**5.3. Positive B-cell CDC crossmatch**

B-cell CDC crossmatching is not as predictive of HAR as the T-cell CDC crossmatch and there has been much controversy about its role [28]. Many centres do not perform B-cell cross‐ matching for cadaveric renal transplantation because of uncertainty about the significance of a positive result. The major limitation is a rate of false positive results of up to 50% [29]. In some cases this reactivity may be due to non-HLA molecules on the surface of the B lympho‐ cytes, including the presence of Fc receptors. While a negative result is reassuring a positive result may mean a transplant is cancelled when it was safe to proceed. Another argument against the routine use of B-cell crossmatching is that antibodies to class II antigens are of less significance in generating antibody-mediated rejection. But recently it has been found that they are not so benign [30].

B-cell crossmatches are often performed as part of the immunologic assessment before live donor transplantation when there is more time to determine the significance of the result. Paired with information about the presence of DSA, determined by more specific means such as antigen-coated beads (Luminex, discussed below) the B-cell CDC cross‐ match results may be more meaningful [31]. If a B-cell crossmatch is positive and there are no detectable antibodies to class I or II antigens, the result may be falsely positive while a positive result in the presence of detectable DSA signifies that the identified DSA may be functionally relevant in that it can activate complement and were associat‐ ed with increased risk of rejection [32]. This has led to the suggestion that the B-cell CDC crossmatch should not be used alone to determine transplant suitability and that it be interpreted only in the light of accompanying Luminex results [31].

overall opinion on the likelihood of immune complications. The area remains controversial

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Virtual crossmatching refers to the comparison of the anti- HLA antibodies of the recipi‐ ent, derived from Luminex, with the HLA of the donor [40]. If there is a DSA present this would represent a positive virtual crossmatch. Antibodies are defined against HLA class I and II antigens. Synthetic microspheres (beads) coated with HLA antigens are commercially available for this testing. Beads may be coated with multiple HLA antigens for screening purposes or a single HLA antigen for defining specificity of antibodies more precisely. For the virtual crossmatch, multiple beads each coated with a single HLA antigen are mixed with recipient serum. Anti-HLA antibodies present bind to the beads and are detected by an isotype-specific (e.g. IgG) detection antibody via flow cy‐ tometry. Unique fluorochromes within the beads mark the HLA antigen specificity of each bead. This technique is as sensitive as flow crossmatching and provides the specific‐

It has long been established that the presence of antibodies that react with human leucocytes portend worse long-term graft survival [42]. This information has been further refined by more sensitive antibody detection systems, particularly Luminex. It has been shown that recipients with DSA have worse graft survival than those with third party anti-HLA Abs (antibodies against HLA antigens that are not donor-specific) who in turn have reduced graft survival compared with recipients without any anti-HLA antibodies [43]. Therefore, the presence of a DSA suggests inferior graft survival compared with no DSA even in the presence of a negative

Luminex testing offers significant advantages over CDC and flow crossmatch in terms of defining the HLA specificity of identified antibodies. The presence of a DSA detected by Luminex in the setting of a negative or positive CDC crossmatch appears to have prognostic importance in terms of graft survival and acute rejection risk; however, there are insufficient data to determine the significance of a DSA with a negative flow crossmatch [40, 44-46].

In each assay, negative control beads provide a minimum threshold for a positive result. Positive results can then be graded as weak, moderate or strong on the basis of the de‐ gree of fluorescence of the positive bead. This result can be scored as a median fluores‐ cence index (MFI) or molecules of equivalent soluble fluorescence. The molecules of equivalent soluble fluorescence of a DSA have been shown to correlate with antibody ti‐

While Luminex testing has added significantly to the understanding of crossmatching, the methodology has some significant limitations that can make interpretation difficult. Limita‐ tions include possible interference by IgM antibodies, variable antigen density on beads, conformational changes to antibodies in the process of binding to the beads, and gaps in the

and no clear recommendation can be made at this time.

**5.5. Virtual crossmatching**

ity of the antibody [41].

CDC crossmatch [44].

tre and predict graft failure [47].

HLA antibody repertoire in bead sets. [45, 48, 49].

### **5.4. The flow crossmatching technique**

A flow crossmatch involves adding recipient's serum to donor lymphocytes and then incu‐ bating them with fluorescein-labelled antibodies against human IgG (antihuman IgG fluores‐ cein isothicyanate [FITC]). This fluorescein-labelled antibody will bind to all the IgG antibodies in the recipient serum. If a DSA in this serum then binds to the donor lymphocytes, it will be detectable by flow cytometry.

Flow crossmatching is performed using the same initial base ingredients as CDC crossmatch‐ ing (i.e. donor lymphocytes and recipient serum) and was first described in 1983 [33]. The two are mixed to allow antibody binding and after washing, fluoresceinated AHG is added to bind attached DSA and hence allow detection by flow cytometry. The read-out may be reported simply as positive or negative or can be further quantitated. Intensity of fluorescence above control, referred to as channel shifts, may be reported while another means of quantitation is to determine the number of dilutions of recipient serum required to generate a negative result.

The subtype of antibody can also be determined by the isotype specificity of the fluorescently labelled detection antibody. Hence if only IgG antibodies are of interest the detection antibody chosen will be of the type that binds only to IgG and not IgM or IgA [34]. Furthermore the subtype of IgG can be elucidated by choosing a detection antibody that binds only to IgG1, 2, 3 or 4. Refining the analysis in this way provides information about the likelihood of comple‐ ment activation *in vivo* as IgG4 does not activate complement.

The role of flow crossmatching in the pre-transplant assessment is controversial. The signifi‐ cance of a positive result is mainly of interest when the CDC crossmatch is negative. In this setting the positive flow crossmatch is likely to be caused by a non-complement fixing antibody, a non-HLA antibody or a low-level antibody that is below the threshold of sensitivity of the CDC methodology. In patients who are not known to be sensitized several studies have suggested that a positive T- or B-cell flow crossmatch was not predictive of increased rejection rates or worse graft survival while in sensitized patients other studies have suggested inferior graft survival [30, 34-39]. A possible reason for this difference is that there would be a higher false positive rate in non-sensitized patients than in sensitized patients given that they are not expected to have a positive result. Another factor determining the significance of the result is the cut-off values used to determine a positive test [34]. These are not applied uniformly between centres and those that apply a very low cut-off value will increase sensitivity at the expense of specificity.

Some transplant clinicians do not use flow crossmatching as part of their pre-transplant assessment and rely on CDC crossmatching along with defining DSA by Luminex, otherwise known as 'virtual crossmatching'. Others contend that flow crossmatching adds important information on the strength of donor-specific antibody reactivity and should be considered in the context of donor-specific antibody results and CDC crossmatching to help develop an overall opinion on the likelihood of immune complications. The area remains controversial and no clear recommendation can be made at this time.
