CD30 Lateral Flow and Enzyme-Linked Immunosorbent Assays for Detection of BIA-ALCL: A Pilot Study

Abstract

Introduction: Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL) commonly presents as a peri-implant effusion (seroma). CD30 (TNFRSF8) is a consistent marker of tumor cells but also can be expressed by activated lymphocytes in benign seromas. Diagnosis of BIA-ALCL currently includes cytology and detection of CD30 by immunohistochemistry or flow cytometry, but these studies require specialized equipment and pathologists' interpretation. We hypothesized that a CD30 lateral flow assay (LFA) could provide a less costly rapid test for soluble CD30 that eventually could be used by non-specialized personnel for point-of-care diagnosis of BIA-ALCL.

Methods: We performed LFA for CD30 and enzyme-linked immunosorbent assay (ELISA) for 15 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. To determine the dynamic range of CD30 detection by LFA, we added recombinant CD30 protein to universal buffer at seven different concentrations ranging from 125 pg/mL to 10,000 pg/mL. We then performed LFA for CD30 on cryopreserved seromas of 10 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas.

Results: Recombinant CD30 protein added to universal buffer produced a distinct test line at concentrations higher than 1000 pg/mL and faint test lines at 250-500 pg/mL. LFA produced a positive test line for all BIA-ALCL seromas undiluted and for 8 of 10 malignant seromas at 1:10 dilution, whereas 3 of 10 benign seromas were positive undiluted but all were negative at 1:10 dilution. Undiluted CD30 LFA had a sensitivity of 100.00%, specificity of 70.00%, positive predictive value of 76.92%, and negative predictive value of 100.00% for BIA-ALCL. When specimens were diluted 1:10, sensitivity was reduced to 80.00% but specificity and positive predictive values increased to 100.00%, while negative predictive value was reduced to 88.33%. When measured by ELISA, CD30 was below 1200 pg/mL in each of six benign seromas, whereas seven BIA-ALCL seromas contained CD30 levels > 2300 pg/mL, in all but one case calculated from dilutions of 1:10 or 1:50.

Conclusions: BIA-ALCL seromas can be distinguished from benign seromas by CD30 ELISA and LFA, but LFA requires less time (<20 min) and can be performed without special equipment by non-specialized personnel, suggesting future point-of-care testing for BIA-ALCL may be feasible.

Keywords: CD30; ELISA; diagnosis; lateral flow assay; lymphoma; screening.

Figure 1

Cytospin cytology of fresh seromas from two patients with pathologically diagnosed BIA-ALCL. Giemsa stain of seroma 61 shows monotonous population of anaplastic cells which are CD30+. Apoptotic bodies of dead tumor cells are seen (arrows). Seroma 62 contains a CD30+ large multinucleate tumor cell and a CD30+ large mononuclear tumor cell surrounded by small CD30 negative lymphocytes and many erythrocytes (600×).

Figure 2

LFA of recombinant CD30 protein at different concentrations produces varied intensity test lines corresponding to different intensities on the color scale. CD30 could be detected at concentrations as low as 250 pg/mL.

Figure 3

LFA of clinical specimens produces positive test line in 3 of 10 undiluted benign seromas (48,52,53) eliminated at 1:10 dilution, while all but 2 (M6, M7) of 10 malignant seromas remain positive at 1:10 dilution.

Figure 4

Representative ELISAs of benign and malignant seromas. (A) Straight line values using manufacturer’s standards. (B) Table of numerical values determined for patient samples. The optical density of sample 1617 is apparently lower than what the lowest standard captured. (C) Bar graph showing CD30 levels below 1200 pg/mL in six undiluted benign seromas, while undiluted BIA-ALCL seroma 1817L contained 2369 pg/mL CD30 and six other BIA-ALCL seromas require dilutions of 1:10 or 1:50 to not exceed 2300 pg/mL. Standard error bars are shown. 1912L and 1912R refer to left and right seromas of the same patient.