Histocompatibility methods

Abstract 

Histocompatibility methods 

Kathryn Tinckam ⁎ 

Departments of Laboratory Medicine and Pathobiology and Medicine, University of Toronto, Toronto ON, Canada M5G 1L5 

Regional Histocompatibility Laboratory, Toronto General Hospital – University Health Network, Toronto ON, Canada M5G 2M1 

Predicting humoral alloimmune potential in transplant recipients is the objective of histocompatibility testing and depends upon accurate 

donor typing and sensitive and specific testing for antibodies to human leukocyte antigen. This review for the transplant clinician will 

describe the evolution and current practices of histocompatibility testing methods for typing, crossmatching and antibody screening. Novel 

methods of measuring T-cell alloimmune potential will also be briefly discussed. Emphasis is given to the clinical applicability and 

limitations of each test, and the collective consideration of all tests in concert, as part of the immunologic risk assessment of the solid organ 

transplant recipient. 

© 2009 Elsevier Inc. All rights reserved. 

1. Introduction 

The landscape of histocompatibility testing in solid organ 

transplantation has changed dramatically in the 40 years 

since crossmatching was first described by Dr Paul Terasaki 

[1]; however, the goal of the testing remains unaltered, that 

is, to provide a reliable estimate of the immunologic risk of a 

transplant recipient in the context of their potential donor(s). 

The nature of this measurement has evolved as techniques 

for human leukocyte antigen (HLA) typing and antibody 

detection have improved in sensitivity and specificity, but 

they nonetheless continue to interrogate the likelihood that 

the recipient immune system will recognize the allograft as 

foreign and initiate inflammatory events resulting in allograft 

damage. In this way, the HLA laboratory estimates risk as a 

complement to the clinical evaluation. Furthermore, HLA 

testing methods are no longer limited to the pretransplant 

period; indeed, antibody assessment as well as newer T-cell 

assays are increasingly studied posttransplant as noninvasive 

predictors of acute and chronic alloimmune complications. 

As such, it is imperative for the clinical service to understand 

the complex and interactive nature of the available 

histocompatibility testing. 

⁎ Tel.: +1 416 340 5142; fax: +1 416 340 3133. 

E-mail address: Kathryn.Tinckam@uhn.on.ca. 

Available online at www.sciencedirect.com 

Transplantation Reviews 23 (2009) 80 –93 

0955-470X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. 

doi:10.1016/j.trre.2009.01.001 

This review, directed to the transplant clinician, will discuss 

currently used methods of HLA typing, antibody screening, 

and crossmatching, with an emphasis on their evolution 

over the last 4 decades, their current clinical utility and 

applicability in 2008, and the factors that must be considered 

in their interpretation. Newer approaches for measuring T-cell 

alloimmune potential, which are being explored in some HLA 

laboratories but are not yet widely clinically utilized, will 

also be briefly discussed. In providing a practical reference of 

histocompatibility testing methods for the clinician, such 

that the basic principles are understood in the context of 

clinical outcomes, the improved communication between the 

clinician and laboratory may facilitate improved immunologic 

understanding of the transplant patient. 

2. HLA typing 

www.elsevier.com/locate/trre 

HLA class I molecules (A, B, and Cw) are found on the 

surface of all nucleated cells in the body. HLA class II (DR, 

DP, and DQ) molecule expression is limited to B-cells, 

antigen-presenting cells, and activated microvascular 

endothelial cells [2,3]. A major initiator of the alloimmune 

response in solid organ transplantation is recognition of 

nonself HLA by recipient T-cells. In response, T-cell 

activation releases proinflammatory mediators with subsequent 

recruitment of the effector cells of the immune system 

[4-17]. The importance of nonself HLA recognition was 

known early in clinical transplantation; the moniker “Tissue

Typing Lab” was used for many years for the HLA 

laboratory because much of the early quantifiable immune 

risk assessment was limited to documenting the number of 

HLA mismatches between a given transplant pair. Currently, 

both serologic and molecular typing methods are routinely 

used in a most HLA laboratories. 

2.1. Serologic typing 

Serologic typing, as the name suggests, requires sera 

containing well-characterized antibodies to a wide range of 

HLA specificities. Although historically laboratories often 

kept large banks of sera from which to make typing reagents, 

it is far more common now to use commercially prepared 

trays which contain sera to all common, and many of the rare 

HLA alleles. Patient lymphocytes are mixed with the various 

sera in the tray wells and incubated with complement and a 

vital dye. If the cell has antigens on its surface to which 

antibody in a particular well are able to bind, then 

complement is activated in those well(s), the membrane 

attack complex forms and inserts into the cell membrane, cell 

death occurs, and the vital dye (often eosin) is taken up into 

the cell [18]. Cell death should therefore occur in any well in 

which the cell surface antigen and serum antibody are 

matched, and those wells are then identified under phasecontrast 

microscopy. Comparing and eliminating the serologic 

specificities of the positive wells assigns the HLA type. 

For example, if 2 wells with sera known to bind (a) A30 and 

(b) A30 + A31 are found to have significant cell death, a 

negative result in a well containing serum binding A31 + A33 

excludes A31 and therefore assigns the type as A30. 

Advantages of serologic typing include obtaining rapid 

results, which is of particular importance in deceased donor 

typing, and also the ability to discriminate “null” HLA 

alleles, which have detectable DNA sequences but no 

antigen expression on cell surfaces, and less immunologic 

relevance. However, finding high-quality sera with sufficient 

specificities to reflect the ever-increasing number of HLA 

alleles [19,20] is progressively prohibitive especially in light 

of increasing clinical interest in HLA-Cw, DP, and DP 

contributions to allograft outcomes. In addition, small 

differences in HLA molecules at the level of the amino 

acid sequence may not be identified using these methods 

despite potential immunologic consequences [21-26]. 

2.2. Molecular typing 

HLA molecules are proteins encoded by DNA regions on 

the short arm of chromosome 6. With their sequences well 

described, HLA typing is becoming increasingly performed 

using molecular methods including sequence specific primer 

PCR (SSP), sequence specific oligonucleotide probes 

(SSOP), and direct DNA sequencing. In SSP, DNA is 

isolated from the subject to be typed and amplified in 

multiple wells, each containing specific primers complementary 

to particular HLA alleles. Only if the DNA is 

complementary to the alleles in a given well will an 

K. Tinckam / Transplantation Reviews 23 (2009) 80–93 

amplification product be formed. The contents of the wells 

are then run electrophoretically through an agarose gel: if 

amplified product is present, then a band will be detected, 

and the HLA typing is assigned by the primers that were 

placed in that particular well. The composition of the primers 

in each well permits positive identification of the HLA 

characteristics. In SSOP, oligonucleotide probes that are 

complementary to the unique segments of the DNA of 

different alleles are mixed with amplified DNA. Unique 

fluorescent tags distinguish those probes that are complimentary 

to the DNA, such that the HLA allele is identified. 

Sequencing determines the exact order of nucleotides in the 

gene of interest, and the HLA type is assigned by comparison 

to published HLA allele sequences [20]. Regardless of the 

specific method, molecular HLA typing offers more precise 

documentation of HLA differences between donor and 

recipient, with many alleles discriminated at all loci of 

interest, and with resolution at the level of the amino acid. 

This may provide a greater understanding of the risk 

associated with different mismatched epitopes between 

donor and recipient [22-27]. 

2.3. HLA typing nomenclature and mismatches 

The documentation of HLA type varies depending on the 

detection method used and is important to review as a direct 

consequence of the different methods described above. 

Historically, because HLA antigens were discovered serologically, 

they were numbered in order of that discovery by 

gene locus, for example, A1, A2, etc, and B7, B8 etc. 

Refinement of serologic methods identified even more 

antigens, previously thought to represent single allotypes, 

which in fact were serologically and genetically unique. For 

example, the B40 antigen was ‘split’ into 2 components 

named B60 and B61, and both are considered part of the B40 

cross reactive group or CREG, which itself is part of the B7 

CREG. Public epitopes are those common to all the members 

of a CREG, whereas private epitopes delineate the individual 

serologically defined antigens. 

However, assignment of serological antigens did not 

represent the true heterogeneity of the HLA system. Early 

studies with mixed lymphocyte culture detected such 

heterogeneity in HLA antigens recognition that was not 

discerned by serology; for example, HLA A2 was found to 

consist of several subtypes stimulating different lymphocyte 

reactivity. DNA sequencing confirms that indeed, multiple 

alleles of each HLA antigen are known to exist despite the 

fact that they may react to a single common typing serum at 

the antigen level. A new molecular typing nomenclature was 

introduced in 1987, where the locus is followed by an 

asterisk, then the first 2 digits describe the type, and then the 

next 2 digits represent a unique allele differing by at least 1 

amino acid difference. 

Frequently, the serological and molecular nomenclature 

correspond (eg, HLA-A⁎0201 has the serological equivalent 

of HLA-A2), but incongruencies do occur. For the purposes 

of solid organ transplantation, it is important for the clinician 

81

82 K. Tinckam / Transplantation Reviews 23 (2009) 80–93 

to recognize which nomenclature system is used by their 

laboratory in the assignment of HLA type as well as in the 

assignment of antibody specificities. Failure to recognize that 

different nomenclature can be used may miss identifying 

donor-specific antibody (DSA). For example, if a donor is 

assigned a typing of B⁎1501 and the donor has an antibody 

defined to B62, the donor specificity of this antibody may not 

be immediately apparent because B62 is a serological 

equivalent of B⁎1501. Such concerns may be easily addressed 

through communication with the HLA laboratory, and in 

addition, several references are readily available [19,20,28]. 

When the typing of a donor and recipient is considered, 

the convention for describing their similarity should reflect 

the “immune burden” that a donor presents to a recipient. 

Originally, number of HLA matches described recipients and 

donors' likeness; however, it is more immunologically 

informative to use mismatches to describe their dissimilarity. 

For example, if a donor is A1,- B8,39 DR1,3 and the 

recipient is A1,24 B39,44 DR1,11, then this is a 0–A, 

2–B,1–DR mismatch or 3 HLA mismatch transplant. 

However, if the donor and recipient typings were reversed, 

then the recipient immune system would see 4 HLA antigens 

(1–A, 2–B,1–DR) as nonself. 

2.4. Using HLA typing in solid organ transplantation 

Before modern era immunosuppression, the impact of 

HLA mismatch on transplant was dramatic and clinically 

significant [29-32]. With current immunosuppressive regimens, 

it is recognized that a most first allografts will have a 

substantial number of HLA mismatches and yet have 

acceptable graft survival; however, large registries still 

show a statistically significant impact of HLA matching on 

deceased donor transplants [33-35]. As graft survival rates 

improve overall, debate exists as to whether organs should be 

allocated according to HLA matching or whether local 

allocation to reduce cold ischemia time should prevail. 

Results from the Collaborative Transplant Study indicate that 

shortening of cold ischemia time does not eliminate the 

effect of HLA matching and argue for consideration of HLA 

type in deceased donor allocation [34]. 

In the setting of re-grafts, repeat Class I and/or Class II 

antigens with a prior donor may have an independent 

deleterious effect on graft survival, underscoring the need for 

accurate donor typing such that risk assessment may be 

properly estimated [36-39]. Furthermore, even matching of 

HLA antigens within a given CREG group may be 

associated with better long term allograft survival [40,41]. 

The development of late antibody mediated outcomes may 

require a diagnosis of DSA, which necessitates knowledge of 

donor typing. 

In addition, for the third of waitlisted patients who have 

preformed antibody to HLA antigens (see HLA antibody 

screening below), accurate donor typing is paramount in the 

identification of lower-risk donors to whom the recipients do 

not have alloantibody, as in acceptable mismatch [21] or 

paired exchange programs [42-44]. Occasionally, patients 

may form antibody to only certain alleles at a given locus, for 

example, antibody to B⁎4402 but not B⁎4401. Molecular 

typing may be used to ensure only those donors with the 

allele of interest are potentially excluded rather than all B44 

donors [45-47]. 

Ongoing work examines whether incompatibilities at 

HLA-Cw, DP, DQ and MICA [48,49], MICB and KIR [50] 

influence graft outcome. Molecular technologies may be 

easily adapted for typing at these loci because the evidence 

surrounding their importance is emerging. 

3. HLA antibody screening 

Transient or persistent sensitization with antibodies to 

HLA, or non-HLA, antigens may occur with exposure 

during pregnancy, blood transfusion, or prior transplant. Up 

to one third of waitlisted patients may have some HLA 

antibodies detected when the most sensitive screening 

methods are used. Preformed antibodies decrease access to 

transplantation with higher rates of positive crossmatches 

and exclusion of donors with unacceptable antigens on the 

basis of antibody screening tests. Indeed, even if the 

crossmatch is negative, permitting transplant to proceed in 

the short term, low titers of antibody directed to donor HLA 

may be associated with high rates of early [51] and late [52] 

antibody-mediated outcomes. Therefore, both sensitive and 

specific analysis of HLA antibodies is necessary to identify 

the risks faced by sensitized recipients and also to explore 

novel approaches for successfully transplanting these 

patients, such as desensitization [53,54], acceptable mismatching, 

and paired exchange [21,42,43]. Repeated 

pretransplant antibody screening forms a most solid organ 

transplant work in most HLA laboratories. 

3.1. Cytotoxic (cell based) antibody screening 

Lymphocytes are gathered from “cell donors” (usually 

20–40 in number) with variable HLA types including all 

common and many rare alleles, and a panel of cells is 

formed. These cell donors are not organ donors per se but 

rather are meant to be representative the HLA antigen 

distribution in the same population from whom deceased 

donors may be (randomly) selected. In this way, the 

percentage of cell donors to whom a given recipient has 

antibodies detected approximates the percentage of potential 

organ donors drawn from that same population to whom the 

recipient would be expected to have a positive crossmatch. 

The test is identical to that of serologic typing except that it is 

now the recipient serum that is mixed with cell donor 

lymphocytes in individual wells along with complement and 

the vital dye. If the serum contains antibodies that bind to the 

cell surface with sufficient density, complement will be 

activated, the cells killed, and the vital dye with dead cells 

may be easily identified (Fig. 1A). If in a panel of 40 cells, 30 

of the reaction wells had significant cell death, the panel 

reactive antibody (PRA) would be reported as 75%.

An obvious limitation of this method is that the PRA 

percentage may numerically change (without a change in 

amount or type of antibody) depending on the cell panel that 

was used in the screening. Commercially made cell panels 

may not necessarily represent the HLA distribution of a 

given region where different racial proportions result in 

different HLA allele frequencies. Furthermore, substantial 

false-positive and false-negative results complicate this test; 

the former in the case of non-HLA antibodies and 

autoantibodies or nonspecific IgM, and the latter as a result 

of its low sensitivity. To activate complement, the antibody 

must be of sufficient density to link complement between Fc 

receptors, and in the setting of low-titer antibody, this may 

not occur and antibody may be missed [55]. Because of 

technical challenges, many centers used cytotoxic PRA 

screening on T-cells only; indeed, many more B-cell 


screening tests have false-positive results, which would 

limit the amount of reliable class II antibody data. Finally, 

detailed specificity analysis is difficult with multiple 

antigens per reaction well; so detailed lists of antibody 

specificities and unacceptable antigens are frequently not 

possible (Table 1). 

Cellular or cytotoxic PRA testing may therefore be best 

thought of as estimating the risk of a given recipient of having 

a positive cytotoxic crossmatch to a potential organ donor 

drawn from a comparable population as the cell panel donors. 

3.2. Solid phase antibody screening 

Clinical syndromes of antibody-mediated damage are 

reported in the absence of detectable antibody by cytotoxic 

screening methods and prompted the development of more 

Fig. 1. Schematic representation of cell-based and solid phase (bead-based) antibody screening. A, A panel of cells in individual wells is used: 2 representative 

wells are demonstrated. Serum is added to each well in the panel. On the left, the antibody in the serum does not bind to the cells, on the right, DSA are present. 

After washing, bound DSA remain and when complement is added, it is activated forming the membrane attack complex and killing the cell with the vital dyeis 

taken up. The wells are viewed on a microscope: no antibody (i) leaves live cells, and when DSA are present, the vital dye identifies the dead cells (ii). B, Beads 

coated with purified or recombinant HLA antigen are combined with serum. In this case, the antibody in the serum is only specific to the bead on the right. When 

fluorescent anti-IgG is added, only the bead with DSA already bound will bind the fluorescent marker. The fluorescence may be measured in comparison to a 

negative control (i) or may be multiplexed in a single reaction with all beads' fluorescence ranked to determine those beads that are positive (ii). 

83


Table 1 

Differences in antibody screening methods 

Antibody identified Cytotoxic antibody 

screening 

Solid phase antibody 

screening 

Class I HLA Ab Yes Yes 

Class II HLA Ab If B-cells are used Yes 

Non-HLA Ab Yes No 

IgM Ab Yes (requires DTT treatment 

of serum to exclude) 

No 

Low-titer Ab No Yes 

Specificities of Ab No Yes 

(single antigen beads) 

Noncomplement 

binding Ab 

No Yes 

Ab indicates antibody. 

sensitive assays. The need to precisely distinguish HLA 

antibody from non-HLA antibody as well as clearly 

discriminate class I and class II antibodies stimulated the 

development of currently available solid phase methodologies. 

These methods use only soluble or recombinant HLA 

molecules rather than lymphocyte targets, which present 

both HLA and non-HLA molecules. The purified molecules 

are adhered to solid phase media, either ELISA [56-58] 

platforms or microbeads [59-61], and then bind only HLA 

antibody in the added recipient serum. Enzyme conjugated 

(ELISA) or fluorescent dye conjugated (microbead) antibodies 

to human IgG are added to detect any HLA-bound 

IgG antibody in the serum. If antibodies were present and 

bound to HLA on the solid phase medium, the optical 

density (ELISA) or fluorescence detection (microbead) 

reflects this. Microbead may be run on a traditional flow 

cytometer (Flow PRA®) or may be multiplexed in a 

suspension array on the Luminex® platform allowing for 

high throughput detection of multiple analytes in a singlereaction 

chamber (Fig. 1B). Both of the microbead-based 

assays are up to 10% more sensitive for lower-titer antibody 

than the ELISA, which in turn is 10% more sensitive than 

antihuman globulin (AHG)-enhanced cytotoxicity–based 

assays for the detection of HLA antibody [55]. By virtue 

of controlling the antigens placed on the beads, these assays 

are specific for HLA antibody only. Furthermore, class I and 

class II HLA antibody may be easily distinguished by using 

class-specific beads, and by manufacturer design, isotype 

detection can be limited to IgG. Finally, precise specificities 

may be determined by using beads that each bind only 1 

unique HLA antigen (Table 1). 

Although the use of these antigen-specific and sensitive 

assays has addressed many of the problems associated with 

the cellular assays, they have their own limitations including 

detection of both noncomplement and complement binding 

antibody simultaneously, and detection of antibody below 

the level associated with a positive crossmatch. Therefore, 

detectable antibody may not always be associated with a 

meaningful clinical outcome, potentially limiting transplant 

with negligible net benefit. In addition, these assays must not 

be considered in isolation as the clinical relevance of non- 

HLA antibodies is explored. The number of HLA alleles 

identified continues to grow, and the full spectrum of unique 

HLA antigens cannot be practically represented on solid 

phase assays. Therefore, donor typing must always be 

reviewed to ensure that all donor alleles are indeed 

represented on the solid phase panel before the absence of 

DSA may be concluded. 

Fluorescence or optical density readouts are continuous 

variables, and considerable controversy exists as to what 

threshold values should be considered positive, resulting in 

sometimes substantial interlaboratory variability [62]. 

Approaches including standardized quantification of fluorescence 

intensity against molecules of equivalent soluble 

fluorochrome may allow for better congruency of antibody 

screening result with better crossmatch prediction [63] and a 

closer association to clinical outcomes [64]. However, 

although ongoing efforts are made to improve standardization, 

it remains important for clinical programs to know what 

thresholds their own laboratory is using and how such 

cutoffs correspond to crossmatch results. 

3.3. Clinical application of antibody screening assays 

Regardless of method, PRA alone has been repeatedly 

associated with poor transplant outcomes [65,66]. Debate 

has existed as to what threshold of PRA percentage should 

be considered “high risk,” but now that specificities may be 

more precisely defined, it is clear that it is the specificity and 

not the percent PRA per se, that defines the clinical risk. 

With PRA output demonstrable as a continuous variable, the 

dichotomous approach of high versus low risk is clearly not 

biologically reflective. A PRA of 5% may confer significant 

risk if the antibody it represents binds to donor antigens. 

Even low-titer antibody may become high-titer antibody if 

stimulated by the appropriate antigen from a donor organ and 

may explain why pretransplant low-titer antibody to donor is 

indeed associated with subsequent posttransplant adverse 

outcomes [67]. Conversely, by defining specificities precisely, 

high PRA patients can now expect comparable longterm 

outcomes as nonsensitized patients provided unsuitable 

donors are avoided [21,68], thereby challenging the concerns 

that organs transplanted into sensitized individuals do not 

achieve their maximum potential survival. Therefore, the 

detection of any HLA antibody must initiate the interrogation 

of specificities, for it is those specificities in conjunction 

with donor typing, rather than a particular PRA percent that 

confers the risk assessment. 

The PRA percentage does remain relevant, but it may 

instead be seen as an estimate of the fraction of potential 

donors that may confer posttransplant humoral immunologic 

risk, and therefore, it represents the ”risk” of donor specificity 

occurring but not necessarily the risk of an immunologic 

event. Calculated PRA is a more standardized approach to 

determining the likelihood that a recipient will have DSA by 

comparing the antibody specificities (determined on solid 

phase assay locally) to the defined frequencies of HLA alleles 

in the population of interest nationally.

Occasionally, because of the high sensitivity of the solid 

phase assays, antibody to donor may still be detected even 

with a negative crossmatch. Data on the significance of these 

findings are conflicting; some demonstrate no impact on 

function [69], whereas more are reporting an impact on 

short-term [51,70] and long-term [52] outcomes. However, 

an equally significant application of solid phase assays has 

been in the determination of which positive crossmatches are 

in fact immunologically relevant (see Section 4.3 below). 

Regardless of assay type, all antibody-screening tests are 

limited by their surrogate status for current or potential 

memory responses. The laboratory's ability to predict a 

future immunologic event is based only upon the serum 

available after patient identification and referral. It is 

immediately seen that a recipient's measurable immune 

history before the time of transplant referral may be 

substantial, but with current laboratory techniques, not 

demonstrable where serum is unavailable. Antibodies may 

wane over time such that routine pretransplant testing reveals 

no antibody, and yet shortly after a repeat stimulus with a 

transplant, a memory response may still occur. As such, 

negative antibody screening alone will never be 100% 

predictive of the absence of humoral alloimmune memory 

potential, and the clinical history of sensitizing events 

remains a critical consideration in the risk assessment of the 

patient without detectable antibodies. 

4. Crossmatching 

The 1969 landmark article by Patel and Terasaki [1] 

unequivocally demonstrated that DSA in the serum of 

recipients at the time of transplant was a major risk factor for 

hyperacute rejection and primary nonfunction. This finding 

was a major driver in the founding of HLA laboratories and 

in the establishment of the T-cell cytotoxic crossmatch as the 

requisite immunologic test before transplant [71] with a 

dramatic subsequent reduction in hyperacute rejection. A 

negative test justified proceeding, but a positive test was 

considered a contraindication. The original study [1] had a 

4% false-negative rate (8/195 negative results had early 

failure) and a 20% false-positive rate (6/30) demonstrating it 

was neither specific nor sensitive enough to define all 

relevant antibodies. In general, the crossmatch identifies 

Table 2 

Differences between crossmatch methods 

whether a recipient has antibodies to a single donor of 

interest, as compared to the PRA, which identifies antibodies 

to a pool of potential donors. Over time, assays have been 

developed to address these limitations [72-84] (Table 2), and 

the improved sensitivity has lead to a critical examination of 

which antibodies identified by more sophisticated techniques 

are predictive of significant clinical outcomes. The solid 

phase antibody screening data must be used in conjunction 

with crossmatch results to help classify them as immunologically 

irrelevant or relevant (high risk of rejection or graft 

loss, or transplant contraindicated) [46] (Table 3). 

4.1. Complement-Dependent Cytotoxicity (CDC) 

crossmatch methods 

The crossmatch is conceptually a simple reaction between 

T lymphocytes and donor serum in the presence of 

complement and a vital dye. The T-cell expresses class I 

HLA as well as non-HLA antigens and therefore acts as an in 

vitro “surrogate” allograft, with the actual allograft expected 

to express the same cell surface proteins on its endothelium. 

As for cytotoxic antibody screening, the result is considered 

positive if a significant proportion of the T lymphocytes 

are killed after the addition of complement, inferring that 

substantial DSA had been bound to the cell surface (Fig. 2A). 

However, similar concerns of low-titer but relevant antibody 

going undetected lead to improvements with this technique of 

increasing sensitivity, including longer incubation times, 

additional wash steps [72], and most commonly, the AHGenhanced 

method [74,76,77,79]. Antihuman globulin, a 

complement fixing antibody to human immunoglobulins, is 

added as a second step and binds any DSA already on the 

lymphocyte (both complement binding as well as noncomplement 

binding DSA), thereby increasing the antibody 

density and improving sensitivity (Fig. 2B). Moreover, the 

lower-titer antibodies detected by this method were associated 

with 36% 1 year allograft loss compared with 18% loss 

in those with a negative test [78]. All these methods may also 

be applied to B-cells, which may identify classes I and II as 

well as non-HLA DSA. 

4.2. Flow Cytometry Crossmatch methods 

First described in 1983 [81], flow cytometry crossmatch 

(FCXM) detects DSA regardless of the ability for 

CDC AHG-CDC Flow 

cytometry 

Cytotoxicity with 

flow cytometry 

HLA Ab detected Yes Yes Yes Yes Yes 

Non-HLA Ab detected Yes Yes Yes Yes No 

IgM detected Yes Yes No No No 

Low titer or noncomplement binding Ab detected No Yes but less sensitive 

than flow cytometry 

Yes Yes Yes 

Ab titer detected Moderate to high Low to moderate Low Low Low 

Ab indicates antibody. 


85 

Solid 

phase


Fig. 2. Schematic representation of the various types of crossmatches. A, In the CDC crossmatch, when DSA is bound to the cell in sufficient density, complement 

is activated, the cell is killed and the vital dye is taken up identifying the dead cells. B, With the AHG modification, antibody may be lower titer and less dense on 

the cell surface, but the additional AHG still allows complement to be activated with subsequent cell death as with CDC alone. C, In FCXM, DSA binds the cell but 

instead of complement, a second fluorescent antibody to human IgG is added. When run through a flow cytometer, the DSA (which may be complement or 

noncomplement binding) may be detected at very low titers, measured as fluorescence on the cells relative to a negative control. D, If complement is added to a 

FCXM with a fluorescent vital dye taken up by complement-killed cells, then the degree of complement activating antibody binding (upper right on the scatterplot) 

may be distinguished from antibody binding without cell death (lower right). E, Lymphocytes are lysed, and the solubilized donor HLA antigen is bound to the 

solid phase. An enzyme-linked secondary antibody that results in a color change detected by a luminometer detects DSA. Only HLA antibodies are detected. 

complement fixation. Recipient serum is incubated with 

donor lymphocytes, which are then stained with a 

fluorochrome-conjugated anti-IgG antibody that remains 

bound only if DSA is initially bound to the cell surface. 

Additional antibodies with different fluorochromes that 

are specific to unique B and T lymphocyte surface 

proteins can be added such that when run through a flow 

cytometer, the B- and T-cells may be easily distinguished 

and individually interrogated for the fluorochrome identifying 

any DSA (Fig. 2C). The output of the flow 

crossmatch is at least semiquantitative (eg, number of 

channel shifts of mean fluorescence above the baseline or 

standardized against molecules of equivalent soluble 

fluorescence beads), although quantification measures 

and thresholds for positivity can vary between individual 

laboratories. Nonetheless, it is less subjective than visual 

assessment of cell death and more biologically representative 

of the continuous nature of antibody amount and 

consequent risk than the dichotomous positive/negative 

result of cytotoxic crossmatches. 

Once again, as for antibody screening, there is considerable 

interlaboratory variability in methods routinely used for 

crossmatching and indeed in concordance of results [85], 

necessitating ongoing efforts to develop approaches to 

improve reproducibility. 

4.3. Crossmatch interpretation 

T lymphocytes express class I HLA and non-HLA 

antigens, and B lymphocytes express both class I and class 

II as well as non-HLA antigens. Therefore, in general, a 

positive crossmatch due to HLA class I antibody will be 

positive on T- and B-cells and due to HLA class II antibody 

will be T-cell negative and B-cell positive. However, 

antibody to irrelevant non-HLA targets may confound 

these results and must be considered to clarify whether a 

crossmatch result is accurately identifying risk (Table 3).

Table 3 

Immunologic relevance of positive crossmatches 

Positive 

crossmatch 

Cause Detected by Immunologically 

relevant? 

T- and B-cell IgG class I HLA 

antibody 

B-cell Low-titer class I 

HLA antibody 

B-cell IgG class II 

HLA antibody 

T and B-cell 

or B-cell 

IgG class I and 

class II HLA 

antibody 

Solid phase testing 

positive for class I 

antibody 


positive for class I 

antibody 


positive for class II 

antibody 


positive for both 

class I and class II 

antibody 

T and/ Autoantibody Autocrossmatch No 

or B-cell 

positive 

T and/ IgG non-HLA Solid phase testing No 

or B-cell antibody negative 

T and/ IgM non-HLA Negative after DTT No 

or B-cell antibody treatment of serum 

T and/ IgM class I or Negative after DTT Unknown—general 

or B-cell class II HLA treatment of serum practice tends to 

antibody 

disregard as 

evidence is poor 

T- and B-cell Thymoglobulin/ 

Alemtuzumab 

Clinical history No 

B-cell Rituximab Clinical history No 

4.3.1. Non-HLA/Autoantibodies 

Historically, a positive crossmatch was considered a 

contraindication to transplantation on the assumption that 

HLA antibody was causative. Of course, cytotoxic antibodies 

to non-HLA antigens on both T- and B-cells may render one 

or both crossmatches positive, as will autoreactive IgM or 

IgG, none of which are immunologically significant [86-92]. 

The autocrossmatch is performed by mixing recipient serum 

with recipient own cells by the same method as the 

allocrossmatch. If the autocrossmatch is positive, the 

allocrossmatch against the donor is invalid without further 

testing; if negative, the allocrossmatch is interpretable. Other 

non-HLA antibodies may be immunologically significant 

[49,93-97] but would not otherwise be detected in lymphocyte 

crossmatches and will not be discussed further. 

4.3.2. Allo-IgM 

It is the development of solid phase assays for antibody 

screening which has now permitted classification of some 

positive crossmatches as immunologically irrelevant when 

not due to IgG HLA antibody [56,57,60,98]. This is 

particularly important in the interpretation of B-cell crossmatches, 

which have a high false-positive rate from non- 

HLA antibody corresponding to immunologically irrelevant 

positive results [99]. Alloreactive IgM antibodies are not 

routinely detected by the manufacturer methods for solid 

phase assays and indeed are of low incidence with apparently 

no impact detected in studies of cytotoxic crossmatch 

outcomes [100-103]. Treating serum with dithiothriotol 


Yes 

Yes 

Yes 

Yes 

(DTT) or heat inactivation will break up the pentameric 

IgM molecule, often reducing the crossmatch to negative; a 

crossmatch that is negative with DTT or heat treatment 

should be considered negative in terms of immunologic risk 

assessment. There are no definitive studies evaluating IgM 

antibody significance in flow crossmatch or solid phase 

testing, but general practice is to discount their presence. 

4.3.3. Low-titer antibody detected only by FCXM 

The practice of FCXM has resulted in the discovery of 

low-titer and/or noncomplement binding antibodies not 

detected by cytotoxic methods. Although not yet adopted 

by all transplant centers, use is increasing with recognition 

that those antibodies detected do predict risk for posttransplant 

rejection and graft loss, not otherwise identified by the 

less-sensitive negative cytotoxic crossmatch. It is generally 

accepted that up to 15% of primary transplants and 30% of 

second transplants may have positive FCXM with negative 

CDC/AHG-CDC crossmatches and that such patients had 

higher rates of early graft loss (b3 months), rejection, and 

worse 1-year allograft survival for both primary [104-109] 

and second transplant [104,105,107,110]. In FCXM in 

particular, it is important to confirm HLA antibodies on a 

solid phase assay [67,98,109,111]; in the absence of HLA 

antibody, a positive FCXM has no impact on graft survival 

[98]. Conversely, a negative flow crossmatch in the 

sensitized patient confirms the absence of low titer DSA 

and more importantly predicts the same graft survival as an 

nonsensitized recipient [68], further underscoring the 

importance of donor specificity rather than PRA as the 

main determinant of posttransplant risk. 

4.3.4. B-cell crossmatches 

B-cell cytotoxic crossmatching became common in the 

1980s to ascertain the presence of class II antibody; however, 

early studies of isolated positive B-cell crossmatches demonstrate 

little impact on outcome [87,112-116]. Later studies 

refute this [117-119], and solid phase testing explains this with 

greater than 75% of isolated B-cell crossmatches being due 

to non-HLA or autoantibodies [99], and in those cases, having 

comparably good outcomes to negative crossmatch recipients 

[109]. Autocrossmatching to exclude positive B-cell 

crossmatch from autoantibody, with solid phase confirming 

class II antibody presence or absence, is imperative. 

A positive B-cell crossmatch with negative T-cell crossmatch 

may also be due to low titer class I antibody because 

class I antigen may be expressed with increased density on 

B-cells compared with T-cells [120]. This may be confirmed 

with solid phase antibody testing and has comparable clinical 

impact to isolated positive FCXM due to HLA antibody 

[117-119,121,122]. 

4.3.5. Historic crossmatches 

The historical serum stored in the HLA laboratory may be 

viewed as a window into immunologic history and memory 

of the patient. Indeed, given the dynamic nature of 

circulating antibodies, patients may be transplanted with a 

87


negative crossmatch using current serum but with a historic 

serum demonstrating a positive crossmatch. Such patients 

have higher rates of early graft loss and diminished graft 

survival [109,123,124] of comparable magnitude to isolated 

positive FCXM recipients, and although not an absolute 

contraindication to transplant per se, the results clearly 

identify increased posttransplant risk. 

4.4. New crossmatch methods 

4.4.1. Cytotoxic antibody identification flow cytometry 

Simultaneous measurement of cytotoxicity (by various 

cell death markers) over a denominator of total (complement 

and noncomplement binding) antibody by flow cytometry 

[125-127] has greater sensitivity than standard CDC assays, 

but their role in refining immunologic risk assessment has 

yet to be demonstrated (Fig. 2D). One cardiac transplant 

study of complement fixation by antibody on solid phase 

beads showed an incremental increase in allograft loss over 

noncomplement fixing antibody [128] (Table 3). 

4.4.2. Solid phase crossmatch 

In this variant, donor lymphocytes are lysed and their 

classes I and II HLA antigens captured on solid phase 

platforms, which may then be used to perform sensitive 

“HLA antibody only” crossmatches specific to a particular 

donor [129,130,131] (Fig. 2E). They have the advantage of 

eliminating monoclonal antibody interference [132] and 

isolating the impact of HLA antibody without further solid 

phase testing, but non-HLA antibody of potential importance 

will not be detected, and detection of noncomplement binding 

HLA antibody may yield results with less clinical significance 

[133]. The overall validity of these relative to 

traditional crossmatching is still to be fully interrogated [134]. 

4.5. Virtual crossmatching 

The virtual crossmatch (VXM), despite its name, is not a 

true crossmatch but rather an application of solid phase 

antibody screening, where the antibody specificities from 

solid phase testing are compared to the donor typing as a 

prediction of the actual crossmatch. If consistently negative, 

this may permit avoiding the preliminary crossmatching, 

reducing cold ischemia in nonsensitized recipients, but if 

HLA antibody are detected, the virtual crossmatching is not 

100% predictive of positive or negative results, and therefore 

in 2009, the prospective crossmatch must still be performed 

to ensure patient safety [68]. 

The VXM may be false positive in the case of very lowtiter/noncomplement 

binding antibody or where the crossmatch 

is less sensitive than the antibody detection method, 

and this may unnecessarily exclude donors. Similarly, 

patients may demonstrate allele-specific antibodies (eg, 

antibody to DRB1⁎0401 but not other DRB1⁎04 alleles) 

[46], which may unnecessarily exclude other DR4 donors. In 

addition, DNA typing may identify null alleles that are not 

expressed as antigens on the cell surface but would be 

excluded by VXM based on typing alone. Alternatively, the 

VXM may be falsely negative, as the ever-expanding list of 

all potential HLA antigens in the population cannot be 

represented on solid phase in its entirety [19,28]. Care must 

be taken to ensure that the donor alleles are completely 

represented on the solid phase panel to report a negative 

VXM. Correlation between VXM and actual crossmatch is 

highly variable depending on the methods used, and the 

operating range must be clarified within each transplant 

center laboratory, until better standardization is achieved. 

5. Immunologic assessment of T-cell 

Pre- and posttransplant HLA laboratory testing has 

traditionally focused on detecting markers of humoral 

pathways; T-cell alloreactivity is as important in the 

alloimmune response, yet its testing has been less developed 

in the clinical laboratory. Mixed lymphocyte culture and 

assays of T-cell precursor frequency are labor intensive and 

do not lend themselves to high-throughput and repeated 

measurements in a waitlist population. Although not yet 

widely used in histocompatibility laboratories, some newer 

assays are currently under evaluation for their clinical 

relevance with preliminary encouraging results and may 

have potential to supplement the current humoral immunologic 

assessments commonly available for transplant patients. 

5.1. ELISPOT 

The ELISPOT assay is analogous to the cytotoxic 

crossmatch, which measures donor-specific B-cell reactivity 

by quantifying antibody, in that it measures donor-specific Tcell 

reactivity (effector and memory cells) by quantifying 

IFN-γ and other cytokine production. Recipient T-cells are 

mixed with either inactivated donor cells (for direct 

presentation of antigen) or peptides of donor HLA (for 

indirect presentation of antigen) in a reaction chamber that is 

coated with antibodies to capture the cytokine of interest. As 

the T-cells recognize the donor HLA epitopes or peptides, 

they produce cytokines that are captured on the plate and 

with the addition of a detecting dye, a “spot” appears for 

every reacting T-cell. Automated programs then count the 

spots to quantify the amount of T-cell alloreactivity. 

Early studies have shown that patients experiencing 

rejection have higher rates of donor-specific IFN-g producing 

cells than those without rejection [135] and indeed may 

predict posttransplant rejection [136-138]. Analogous to 

PRA, the test may be expressed as the percent of panel 

T-cells that induce positive ELISPOT (“PRT” assay). Up to 

30% of patients with high PRT may be discordant with high 

PRA, effectively demonstrating the potential divergence 

between T- and B-cell alloreactivity and the potential 

importance of measuring both pathways [139]. The ELI- 

SPOT may be customized to measure multiple different 

cytokines allowing for interrogation of different T-cell 

activation pathways (eg, Th0 vs Th1). However, the method 

requires long incubation times over several days and is

estricted by moderate reproducibility and lack of commercial 

availability, currently limiting its widespread use. 

5.2. Measurement of nonspecific ATP production by T-cells 

(Cylex Immuknow) 

Phytohemagglutinin may be used to stimulate recipient 

T-cells, and the amount of adenosine triphosphate produced 

is measured in a luminometer. The assay is therefore antigen 

and donor nonspecific and is instead a measure of the general 

state of activation of the T-cells. It too requires overnight 

incubation but is commercially available. A recently 

published meta-analysis of 504 organ transplant recipients 

showed that those patients with low levels of responsiveness 

(ie, overimmunosuppressed) were 12 times more likely to 

develop an infection, in comparison to those with a high 

response score (under immunosuppressed) who were 30 

times more likely to develop cellular rejection [140]. 

Prospective clinical relevance requires confirmation before 

widespread adoption into practice. 

5.3. Soluble CD30 measurement 

CD30 plays a role in alloimmune responses [141], and 

activated T-cells release a soluble form reflecting T-cell 

activation state, which can be measured using ELISA. Also 

donor nonspecific, it has nonetheless been shown to be a 

predictor of graft loss independent of sensitization and with 

an additive effect to those who were sensitized, underscoring 

the importance of considering T- and B-cell immunity 

together [142,143]. 

6. Posttransplant testing 

All of the above testing methodologies are routinely and 

systematically applied pretransplant; however, a major 


immune activating event is the transplant itself with 

subsequent alloimmune responses that can be measured 

posttransplant. Recently, there has been increased interest in 

the posttransplant measurement of alloantibody in particular 

with strong associations between the presence of posttransplant 

antibodies and acute and chronic pathology and graft 

loss in heart [144,145], lung [146-148], and kidney 

transplantation [149-153]. However, a major limitation to 

all these studies thus far is that measurements of antibody 

have not been systematic at serial time points such that the 

temporal relationship between markers and outcomes of 

interest may be known to guide timely interventions. 

Ongoing studies are required to fully investigate these 

relationships and guide posttransplant testing protocols 

analogous to those currently practiced pretransplant. 

7. Conclusions 

In summary, HLA laboratory testing in 2008 results in 

accurate donor and recipient typing, sensitive and specific 

screening for HLA and non-HLA antibodies, and precise 

crossmatching methodologies to more closely describe 

humoral immunologic risk. Donor-specific antibodies to 

HLA and non-HLA may be semiquantitatively ranked by 

strength such that risk may be more accurately viewed as a 

biological continuum rather than dichotomous (Fig. 3). 

Higher titer antibodies may confer immediate risk such that 

aggressive therapies or acceptable mismatch strategies are 

required to permit safe transplant. Lower-titer antibodies 

may identify patients who require altered immunosuppression 

or closer follow-up. Solid phase testing determines the 

relevance of cell-based assays in clinical practice. Risk 

continues to evolve posttransplant, and the utilization of 

HLA testing in this period must be systematically evaluated. 

Fig. 3. The immunologic risk continuum. Using crossmatch tests of differing sensitivity and confirming relevance of the results with solid phase testing allow for 

immunologic risk to be viewed as more biologically continuous. Antibody increases toward the right, with positive tests of decreasing sensitivity, and with 

corresponding increased risk. Antibody decreases to the left with increasingly sensitive negative tests and therefore lower immunologic risk. However, immune 

memory potential still cannot be fully measured at this time, and consequently, no patient can be truly classified as risk-free. Similarly, even the highest risk 

patients may have some opportunity for transplant with modified immunosuppression and monitoring. 

89


Each method outlined in the categories above has inherent 

strengths and limitations, and as such, no one test is intended 

to function in isolation as the single predictor of transplant 

immunologic risk. Human leukocyte antigen typing identifies 

potentially appropriate donors for highly sensitized 

patients, who in turn must have precise antibody specificities 

documented to make the best utilization of modern typing 

methods. Antibody screening for HLA antibody alone may 

miss clinically relevant non-HLA antibodies; therefore, 

cellular-based assays continue to have a role, as do novel 

solid phase methods. Crossmatch results do identify DSAs, 

but their interpretation is most accurate and predictive of 

clinical outcomes when the results of solid phase antibody 

testing is considered concurrently. A complete estimate of 

risk must collectively consider donor and recipient HLA 

typing, all methods of antibody detection, and new methods 

of T-cell alloreactivity to assess risk more accurately on a 

biological continuum. 

The HLA laboratory has evolved from a “crossmatching 

laboratory” to one that provides sophisticated risk assessment 

consultation for the clinician. Newer methods addressing 

measures of T-cell alloimmunity and B-cell memory 

must be fully investigated and considered in the context of 

clinically relevant outcomes. At the intersection of clinical 

patient data with immune testing opportunities, and with 

expertise in quality assurance and reproducibility, and 

complex genetic and protein platforms, the HLA laboratory 

remains the ideal setting for the ongoing translation of 

evolving immunologic assays into clinical practice. 

The author reports no personal or financial relationship 

with any company or entity that might bias her work or is 

related to the content of this paper. 

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