Automated Immunostaining Platform in Cytology.

BACKGROUND: In the modern era, immunocytochemistry (ICC) is an essential part of investigations. There is now an increasing workload of ICC in laboratories. Automated immunostaining platform may help the laboratories to deal with such a rising burden of ICC.
SUMMARY: In this brief review article, we have described the various aspects of automated ICC, particularly the benefits, limitations, types of the system, working manner, and comparison of different commercially available systems. Copyright:

INTRODUCTION

There is now an increasing need for categorization of the tumors, finding the primary origin of malignancy and also various other data that clinicians expect from the cytology preparations for the personalized management of the individual cases. Therefore there is an increasing demand for immunocytochemistry (ICC). The discovery of heat-induced epitope retrieval led to the utility of a large number of primary antibodies.[12] ICC is performed on cell block sections of fine-needle aspiration cytology (FNAC) as well as on variously prepared cytology preparations. With the increasing demand of ICC in the cytology, the automated immunostaining platform (AIP) is now rapidly replacing the manual ICC.[3]

In this brief review article, we have described the various aspects of automated ICC, particularly the benefits, limitations, types of the system, working manner, and comparison of different commercially available system.

ADVANTAGES OF AUTOMATED IMMUNOCYTOCHEMISTRY

The AIP has the following advantages:

Consistency: The technique gives consistent results irrespective of the personnel who are handling the machine. It is crucial when the laboratory deals with a large number of samples.

Skill: AIP does not require experts to perform ICC as the machine handles all the technical steps involved in the technique.

Time: Automation saves the technologist's efforts as well as time as he can perform other tests while the ICC is automatically run.

Error: The chances of human error are decreased as compared to manual ICC.

Immunocytochemistry (ICC) and immunohistochemistry (IHC)

ICC and IHC can be performed simultaneously in the same batch. It is essential in those laboratories where both cytology and histology samples are processed together for ICC.

Reagents supervision

The AIP has the self-monitoring programmed facility. The system keeps a regular sensor-based check on the level of bulk reagents, waste disposal, staining capacity of the kit installed, the expiry date of the barcoded kits, incubation temperatures as well as the count of the number of slides put for staining.

LIMITATIONS OF AUTOMATED ICC

Despite so many advantages, the automated ICC may have many limitations.

An unknown technique to the newer technologists

When a laboratory introduces AIP, the technicians may feel difficulty in the initial period to handle the more modern technology.

Skill to handle problems in running the test

In the case of the non-functioning of the machine, the technicians may not be able to handle the manual ICC.

Cost

AIP is an expensive technique as the instrument cost and running cost are relatively high due to the patented consumables.

Maintenance

The AIP requires regular maintenance, service, and decontamination schedule. These steps are mandatory to maintain the quality of results.

Repair

Every machine has a specific period under which it functions perfectly well; however, after that, it needs more frequent repairing and maintenance due to the routine wear and tear of the machine. The repair time may affect the timely dispatch of ICC results.

Skill development

The technical skill of the technicians fails to develop as the enclosed machine is running all the steps. Most of the work in the tertiary care centers becomes machine dependent which may result in insufficient skill development in contrast to the more intellect-based manual staining standardization protocols.

OPERATING MODES OF THE AUTOMATED IMMUNOSTAINING PLATFORM

The AIP operates in two different systems: open system and closed system.

Open system

The antigen retrieval buffers, incubation timings of primary as well as the secondary antibody, treatment with wash buffers, etc., can be manipulated in case of open operating modes of IHC staining.[2] This system allows flexibility according to the requirements and availability of the reagents. This system is more useful in research-related work. The open system permits customized staining methods that can be conveniently standardized by the experienced technologists.

Closed system

All the steps involved in closed operating modes, that is, deparaffinization, use of wash buffers, antigen retrieval etc., are performed by a single unit.[2] Antigen retrieval systems etc., are not disclosed by the manufacturers as they are available under their commercially available patented consumables. The closed systems more or less mimic the standard staining protocols. The manufacturers provide ready-to-use primary antibodies. However, there is a flexibility to use third-party primary antibodies in some of the systems. The incubation timings and dilutions can also be varied accordingly. Such kind of systems is more successful in case of clinical settings as these give reproducible and consistent results irrespective of the technical personnel posted in ICC staining laboratory.

Slide loading

There are two types of slide loading in automated ICC:

Batch loading: In this system, the slides for ICC are loaded in a batch. The ICC program is set for the whole batch, and no slides can be loaded in between the running of the program.

Continuous loading: In this type of machine, the cases are loaded in the system for ICC. In between the program, one can run other cases for different sets of ICC. It is an advanced system as the technical staff need not wait for the completion of the first program.

Table 1 shows the comparison of the open and closed systems.

Table 1

The comparison of the open and closed systems

Characteristics	Open system	Closed system
Reagents	Flexibility of reagent usage	Patented consumables mandatory
Protocols	Can be customized	Standardized protocols
Expenses	Relatively less expensive	Highly expensive
Skill	Involves a lot of skill	Does not involve much skill
Results	Consistency varies according to the skill of the technical personnel	Gives consistent results Irrespective of personnel involved
Application	Research based	Clinical laboratory

Commercially available systems

Presently in the market, different commercially available automated ICC are available. Table 2 provides a detailed comparison of the various systems.[4567891011]

Table 2

Comparison of different automated immunostaining machine

Features	BOND RX[4]	AIHS 660[5]	Benchmark XT/ULTRA[6]	Intellipath FLX[7]	Autostainer Link 48[8]	IHC Stainers360/720[9]	NANOMtrx 300[10]	Wave RPD[11]
Company	Leica	AMOS SCIENTIFIC	Roche	Biocare medical	Agilent	Thermo Scientific	Biogenex laboratories	Celerus diagnostics
Operating mode	Open	Closed	Closed	Open	Closed	Open	Closed	Closed
Slide baking	Yes	No	Yes	No	No	No	Yes	Yes
Dewaxing capability	Yes	Yes	Yes	No	Separate module	Separate module	Yes	Yes
Counterstaining	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Dehydration and clearing	No	No	No	No	No	No	Yes	No
Coverslipping	No	No	No	No	No	No	No	No
Slide capacity	30	48	30	50	48	36/84	30	16
Reagent Dispensing System	Covertile	Solvent Membrane	Liquid Coverslip vortex mix	Open slide	Open slide	Open slide	Open slide	Dynamic capillary mix
Additional tests or special features	ISH, FISH, IF, Multipex IHC	Can also be performed on frozen sections, minimum dispensing of 50 microlitres	ISH, FITC	Simultaneous Multiplex IHC Capacity, continuous random access/5 simultaneous and independent batches can be run	Connects up to three instruments and three PT links to one computer	Multiple sizes available to accommodate the needs of any size laboratory	ISH and special stains, industry smallest footprint, gentle wash and blow dry steps	Rapid IHC in less than 1 h, a read/writable digital memory chip track usage of consumables./plug and play reagents
Reagent Dispenser	Rinsed probe tip	Reagent dispensing Teflon coated nozzle	Disposable dispensers	Rinsed probe tip	Rinsed probe tip	Rinsed probe tip	Rinsed probe tip	Disposable cartridge
Configuration	Floor	Benchtop	Floor	Benchtop	Benchtop	Benchtop	Benchtop	Benchtop
Robotic movement
	Matrix/array	Matrix/array	Rotary	Matrix/array	Matrix/array	Matrix/array	Matrix/array	Matrix/array
Waste segregation/hazardous/non-hazardous	Yes	No	No	Yes	Yes	Yes	Yes	Minimal excess reagent is collected in the self-contained disposable wicking cassette

ISH=In situ hybridization, FIS=Fluorescence in situ hybridization, FITC=Fluorescein isothiocyanate, IF=Immunofluorescence

ANTIGEN RETRIEVAL

Antigen retrieval is an essential part of ICC. Many automated ICC systems have the facility to have antigen retrieval by applying heat or by enzymatic treatment. Presently most of the commercially available system has the facility of antigen retrieval. Some systems heat the slides [Table 2] and perform the antigen retrieval. The automated ICC systems also use the enzymatic retrieval system, and only the proprietary enzymatic/buffer product can be used in such a system. The company uses strict conditions, and the reproducibility of the antigen retrieval technique is high in such a machine. The additional cost of the proprietary reagent is balanced with the quality of the antigen retrieval and the shorter duration of the procedure.

REAGENTS DELIVERY SYSTEMS

There are two mechanisms to deliver the reagents to the slides.

Rotary system

Here, the slides and the reagent delivery systems are horizontal in position, and the reagent delivery system is just above the circular disc holding the slides. The reagent delivery platter rotates over the slide platter and directly delivers the reagents on the respective slides.

Matrix system

Here, the slides are arranged in rows and columns. The robotic arm moves and delivers the reagents to the respective slide.

The comparison of the two systems is mentioned in Table 3.

Table 3

The comparison of the matrix and rotary system of reagent delivery

Features	Matrix/array system	Rotary system
Position of the slide and delivery system	Rows and columns	Horizontal and parallel to each other
Chance of contamination	More	Very less chance
Speed	Relatively, slow. However, it may be faster by using more than one robotic arms	Relatively fast
Capacity	Large capacity of slides accommodate	Relatively less number of slides

DELIVERY OF THE REAGENTS

The delivery of the reagents to the slides are done by following different methods:

Capillary action method

Here, the reagents are delivered by the capillary method. The slides are kept in a vertical position, and the gap between the slide and its cover forms a capillary action that sucks the reagents.

Disadvantages: If the distance between the slide and the cover is not optimal, then the capillary force may not be generated, and adequate reagents may not be distributed.

Direct dispensing

In this system, the slides are placed in a horizontal position, and the reagents are put directly over the slides. There are mainly two ways:

Rotary type: Here, the particular volume of the reagents are put on the slides either by print cartridge like or by syringe-like action.

Probe type: Here, the predetermined volume of the reagents are delivered to the slide by probe type dispenser.

Disadvantages: There is a remote chance of contamination of the reagents in the probe-type dispenser.

REAGENT COST DUE TO PROPRIETARY AGENT

In certain closed systems, one has to use only the selected reagents and pre-diluted antibodies made by the companies. It has the following advantages 1) easy to standardize, 2) time saving, and 3) less technical skill requirement. However, the proprietary reagents are costly as they have to be purchased only from the respective companies. It may increase the total expenditure unnecessarily high.

IMMUNOCYTOCHEMISTRY (ICC) VERSUS IMMUNOHISTOCHEMISTRY (IHC) IN THE AUTOMATED IMMUNOSTAINING PLATFORM

The differences in the specific phases of the ICC procedure in comparison with IHC concerning the AIP are evaluated below.

Pre-analytic phase

A wide variety of sample collection and fixation techniques are used in cytology preparations.[12] It has been noted that the quality of the staining does not differ significantly in those various preparations.[13]

Cytology preparation

Cell block section[1213]

Cytospin preparation[13]

SurePath preparation[14]

Thin Prep preparation[15]

Direct smear.

Fixation

The following fixatives are used for ICC and can be used in AIP

The 10% neutral buffered formalin for cell block section[1213]

95% alcohol

Cytology material fixed in “ThinPrep media and postfixed in formalin”.[12]

Cytology material fixed in “SurePath media”[12]

Air-dried slide[12]

Destained hematoxylin and eosin-stained or Papanicolaou's stained slide.[16]

Analytic phase

Control of ICC

It is mandatory to use the same type of positive/negative control in ICC of the cytology specimen. It means that in the case of ICC of cytology smear, one should use the smear as a control slide, and formalin-fixed paraffin-embedded tissue section should be avoided for the control of cytology smear. In many cases, the cytology preparation may have internal normal positive or negative control.[17] The validation means the validating the protocol and result of a test. Overall the IHC requires at least ten positive and negative control cases for validation as recommended by the College of American Pathologists (CAP).[17] There is no such suggestion for ICC; however, the same number of positive and negative control cases are suggested by us for the validation of ICC. Alternatively, the laboratory director should be responsible for deciding the number of positive or negative cases for a particular test to validate as suggested by CAP.[17]

Antigen retrieval

Many cytological preparations are not fixed in buffered formalin so it may appear that the epitope retrieval may not be needed. However, it is preferable to have an antigen retrieval protocol in both the smear and other cytology preparations to get the optimum result.[18] Majority of the people used the heat-induced AR technique to get the optimal result. Usually, 20–40 min heating in the buffered solution of the cytology slides provides the optimum result. In automated immunostaining protocol, one should have the proper AR protocol for cytology preparation.[1819] The AIPs usually use the already prepared commercial antigen retrieval solution, and the user should obey the company manual. Overall the flexibility of AR is minimal in the automated platform. Any change of the reagents and protocol for the cytology preparations needs strict validation, as suggested by the CAP.[17]

Antibody dilution and incubation

In the case of closed system, the primary antibody is already diluted for ICC, and there is no flexibility to change the dilution. However, in the open system the dilution of the primary antibody can be altered to standardize a single protocol in the laboratory.[20] It has been noted that the automated platforms usually need more concentrated antibody than the manual system.[20] The requirement of the higher concentration of the primary antibody may be due to overall less incubation time in the automated platform than that of manual system.

Incubation time is a vital step in automated ICC. The overall incubation time of the frozen section is noted lesser than FFPE tissue section.[21] The cytology smear or section should be properly standardized in each laboratory.

Detection kit and counterstaining: The antigen-antibody detection kit and also counterstaining are often different in ICC.

Post analytic phase

Reagent trapping[22]

During the examination of the ICC of cytology preparation, one should be careful about the interpretation of the three-dimensional clusters of cells as the reagents may be trapped in the groups of cells and may give false-positive results. Careful monolayer preparation of the cytology sample may avoid the reagent trapping.

Cell fragility and background staining[22]

The cellular cytoplasmic content of the antigen may come out from the fragile cells and may produce dirty background staining in case of cytology smears and other cytology preparations.[22]

Table 4 highlights the comparison between ICC and IHC.

Table 4

The comparison of immunocytochemistry and immunohistochemistry

Features	Formalin fixed paraffin embedded section (FFPE) and cell block section	Cytology preparations
Programming/protocol to be run	For FFPE section	For Non-paraffin preparations/smear
Bar coding	Specific according to the protocol	Specific according to the protocol
Controls for standardizations	Preferably FFPE section	Preferably smears
Antigen retrieval time	Standard machine protocol	Antigen retrieval gives better result
Antibody incubation time	More time needed	Less in comparison to IHC/CB
Antibody concentration	More concentration needed	Less in comparison to IHC/CB
Cellular variability	Remains same in all subsequent tissue sections	May not show same amount of cellularity in case of cytosine and LBC preparations

IHC=Immunohistochemistry. CB=Cell block. LBC=Liquid-based cytology

FACTORS INFLUENCING THE CHOICE OF THE AUTOMATED ICC SYSTEM

The workload of ICC: High throughput machines are more useful in case of laboratories where the workload of ICC is high. The laboratory should choose the system that can deal with a large number of slides in less time.

Application of third-party antibodies: The machines which allow the compatibility with third-party primary antibodies are preferred over the devices which are compatible with only in-house primary antibodies. The flexibility to purchase the reagents or primary antibody from different vendors may reduce the running cost of ICC significantly.

User friendly: The running of the system should be user friendly so that the cytologists can run the machine smoothly. The system can be stopped whenever necessary at the time of running ICC so that one can replace the reagents or modify the incubation period etc.

Facilities for the other tests: The facility of performing other tests like in situ hybridization and reagent chilling chambers for heat-labile tests is a limiting factor in the choice of purchase of AIP.

Counterstaining: It is preferable to have automated counterstaining facility after ICC.

Back up service: Frequency of response to the complaint by the expert service staff available in a particular region also plays a vital role while selecting the AIP.

The budget of the purchaser: The cost of the AIP is also one of the most critical factors while purchasing the AIP. Here, one should also take care of the maintenance cost, and reagents cost.

Space and infrastructure: the installation space and infrastructure available to support a particular AIP.

Clinical or research-based setting: The research work involved in immunostaining inclines the purchaser to buy an open system as compared to the closed operating system, which remains the first choice for diagnostic purposes.

Through-put rate per hour: The throughput rate per hour is an important area. Most of the instrument takes approximately 6 h to complete the entire batch. In a continuous system of slide loading, there is an additional facility of loading the slide in between the operation procedure. It helps to perform the urgent immunostaining between the staining procedure.

Laboratory information system[ LIS is an integral part of most of the AIPs. The barcoding and radiofrequency identification help to locate every slide with full details. The data of AIP is readily transferable to LIS. Now many large laboratories have introduced the whole slide imaging (WSI). The whole slide images of the stained slides may be included in the LIS for better interpretation.

LOCAL EXPERIENCE

We are using AIP in our laboratory for the last five years. In each year, we did near about 4000 different immunostaining on cell block sections. We used 80 different antibodies in this automated ICC platform. The common antibodies were CK7, TTF1, CK20, p63, WT1, PAX8, LCA (CD45), p53, PD-L1, ALK (D5F3), ROS, CD3, and CD20. There are several substantial impacts after the installation of the automated ICC platform. These include: 1) The remarkable improvement of the quality of the immunostaining, 2) the consistently good quality of the staining, 3) remarkable shortening of the turn over time, 4) the reduction of the skilled workforce time and 5) increased cost of the immunostaining. We guess that there are little advantages of automated ICC platform in a small laboratory with less than 20 immunostaining per day. In addition, the open system with the use of free choice of antibodies may reduce the cost of the immunostaining. In large laboratories, the LIS should be connected with the automated ICC platform.

CONCLUSION

AIP is almost an inevitable part of a standard laboratory. There is no such best system in this area. Every laboratory may have its priorities, and according to their need, they should choose the system. The cost and quality are the main concerns of AIP. The strict quality control is mandatory in AIP. Due to the growing need for laboratory automation, it is expected that the automated ICC system is connected with the laboratory information system. If possible, the stained slides should be digitized and analyzed.

Statement of ethics

This is a review article and there is no ethical issue related to the work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.