Mollison’s Blood Transfusion in Clinical Medicine - part 8 pptx

Bone marrow was the original source of progenitor cells for haematopoietic grafting, but mobilized peripheral blood and cord blood have gradually sup-planted marrow as a source of PC.. N

Transfusion of haematopoietic cells

The object of transfusing allogeneic haematopoietic

progenitor cells is to establish a permanent graft of

transfused progenitor cells in the recipient The fate

of allogeneic progenitor cells infused into the venous

circulation depends on their ability to traffic to sites of

haematopoietic tolerance (‘microenvironment’) and on

managing two immunological phenomena: (1) the

rejec-tion of donor progenitor cells by the host

immuno-logical response and (2) an immunoimmuno-logical reaction

of grafted immunologically competent cells against

the host: GvHD Both of these reactions depend on the

degree of histocompatibility between donor and

recipi-ent and also on the immunological competence of the

recipient Engraftment and kinetics also depend on

patient age, disease status, the preparative regimen,

GvHD prophylaxis and the cellular content of the graft

Bone marrow was the original source of progenitor

cells for haematopoietic grafting, but mobilized

peripheral blood and cord blood have gradually

sup-planted marrow as a source of PC The engraftment

potential of the component is commonly designated

in terms of mononuclear cells that express the CD34

antigen, the cluster designation of a transmembrane

glycoprotein present on haematopoietic progenitor cells

(Krause et al 1996), although accessory cells in the

graft clearly play an important role (Ash et al 1991).

Cells that express CD34 include lineage-committed

haematopoietic progenitors, multipotent progenitors

and possibly pluripotent stem cells as well Flow

cytometric assays are used to quantify CD34+ cells

in both the donor and the component However,

problems with interlaboratory accuracy and

repro-ducibility, especially of different PC sources, have been

notorious, even with the adoption of a standardized

technique (Sutherland et al 1996; Keeney et al 1998).

Peripheral blood-derived progenitor cells

Peripheral blood-derived progenitor cells (PBPCs)

were reported to circulate in mammalian blood as

early as 1909 (Maximow 1909), and the ability of

cir-culating cells to repopulate a lethally irradiated animal

was demonstrated in a parabiotic rat model in 1951

(Brecher and Cronkite 1951) However, circulating

haematopoietic progenitor cells were not confirmed in

human blood until the 1970s (McCredie et al 1971).

Collection of PBPCs obtained from peripheral blood

by leucapheresis (see Chapter 17) has now all butreplaced infusion of bone marrow PBPCs have theadvantages of engrafting more rapidly and sparing thedonor a general anaesthetic, which result in lower

morbidity and cost (Kessinger et al 1989; Azevedo

et al 1995; Bensinger et al 1995; Korbling et al 1995; Schmitz et al 1995) Allogeneic PBPCs have a higher

CD34+cell content than does marrow, which, pendent of stem cell source, increases patient survivalwhile reducing transplant-related mortality and relapse

inde-(Mavroudis et al 1996; Bahceci et al 2000; Zaucha

et al 2001) A theoretical argument against the use

of PBPCs is the greater number of ‘T’ lymphocytes that contaminate these collections, compared with thenumber of T cells in bone marrow, suggesting the possibility of an increased risk of severe acute GvHD.Indeed, although the risk of acute GVHD after PBPCs

is similar to that observed among historic bone

mar-row transplant (BMT) controls (Pavletic et al 1997; Przepiorka et al 1997), the probability and severity

of chronic GVHD (cGVHD) appear to be increased

(Bacigalupo et al 1996; Flowers et al 2002).

Liquid storage and cryopreservation

Collection of allogeneic PBPCs is ordinarily scheduled

to coincide with the conclusion of the patient’spreparatory regimen, so that the graft can be infusedwhile fresh Most centres opt to transfuse the cells assoon as possible Refrigerated storage of unmanip-ulated mobilized collections at 2– 6°C for 24 h and as

long as 72 h results in little detectable loss of the in vitro functional properties (Beaujean et al 1996; Moroff et al 2004) PBPCs, like bone marrow, can

be cryopreserved by slow cooling (1–3°C /min) in thepresence of the cryprotectant dimethylsulphoxide(DMSO), variable amounts of plasma, with or withouthydroxyethyl starch (Hubel 1997) Grafts can be stored

at –80°C, but are usually placed in liquid nitrogen at–140°C or colder, at which engraftment potential ispreserved for years

Cryopreserved grafts are thawed in a waterbath at37–40°C and infused through a 170-µ filter Prolongedpost-thaw storage is inadvisable, as prolonged expos-ure to 10% DMSO may harm the cells Storage up

to 1 h does not reduce viability or colony-formingactivity (Rowley and Anderson 1993) Rapid infusion

of DMSO has been associated with flushing, nausea,vomiting, diarrhoea and hypotension, probably the

result of histamine release Reversible encephalopathy

has been reported when doses have approached 2 g/kg,

so that caution is advisable when large volumes of

PBSCs are thawed (Dhodapkar et al 1994) The graft

can be washed free of cryoprotectant, but progenitor

cells may be lost in the process

Cord blood progenitor cells

Umbilical cord blood is a rich source of progenitor

cells (Knudtzon 1974; Broxmeyer et al 1989a) The

use of cord blood progenitor cells (CBPCs) has

import-ant real and potential advimport-antages The number of

donors is unlimited, procurement is easy and

inexpens-ive and the cells can be HLA typed and preserved in

liquid nitrogen Human CBPCs with high proliferative

capacity and NOD/SCID mouse engrafting ability can

be stored frozen for > 15 years, and probably remain

effective for clinical transplantation (Broxmeyer et al.

2003) In addition, because many of the functions of

the immunologically competent cells in cord blood are

not fully developed, the chance of their inducing

GvHD appears to be diminished (Szabolcs et al 2003).

Even after the transplantation of CBPCs from unrelated

donors, mismatched for two, or as many as three, HLA

antigens, the risk of severe GvHD seems to be low

(Wagner 1995)

Umbilical cord blood banking

Umbilical cord blood is collected by either the

obstetri-cian or the midwife in utero during the third stage of

delivery or ex utero after delivery of the placenta by

trained nurses or technologists (Wall et al 1997;

Fraser et al 1998) Collection volume and cell yield

appear to be similar with both methods (Lasky et al.

2002) A maternal blood specimen is screened for

markers of transmissible disease, and a sample from

the unit is cultured, HLA typed, analysed for cell

count, viability and in many instances CD34+ cell

number and colony count by culture Suitable units

are processed to remove red cells and plasma and are

frozen at a controlled rate and stored in liquid nitrogen

(Armitage et al 1999).

Related donor transplants

The first successful transplantation of CBPCs from

an HLA-identical sibling was given to a patient with

Fanconi’s anaemia (Broxmeyer et al 1989a) In 44

paediatric transplantations of CBPCs from siblingdonors, patients receiving HLA-identical or 1-antigenmismatched grafts showed an actuarial probability ofengraftment of 85% at 50 days after transplantation;there were no instances of late graft failure (Wagner

et al 1995) The median total nucleated cells per

kilogram (TNC/kg) was 5.2× 107 The probability

of GvHD at 100 days post transplant was 3% and ofchronic GvHD at 1 year was 6% The probability ofsurvival with a median follow-up of 1.6 years was72% Among 102 children with acute leukaemiatransplanted by the Eurocord collaborative investiga-tors, 42 received a graft from a related cord blooddonor; 12 of these were HLA mismatched (Locatelli

et al 1999) Nucleated cell dose (> 3.7 × 107/kg) related with engraftment; two-year survival was 41%.Rocha and co-workers (2000) compared 113 recipi-ents of HLA-identical sibling CBPC transplants formalignant disease with records of 2052 siblings trans-planted with bone marrow between 1990 and 1997.Although the umbilical cord blood (UCB) had asignificantly longer delay in recovery of neutrophil and platelet reconstitution, no significant difference insurvival and a significantly lower risk of GvHD andchronic GvHD was reported in the CBPC group Bonemarrow recipients received nearly 10-fold the totalnucleated cells per kilogram body weight (TNC/kg)

cor-Of 44 children with non-malignant conditions saemia, sickle cell disease), two-year survivals were

(thalas-79% and 90% respectively (Locatelli et al 2003) One

child with sickle cell disease and seven with saemia failed to sustain engraftment Four childrensuffered acute grade II GvHD

thalas-Unrelated donor transplants

Several thousand CBPC transplants have been formed and more than 100 000 umbilical cord com-ponents are available worldwide With the growth ofpublic (‘unrelated’) CBPC banks, the number of CBPCtransplants from unrelated donors now exceeds thatfrom related donors In one early series, a high rate

per-of engraftment (23 out per-of 25 cases) was observed inchildren infused with allogeneic CBPC despite thedonor–recipient pair discordance of 1–3 HLA antigens

(Kurtzberg et al 1996) A retrospective analysis of 562

unrelated CBPC transplants found that engraftmentexceeded 80% and survival rate was 61%; pre-freeze

cell count of the graft ranged from 0.7 to 10 TNC/kg

(Rubinstein et al 1998) The number of nucleated cord

blood cells that were transfused per kilogram of the

recipient’s weight emerged as the main influence on

engraftment A retrospective analysis of 537 paediatric

CBPC transplants from the Eurocord Registry,

includ-ing 138 related transplants and 291 unrelated donors,

reported similar results (Gluckman and Locatelli

2000) Laughlin and co-workers (2001) reported

CBPC transplants in 68 adult recipients who received

a median of 2.1× 107 TNC/kg TNC number per

kilogram correlated with rapidity of engraftment and

high CD34+ number was associated with event-free

survival Overall survival (22 months) was 28% As

expected from the experience with bone marrow

transplantation, GvHD is significantly higher in the

setting of grafts from unrelated donors and depends as

well upon the age of the recipient, the degree of

histo-compatibility between donor and recipient, the nature

of the preparatory regimen and a variety of other

fac-tors In this series, grades III and IV GvHD occurred in

20% and chronic GvHD in 36%

Reconstitution of adult recipients of cord blood

CBPCs have so far been used primarily for children

and doubt has been expressed as to whether the

num-ber of progenitor cells in cord blood from a single

donor will be sufficient to repopulate the majority of

large adults who require a transplant Most analyses

indicate that the key clinical outcomes (days to

neutrophil engraftment, platelet engraftment, severe

GvHD and disease-free survival) are all superior in

younger patients; age-related outcomes are widely

attributed to the number of nucleated cells in a single

unit of cord blood (Laughlin et al 2001) There is,

as yet, no quantitative assay for the progenitor cell

subset that has the capacity for long-term bone

mar-row repopulation On the other hand, the number of

progenitor cells that can be assayed (GM,

CFU-GEMM, etc.) is large enough, suggesting that the

number of the more primitive progenitor cells may be

sufficient (Broxmeyer 1995) There is evidence that the

total cellular content of placental cord blood (PCB)

grafts is related to the speed of engraftment, though

the total nucleated cell (TNC) dose is not a precise

pre-dictor of the time of neutrophil or platelet

engraft-ment It is important to understand the reasons for the

quantitative association and to improve the criteria for

selecting PCB grafts by using indices that are more

precisely predictive of engraftment (Rubinstein et al.

1998) The post-transplant course of 204 patients whoreceived grafts evaluated for haematopoietic colony-forming cell (CFC) content among 562 patientsreported previously were analysed using univariateand multivariate life-table techniques to determinewhether CFC doses predicted haematopoietic engraft-ment speed and risk for transplant-related events moreaccurately than the TNC dose Actuarial times to neutrophil and platelet engraftment were shown tocorrelate with the cell dose, whether estimated as TNC

or CFC per kilogram of recipient’s weight CFC ation with the day of recovery of 500 neutrophils/µlwas stronger than that of the TNC In multivariatetests of speed of platelet and neutrophil engraftmentand of probability of post-transplantation events, the inclusion of CFCs in the model displaced thesignificance of the high relative risks associated withTNC The CFC content of PCB units is associatedmore rigorously with the major covariates of post-transplantation survival than is the TNC and is, therefore, a better index of the haematopoietic content

associ-of PCB grafts (Migliaccio et al 2000) A positive

cor-relation between CD34+cells and circulating day-14colony counts (CFU-GM) has been reported suggestingthat with umbilical cord progenitor cells (UCPCs), aswith PBPC, CD34 is a reliable measure of haematopoi-

etic potential (Payne et al 1995; Siena et al 2000).

Data from 102 patients identified CD34+cell dose asthe only factor that correlated with rate of engraftment

(Wagner et al 2002) Studies from Spain and Japan

of small numbers of adults with haematological nancies report promising rates of engraftment and

malig-disease-free survival (Sanz et al 2001; Ooi et al 2004).

Progenitor cell expansion

If the number of progenitor cells in cord blood proves

to be scarcely sufficient for repopulation in manyadults, the possibility of expanding the number by

culture in vitro has been proposed (Apperley 1994; Broxmeyer et al 1995) and several groups are devel- oping methods to do so (Kogler et al 1999; Pecora

et al 2000; Jaroscak et al 2003) Whether the most

important primitive progenitor cells are expanded by

culture cannot be established in vitro As yet, no

evidence has confirmed that increase in engraftmentkinetics or expansion of stem cells has been achieved,

and the possibility of increased frequency of GvHD

with some expansion methods has been raised (Shpall

et al 2002; Jaroscak et al 2003).

Plasma from cord blood has been found to increase

the self-renewal capacity of stem cells in vitro (Carow

et al 1993) Cord blood plasma, but not plasma from

adults or fetal calf serum, had this effect and cord

blood plasma also increased the expansion in vitro of

the number of progenitor cells induced by growth

fac-tors (Bertolini et al 1994) Furthermore, CBPCs fully

retain this expansion potential after cryopreservation

(Bertolini et al 1994).

Use of multiple cord blood collections

Because limited cell dose compromises may

comprom-ise the outcome of adult UCB transplants, multiple

cord blood units have been combined to augment

the dose Zanjani and co-workers (2000) have

trans-planted human UCB from multiple donors in a fetal

sheep model Short-term donor engraftment derived

from both donors, but for long-term haematopoiesis, a

single donor predominated

Multidonor human UCB transplants using up to

12 units have been published (Ende and Ende 1972;

Shen et al 1994) Weinreb and co-workers (1998)

reported that a unit that was partially HLA matched

predominated in a recipient who received a

combina-tion of 12 units Another patient with advanced acute

lymphocytic leukaemia received a mismatched,

unre-lated UCB transplant using units from two donors and

achieved a complete remission with double chimerism,

which persisted until relapse (De Lima et al 2002).

Barker and co-workers (2005) have augmented graft

cell dose by combining two partially HLA-matched

units Twenty-three patients with high-risk

haemato-logical malignancy received 2 UCB units (median

infused dose, 3.5× 107 NC/kg) and 21 evaluable

patients engrafted at a median of 23 days At day 21,

engraftment was derived from both donors in 24%

of patients and a single donor in 76% of patients

One unit predominated in all patients by day 100

Although neither nucleated or CD34+cell doses nor

HLA match predicted which unit would predominate,

the predominating unit had a significantly higher

CD34+dose The result is similar to the predominant

lymphocyte chimerism that persists in trauma patients

who receive multiple blood transfusions (Lee et al.

1999)

Law, ethics, related banks and genetic selection

Controversy continues regarding the propriety ofrelated (‘private’) CBPCs for which the family pays tohave the infant’s cells cryopreserved for future use, ascontrasted with unrelated (‘public’) banks, in which

donated cords are stored for general use (Burgio et al.

2003) Both systems have their adherents and theyshould not be mutually exclusive Although mostrelated banks with commercial origins have soughtparticipation from expectant mothers who agree topay for storage of a cord from their newborn infant,others have been supported by federal grants (Reed

et al 2001) The infrequent utilization of a related

cord blood unit does minimize its utility The ity that the cord blood will be of use in a family with

probabil-no history of blood or genetic disease is low (estimated

at 1/200 000); moreover, one’s own stem cells may beimmunologically less potent than those of an unrelateddonor for treating neoplastic diseases However, several such transplants have been performed success-fully and prohibiting such storage despite appropriateinformed consent seems curiously patronizing Thelegal issues regarding property rights have been dis-

cussed (Munzer 1999) In vitro fertilization and

pre-natal genetic diagnosis to select an embryo donor onthe basis of specific, desirable disease and HLA charac-teristics have been used successfully to treat a child

with Fanconi’s anaemia (Grewal et al 2004).

Effect of ABO incompatibility of grafted cells

As ABO and HLA antigens are inherited ently, ABO incompatibility may occur in 20 – 40% ofHLA-matched allogeneic haematopoietic stem celltransplants ABO incompatibility between donor pro-genitor cells and the recipient’s plasma is not a barrier

independ-to successful transplantation (Sindepend-torb et al 1977; Buckner et al 1978) In a series of 12 subjects who

received major ABO-incompatible marrow, not onerejected the graft and the incidence of GvHD was nohigher than in subjects who received ABO-compatible

marrow (Hershko et al 1980).

With major ABO-incompatible marrow grafts,defined as incompatibility of donor ABO antigens withthe recipient’s immune system, steps must be taken toprevent an acute haemolytic reaction due to lysis ofincompatible red cells contained in the progenitor cellgraft To avoid haemolysis, grafts are purged of red

cells A satisfactory method has been described

(Warkentin et al 1985) An alternative method of

removing red cells from marrow uses a cell separator

(Blacklock et al 1982) When PBPC or CBPC are used,

the number of contaminating red cells is small

Delayed donor red cell engraftment and pure red

cell aplasia are well-recognized complications of major

ABO-incompatible haematopoietic stem cell

trans-plantation (Hows et al 1983; Sniecinski et al 1988;

Stussi et al 2002; Griffith et al 2005) Donor red

blood cell chimerism is delayed as long as three-fold

(median 114 days) following reduced-intensity

non-myeloablative compared with non-myeloablative

condi-tioning for transplant and the delay correlates with the

recipient anti-donor isohaemagglutinin titre (Bolan

et al 2001a) Late-onset red cell aplasia, most likely

related to delayed lymphoid engraftment, may occur

(Au et al 2004) In some patients, thrombopoiesis may

be delayed as well (Sniecinski et al 1988).

After transplants of major ABO-incompatible grafts,

the direct antiglobulin test (DAT) may turn positive

after about 3 weeks If substantial numbers of donor red

cells enter the circulation, transient immune-mediated

haemolysis may result (Sniecinski et al 1987) Anti-A

and anti-B may remain demonstrable in the recipient’s

plasma for some months and the DAT may remain

positive during this time In patients with minor

ABO-incompatible transplants, defined as those in

which the recipient antigens are incompatible with the

donor’s immune system, haemolysis may develop 1–

2 weeks after transplantation owing to lysis of

ABO-incompatible recipient cells as the donor immune

lymphocytes engraft (Hows et al 1986) This type of

haemolysis has been seen only in patients receiving

ciclosporin and prednisone GvHD prophylaxis, and

may not develop in patients receiving methotrexate

(Gajewski et al 1992) Massive immune haemolysis

may occur, and fatalities can be avoided by early,

vigorous donor-compatible red cell transfusion until

haemolysis subsides (Bolan et al 2001a) Reactions

are most common and severe when the donor is group

O and the recipient group A, but neither blood group

nor agglutinin titre reliably predict clinical severity In

some of the patients, haemolysis caused by anti-A or

anti-B (or both) destroys transfused group O cells,

probably as a result of activated complement

compon-ents affixing the group O cells (bystander haemolysis).

Haemolysis has also been observed when the donor

lymphocytes produce anti-D, etc (see Chapter 11)

Special consideration of the ABO group of ents transfused to patients receiving ABO-incompatiblegrafts should begin with the initiation of the prepar-atory regimen to ensure that blood is compatible with both donor and recipient (Table 14.1) With bi-directional (major–minor) incompatibility, red celltransfusions should be limited to group O Plateletconcentrates administered to adults may be of anyblood group, although plasma reduction may be pru-dent, especially for large-volume group O platelets.Plasma-compatible platelets should be used for infantsand children Some centres use the soluble antigenscontained in plasma to neutralize isohaemagglutinins

compon-As intravenous immunoglobulin contains variabletitres of red cell antibodies, especially of anti-A, somecentres screen for alloantibodies, whereas others avoidhigh-dose IVIG for group A recipients during the post-transplant period

Donor lymphocyte infusionLymphocytes have been studied more often as bloodcomponent contaminants responsible for adverseeffects than as therapeutic cells However, some ostens-ibly adverse effects of mononuclear cell infusions can

be exploited for therapeutic benefit The mechanismsinvolved in TA-GvHD (see Chapters 13 and 15) areprobably responsible for the graft-versus-malignancyeffect in allogeneic stem cell transplantation Studies inanimal models are consistent with the observation byBarnes and Loutit (1957) that transplanted bone mar-row has immune activity against residual leukaemia

(Kloosterman et al 1995) Clinical experience with

haematopoietic transplantation has been consistent withthe presence of antileukaemic activity also in humans,now commonly referred to as the graft-versus-leukaemiaeffect (GvL) The term ‘adoptive immunotherapy’ was coined by Mathé (1965) who used both marrowtransplants and leucocyte infusions to treat acuteleukaemia Kolb and co-workers (1990) provideddirect clinical evidence for GvL: the transfusion ofdonor lymphocytes in conjunction with the adminis-tration of alpha-interferon (IFN-α) induced cytoge-netic remission in three patients with CML in relapsefollowing allogeneic bone marrow transplantation.Numerous independent studies confirm the GvL effect

in CML (Slavin et al 1992; Bar et al 1993; Porter et al.

1994) In both European and North American istries, more than 90% of the patients received original

reg-grafts and subsequent donor lymphocyte infusion

(DLI) from related donors, typically from an

HLA-identical sibling The results reported by the European

Group for Blood and Marrow Transplantation

(27 centres, 135 patients, 75 evaluable with CML) are

similar to those reported by the North American

Multicenter Bone Marrow Transplantation Registry

(25 centres, 140 patients, 55 evaluable with CML):

DLI-induced clinical remission at a rate approaching

80%, and molecular remission (inability to detect

bcr-abl mRNA transcript using polymerase chain reaction)

in nearly all patients entering clinical remission (Kolb

et al 1995; Collins et al 1997).

Infusion of small numbers of lymphocytes (107)

(‘bulk dose’) usually suffices, and excess cells collected

by leucapheresis are often aliquoted and stored for

repeated treatment if needed The host’s circulation,

which often contains a mixture of both donor and host

cells during chronic phase relapse, typically converts

to cells of only donor origin The time to remission

ranges from 1 to 9 months, with a mean of about

3 months (Kolb et al 1995; Collins et al 1997) Nearly

all responses are seen within 8 months after DLI, andthe probability of remaining in remission at 2 and

3 years is 90% and 87% respectively (Kolb et al 1995; Collins et al 1997) Although late relapses occur and

toxicity may be significant, DLI efficacy is durable insurviving patients with CML: 26 out of 39 (67%) pati-ents were alive at follow-up with 25 (96% of survivors)

remaining in complete remission (Porter et al 1999).

Separating graft-versus-leukaemia from graft-versus-host-disease

GvHD occurring after DLI correlates strongly withantileukaemic response However, the GvL effect andGvHD may be separable, and GvHD may not be

required for durable disease remission (Weiss et al 1994; Rocha et al 1997; Slavin et al 2002) Murine

studies suggest that the rate of GvHD is inversely

* Restrictions for ABO- and/or Rh-incompatible transplant recipients supported with blood components during pre-transplant conditioning and during the post-transplant period.

†Use any ABO group for platelet support for adults Use plasma-compatible

components for children.

‡ Plasma may be transfused to neutralize isohaemagglutinin(s).

§ Graft plasma depleted, no plasma neutralization required.

¶ Rh-positive components initiated on the day of transplant.

** Rh-negative platelets preferred.

†† Rh-negative red cells preferred during the pre-transplant conditioning regimen and post transplant.

Table 14.1 ABO and/or

Rh-incompatible progenitor cell:

transplant transfusion restrictions.

proportional to the interval between transplant and

DLI (Johnson et al 1999) These considerations have

popularized escalating dose DLI regimens (Dazzi et al.

2000) Although probability of achieving remission

in relapsed CML does not differ, the escalating dose

regimen is associated with a lower incidence of GvHD

DLI is typically initiated early in disease as soon as

disease recurrence is anticipated, and a starting dose

of 105T cells/kg is escalated 10-fold at 2- to 4-week

intervals (Weiss et al 1994) Efforts to modify the

composition of donor-derived lymphocytes (DDLs)

have focused on selective CD8-positive T-cell depletion,

which appears to be more effective than non-selective

T-cell depletion in reducing GvHD while preserving

GvL (Soiffer et al 2002).

Target antigen as the primary determinant of

efficacy and toxicity

Falkenburg and colleagues (1999) reported the first

successful treatment of relapsed accelerated CML using

in vitro-expanded leukaemia-specific lymphocytes.

Presumably, cell selection and culture restored the

anti-tumour activity and specificity against leukaemic

cells that weakened with disease progression (Smit

et al 1998) Successful salvage therapy of a child with

previously DLI-resistant CML by using DDL pulsed in

vitro with a mixture of normal irradiated lymphocytes

obtained from the child’s parents has been reported

Efficacy and toxicity in viral diseases

Walter and co-workers (1995) have used in

vitro-stimulated, culture-expanded, CMV-specific

donor-derived cytotoxic T cells to successfully reconstitute

cellular immunity against CMV in 11 out of 14

allo-geneic marrow transplant patients The DLI therapy

consisted of four escalating cell doses (0.33, 1.0, 3.3

and 10.0 × 108cells) administered at weekly intervals

beginning at days 30 – 40 after transplantation

DLI-associated toxicity, CMV disease and CMV viraemia

were not observed The results have been confirmed in

similar studies (Einsele et al 2002; Roback et al 2003).

Epstein–Barr virus-related lymphoproliferative

disorders

The incidence of Epstein–Barr virus-related

lympho-proliferative disorders (EBV-LPDs) occurring in T

cell-depleted transplants has been estimated at 6 –12%,and secondary lymphomas occurring in this clinicalsetting respond readily to DLI at a dose approximately10-fold smaller than that typically used for activityagainst the primary leukaemia Sustained clinicalremissions have been achieved with only mild GvHD,and patients have often required no additional mainten-

ance therapy (Papadopoulos et al 1994; Wagner et al.

2004) EBV-specific lymphocyte infusions have cessfully treated EBV-LPD and EBV-positive Hodgkin

suc-disease (Rooney et al 1998; Bollard et al 2004).

The transfusion of plasma components

Fresh-frozen plasmaFresh-frozen plasma (FFP) is plasma obtained from asingle donor by normal donation or plasmapheresisand frozen within 6 h of collection to a temperature

of –30°C or below FFP contains all circulating lation factors in the concentration present in freshplasma, and haemostatic activity is maintained for ayear or longer, depending upon the storage temper-ature Once thawed, FFP must be stored at 4 ± 2°C for

coagu-no longer than 24 h before infusion FFP must coagu-not berefrozen, but once thawed (or after 1 year of storageand thaw), it can be used as single-donor plasma, i.e.not to replace labile coagulation factors, for as long

as 5 weeks The concentration of coagulation factor,the citrate concentration and the volume of each unitmay vary depending on the characteristics of the donorand of the collection In 51 units collected by apheresisfrom plasma donors, factor concentrations at the fifthand ninety-fifth percentile measured: V (690–1270 units/l); VII (830 –1690 units/l); fibrinogen 1800–3700 µg/l;

antithrombin (920 –1290 units/l) (Beeck et al 1999).

Risks of fresh-frozen plasma

Allergic reactions may occur after transfusion of FFP,

of which the most serious is severe anaphylaxis, whichmay develop in IgA-deficient patients with class-specific anti-IgA (Chapter 15) Such reactions are rare.Transfusion-related acute lung injury (TRALI) mayoccur when the FFP contains strong leucocyte antibod-ies (see Chapter 15) The other main risk of treatmentwith FFP is the transmission of infectious agents, par-ticularly viruses such as hepatitis B and C viruses, HIV,parvovirus and West Nile virus Owing to donor selec-

tion and the availability of methods of inactivating

viruses that are used to treat whole plasma in some

countries, the risk of transmitting viruses has greatly

decreased However, the problem of inactivating

non-lipid-enveloped viruses and the transmission of

non-viral agents remains (Chapter 16) Therefore, FFP

should still be used only when no safer alternative

exists (Shimizu and Robinson 1996)

Precautions to be taken before infusion

FFP containing potent anti-A or anti-B agglutinins or

haemolysins, or FFP that has not been tested for their

presence, should not be given to recipients with

corres-ponding red cell antigens Fresh plasma, which is now

rarely used, may contain red cells, so that appropriate

measures should be taken to prevent immunization of

D-negative women of childbearing age There is no

credible evidence that FFP presents such a risk

Indications for fresh-frozen plasma:

overused and abused

There is no justification for the use of FFP as a volume

expander because safer alternatives (colloids and

crystalloids) are available

Factor V deficiency No concentrate of factor V is

available and FFP can be used as a source of factor V

Cryoprecipitate-poor plasma contains 80% of the

amount of factor V in FFP and can be used as an

alternative for FFP (Hellings 1981)

Severe liver disease The liver is the major site of

syn-thesis of coagulation factors II, V, VII, IX, X, XI, XII

and fibrinogen as well as of factors with potential

antithrombotic activity such as proteins C, S and

antithrombin Patients with severe liver disease may

experience defects in factor synthesis and increased

factor degradation that can result in generalized

bleed-ing Unfortunately, studies regarding the predictability

of bleeding and its most effective management by

transfusion in the presence of different degrees of

hep-atic impairment are old, poorly documented or both

Most recommendations rely upon expert opinion

What seems clear is that no single coagulation assay

predicts bleeding (Spector and Corn 1967)

Prolonga-tions of the prothrombin time (PT) and activated

partial thromboplastin time (aPTT) are the most

fre-quent abnormalities among the commonly performedclotting tests in patients with liver disease, and mayreflect impaired protein synthesis, vitamin K deficiency

or even disseminated intravascular coagulation (DIC).The presence of an abnormal test does not necessitateintervention, especially in the non-bleeding patient.Furthermore, in 30 patients with chronic liver disease,

a moderate-dose plasma infusion (12 ml/kg or about

4 units) did not return the PT and aPTT to normal

(Mannucci et al 1982) In the bleeding patient, doses

calculated to bring coagulation factor levels to the20–30% range (20 ml/kg or 6–7 units) may be required

as frequently as every 4 – 6 h to correct the abnormal

coagulation tests (Spector et al 1966) The routine use

of FFP as prophylaxis for excessive surgical bleeding inpatients with severe liver disease finds few supportersand less evidence of benefit (Oberman 1990)

Treatment of acquired deficiencies of factors II, VII, IX and X due to treatment with

anticoagulants: warfarin reversal

The major risk of anticoagulant therapy is rhage For patients treated with the oral vitamin Kantagonists, the annual risk of severe haemorrhage

haemor-ranges from 1–5% (Levine et al 2001) The intensity

of anticoagulation (including poor control), its tion and, in some studies, advanced age and cerebrov-ascular disease all increase the bleeding risk (Landefeldand Goldman 1989) For the bleeding patient or thepatient at extreme risk, urgent reversal of vitamin Kantagonists can be achieved with plasma infusion tobring factor levels to 30 – 40% The volume of plasmacan be calculated easily based on the patient’s bodyweight but as 6 units or more (1500 ml) may be required

dura-to reverse anticoagulation in an adult, volume erations may make a prothrombin complex concentrate(PCC) the preferred infusion (Schulman 2003) Recom-binant VIIa has also been used in this situation (Deverasand Kessler 2002) (see Chapter 18) Intravenous vita-min K1, the specific warfarin antagonist, may require

consid-12 h or more to be fully effective (Nee et al 1999).

Disseminated intravascular coagulation:

a vehicle on the road to multi-organ dysfunction syndrome

Disseminated intravascular coagulation is a condition

in which the intravascular activation of the clotting

cascade leads to the final common pathway of

sus-tained and excessive thrombin generation Liberated

thrombin and proteolytic enzymes bring about the

intravascular production of fibrin and deposition of

platelets, with activation of the fibrinolytic system and

an increased level of fibrin degradation products

(FDPs) (Levi et al 2001) In mild DIC the platelet

count and the levels of clotting factors may be normal

due to compensatory increases in production As DIC

becomes more severe, the levels of clotting factors

and platelets fall, and a state that may be described as

decompensated DIC may lead to multi-organ

dysfunc-tion syndrome (MODS)

DIC may be precipitated by a wide variety of stimuli,

most related to the entry of tissue thromboplastins into

the circulation, for example after abruptio placentae,

crush injury, head trauma and snake envenomation

Other conditions associated with DIC include

infec-tions, malignancies, amniotic fluid embolism, giant

haemangioma and intravascular lysis of incompatible

red cells (Levi et al 2004).

The cardinal principle of treatment of DIC remains

elimination of the underlying cause as, once this

has been accomplished, haemostasis usually returns to

normal When the underlying cause cannot be treated

effectively, uncontrollable bleeding may result The

transfusion of blood may be essential and the

replace-ment of clotting factors has to be considered This

replacement should be guided by coagulation assays

and fibrinogen levels If levels of clotting factors are

severely reduced (< 25%), FFP may be given and if the

fibrinogen concentration falls below 60 mg /dl,

cryo-precipitate may be helpful An initial dose of 10 bags,

to provide 4 – 6 g of fibrinogen, has been suggested

(Prentice 1985) Despite the theoretical objection of

adding ‘fuel to the fire’, the administration of

fibrino-gen does not seem to be particularly dangerous

Thrombotic thrombocytopenia purpura

Before the mechanisms involved in thrombotic

throm-bocytopenia purpura (TTP) were suspected, a plasma

factor was postulated to correct the syndrome

charac-terized by microangiopathic haemolysis and

throm-bocytopenia (Upshaw 1978) Relapses in chronic TTP

were reversed or prevented by infusions of small

volumes of FFP or cryoprecipitate-depleted FFP or by

plasma infusion combined with plasmapheresis (Byrnes

and Khurana 1977; Bukowski et al 1981) The plasma

factor is not destroyed by the solvent detergent ment of FFP used to inactivate lipid-encapsulated

treat-viruses (Moake et al 1994) In the majority of cases,

the plasma factor relates to the activity of a proteinase that cleaves unusually large multimers ofvWF that are associated with the TTP thrombi (Asada

metallo-et al 1985; Tsai 1996) (see Chapter 17).

Cryoprecipitate-depleted fresh-frozen plasma (cryosupernatant)

Cryosupernatant is plasma from which about one-half

of the fibrinogen, factor VIII and fibronectin has been removed as cryoprecipitate The product is alsodepleted of the largest multimers of vWF, which sediment in the cryoprecipitate fraction and whichmay be partly responsible for platelet aggregation inTTP (Moake 2004) In some circumstances cryosuper-natant may be more effective than FFP in the treatment

of TTP Seven patients with TTP who failed to respond

to intensive plasma exchange with whole plasmaresponded to plasma exchange with cryosupernatant

(Byrnes et al 1990).

Cryoprecipitate

When plasma is fast frozen and then thawed slowly at

4 – 6°C, the small amount of protein precipitated isrich in fibrinogen, factor VIII, vWF and factor XIII.After decanting almost all of the supernatant plasma,the precipitated protein can be dissolved by warming

to yield a small volume of solution The introduction

of cryoprecipitate revolutionized the treatment ofhaemophilia by providing a highly effective, conveni-ent, readily available source of factor VIII Moderntreatment has moved away from cryoprecipitate topathogen-inactivated factor VIII concentrate and torecombinant factor VIII Cryoprecipitate is used now

as a source of factor VIII and vWF only if safer trates are not available

concen-Cryoprecipitate, containing approximately 200 –

250 mg of fibrinogen in a volume of 10 –15 ml, pared from a single donor, is used primarily as a source

pre-of fibrinogen The most common indication remains

as a replacement for fibrinogen consumed in DIC,although it has been used as a topical haemostaticagent as well (fibrin glue) (Reiss and Oz 1996).Commercial fibrin sealants are safer, better standard-ized and more effective, and avoid the potential risk of

immunization to contaminant factor V that has been

reported when bovine thrombin is used to activate

topical cryoprecipitate (Rousou et al 1989; Rapaport

et al 1992; Atrah 1994) (see also Chapter 18).

Thawing by microwave is rapid and preserves

fibrino-gen concentration (Bass et al 1985).

Cryoprecipitate also contains about 60% of the

vWF and 20–30% of the factor XIII of the original unit

of FFP, but the component in not often used as a source

of these proteins

‘Contaminants’ in cryoprecipitate Cryoprecipitates

contain about 30 –50% of the original fibrinogen and

have about the same titre of anti-A and anti-B as that

of the original plasma unit (Rizza and Biggs 1969; Pool

1970) Because of the risk of haemolysis, neonates

should receive only ABO-compatible cryoprecipitate

Plasma fractionation

The transfusion of whole plasma is unnecessary and

usually inefficient if recipients require only a single

protein, for example factor VIII Plasma contains

hun-dreds of different proteins, many of which are obvious

candidates for replacement therapy, whereas others

are well characterized physicochemically, but of

un-known function Commercial plasma fractionation uses

dilution, pasteurization and nanofiltration to remove

and inactivate most viruses, although no product can

be guaranteed ‘pathogen free’ The immunoglobulin

(Ig) fraction (predominantly IgG) separated from

whole plasma by alcohol fractionation was at first

considered virtually free of the risk of transmitting

viral hepatitis However, HCV has been transmitted

by both IVIG and anti-D Ig (Bjoro et al 1994; Meisel

et al 1995; Power et al 1995).

The most widely used method of fractionating

plasma is still the cold alcohol precipitation technique

described by Cohn and colleagues (1944) or

modifica-tions thereof (Kistler and Nitschmann 1962) Cohn

fractionation relies on changes in ethanol

concentra-tion and pH for bulk precipitaconcentra-tion of different protein

fractions An example of a fractionation scheme is

shown in Fig 14.6 Ethanol is removed by

lyophiliza-tion or by ultrafiltralyophiliza-tion Alcohol fraclyophiliza-tionalyophiliza-tion is now

combined with glycine precipitation or polyethylene

glycol, and with other separation methods such as

chromatography to isolate specific proteins, such as

coagulation factors and protease inhibitors

Albumin

Albumin is available for clinical use either as humanalbumin in saline containing 4%, 4.5%, 5%, 20% or25% protein, of which not less than 95% is albumin,

or as plasma protein fraction (PPF), available only as

a 5% solution, of which at least 83% is albumin Compared with albumin, most preparations of PPFcontain larger amounts of contaminating proteins.Hypotensive reactions attributed to pre-kallikreinactivator and acetate have been observed with PPF,

but not with albumin (Alving et al 1978; Ng et al.

1981) For these reasons, most clinicians find little reason to select PPF when an albumin solution is indicated

Albumin preparations are pasteurized by heat treatment at 60°C for 10 h and filtered When pre-pared in this way, the fraction has proved free of transfusion-transmitted agents such as hepatitis virusesand HIV

Although albumin contributes 75–80% of the loid osmotic pressure of the plasma, subjects with agenetically determined total absence of plasma albu-min, in whom the colloid osmotic pressure of plasma isbetween one-third and one-half of normal, may becompletely asymptomatic (Bearn 1978) Such subjectsshow an increase in various plasma globulins and aslight decrease in blood pressure, changes that areregarded as compensatory The indications for infu-sions of albumin in hypovolaemic patients are discussed

col-in Chapter 2

Recombinant albumin

Recombinant albumin has been synthesized in yeast,

in Saccharomyces cerevisiae or Pichia pastoris, and

appears to be similar to the plasma-derived protein

(Dodsworth et al 1996) A 20% solution (Recombumin

20%, Aventis Behring) prepared as a pharmaceuticalexcipient has been tested for safety in doses up to

65 mg in some 500 subjects It is uncertain when, ifever, recombinant albumin might be commerciallyavailable as a product for transfusion

Fibrinogen

The rate of disappearance of injected fibrinogen hasbeen studied by giving infusions to patients with thevery rare condition, hereditary afibrinogenaemia: in

two cases, one-half of the injected fibrinogen

dis-appeared during the first 24 h, presumably due to

mix-ing with the protein ‘pool’; thereafter the fibrinogen

disappeared with a T1/2of 4 days (Gitlin and Borges

1953)

In clinical practice, hypofibrinogenaemia is most

often encountered as one feature of the syndrome of

DIC; in this condition the transfusion of fibrinogen is

seldom indicated Purified fibrinogen prepared by

frac-tionation of pooled plasma, unless virally inactivated,

carries a high risk of the transmission of viral diseases

such as hepatitis and no commercial fractionation

con-centrate is licensed

Factor VIII (anti-haemophilic factor)

Factor VIII levels in haemophiliacs

Severely affected patients with haemophilia A have nodetectable factor VIII activity in their plasma and suf-fer from repeated episodes of spontaneous bleeding,particularly in the large joints and muscles Patientswhose factor VIII activity is 1–5% of normal, about10% of affected individuals, are defined as ‘moderatehaemophilia’ and have infrequent attacks of bleeding.Those with levels exceeding 5%, some 30 – 40% ofpatients, are mildly affected and seldom if ever suffer

5000 or more litres of pooled frozen plasma

Cryoprecipitation

Cryosupernatant

Factor VIII Factor XIII

Albumin

Immunoglobulin Cold ethanol fractionation

Cold ethanol precipitation

Fig 14.6 The various blood products

(in squares) obtained by stepwise fractionation of large pools of fresh-frozen plasma using different cryoprecipitation, ethanol precipitation and adsorption procedures.

spontaneous bleeding Intracranial haemorrhage is the

most common cause of death from bleeding, is

spontan-eous in about 50% of cases and should always be

con-sidered in patients with haemophilia who complain of

headache

Treatment with factor VIII

In severe haemophilia A, treatment with factor VIII

must be provided as soon as possible after bleeding has

occurred The dose depends on the kind of

haemor-rhage The aim of initial ‘episodic’ therapy in the case

of haemarthrosis or serious bruising is to raise the

fac-tor VIII level to 30 –50% of normal; if a haematoma

has developed, the level should be raised to 50%, and

in case of gastrointestinal bleeding, to > 50% The

level should be raised to 100% if there is spontaneous

intracerebral haemorrhage or head trauma (Furie et al.

1994) One unit of factor VIII is the amount of factor

VIII activity in 1 ml of normal plasma For example, as

the plasma volume is about 50 ml/kg, 3500 units must

be given to a recipient of 70 kg to increase the level to

100% Regardless of the source of factor VIII, the

plasma level reached after administration is only about

70% of the expected level and this must be taken into

account in calculating the dose required The half-life

of factor VIII is 8–12 h Thus if one-half of a dose is

given at 12-h intervals after the initial dose, the level is

kept relatively constant (Furie et al 1994).

Prophylactic administration of factor VIII

The incidence of bleeding can be all but abolished and

even arthropathy can be prevented if factor VIII is

administered prophylactically from a very early age so

as to maintain the factor VIII concentration above 1%

of normal (Nilsson et al 1994) Once joint damage

has occurred, it cannot be reversed by prophylactic

treatment (Manco-Johnson et al 1994) The amount

of factor VIII needed for universal application of

pro-phylactic treatment is impractically large Short-term

prophylaxis (3 months of bi-weekly infusions

calcu-lated to keep trough values at 1–3%) should be

con-sidered for patients with frequent haemorrhages or

with chronic synovitis, especially if active rehabilitation

is considered (Kasper et al 1989) With preoperative

prophylaxis, surgical mortality from haemorrhage

approaches zero for most procedures (Kitchens 1986)

The initial infusion is routinely given several hours

prior to surgery and the factor VIII level confirmedbefore induction of anaesthesia The duration of post-operative infusions depends on both the nature of the

procedure and the clinical course (Kasper et al 1985).

Continuous postoperative factor infusion has become

an increasingly popular strategy to maintain constantfactor levels (> 50%) and consume less factor concen-trate, although experience with this technique is stilllimited

Factor VIII levels of healthy donors: maximizing collection potential

The factor VIII activity in group A donors is on average 8% higher than in group O donors, and thelevel in males is about 6% higher than in females(Preston and Barr 1964) Strenuous exercise produces

an almost immediate increase in factor VIII levels, ing for at least 6 h (Rizza 1961) These observationscarried greater importance when cryoprecipitate wasused as a source of factor VIII

last-DDAVP (1-deamino-8-D arginine vasopressin, alsoknown as desmopressin acetate), a synthetic derivative

of vasopressin, injected intravenously, produces a rapidrelease of vWF into the circulation Although vWF is acarrier protein for factor VIII, factor VIII and vWF

may not increase concurrently (Cattaneo et al 1994; Castaman et al 1995) DDAVP is the treatment of

choice in patients with mild haemophilia with factorVIII levels of > 10%, but should not be used in childrenunder 1 year of age because of the risk of hypona-

traemia (Weinstein et al 1989; see also Chapter 18).

DDAVP is used primarily for therapeutic purposes.However, if DDAVP (0.2 µg/kg) is injected into blooddonors 15 min before venepuncture, the yield of factorVIII in fractions prepared from the resulting plasma

is increased two-fold (Nilsson et al 1979; Mannucci

1986) DDAVP can also be administered intranasallyand is effective within 1 h with minimal side-effects

revolution-added assurance that emerging agents would not

evade inactivation technology

Choice of factor VIII concentrate

Factor VIII can be administered as cryoprecipitate, as

plasma-derived factor VIII concentrate or as

recombi-nant factor VIII Factor VIII activity is well maintained

at –30°C to – 40°C for cryoprecipitate or at 4°C

for lyophilized products Commercial plasma-derived

factor VIII is now treated with at least two methods

for inactivating transfusion-transmitted viruses, and

no documented transmissions of the lipid-encapsulated

viruses HIV, HBV or HCV have been reported since

1985 Non-enveloped viruses such as hepatitis A and

parvovirus B19 may still be transmitted by plasma

fractions (Mannucci 1992; Santagostino et al 1997).

Both plasma-derived and recombinant products are

labelled for factor VIII potency and all preparations

appear to be equally effective when assessed by

post-administration factor levels Patient age, susceptibility

and product cost still largely determine the choice of

treatment

Purified factor VIII concentrates

Intermediate-/high-purity concentrate Factor purity

is commonly defined as specific activity (International

Units of clotting activity per milligram of protein)

Most fractionation centres use large pools of plasma

(5000 –30 000 donations) to prepare products of

inter-mediate purity (< 50 units/mg) and high purity (> 50

units/mg) The primary procedure is cryoprecipitation,

but additional fractionation steps are undertaken to

give a higher potency, stability and solubility than are

obtained with the freeze-dried cryoprecipitate

Ultrapure concentrate Concentrates purified by

using affinity chromatography with monoclonal

anti-bodies against factor VIII have a specific activity of

factor VIIIC of > 3000 iu/mg protein In a group of

patients treated with this product for more than

24 months, clinical efficacy, T1/2and recovery were

excellent (Brettler et al 1989) One oft-stated

advant-age of purified high-potency concentrates is a less

pro-nounced effect on the immune system of the patients,

documented primarily in HIV-positive patients in

whom the CD4+cell count declines less rapidly after

treatment with high- than after intermediate-purity

concentrates (de Biasi et al 1991; Hilgartner et al 1993; Seremetis et al 1993) No increase in AIDS-

associated infections or decrease in survival has been

documented (Goedert et al 1994) No difference in

effect on the immune system in HIV-negative patientshas been established Suspicion that ultra high-purityconcentrates (and recombinant concentrates) maymore easily induce factor VIII inhibitors, particularly

in children, have not been confirmed by prospectivestudies (Bray 1994; Peerlinck 1994) The relationshipbetween mutation type and inhibitor development isprobably far more important As many as 35% ofpatients with ‘severe molecular defects’, intron 22inversions, large deletions or stop mutations develop

an inhibitor, whereas few with small insertions or

dele-tions do so (Oldenburg et al 2002).

Recombinant factor VIII Recombinant clones

encod-ing the complete 2351-amino-acid sequence for humanfactor VIII have been isolated and used to produce fac-tor VIII in cultured mammalian cells The recombinantprotein corrects the clotting time of plasma fromhaemophiliacs and is virtually indistinguishable from

plasma-derived factor VIII (Wood et al 1984).

Clinical trials have shown that in vivo recovery and

T1/2of recombinant factor VIII (r-factor VIII) are notsignificantly different from those of plasma-derivedfactor VIII The r-factor VIII Recombinate (BaxterBioscience) and Koginate (Cutter Biological/Miles)have now been used successfully in the treatment ofbleeding episodes and for prophylaxis The observedincidence of inhibitor formation is similar to studies ofpreviously untreated patients (PUPs) receiving plasma-derived FVIII Long-term trials demonstrate the safetyand efficacy of r-FVIII in chronic treatment of

haemophilia A (Lusher 1994; White et al 1997).

Preparations of the early recombinant proteins usedplasma-derived reagents during manufacture How-ever the most recent generation is processed and for-mulated without the addition of human or animalplasma additives

Animal factor VIII concentrates Concentrates

pre-pared from bovine or porcine plasma have 100 timesmore factor VIII activity per milligram of protein thannormal human plasma The original preparations were immunogenic and could as a rule be used effect-ively for only 7–10 days, following which antibodiesagainst the animal protein developed Polyelectrolyte-

fractionated porcine factor VIII concentrate (PE porcine

VIII) appears to be considerably less antigenic and

contains negligible amounts of platelet-aggregating

factor (Kernoff et al 1984).

Factor VIII inhibitors (antibodies)

Factor VIII inhibitors are found either as isoantibodies

in 15–35% of haemophilia A patients following

treat-ment with factor VIII-containing materials or, more

rarely, as autoantibodies in non-haemophiliacs The

titre of the antibody measured in Bethesda units (Bu)

determines both risk and management About one-half

of the antibodies in haemophiliacs are of low titre

and transient Patients with factor VIII inhibitors are

relatively refractory to treatment with factor VIII and

must be given very high doses to secure a response

Concentrates of factor VIII are effective if the inhibitor

titre is less than 20 Bu/ml; with higher titres, factor VIII

concentrates alone are ineffective Haemophiliacs with

factor VIII inhibitors may show a rise in inhibitor titre

following the infusion of factor VIII; in such patients

factor VIII concentrates are not effective after 5–7 days

of treatment (Blatt 1982)

In treating major haemorrhage in haemophiliacs

with inhibitors, provided that the inhibitor titre does

not exceed 20 Bu/ml, human factor VIII concentrate

can be used initially In average-sized adults (70 kg),

5000 units of factor VIII are given initially, followed

by 500–1000 units per hour Thereafter, the dose is

adjusted according to the factor VIII level (Blatt 1982)

In patients with inhibitor titres exceeding 20 Bu/ml,

activated prothrombin complex concentrates appear

to be effective, but have an increased risk of

throm-boembolic complications (Hilgartner et al 1990;

Tjonnfjord et al 2004) These concentrates bypass

the need for factor VIII in a manner not thoroughly

understood Recombinant activated factor VII (rFVIIa)

has been found effective in patients with antibodies

against factor VIII (see also Chapter 18) (Hedner and

Kisiel 1983; Abshire and Kenet 2004) Standard

dos-ing of rFVIIa (90 µg/kg) allows binding of FVIIa to the

surface of activated platelets and can directly activate

factor X in the absence of tissue factor Experience with

bolus dosing suggests that higher dosing (> 200 µg/kg)

may be more efficacious in treating haemophilia patients

In patients who require very large amounts of

fac-tor VIII, animal facfac-tor concentrates have been used

successfully (Kernoff et al 1984) Four haemophiliacs

with factor VIII inhibitors received repeated infusions

of porcine VIII for periods up to 27 days with tory results An antibody response was detected inonly one out of the four patients Equally good results

satisfac-were obtained in a later trial (Hay et al 1990) The

newer treatments should render animal factor trates a historical curiosity

concen-In about 50% of patients with high-titre inhibitorsand up to 90% of those with titre < 5, immune toler-ance can be induced by desensitization regimens that

involve daily factor VIII infusions (Ewing et al 1988).

Treatment may be required for weeks or months andinfusions of factor VIII are sometimes combined withshort courses of immunosuppressive agents such ascorticosteroids, cyclophosphamide and IVIG (Kasper

et al 1989; Mariani et al 1994).

Treatment with IVIG is beneficial in some patientswith haemophilia A and inhibitors and particularly inpatients with factor VIII autoantibodies The effect

may be due to anti-idiotype antibodies (Sultan et al 1994; Schwartz et al 1995).

Transfusion in patients with von Willebrand disease

Von Willebrand disease is a common inherited somal dominant bleeding disorder characterized byeasy bruising, epistaxis, bleeding with dental proced-ures and gastrointestinal haemorrhage In the majority

auto-of patients, bleeding results from decreased vonWillebrand factor (vWF), a protein carrier of factorVIII that mediates platelet–platelet interaction andplatelet binding to vascular subendothelium (Ruggeriand Ware 1993) The vWF gene is located on chromo-some 12, and numerous polymorphisms and mutationshave been reported; the von Willebrand syndromes arenow classified by molecular, functional and clinicalcriteria (Sadler 1998)

Appropriate treatment depends on the specific type

of vWD Type 1 vWD, the most common form, usuallypresents with mild or moderate bleeding Laboratorydiagnosis shows low vWF antigen, activity (ristocetincofactor) and factor VIII levels, as well as a prolongedbleeding time Such patients usually respond well to

an infusion of DDAVP (0.3 µg/kg) with release ofsufficient vWF and factor VIII from tissue storeswithin 30 min to elevate these levels several fold forabout 4 h (Scott and Montgomery 1993) Some of theless common vWD variants do not respond well to

DDAVP (Ruggeri et al 1980; Sutor 2000) For the

10–20% of patients with vWD who do not respond

to DDAVP and for patients who become refractory

(‘tachyphylactic’) with repeated treatment given over a

long period of time (Rodeghiero et al 1992),

replace-ment therapy is available in several forms Patients

who require treatment with vWF should be given

high-purity, solvent detergent-treated vWF concentrate

(Pasi et al 1990; Burnouf-Radosevich and Burnouf

1992) If this is not available, factor VIII concentrate

which contains vWF should be used instead (Cohen

and Kernoff 1990) Cryoprecipitate, although rich in

vWF, has generally not been treated to reduce the risk

of viral transmission, and is therefore the least

desir-able of the availdesir-able components For planned surgery,

the products should be given a few hours before

opera-tion and, if factor VIII or ristocetin cofactor is used to

estimate effectiveness, the level should be measured

immediately before the operation is started; if the

level is not high enough (50 –100%), more concentrate

should be given Repeated infusions every 12 h may be

necessary for a week or more

Treatment of factor IX deficiency (haemophilia B

or Christmas disease)

Patients with haemophilia B are treated with factor

IX concentrate The administration of crude factor IX

concentrates derived from cryoprecipitate supernatant

(‘prothrombin complex concentrate’) was associated

with thrombotic complications (Kohler 1999), but

highly purified, virus-inactivated factor IX

concen-trates, based either on a combination of three

conven-tional chromatographic steps (Burnouf et al 1989) or

on immune-affinity chromatography with monoclonal

antibodies against factor IX, are now available (Kim

et al 1990; Tharakan et al 1990) Recombinant factor

IX is also available Some patients achieve only 80% of

the predicted plasma level and interpatient variability

is wide when this product is used, so that baseline

recovery measurements are important to ensure

ade-quate treatment Treatment with highly purified or

recombinant product avoids the side-effects

men-tioned above (Kim et al 1990) Each unit of factor IX

administered raises the plasma concentration by 1%

The T1/2of plasma factor IX is 20 h As in haemophilia

A, the dose needed is determined by the kind of

bleed-ing (see above) (Furie et al 1994) If no factor IX

concentrate is available, PCC (see below) can be used

instead Treatment with PCC may lead to thromboticdisorders and DIC (Aronson and Menache 1987).Treatment with factor IX concentrate may induceinhibitor formation, although this is less frequent thanthe formation of factor VIII inhibitors in patients with

haemophilia A (Knobel et al 2002) The treatment of

patients with factor IX inhibitors is essentially thesame as that of patients with factor VIII inhibitors (seebelow)

Prothrombin complex concentrates (concentratescontaining factors II, VII, IX and X)

Concentrates containing these vitamin K-dependentfactors were originally produced for the treatment ofinherited factor IX deficiency but are also used foracquired deficiencies of factors II, VII, IX and X, forexample in patients with liver disease or warfarin over-dose As described above, PCCs have also been usedfor treating minor bleeding episodes in haemophiliacswith inhibitors The concentrates should be adminis-tered rapidly, and immediately after reconstitution.PCCs are thrombogenic and their use remains con-troversial except in the treatment of haemophilia B,although, even here, purified factor IX concentrate haslargely replaced them (see above)

Use of some other coagulation factors

Factor VII

Fresh-frozen plasma can be used to treat factor VIIdeficiency; however, the need for frequent infusionsand the risk of viral transmission have all but elimin-ated its use for this indication Plasma-derived factorVII concentrate has been used successfully to treatpatients with hereditary factor VII deficiency (Dike

et al 1980) Long-term prophylaxis in children has also been successful (Cohen et al 1995) Because of

its short half-life (3 – 4 h), frequent infusions are necessary, especially for surgery, although levels of

15 –25% of normal are sufficient for this indication.Recombinant factor VIIa has been found to be effect-ive in haemophiliacs with antibodies to factor VIII and in some subjects with poorly controlled massive orlife-threatening haemorrhage (see above and Chapter

18) (Hedner et al 1988; Schmidt et al 1994) rFactor

VIIa is the treatment of choice when no

pathogen-reduced VII concentrate is available (Lusher et al 1998).

Factor XIII

Pasteurized factor XIII concentrates prepared from

either plasma or placenta are used to treat patients

with factor XIII deficiency, a condition that can be as

dangerous as haemophilia A or B (Smith 1990) Levels

as low as 5% are sufficient to control bleeding Because

the half-life of factor XIII is measured in weeks,

infusions may be given every 14 –21 days In the USA,

where no factor XIII concentrate is licensed, FFP and

cryoprecipitate are the components of choice

Protein C

Protein C is a serine protease zymogen, which is

activ-ated by thrombin Activactiv-ated protein C interferes with

the activated forms of factors V and VIII; it requires a

cofactor, protein S Activated protein C also stimulates

fibrinolysis by neutralizing the inhibitor of tissue

plasminogen activator Protein C deficiency, whether

hereditary or acquired as, for example, in severe liver

disease, leads to venous thrombosis Protein C

con-centrates are now available: a vapour-treated protein

C concentrate that has been shown to be effective

for long-term therapy in an infant with severe

protein C deficiency (Dreyfus et al 1995) and an

immunoaffinity-purified, activated protein C

con-centrate, virus inactivated by chemical treatment

(Orthner et al 1995) Recombinant human activated

protein C has anti-inflammatory and profibrinolytic

properties in addition to its antithrombotic activity

One recombinant formulation, drotrecogin alpha,

produced dose-dependent reductions in the levels of

markers of coagulation and inflammation in patients

with severe sepsis In a randomized trial, treatment

with drotrecogin alfa activated significantly reduced

mortality in patients with severe sepsis, but seemed to

be associated with an increased risk of bleeding

(Bernard et al 2001) The mechanism of the

anti-inflammatory effect is poorly understood, but may

involve inhibition of tumour necrosis factor

produc-tion by blockade of leucocyte adhesion or interference

with thrombin-induced inflammation

C1 esterase inhibitor (C1 inh.)

Hereditary functional deficiency of C1 inh is due to

either a deficiency or a dysfunction of the protein

Acquired deficiencies of C1 inh also occur This

pro-tease inhibitor is involved in the regulation of severalproteolytic systems in plasma, including the comple-ment system, the contact system of intrinsic coagula-tion and kinin release and the fibrinolytic system

(Cugno et al 1990) Functional deficiency of C1 inh.

permits production of vasoactive peptides that altervascular permeability and cause angioneurotic oedema,

a serious, potentially fatal syndrome characterized byattacks of swelling of the subcutaneous tissues andmucous membranes of the face, bowel and upper air-way Mortality rate in affected kindred approaches30% The pathogenesis of angioedema is not com-

pletely understood (Baldwin et al 1991).

Pasteurized C1 inh concentrates are now availableand acute attacks of angioedema respond within hours

of treatment (Brummelhuis 1980; Gadek et al 1980;

Bork and Barnstedt 2001) Long-term prophylaxis withC1 inh concentrate in hereditary as well as acquiredC1 inh deficiency has also been successful (Bork and

Witzke 1989; Waytes et al 1996) Activation of the

complement and contact systems occurs in septicshock, together with a decrease of plasma C1 inh lev-els Preliminary results show that complement andcontact activation can be diminished by treatment

with high-dose C1 inh concentrate (Hack et al 1992).

Immunoglobulins

IgG

Following i.v injection, IgG distributes between theintravascular and extravascular compartments; equi-librium is reached in about 5 days (Cohen andFreeman 1960) The daily movement of IgG from theintravascular to the extravascular compartment isequivalent to about 25–30% of the plasma IgG and isbalanced by a similar transfer in the opposite direction.When equilibrium has been attained, the plasma

level declines with a T1/2of 21 days The fractional rate

of catabolism is largely independent of plasma IgGconcentration so that the total IgG turnover variesdirectly with the plasma IgG level The rate of IgG syn-thesis is the primary factor determining the serum IgGlevel (Schultze 1966) The catabolism of IgG has alsobeen studied by injecting HBs antibody in high titre,with disappearance of antibody followed with a very

sensitive radio-immunoassay The T1/2 of these IgGantibodies was calculated to be 19.7 days (Shibata

et al 1983) Catabolism may be enhanced by fever,

burns and infection For further information,

includ-ing the turnover rates of IgG subclasses, see Chapter 3

Intravenous infusion of the ‘standard’ 16% Ig

preparations prepared for intramuscular injection may

produce severe reactions (see Chapter 15) This

pre-paration should not be administered intravenously

Following i.m injection of IgG, the protein passes

via the lymphatics into the bloodstream Analysis of

plasma concentration curves is relatively complex as

the influx into the plasma from the site of injection is

offset by efflux from the plasma into the extravascular

space and also by catabolism In a study in which 125

I-labelled IgG was used, the average fraction of the dose

cleared per day from the site of i.m injection, after

injecting 2 ml of solution into the deltoid muscle, was

estimated to be about 0.37 Plasma levels were almost

maximal at 2 days, and corresponded to about 40% of

the values that would have been attained immediately

after i.v injection of the same doses (Smith et al 1972;

see also Table 10.1) In a single case, surface counting

over the site of injection showed that approximately

45% of the total injected dose was cleared per day

(Jouvenceaux 1971) In two normal subjects following

the injection of 10 ml of 16% Ig into the gluteal region,

plasma levels corresponded to 32% of the injected

dose on day 5 in one case and 20% on day 7 in the

other (Morell et al 1980) In retrospect, uptake may

have been poor in these two cases because the

injec-tions were made into fatty tissue rather than into

muscle One survey indicated that when injections are

given into the gluteal region, few female patients and

fewer than 15% of male patients receive an i.m

injec-tion (Cockshott et al 1982).

Following subcutaneous injection of IgG into the

buttock, the rate of uptake is distinctly slower than

after i.m injection and maximum plasma levels have

still not been attained 5 days after injection (Smith

et al 1972).

Composition of IVIG preparations: not all are

created equal

Several Ig preparations containing fully functional

immunoglobulins are now available for i.v use These

are generally 5% or 10% solutions but concentrations

range from 3% to 12% One product is prepared by

DEAE fractionation, followed by treatment of the

IgG-containing fraction at pH 4 with a low

concentra-tion of pepsin; the IgG molecule is not cleaved, but

high-molecular-weight aggregates responsible for phylaxis and some of the other adverse reactions aredispersed (Jungi and Barandun 1985) If Ig for i.v use

ana-is to be stored in the liquid state, pH must be kept low

to maintain purity and stability The low pH of theproduct may be responsible for pain, erythema andeven phlebitis sometimes experienced at the injectionsite Freeze-dried preparations can be reconstitutedimmediately before use at a pH of 6.6, thus avoidingthe above side-effects

Stringent requirements for IVIG have been set by theCommittee for Proprietary and Medicinal Products(http://pharmacos.eudra.org) Human Ig preparationscontain very little IgM, but variable amounts of IgA.Some two dozen commercial preparations are availableand vary in physicochemical characteristics includingconcentration, volume, osmolality, sodium and sugarcontent (Lemm 2002)

Changes in immunoglobulin preparations

on storage

Although most IgG antibodies show no obviouschange in potency over a period of several years in Igpreparations kept at 4°C, the concentration of anti-Ddiminished at about 8% a year over 2– 4.5 years in

28 preparations tested (Hughes-Jones et al 1978).

There was no evidence of appreciable breakdown

of IgG molecules in the preparations in the sameperiod Low levels of immune complexes and variableamounts of IgG dimer are found in commercial pre-parations derived from large numbers of donor plasmacollections The proportion of dimer increases overmonths of storage and is enhanced by low temperature

(Ordman et al 1944) A relatively large dose (450 mg)

of Ig provides some protection for periods up to 6 weeks

(Kekwick and Mackay 1954, p 58) In the prophylaxis

of hepatitis A, a single dose of Ig (750 mg) may protect

for about 5 months (Pollock and Reid 1969)

Hyperimmune Igs prepared from selected donors

with high titres of the relevant antibodies are used in

the prophylaxis of hepatitis B, diphtheria, tetanus,

rubella, herpes zoster, rabies, measles and infection

with CMV, pseudomonas and other agents Anti-D Ig

is used in the prevention of primary Rh D

immuniza-tion (see Chapter 10)

Anti-HBs Ig in very high doses can prevent

reactiva-tion of hepatitis in 65–80% of HbsAg-positive patients

receiving liver transplant grafts (Terrault and Vyas

2003) Monoclonal anti-HBs is available Specific

anti-pertussis toxoid Ig in high titre has been shown in

a randomized trial to reduce significantly the number

of ‘whoops’ in 47 children with less than 14 days of

disease before therapy Duration of whoops post

treat-ment was 8.7 days for patients with whooping cough

(Granstrom et al 1991).

AIDS A decreased incidence of bacterial infections

and sepsis and an improved survival rate have been

observed in infants with congenital AIDS receiving

monthly IVIG (Calvelli and Rubinstein 1986) The

beneficial effect of IVIG has been confirmed in children

with advanced AIDS receiving zidovudine The

bene-fit, however, was only apparent in children who

were not receiving trimethoprim-sulphamethoxazole

as prophylaxis (Spector et al 1994) In one study,

pro-phylactic administration of IVIG to premature infants

significantly reduced the risk of infections (Baker et al.

1992), but in another similar study, in which a

differ-ent preparation was used, IVIG had no effect (Fanaroff

et al 1992) No recent study has evaluated IVIG

therapy in children with AIDS receiving highly active

antiretroviral agents (HAARTs), and the use of IVIG

should probably be restricted to children who develop

recurrent infections despite the administration of

HAARTs and prophylactic cotrimoxazole

Replacement therapy for immunodeficiency

syn-dromes Patients with either congenital or acquired

hypogammaglobulinaemia with IgG levels of less than

2 g/l are candidates for treatment Intravenous

infu-sion is usually preferred, because large amounts of Ig

have to be given that are poorly tolerated when given

by repeated i.m injection Although subcutaneous

injection of Ig preparations for i.m use has also been

satisfactory (Roord et al 1982), the ease and ready

acceptance of intravenous administration has largelyreplaced all other regimens Patients with X-linkedagammaglobulinaemia, severe combined immuno-deficiency (SCID), Wiskott–Aldrich syndrome amongothers have benefited Dosage has been determinedempirically, although prospective unblinded studiesconfirm their effectiveness (Buckley and Schiff 1991).The recommended intravenous dose is 300–400 mg/kgbody weight once per month If the response is unsatis-factory, the dose can be increased to attain a troughlevel of 400 –500 mg/dl

Neonatal sepsis Promising results in treating

neo-natal sepsis, particularly in premature infants, led to the recommendation that infants weighing less than

1500 g at birth should be given 0.5 g of Ig daily for

6 days, as a routine measure (Sidiropoulos et al 1981).

In a randomized study, 133 newborn infants weredivided into two groups based on whether the dura-tion of gestation was shorter or longer than 34 weeks.The infants were assigned to receive either 500 mg/kgIVIG weekly for 4 weeks or no therapy Septicaemiaand infection-related deaths were significantly less fre-quent in the group of infants born before 34 weeks

who had received IVIG (Chiroco et al 1987) In

another prospective randomized study, 753 prematureinfants with early onset sepsis were randomly assigned

to receive a single injection of IVIG (500 mg/kg) oralbumin (5 mg/kg) At 7 days, none of the infants whoreceived IVIG but five of the control subjects had died

(P< 0.05) However, at 56 days, there was no ence in death rate A single infusion may be insufficient

differ-to reduce infection-related mortality for more than afew weeks No serious side-effects occurred in any of

the treated infants (Weisman et al 1992).

Infection in adults Selected Ig preparations

contain-ing antibodies in high titre may have a role in severeviral and bacterial infection (Sawyer 2000) Trials

with anti-Pseudomonas Ig prepared from plasma from vaccinated volunteers reduced the number of Pseudo- monas infections and mortality in children and adults with severe burns (Jones et al 1980) The protective

effect of human IVIG preparations in bacterial

infec-tion has been shown clearly in mice (Imaizumi et al.

1985) However, results of other trials in patients withsevere burns or multiple injuries have been less con-

vincing (Berkman et al 1990).

Chronic lymphocytic leukaemia

Hypogammaglobulinaemia is common in patients

with chronic lymphocytic leukaemia (CLL) and

response to immunization is impaired The incidence

of infections was reduced by 50% in 42 patients with

CLL who received IVIG (400 mg/kg every 3 weeks)

compared with 42 patients receiving a placebo

(Cooperative Group for the Study of Immunoglobulin

in Chronic Lymphocytic Leukemia 1988) This result

has been confirmed (Griffiths et al 1989) A dose of

250 mg per kilogram appears to be equally effective

(Gamm et al 1994).

Multiple myeloma In two prospective studies, a

substantial reduction in bacterial infections has been

observed in patients treated with IVIG (Schedel 1986;

Lee et al 1994).

Kawasaki syndrome Kawasaki syndrome, a

child-hood vasculitis thought by some to be associated with

infection by a retrovirus, responds to treatment with

high-dose IVIG (400 mg/kg per day for 3 days)

com-bined with aspirin (Nagashima et al 1987) Fever

resolves in more than 85% of patients within 48 h and

coronary aneurysm formation is prevented (Burns

et al 1998) Although the mechanism of action is

unknown, IVIG reduces nitric oxide production and

the expression of inducible nitric oxide synthase,

factors associated both with vascular smooth muscle

relaxation and with aneurysm formation (Fukunishi

et al 2000).

Immune thrombocytopenic purpura The chance

observation that IVIG raised the platelet count in

immunodeficient children with severe

thrombocytope-nia has led to its widespread use in the treatment of

immune thrombocytopenic purpura (ITP), first in

chil-dren and subsequently in adults (Imbach et al 1981;

Fehr et al 1982) The mechanism of action in this and

in other autoimmune disorders is unknown, but may

involve anti-idiotype antibodies, regulatory effects on

lymphocytes and macrophages or interference with the

effects of complement activation (Tankersley et al.

1988; Mollnes et al 1997; Hansen and Balthasar

2004) Because children with acute thrombocytopenic

purpura are at risk for life-threatening haemorrhages,

treatment with IVIG has been advised when the

platelet count falls below 10× 109/l (Blanchette and

Turner 1986) In adults, treatment with IVIG has beenrecommended for patients who are bleeding or as pro-phylaxis for surgery, because infusions usually induce

a significant, albeit short-term rise in the platelet count

(Oral et al 1984) The recommended dose is the

origi-nal empirical schedule of 400 mg/kg per day for 4 days

or 1 g per kilogram per day The injection of relativelysmall amounts of anti-Rh D Ig has also been successful

in inducing remissions in ITP In D-positive adults,750– 4500 µg of anti-D was given in one series (Salama

et al 1986); in another, all of 13 D-positive patients

given 2500 µg responded, and a single D-negative

patient failed to respond (Boughton et al 1988).

Success has also been achieved by giving a single dose

of 4 ml of D-positive red cells coated in vitro with

100 µg of anti-D (Ambriz et al 1987) These

observa-tions suggest that anti-D produces its beneficial effects

by causing D-positive red cells to bind to, and thusblock, Fc receptors on macrophages Remission hasbeen reported in one D-negative pregnant woman whohad failed to respond to steroids and who was given

120 µg of anti-D intravenously (Moise et al 1990).

The effective agent in anti-D Ig may be HMW IgGpolymers rather than anti-D Preparations of anti-D Igcontain a substantially higher proportion of HMWIgG polymers than non-specific Ig preparations andthese polymers are more effective in blocking Fc

receptors (Boughton et al 1990) In a randomized

study, the effect of intravenous anti-D on the plateletcount in childhood acute ITP has been found to be

inferior to that of IVIG (McMillan et al 1994);

however, one treatment occasionally proves effectiveafter the other has failed IVIG and anti-D may work

through different mechanisms (Cooper et al 2004).

Other autoimmune diseases

IVIG has been used successfully in treating patients

with autoimmune neutropenia (Pollack et al 1982;

Bussel and Lalezari 1983) For the effect of IVIG inautoimmune haemolytic anaemia, see Chapter 7.IVIG has also been used in treating patients withother autoimmune diseases, among them myastheniagravis, the Guillain–Barré syndrome, multiple sclerosisand chronic demyelinating polyneuropathy (Knezevic-Maramica and Kruskall 2003) In patients with factorVIII or factor IX inhibitors, the antibody titre decreasesfollowing the administration of IVIG (Nilsson and

Sundqvist 1984; Sultan et al 1994).

Alloimmune diseases High-dose IVIG has been used

in Rh D haemolytic disease (see Chapter 12), in

neona-tal alloimmune thrombocytopenia (see Chapter 13)

and in post-transfusion purpura (see Chapter 15)

Bone marrow transplantation

IVIG has been shown to reduce the incidence of

septi-caemia, interstitial pneumonia, fatal CMV disease,

acute GvHD and transplant-related mortality in adult

recipients of related marrow transplants (Siadak et al.

1994) The mechanism responsible for this effect is not

known (Gale and Winston 1991; Sullivan et al 1991).

A meta-analysis of 12 randomized, controlled trials of

prophylaxis in bone marrow transplantation revealed

a significant reduction in fatal CMV infection;

CMV-interstitial pneumonia, non-CMV pneumonia and

transplant-related mortality (Bass et al 1993)

Con-tinued administration after day 90 does not appear

to reduce late-occurring infections or chronic GvHD

(Sullivan et al 1996).

Adverse reactions

Adverse reactions occur in as many as 15% of

admin-istrations (Boshkov and Kelton 1989) A commonly

recognized complex of symptoms including flushing,

headache, nausea and fever has been attributed to the

presence of IgG aggregates and complement fixation

by IgG dimers The syndrome was first reported with

i.m administration, but clearly occurs with IVIG,

especially when infusion is rapid (Nydegger and

Sturzenegger 1999) An anaphylaxis-like syndrome

that includes chills, arthralgias, flank pain, urticaria

and circulatory collapse was reported with the first

IVIG preparations, but occurs less commonly with the

low pH pepsin treatment described above (Barandun

and Isliker 1986; Tankersley 1994)

Passive antibody transfer is the intended

con-sequence of treatment with immunoglobulin

pre-parations; however, some passive antibodies cause

diagnostic uncertainty, whereas others such as red cell

alloantibodies may cause haemolysis Persistent

meas-urable titres of anti-HBs and anti-CMV in particular

may persist for months, although the titre will fall

over time and evidence of virus by other assays will be

lacking (Lichtiger and Rogge 1991) Antibodies to

numerous red cell antigens have been reported in up

to one-half of commercial Ig preparations in the past,

although this is a less common fining in the newer

preparations (Niosi et al 1971; Nydegger and

Sturzenegger 1999) Both passively acquired DAT andhaemolysis occur after infusion of passive antibody

(Copelan et al 1986; Moscow et al 1987) In a series

of 47 patients who received high-dose IVIG as laxis for cytomegalovirus infection following bonemarrow transplantation, almost one-half were found

prophy-to have an acquired DAT, and a quarter a positive indirect antiglobulin test (IAT) caused by passive

anti-A, -B, -D and -K (Robertson et al 1987) Most

findings appeared within 1 week of initiation of IVIGtherapy Transient neutropenia, presumably as a result

of passive neutrophil antibody, has been reported as

well (Tam et al 1996).

Thromboembolism, including venous thrombosis,

pulmonary embolism, myocardial infarction, strokeand hepatic veno-occlusive disease, has been associatedwith IVIG infusion (Go and Call 2000) The mechan-ism is unclear; however, changes in viscosity, proco-agulant contaminants and platelet activation have allbeen implicated Manufacturers and regulatory agen-cies have called specific attention to this complication(Dalakas and Clark 2003)

Renal dysfunction, including acute renal failure and

17 fatalities, has been reported to the FDA (Epsteinand Zoon 2000) The pathological appearance of theproximal renal tubules of the kidney has suggested

‘sucrose nephropathy’, so that sucrose added to someIVIG preparations has been implicated as the cause

(Cayco et al 1997) Patients with underlying renal

disease, especially the elderly and those with diabetesmellitus sepsis, monoclonal gammopathies and volumedepletion seem particularly susceptible to renal failure

Aseptic meningitis has been reported as a

dose-related complication of IVIG infusion, particularly in

patients with pre-existing migraine (Sekul et al 1994).

Case reports of recurrent migraine and seizures havebeen published and may be causally related, but themost intriguing is that of a patient who suffered recur-rent episodes of hypothermia with IVIG infusion

(Duhem et al 1996).

Transmission of HCV through IVIG has been

referred to above (Bresee et al 1996).

Novel intravenous immunoglobulins

By hybridoma technology, genetic engineering andchemical methods, novel specific monoclonal antibody

preparations now constitute a significant proportion

of biopharmaceutical products in development Several

chimeric and humanized monoclonal antibodies are

now licenced therapeutics (Roque et al 2004).

Antithrombin

Hereditary AT deficiency occurs in at least two forms:

in one, the level of antithrombin is low (about 50% of

normal), and in the second, antithrombin is

function-ally deficient In both cases the deficiency of this

natural anticoagulant is associated with a high risk of

venous thrombosis The first event often presents in

adolescence or young adulthood (Demers et al 1992).

Antithrombin inactivates five of the activated

coagula-tion factors This funccoagula-tion of antithrombin at several

levels of the coagulation pathway probably explains

why an apparently modest decrease in antithrombin

activity, as in patients with familial low antithrombin

levels, leads to a thrombotic tendency (Abilgaard 1984)

Heat-treated antithrombin concentrates are

avail-able with an initial 50% disappearance time of 22 h

and a biological half-life of 3.8 days, and are indicated

for the prevention or treatment of thromboembolic

disorders in patients with hereditary antithrombin

deficiency (Lechner et al 1983; Menache et al 1990;

Lebing et al 1994) A recombinant human

antithrom-bin has been used in congenitally deficient patients

who require surgery; the optimal dosing regimen

remains to be defined (Konkle et al 2003).

Acquired AT deficiency has several causes

Admin-istration of antithrombin may benefit patients with

cirrhosis who are to undergo surgery and patients in

hepatic coma or pre-coma (Lechner et al 1983)

In DIC, in which antithrombin levels are often low,

treatment with antithrombin concentrate may help,

particularly when treatment is started early and enough

concentrate is given to maintain a plasma level of at

least 100% of normal (Lammle et al 1984; Gabriel

1994; Schwartz 1994)

Fibronectin

Plasma fibronectin, an opsonic glycoprotein that may

play a role in wound healing, infection and vascular

integrity, enjoyed a short but enthusiastic vogue as a

therapeutic agent when administered in the form of

cryoprecipitate (Saba et al 1978; Saba and Jaffe 1980).

Treatment of trauma and burn patients deficient in

plasma fibronectin with cryoprecipitate purportedly

resulted in clinical improvement (Saba et al 1986).

However, a controlled trial of fibronectin found nobenefit for patients with severe abdominal infections

(Lundsgaard-Hansen et al 1985) Similarly, patients

with septic shock or severe injury showed no evidence

of improvement after treatment with fibronectin

(Rubli et al 1983; Hesselvik et al 1987; Mansberger

et al 1989).

α1-Antitrypsin

α1-Antitrypsin (α1-AT) is a major serine endopeptidaseinhibitor in human plasma, which inhibits neutrophilelastase, an enzyme involved in the proteolysis of connective tissue, especially in the lung Hereditarydeficiency of α1-AT may lead to progressive emphy-sema Clinical trials have suggested that replacementtherapy in deficient patients may restore the con-centration of α1-AT in plasma and thereby limit the

development of emphysema (Gadek et al 1981).

Concentrates of α1-AT, which can be treated at 60°Cfor 10 h, are available Weekly injections of 4 g for

6 months were given to 21 patients homozygous for

the deficiency allele P1Z (Wewers et al 1987) Peak

levels in plasma were above the normal upper range.After a rapid decline during the first 2 days after infu-sion, corresponding to redistribution of α1-AT into theintravascular space, there was a slower rate of declineconsistent with the normal 4- to 5-day half-life ofplasma α1-AT The lowest levels before the next injec-tion were always above the threshold level Diffusion

of the infused material across the alveolus and asignificant increase in elastase activity in epithelial lin-ing fluid could be demonstrated Similar results were

obtained in another study (Konietzko et al 1988).

There are, however, still unanswered questions withregard to replacement therapy with α1-AT Whethersuch therapy actually prevents the development or thefurther progress of emphysema remains unknown and

would require a large randomized trial (Dirksen et al.

1999) Neither has the question as to which deficientpatients should be treated been answered Consideringthe number of deficient patients (estimated at 70 000

in the USA), long-term demand cannot be met by α1

-AT produced from plasma Recombinant α1-AT wouldsurely be needed and such products are in develop-ment The American Thoracic Society and EuropeanRespiratory Society have reviewed this subject (2003)

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documented the frequency of fever as 16.8%, 12.4% ifpatients with fever in the week prior to transfusion

were excluded (Lane et al 2002) The study

invest-igators found that fever associated with transfusionwas recorded about four times as often as the hospitalattending staff reported it using a voluntary trans-fusion reaction form Fever exceeding 2°C occurred in3.1% of transfusions Because patients with AIDS areparticularly susceptible to infection and possibly morelikely to develop fever during the course of trans-fusion, the true rate of febrile reactions following red cell transfusion probably lies between these two values In contrast, fever occurs in as many as 30%

of platelet transfusions, a striking disparity that mayreflect platelet-specific factors as well as the effects

of inflammatory cytokines, chemokines and bacterialpyrogens (Chapter 16) that accumulate in platelet con-centrates over the course of room temperature storage

(Mangano et al 1991; Heddle 1999) In a study of 598

leukaemia patients who received 8769 transfusions,fever occurred in 4.4% of patients, but rose to morethan 22% if chills with rigors were included in the

definition of these reactions (Enright et al 2003) Only

2.2% of platelet transfusions resulted in a moderate orsevere reaction of any kind

Leucocyte antibodies in febrile reactions

The possibility that leucocyte antibodies might causetransfusion reactions was suggested by the association

of potent leucoagglutinins in the serum of patientswho had received multiple transfusions and who suf-fered febrile reactions (Brittingham and Chaplin 1957;Payne 1957) Evidence confirming this association was

of transfusion

15

Adverse effects due to overloading the circulation are

discussed in Chapter 2, reactions caused by red cell

incompatibility in Chapter 11, and transmission of

infectious agents in Chapter 16 The present chapter

includes reactions due to incompatibility of white

cells, platelets or plasma components Cases in which

the incompatible antibody is present in transfused

plasma, or is made by grafted lymphocytes, are

dis-cussed, as well as those in which the antibody

con-cerned is made by the recipient Non-immunological

reactions, such as those due to the presence of

cytokines in stored blood components and those due

to citrate anticoagulant and iron overload, are also

described

Reactions due to leucocyte antibodies

Febrile reactions due to antibodies in the recipient

Frequency of febrile reactions

The frequency of febrile reactions to transfusion

depends on the type of blood component, its storage

conditions and a variety of factors specific to the

recipi-ent Of nearly 100 000 units of whole blood and red

cells transfused from one blood centre in 1980, less

than 1% was reported to result in a febrile reaction,

and only 15% of recipients who were subsequently

transfused experienced a second episode of fever

(Menitove et al 1982) This oft-cited statistic is

undoubtedly too conservative because of an

under-reporting bias in the study design A prospective study

of 531 HIV-infected and AIDS patients who received

3864 red cell units during 1745 transfusion episodes

published subsequently (van Loghem et al 1958) The

role of leucocytes in causing transfusion reactions

was shown clearly in a study of five patients who had a

history of severe febrile reactions following blood

transfusion and whose serum contained

leucoagglu-tinins; transfusion of a fraction of blood containing

more than 90% of the buffy coat produced a severe

febrile reaction, but transfusion from the same unit of

red cells and plasma with less than 10% of the buffy

coat caused no reaction (Fig 15.1) The severe

reac-tions were characterized by flushing within 5 min of

the start of transfusion and a sensation of warmth The

patient then felt well for 45 min; about 60 min after the

start of the transfusion, temperature spiked and a

severe febrile reaction began (Brittingham and Chaplin

1957) In complementary observations, Payne (1957)

found leucoagglutinins in the serum of 32 out of 49

patients with a history of febrile transfusion reactions

Moreover, in 13 out of 15 patients receiving repeated

transfusions, leucoagglutinins appeared at about the

time the patients developed the first transfusion

reac-tions Blood containing less than 0.2 × 109leucocytes

per litre provoked no reaction in these patients

In a detailed study of a single subject 0.4 × 109

leuco-cytes (the number present in 50 ml of normal blood)

would not produce a reaction whereas the injection of

1.5 × 109or more would do so regularly The reactions

produced by 1.5 × 109leucocytes were mild when the

titre of the leucoagglutinins was low, but severe at a

time when the titre was high (Brittingham and Chaplin

1961) From another study of eight patients, the least

number of leucocytes required to produce a reaction

varied from 0.25 × 109to more than 25 × 109 Thedegree of temperature elevation was related to thenumber of incompatible leucocytes transfused (Perkins

et al 1966).

These early studies indicate that leucocyte-poorblood prepared for transfusion to patients who havehad febrile reactions due to leucoagglutinins shouldcontain fewer than 0.5 × 109leucocytes or about 10%

of the number contained in a fresh unit of whole blood.Current component filtration technology achieves leucocyte levels that are lower by several orders ofmagnitude

Features of febrile reaction

By convention, an increase in body temperature of 1°C

or more (body temperature > 38°C) that occurs during

or within several hours of transfusion merits ation as a transfusion-related event In practice, trans-fusion reactions related to leucocyte antibodies mayinclude an array of signs and symptoms in addition tofever, including dyspnoea, hypotension, hypertensionand rigors; placing them in the ‘febrile’ reaction cat-egory is convenient, but somewhat arbitrary Patientswho develop febrile reactions related to leucoagglu-tinins usually do not start to feel cold for at least

evalu-30 min after the transfusion has started, and signs maynot develop for 60–80 min (see Fig 15.2) However,non-specific symptoms such as chilliness, nausea andheadache may precede the onset of chills and fever.Antibodies bind to the transfused leucocytes and theresulting complexes bind to and activate monocytes,

Buffy-rich (fraction II)

Fibrinolysis observed

Infusion begun

2 1

o F)

Fig 15.1 Effect of transfusing the

buffy coat to a patient whose serum

contained leucoagglutinins Two

fractions were prepared from fresh

blood: fraction I, containing many

red cells with very few white cells and

platelets and fraction II, containing a

few red cells and most of the plasma,

platelets and white cells Transfusion

of this second fraction produced a

very severe febrile reaction whereas

transfusion of the first ‘buffy-poor’

fraction produced no fever From

Brittingham and Chaplin (1957) with

permission.