Median channel shift less than the cutoff in flow cytometric crossmatch: Not to be ignored!

Sir,

The presence of donor-specific antibodies in the patient's serum is well known to be a factor in the outcome of a renal transplant recipient. There may be multiple causes of the formation of the antibodies including previous transplant, pregnancy, transfusion, or even infections.[1] Flow cytometric crossmatch (FCXM) is a sensitive, quantitative, and objective, yet cost-effective method for detection of donor-specific antibodies.[2] Although it lacks the specificity that would be offered by a Luminex single antigen bead (SAB) assay, it is a useful screening method. In a country like India where Luminex SAB assay is not yet a standard of care due to its cost implications and the majority of the transplants are with living donors, it is extremely important to identify the clues provided by FCXM results.

The standard FCXM is performed by separating the donor lymphocytes and incubating them with patient serum, along with negative and positive controls. This is followed by incubation with fluorescent-labeled anti-human immunoglobulin (Ig), which is specific for Fc part of IgG and antibodies to identify B- and T-cells.[3] The analysis includes calculating the median channel shift (MCS) of median fluorescence intensity (MFI) of the test serum vis-à-vis negative control. A cutoff is set-up by each laboratory individually which in our laboratory is set by testing number of normal sera and the cutoff point is equal to 3 standard deviation (SD).[4] The normal sera were obtained from AB-positive, nonsensitized males, which were also tested on Luminex to confirm the lack of any human leukocyte antigen (HLA) antibody. Using these sera, the cutoff was calculated to be 50 for T-cells and 80 for B-cells, using the 3SD rule. A test is only reported when positive control works satisfactorily for that particular sample. All quality control measures are taken in keeping with the accreditation guidelines vide ISO 15189:2012 as this test is accredited by the National Accreditation Board for Laboratories. Some laboratories also tend to use the 10% cutoff by considering the fluorescence index (FI), which is the percentage shift in the test serum as compared to the positive control ([test serum MFI − negative control MFI]/[positive control MFI − negative control MFI] × 100).[5]

Most of the negative FCXMs results that we get have a shift less than or equal to the negative control while the positive cases are well beyond the cutoff. We use the commercial controls as negative sera, every new lot of which is verified by testing them with Luminex SAB assay. Commercial positive controls are run as quality control measure with every test to prove the validity of the test and also meet the requirement by accreditation authorities. We rarely find cases where there is a shift, which is less than that of the cutoff. These cases are usually the ones that lie in the gray zone and an immunologist may be tempted to call them negative. We hereby report two renal transplant cases which had a shift less than the cutoff for FCXM, and which on further evaluation revealed weak donor-specific HLA antibodies (DSA) as detected by Luminex SAB assay. Both these cases were worked up in the mid of 2015.

The first case was a 49-year-old female for whom daughter was the donor and the FCXM revealed T-cell MCS of 29 and B-cell MCS of 36 as compared to the negative control [Figure 1]. The complement-dependent cytotoxic (CDC) crossmatch was negative while there was panel reactive antibody (PRA) of 7% for class I. On further evaluation using Luminex SAB assay, DSA was identified against HLA-B*08, which was donor-specific. However, its MFI was only 1422 (cutoff 1000). Thus, the MCS for T- and B-cells, although less than the cutoff, was significant. The low titer of antibody also explains the negativity of CDC. This antibody could have been missed if the FCXM had been reported as negative. The Clinical and Laboratory Standards Institute guidelines also state that isolated T-cell positivity could be due to either non-HLA antibodies or weak Class I antibodies, which was the scenario in this case.

Figure 1.

Flow cytometric crossmatch graph showing T-cell median channel shift of 29 and B-cell median channel shift of 36.

The second case was that of a 43-year-old patient for whom sister was the donor. The CDC crossmatch was negative while the FCXM showed an MCS of 42 beyond the negative control which was less than the cutoff for B-cells [Figure 2]. The FI was only 1.8%. The FlowPRA was also negative. On performing Luminex SAB assay, DSA was identified against HLA-DRB1*14, which was again donor-specific, and had the MFI of 1519.

Figure 2.

Flow cytometric crossmatch graph showing a median channel shift of 42 for B-cells.

Both these patients underwent plasma exchange and were transplanted when their DSA MFI was <1000 and FCXM was negative. In both the cases, the posttransplant period was uneventful.

These two cases clearly show the significance of reporting/alerting the nephrologist of even a slight MCS in FCXM, which may be less than that of the set cutoff for the laboratory. Such cases warrant a further analysis using SAB assays. Most cases that are negative would have the MFI less than that of the negative control. There have been studies that have analyzed and reported the increased chances of acute humoral rejection in FCXM-negative and DSA-positive cases.[6] The negativity in the FCXM cases could have been due to the shift being less than that of the set cutoff. We are also planning to change the cutoff level to 2SD, instead of the current 3SD level, as the same would help us identify more such cases with weak HLA antibodies.

We would like to state that any shift beyond the negative control (regardless of the cutoff) in an FCXM should warrant a further evaluation, preferably using a Luminex SAB assay.

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Conflicts of interest

There are no conflicts of interest.