as plasma inhibits the inac-

tivation of virus (Moroff et al 1992) The addition of

agents that neutralize oxygen radicals further protects

the platelet membrane (Margolis-Nunno et al 1994).

Some psoralen derivatives also limit platelet damage(Goodrich 1994) Moreover, the derivatives do notappear to be mutagenic in the absence of UVA

(Wollowitz et al 1994; Yerram et al 1994) The

potential mutagenicity of psoralen is also avoided byabsorbing excess agent on a ligand, C18, fixed on sili-con as used for S–D-treated plasma (Margolis-Nunno

et al 1995) Extensive preclinical toxicology studies

for S-59-treated platelets showed no CNS, cardiac orreproductive toxicity, and no evidence of genotoxicity

or phototoxicity In a heterozygote p53 knockoutmouse model, exposure to S-59-treated plasma over

6 months or 26 weeks did not produce excess genicity This system (INTERCEPT) is licensed inEurope as a medical device

carcino-Three clinical trials in 166 thrombocytopenicpatients were conducted in Europe, two with buffycoat platelets and one with apheresis platelets Thesestudies demonstrated that, for equal platelet doses,INTERCEPT platelets provided similar platelet count

increments to conventional platelets; the adverse

reac-tion profile appeared comparable (van Rhenen et al.

2003) A USA phase III study evaluating haemostatic

efficacy and safety of transfusions of apheresis platelet

concentrates randomized 645 patients to receive

either S-59 photochemically treated or conventional

platelets for up to 28 days Although the mean 1-h

post-transfusion platelet corrected count increment

(CCI) (11.1 × 103treatment vs 16.0 × 103control),

average number of days to next platelet transfusion

(1.9 treatment vs 2.4 control) and number of platelet

transfusions (8.4 treatment vs 6.2 control) differed,

the trial demonstrated equivalence in prevention and

treatment of grade 2 and higher grade bleeding

accord-ing to WHO criteria (McCullough et al 2004).

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