A novel automated IHC staining system for quality control application in ALK immunohistochemistry testing

Abstract

The establishment of positive and negative controls in immunohistochemistry (IHC) screening for anaplastic lymphoma kinase (ALK) rearrangements is essential in the treatment of lung adenocarcinoma. However, positive control of patient tissue is rare and comes with ethical issues. A novel automated solution for ALK IHC quality control management was investigated by comparison with the established D5F3 antibody on the VENTANA system in 87 lung adenocarcinoma specimens with known ALK status re-analyzed by fluorescence in situ hybridization. The BP6165 concentrated antibody on the LYNX480 PLUS platform demonstrated excellent sensitivity and specificity (98.30% and 100%, respectively) in 87 biopsy specimens. The ALK controls in liquid form (CLFs) applied in an automated way showed a more regular circular shape and better cell distribution than those applied manually. In addition, the novel controls can show changes in the same pattern as tissue controls under different antibody concentrations and antigen retrieval conditions. The automated solution for ALK IHC quality control management provides a convenient solution without the consumption of scarce tissue for IHC testing in day-to-day pathology practice. The availability of standardized protocols for the detection of ALK rearrangements using the BP6165 concentrated antibody on the LYNX480 PLUS platform will expand the number of laboratories that can reliably and consistently determine the eligibility of patients with lung adenocarcinoma for treatment with ALK tyrosine kinase inhibitors.

Keywords: anaplastic lymphoma kinase gene ALK; automated; controls in liquid form (CLFs); lung adenocarcinoma; quality control; rabbit monoclonal antibody.

FIGURE 1

Optimized protocols for ALK IHC assays using the BP6165 concentrated antibody on the LYNX480 PLUS platform.

FIGURE 2

ALK staining patterns on lung adenocarcinoma samples using the BP6165 concentrated antibody on the LYNX480 PLUS platform (A, C) compared with the ALK (D5F3) CDx on the VENTANA BenchMark XT platform (gold standard) (B, D).

FIGURE 3

(A1–A5) ALK positive controls in liquid form manually pipetted (original magnification: ×4). (B1–B5) ALK negative controls in liquid form manually pipetted (original magnification: ×4).

FIGURE 4

(A1–A5) ALK positive controls in liquid form automatically added on the LYNX480 PLUS platform (original magnification: ×4). (B1–B5) ALK negative controls in liquid form automatically added on the LYNX480 PLUS platform (original magnification: ×4).

FIGURE 5

The controls in liquid form setting schemes on the LYNX480 PLUS platform. (A) Setting one positive and one negative control in liquid form away from the label end of the slide; (B) Setting one positive and one negative control in liquid form near the label end of the slide.

FIGURE 6

(A1–A3) Lung adenocarcinoma samples stained under different antigen retrieval conditions (original magnification: ×20). (A1) Antigen retrieval time of 0 min shows a negative staining result. (A2) Antigen retrieval time of 20 min shows a weak staining result. (A3) Antigen retrieval time of 60 min shows an optimal staining result. (B1–B3) ALK positive controls in liquid form stained under different antigen retrieval conditions (original magnification: ×20). (B1) Antigen retrieval time of 0 min shows a negative staining result. (B2) Antigen retrieval time of 20 min shows a weak staining result. (B3) Antigen retrieval time of 60 min shows optimal staining result. (C1–C3) ALK negative controls in liquid form stained under different antigen retrieval conditions (original magnification: ×20). Antigen retrieval times of 0 min (C1), 20 min (C2) and 60 min (C3) all show standard negative staining results.

FIGURE 7

IHC staining results of lung adenocarcinoma samples, ALK positive and negative cell controls by applying different antibody concentrations. (A1–A4) Lung adenocarcinoma samples stained with BP6165 at different concentrations (original magnification: ×20). (A1) The highest concentration with an antibody dilution rate of 1:25 shows nonspecific staining on lymphocytes; (A2) antibody dilution rate of 1:200 shows an optimal staining pattern; (A3) antibody dilution rate of 1:1,600 shows weaker staining than the optimal staining; (A4) dilution rate of 1:6400 shows even weaker staining. (B1–B4) ALK positive controls in liquid form stained with BP6165 at different concentrations (original magnification: ×20). (B1, B2) Both antibody dilution rates of 1:25 and 1:200 show a standard staining pattern; (B3) antibody dilution rate of 1:1,600 shows a lower positive rate, away from the standard staining pattern; (B4) antibody dilution rate of 1:6400 shows even weaker staining. (C1–C4) ALK negative controls in liquid form stained with BP6165 at different concentrations (original magnification: ×20). (C1) antibody dilution rate of 1:25 shows “background staining;” (C2–C4) dilution rates of 1:200, 1:1600, and 1:6400 show standard negative results.