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ORIGINAL PAPER 4A-S14-3

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Blackwell Publishing Ltd

Effect of recipient immune status on the persistence and clinical consequences of transfused leucocytes

William Reed

1,2

, Garth H. Utter

3

, Tzong-Hae Lee

1

& Michael P. Busch

1,2

1

Blood Systems Research Institute (BSRI), 270 Masonic Avenue, San Francisco, California, USA

2

University of California San Francisco, Department of Laboratory Medicine, San Francisco, California, USA

3

University of California Davis Medical Center, Department of Surgery, Sacramento, California, USA

The presence of a population of allogeneic cells underlies and partially defines bothmicrochimerism (MC) and graft-versus-host disease (GVHD). Although the relation-ship between these two apparently distinct clinical entities is not well understood, itis plausible that progenitor cell dose, histocompatibility, recipient immune status andgenetic profile all help determine whether allogeneic cells die, persist as a minorpopulation or proliferate in a GVHD response. Although MC has been described, at lowlevels, in association with pregnancy and autoimmune disease, much higher levels ofMC have recently been found in association with blood transfusion. This transfusion-associated microchimerism (TA-MC) has thus far been identified only in patientsmultiply transfused for severe traumatic injury, which is well documented to inducebroad short-term suppression of both the innate and adaptive arms of the host immunesystem. In this review, we will summarize the limited available data concerning theclinical, immunologic and genetic settings in which TA-MC and GVHD develop andexamine the factors that may influence their development. We will not addresshumoral alloimmunization as a result of transfusion here because its relationship todonor leucocyte persistence is unknown.

Key words:

graft-versus-host disease, immune suppression, microchimerism, trans-

fusion, trauma.

Introduction

Microchimerism has recently been described in associationwith pregnancy, autoimmune diseases and twinning [1–3]. Ithas even been suggested that fetal progenitor cells may crossthe placenta and function in the repair of damaged maternaltissues [4]. Despite these intriguing reports, the levels of MCthey describe are extremely low and whether MC actuallyplays a beneficial or pathogenetic role (or

no

physiologic roleat all) in pregnancy or autoimmune disease remains unknownand controversial.

Our research group has focused on investigating the fateof donor leucocytes following transfusion [5]. Our studies,using methods derived from nucleic acid testing (NAT) ofhuman leucocyte antigen (HLA) and viral polymorphicsequences, developed and applied modern molecular toolsspecifically for the detection and quantification of allogeneiccells against the much greater background of self cells.Investigating several clinical transfusion settings, we weresurprised to discover that patients transfused for severetraumatic injury regularly developed high-level long-termtransfusion-associated microchimerism (TA-MC), but thosepatients receiving blood for conditions such as sickle celldisease, HIV, or cardiac or orthopedic surgery did not. Ofconsiderable importance, the MC levels associated withtransfusion are on the order of 1% whereas those associatedwith autoimmune disease have been on the order of0·001%. Whether this difference in magnitude is alsoassociated with qualitative differences in clinical conse-quences is not known.

Correspondence

: Michael P. Busch, MD, PhD, Blood Systems Research Institute, 270 Masonic Avenue, San Francisco, CA 94118, USAE-mail: mbusch@bloodsystems.orgThis project was supported by grants from Blood Systems Foundation and NHLBI Special Center of Research (SCOR) grant in Transfusion Medicine #P50-HL-54476

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Because the technical problems associated with reliablecharacterization of low-level MC are so significant for thefield, we have devoted considerable resources to the develop-ment and validation of these methods with the aim of achievinghigh sensitivity, high specificity and having available assaysbased on multiple genetic loci that are informative for MCand able to corroborate one another [6]. These genetic lociinclude Y-chromosome, HLA class I and class II and a group ofinsertion-deletion (InDel) polymorphisms that are commonin the population. The InDel polymorphisms are particularlyattractive as allogeneic markers because their alleles arecharacterized by two to three base pair differences and arenot genetically linked to either sex determining genes orthe immune response genes making them nearly ideal forsophisticated molecular analysis of MC. A review for ISBTlast year focused on the development and validation of thistechnology for the detection and characterization of MC [7].

Notwithstanding the efforts in assay development andvalidation, a pivotal question in the TA-MC field is why theallogeneic cell population persists and expands in somepatients whereas being cleared in others. This question is alsoapplicable to graft-versus-host disease, whether caused bytransfusion, for which the exposure to allogeneic leucocytesis unintended, or by haematopoietic stem cell (HSC) trans-plantation, for which it is deliberate. Factors that may berelevant to these questions include blood product character-istics, the nature of the immune compromise associatedwith traumatic injury, donor–recipient histocompatibility,progenitor cell dose and genetic polymorphisms such asfunctionally significant cytokine allelic differences that mayinfluence the persistence or rejection of infused leucocytes inthe recipient. In addition, there may be other factors or inter-actions requiring a confluence of factors that will prove evenmore difficult to analyse. In this review, we will focus on theimmunologic data available from the relatively modest studieswe have conducted to date and also describe what we considerto be important open questions in the field and what studieswe are currently designing to address them.

Traumatic injury and immunosuppression

An immune deficit has long been recognized in injured patientsand is not controversial. It appears likely that this immunedeficit is a necessary but not sufficient precursor for the estab-lishment of TA-MC in the injured patient. Interestingly, thisdeficit does not appear to result in graft-versus-host disease(GVHD) simply because, in contrast to TA-MC, GVHD isthought to be extremely uncommon among injured patients.

Short-term immunosuppression and its infectious com-plications are an important cause of death in trauma patients[8,9]. Early studies have shown impaired function of T cells,B cells and monocytes. Studies in mice using stimuli such asphytohemagglutinin (PHA), pokeweed mitogen or bacterial

lipopolysaccharide have demonstrated suppressed secretionof Th1 cytokines such as IL-2 and IFN-gamma and increasedsecretion of the Th2 cytokines IL-4 and IL-10 at days 5–10 aftertrauma, with concomitant decreases in T-cell proliferativeresponses [10–14]. These murine observations have beencorroborated in humans [15–19]. In addition to defects inT-cell responses, decreased IgG or IgM production has beenobserved. In spite of a monocytosis, monocyte function issuppressed, along with decreased HLA expression and IFN-gamma secretion after stimulation [17,20,21].

Early studies also pointed to increased macrophageproduction of prostaglandin E

2

[22,23], IL-6 [24,25] or IL-10[26] as the mechanism of T-cell suppression. Additionally,toll-like receptor (TLR)-2 and TLR-4 stimulation leads toincreased IL-1, IL-6 and TNF-alpha secretion by macrophages[27]. Natural killer (NK) cells may play a role in suppressingT-cell responses, as blockade of these cells prior to burn injuryprevents suppression of T-cell responses [28]. Neutrophils arealso affected by trauma, secreting increased levels of IL-10after stimulation [29]. Serum from trauma patients cansuppress T-cell responses from normal individuals, suggest-ing that a soluble factor may contribute to the immune sup-pression [30–32]. At the molecular level, patients’ peripheralblood mononuclear cells also have decreased levels of thenuclear transcription factor NF-kappa-B, revealing a defectin an important pathway of T-cell activation [33].

The theme of these studies implicates Th2-type cytokinesin the suppression of T-cell immune response post-trauma.Corroborating this, administration of the Th1-biasing cyto-kine IL-12 has decreased mortality after infectious challengein traumatized mice [16,34]. Additionally, blockade of the Th2cytokine IL-10 improved survival after infectious challenge[14]. Thus, available evidence supports the hypothesis thattrauma predisposes the immune system to a Th2-type response,which blocks the ability of T cells to proliferate and secreteTh1 effector cytokines. These findings may also be directlyrelevant to the establishment of TA-MC in the transfusedinjured patient. More detailed assessment of immunologicparameters, especially studies of killer cell immunoglobulin-like receptor (KIR) and T-regulatory cells, and how they varyover time, is needed in prospective studies of TA-MC in orderto develop a more detailed understanding of the immunedeficit associated with injury and how it relates to TA-MC.

Frequency, kinetics and donor-recipient factors in TA-MC

In order to confirm Schechter’s historical observationsregarding the proliferation of donor leucocytes shortly aftertransfusion [35,36], we studied the fate of donor leucocytesamong women undergoing elective surgery who received arelatively large number of allogeneic cellular blood trans-fusions using the only available polymerase chain reaction

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(PCR) test at the time, a Y-chromosome-based assay [37]. Tenpatients who sustained traumatic injury were recruited toaugment the analysis. Although none of the elective surgerypatients had prolonged male donor cells present, 7 of the 10trauma patients showed survival of donor leucocytes formore than 6 months post-transfusion. Furthermore, multipleimmunophenotypic lineages of male donor cells were observed,including T-lymphocytes (CD4

+

, CD8

+

), myelomonocytes(CD15

+

) and B-lymphocytes (CD19

+

). Mixed lymphocytecultures suggested that the cells of the recipients had dimin-ished alloreactivity to the cells of the donor implicated as thesource of TA-MC by HLA typing but not to those of the otherdonors.

These unexpected findings warranted confirmation. Wesubsequently enrolled a prospective cohort of 45 severelyinjured patients who were transfused with at least two units ofnon-leukoreduced blood [38]. Blood from these patients wastaken at hospital discharge. Twenty-four of the 45 subjects(53%) had evidence of TA-MC using the HLA-based allele-specific PCR detection system available at that time. Althoughthe timing of blood sampling could not exclude the possibilitythat normal survival kinetics explained the persistence ofdonor leucocytes in some of the subjects, 3 of the 24 patientswho had evidence of TA-MC had not received any blood forat least 2 weeks prior to sampling. This amount of time effec-tively excluded the possibility that the typical proliferationof donor leucocytes post-transfusion explained all of theresults. Although the group of patients was small, we studiedthe clinical and blood product characteristics we thoughtlikely to be associated with development of TA-MC. Therewere few clinical predictors: neither the number of bloodtransfusions, gender of recipients, measures of injury severitynor the proportion of recipients that underwent splenectomyvaried between those who did and did not develop TA-MC.One factor that seemed to be important was the age of thetransfused unit implicated in TA-MC with the freshest unitsbeing implicated; this observation is consistent with studiesshowing that the ability to induce the activation marker CD-69 declines with storage time in donor blood [39]. The largemajority of instances of TA-MC had evidence of only one ortwo minor-type HLA-DR alleles, suggesting that TA-MCinvolves only one donor despite some patients receivingblood products from a multitude of donors.

This study also included a second smaller group of 13trauma patients transfused with leucoreduced blood fromwhom we obtained a blood sample immediately upon arrivalto the hospital (prior to any transfusion) as well as near the timeof hospital discharge. Seven of these patients had evidenceof TA-MC after transfusion, whereas all pretransfusionsamples were negative, indicating that the PCR resultswere unlikely to be attributable to any source other thantransfusion, such as contamination of specimens or micro-chimerism of maternal-fetal origin. This result also suggests

that leucoreduction would not be a suitable strategy to preventTA-MC.

This study, because it was co-ordinated closely between ablood centre and a hospital trauma service, allowed us to testcryopreserved recipient samples drawn shortly after injury(and prior to transfusion) as well as those drawn prior tohospital discharge for evidence of immunologic responsive-ness to PHA [40]. In doing this, we wanted to confirm previousreports of global immunosuppression associated with trauma.Among transfusion recipients who went on to develop TA-MC, lymphocyte responsiveness was initially at lower levelsand recovered less by the time of hospital discharge thanamong transfused patients who did not develop TA-MC.These immunologic changes were similar for patientstransfused with leucoreduced and non-leucoreduced bloodcomponents. In a comprehensive recall of 646 of the 656blood donors for these patients, we were able to performmixed lymphocyte reactions (MLRs) between each patient’spretransfusion lymphocytes and lymphocytes from each oftheir blood donors. For patients who went on to developmicrochimerism, a single donor could be identified whoselymphocytes showed bidirectional hyporesponsiveness withlymphocytes from the patient who received that donor’s unit.Although the microchimeric donor could not be definitivelyidentified from the limited HLA typing data available, in eachcase the low-resolution HLA type of the implicated donormatched the DR type of the chimeric cells detected by PCRtargeting HLA-DR alleles, suggesting strongly that the hypo-responsive donor was indeed the source of the chimeric cells.

Follow-up of survivors 2 to 3 years postinjury revealedthat 5 out of 20 subjects who tested positive for TA-MC athospital discharge demonstrated evidence of long-term TA-MC, still likely involving only the same single donor [41].Remarkably, the proportion of circulating leucocytes withdonor DNA appeared to increase from levels similar to thoseassociated with autoimmune diseases (10

−

4

–10

−

6

) at hospitaldischarge to 0·4–4·9% at a median of 25 months later. Eachof the five units of blood implicated in long-term TA-MC hadbeen stored for less than 14 days from collection to transfu-sion, and was transfused within 48 hours of a life-threateningepisode of haemorrhage.

We subsequently conducted an analysis of TA-MC follow-ing postinjury transfusion with exclusively leucoreducedblood products. As a post hoc analysis of a subgroup ofpatients who participated in a single-centre randomized con-trolled trial evaluating non-leucoreduced vs. leucoreducedblood, we evaluated 67 patients who had been hospitalizedfor traumatic injury at a different hospital than the previoustrauma patients we had studied [42]. At a median follow-upof 8 months, 9 of 32 patients in the non-leucoreduced group(28%) and 13 of 35 patients in the leucoreduced group (37%)developed TA-MC (

P

= 0·43). Thus, although leucoreductionremoves the vast majority of donor leucocytes, it does not

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prevent or reduce the likelihood of developing TA-MC. Thisresult confirmed the prior unexpected result showing thatleucoreduction did not prevent TA-MC. Although leucore-duction typically removes several logs of passenger leuco-cytes, it is possible that a progenitor or stem cell present inperipheral blood of the donor has different surface chargeproperties associated with its developmentally immaturestate and that these altered charge properties allow the cellsto selectively pass through a leucoreduction filter.

Additional studies are currently in progress, funded byBlood Systems Foundation and the National Heart Lung andBlood Institute, to define the kinetics and durability ofpostinjury TA-MC. In one recent study, we sampled the bloodof a group of US military veterans previously injured in com-bat during the World War II: Korean, and Vietnam conflictswho reported a history of transfusion at the time of injury.Preliminary results show that 27 of 168 subjects (16·1%) (butnot control blood donors of similar age and gender) haveevidence of enduring TA-MC, with similar prevalence amongveterans across conflicts (unpublished data).

Histocompatibility, engraftment and recipient cytokine polymorphisms

Although one might presume that as with graft-versus-hostdisease, donor-recipient compatibility at HLA could be theprimary determinant of TA-MC as it is in HSC transplantationand TA-GVHD, we have not been able to marshal evidence tosupport this hypothesis. Although the reason for this is notknown (and could possibly involve a detection bias fromusing HLA-DR–based assays as a means of detecting TA-MC),it may represent an important clue as to the biology of TA-MC. Although the expanding cell population and long-termsurvival of donor leucocytes along with the apparent re-presentation of multiple immunophenotypic lymphocytesubsets suggest that the recipient may be engrafted withrecipient HSCs, it is also possible that something short of truehaematopoietic engraftment has taken place and that thisphenomenon is not as sensitively dependent upon histo-compatibility. One possibility is that TA-MC may be largelythe result of homeostatic proliferation of T cells [43]. Thealternative explanation is that the transfusion recipient hasexperienced haematopoietic engraftment by the small numberof HSC in a fresh unit of transfused blood. Some precedentfor this idea may be found in work of Sharkis [44], who demon-strated in a murine system that multiorgan, multilineageengraftment can be established reproducibly by a singlehaematopoietic precursor cell. We believe that the extent ofengraftment is a fundamental question regarding the natureof TA-MC that must be addressed. If transfused traumapatients have indeed become stably engrafted at the HSClevel, it would suggest that engraftment is possible, at leastin the unique clinical setting of traumatic injury, without

chemotherapy or irradiation; this may have relevance for avariety of clinical problems. Moreover, if the chimeric lym-phocyte population is polyclonal, it is more likely that itsconstituent cells function immunologically within therecipient. We are currently planning experiments using largenumbers of mononuclear cells collected by apheresis frompatients with long-term high-level TA-MC to determinedefinitively whether they are engrafted at the HSC level andwhether their chimeric lymphocyte populations are oligo- orpolyclonal. Our murine transfusion studies show that in amurine transfusion model system, transfused donor blood iscapable of repopulating lymphocyte subsets in immunologicknockout recipients and that the lymphocytes function withrespect to antigen recall and viral clearance [45,46].

Potential mechanisms underlying the development of TA-MC include genetic heterogeneity in the recipient. We havenoted in previous discussions that the classical measures ofhistocompatibility do not seem to explain the phenomenonof TA-MC although the data sets analysed thus far are smalland non-classical HLA, KIR receptors or other factors mayplay an as yet unappreciated role. Still, because only a subsetof massively transfused severe trauma patients developslong-term microchimerism, heterogeneity in the developmentof tolerance must also exist. Cytokine single nucleotidepolymorphisms have been found relevant to the outcome insolid organ transplantation with associations noted for TNF-alpha (–308), IL-10 (–1082), IFN-gamma (+ 874) and TGF-beta

1

(codon 25) [47,48]. TNF-alpha is a pleiotropic cytokinethat can activate, differentiate and kill immune cells; it alsoacts as a critical mediator of the inflammatory response andis present at increased levels in some individuals as a result ofa G to A transition polymorphism in its promoter at position–308 [49]. IL-10, described as an anti-inflammatory cytokineand B-cell proliferation factor, has an A to G transitionpolymorphism at position –1082, which is associated withincreased IL-10 production [50]. IFN-gamma is a cytokineimportant in both innate and adaptive immunity and isreported to contain a polymorphic repeat cytosine adenosine(CA) microsatellite of variable length in its first intron. Inthis microsatellite, the presence of 12 CA repeats is associ-ated with increased IFN-gamma production and is linked toan adjacent 5

′

-A to T transition polymorphism at position+874 [51]. TGF-beta

1

primarily acts to inhibit proliferationand activation of immune cells and is present at decreasedlevels in individuals with a G to C transition polymorphismin codon 25 (position +915) [52]. Because immunologicmechanisms of solid organ rejection may be similar to thosethat prevent TA-MC in normal individuals, it is possible thatpolymorphisms in these genes may also influence the rate ofdevelopment of TA-MC in transfused trauma patients. Recentdata from our laboratory suggest that the TNF-alpha (–308)mutation may be associated with the development of TA-MC(unpublished data).

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GVHD and TA-MC

Although the relationship between GVHD and TA-MC is notfully understood, it is clear that both entities are partiallydefined by the presence of an allogeneic cell population inthe peripheral blood. Because TA-MC has only recently beennoted in transfused trauma patients, it is unclear whether itexists as a distinct clinical entity or whether it might exist ona progressive continuum, sometimes being cleared from thecirculation, sometimes persisting in a clinically benign stateand occasionally evolving into GVHD. It is also possible thatthere may be milder forms of GVHD that go undiagnosed andfrom which the recipient usually recovers. We are currentlyconducting collaborative studies with the US Military and theArmed Forces Institute of Pathology to determine whetherseverely injured and transfused soldiers who died with un-explained multiple organ dysfunction have evidence of anallogeneic cell population in their peripheral blood or affectedtissues. To our knowledge, this would be the first time adiagnosis of GVHD has ever been explicitly pursued in thispopulation. In this section, we will briefly review the epide-miology and pathophysiology of GVHD in order to allow acomparison with what is known about TA-MC. Much of thematerial is drawn from our recent textbook chapter, whichgoes into greater detail for the interested reader, includingdetailed discussion of the clinical characteristics, preventionmeasures and treatment of transfusion-associated GVHD(TA-GVHD) [53].

Clinical GVHD was first described by Shimoda [54] in 1955as ‘postoperative erythroderma’ in a case report of what waslater identified as TA-GVHD. The first accounts of recognizedTA-GVHD were published in the mid-1960s by Hathawayand associates [55,56]. Subsequently, TA-GVHD wasdescribed in immunosuppressed patients [57] and later alsoin immunocompetent transfusion recipients who wererecognized as being at risk because of a relatedness in HLAantigens to the blood donor [58,59]. Transfusion-associated-GVHD remains a rare but distinct threat to at-risk recipientsbecause of its severe course and resistance to therapy.Recognition of susceptible recipients who can be protected byselecting gamma-irradiated blood for transfusion is essential.

Graft-versus-host disease after transplantation was firstrecognized in 1959 in a report of ‘secondary-like disease’afflicting leukaemia patients who had received bone marrowtransplants [60]. We now recognize acute GVHD and chronicGVHD as clinically distinct entities in transplant patients,although some overlap may occur between the two conditions[61,62]. Graft-versus-host disease is most common in bonemarrow transplant recipients but also occurs in recipientsof solid organ transplants, particularly liver transplants[63,64]. Disease severity is extremely variable, but both acuteand chronic GVHD following transplantation can often beprevented or treated effectively [65].

The simplified model of pathogenesis that has emergedsuggests that immunocompetent alloreactive donor lym-phocytes that are not cleared because of a compromised hostimmune system, the cells go on to proliferate in the recipient.This is followed by attack and destruction of host tissues intarget organs, including liver, intestines, skin and, perhaps,lung [66]. Supporting the validity of this model in TA-GVHDis a positive correlation between the number of transfusedviable donor lymphocytes and the degree of immunosup-pression in the host on one side and the likelihood ofoccurrence and severity of TA-GVHD on the other [60]. Thethreshold dose of lymphocytes to incite TA-GVHD in humansis not precisely known, but case reports suggest that leucore-duction by current filtration (usually to < 5–6

×

10

6

whiteblood cells per 300 ml of blood component) is not sufficientto prevent the disease [67–69].

Occurrence of TA-GVHD in immunocompetent recipientshas been explained by a so-called one-way HLA match inwhich the donor cells are homozygous for an HLA type forwhich the recipient is heterozygous [59]. As a consequence,the recipient immune cells do not recognize the donor cellsas foreign and fail to mount an immune response that wouldnormally clear the donor cells. The latter, however, respondto the mismatched haplotype and incite the GVH reaction,which proceeds as aggressively as in immunocompromisedpatients. It is interesting to note that here, as in TA-MC,GVHD is estimated to occur less often than would be predictedby the occurrence of random HLA haplotype compatibility inthe population. This may be the result of under-diagnosis butit also may reflect the need for additional cofactors, such asage of the blood or the presence of specific recipient genetictraits.

Refinements in the basic model suggest a complexinteraction of recipient and host immune cells and emphasizethe notion that the latter is not mere passive bystanders butparticipates in and maintains the GVH reaction throughdysregulated cell activation and cytokine release. RecipientT-helper subset 1 (Th1) cytokines interleukin-2 (IL-2) andinterferon-beta and proinflammatory cytokines IL-1 andtumour necrosis factor beta (TNF-beta) appear to promote thedeleterious effects of GVH, whereas Th2 cytokines IL-4 andIL-10 are thought to have a down-regulating effect [70].

New insights have also been achieved into the roles of lym-phocyte subpopulations in the pathophysiology of GVHD. Amouse model provided evidence that T cell subsets playopposite roles in the disease process, with recipient CD8 cellsproviding protection whereas recipient CD4 cells promote aGVH response [71,72]. According to the authors, patientswith impaired CD8 and NK cell function are at increased riskfor TA-GVHD, especially if they receive HLA haploidenticalblood. Interestingly, this hypothesis provides one explanationfor the clinical observation that human immunodeficiencyvirus (HIV)-infected persons do not develop GVHD following

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allogeneic transfusions despite impaired cellular immunity.In these patients CD4 counts drop early but CD8 countsremain high until late in the disease course. Infection ofinfused donor CD4 cells by HIV, followed by attack of theinfected cells by cytolytic CD8 cells, may provide an additionalexplanation for the apparent absence of GVHD in patientswith acquired immunodeficiency syndrome (AIDS) [73].

With regard to the effector cells that cause tissue damagein GVHD, evidence from studies of patients with TA-GVHDsuggests that donor T cells participating in the diseaseprocess are clonally restricted in expression of T-cell receptorrepertoires [74] and are composed of a variety of CD4- andCD8-positive clones that may cause tissue damage throughdifferent mechanisms, including direct and indirect cyto-toxicity [75]. The latter finding may explain the oftencharacteristically sparse lymphocytic infiltrate of target tissuesin GVHD. Indirect cell lysis may occur through an apoptosis-inducing mechanism or through cytokine release (TNF-beta,IL-1) followed by attraction of platelets and neutrophilscausing cell damage by release of free radicals and elastases.

Summary and future research directions

Microchimerism has been recently described in associationwith a number of specific allogeneic exposures includingpregnancy, twinning, transplantation and blood transfusion.Both recipient immunosuppression as a result of injury andgenetic variation, such as functionally important cytokinepolymorphisms, may influence the establishment of TA-MC.In the setting of traumatic injury, leucocytes from a singleblood donor may persist months to years at a level that risesover time to as much as 3–4% of the recipient’s total circu-lating leucocytes. This TA-MC phenomenon may affect 1 in10 transfused trauma patients who will develop high-level,persistent TA-MC. It is important for future research todetermine whether persistent TA-MC occurs in other clinicalsettings where tissue injury is prominent, the immunologicalmechanisms involved and the extent of haematopoietic andimmunological engraftment. The latter question is especiallyrelevant as viable donor lymphocytes from transfused cellu-lar blood components may also cause TA-GVHD. The modestdirect evidence available to date suggests that there are anumber of variables involved in determining the establishmentand clinical behaviour of both TA-MC and GVHD and that anas yet unknown confluence of these variables may need tobe present to create conditions necessary and sufficient forTA-MC and/or GVHD.

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