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"The Art of War" against cancer

The military has a cloud: knowing ourselves and knowing each other.

The battle between mankind and cancer has already begun. In this war without smoke, mankind has advanced very hard, but has never retreated and has been moving forward. Under the continuous efforts of experts from all walks of life both at home and abroad, people began to unveil the mystery of cancer from one layer to another, from understanding the mechanism of disease, preventing in advance, to accurate diagnosis and effective treatment, and constantly innovating and optimizing theory, technology and methods. And as a sword to help this battle win early.

In order to win this battle between mankind and cancer, it is very important to know ourselves and know each other. Before firing, you must know where the enemy is and not to injure civilians. Immunohistochemistry (IHC) is a scout that combines fluorescent or colorimetric chemicals on antibodies, using immunological principles to specifically detect binding between antigens and antibodies. Whether there is a target antigen in the tissue, if the target antigen is on cancer cells, this way can be used to understand where the enemy cancer cells are.

Immunohistochemical staining has the advantages of high specificity, high sensitivity, simplicity, and low cost. Therefore, it is widely used in hospitals and research institutes, and it is usually screened for cancer by specific tumor markers. With the development of the times, the manual dyeing commonly used in the initial stage of immunohistochemical staining is prone to poor stability, uneven dyeing, edge effects and non-specific background. At present, immunohistochemistry technology is entering a new stage of development - automated immunohistochemical staining technology.

Automated immunohistochemical staining began in the 1880s and has been around for 20 years. The first automated immunohistochemical staining machine placed reagents on a rotating disk and used a computer-controlled microfluidic transport system for staining. In recent years, automated immunohistochemical staining technology has developed rapidly. After several updates, automatic, immunohistochemical and in situ hybridization staining have been integrated into the same platform. The interface is simple and can be directly connected to the laboratory information system. , greatly optimized the workflow of the pathology department.

At present, automated immunohistochemical staining systems are mainly based on one of the following four dyeing techniques.

OpenIndividual Slide Staining

This technique arranges the slices in parallel, using a liquid dispenser to dispense reagents to different areas of the slice. Depending on the location and size of the tissue, one or more zones can be selected to add reagents, the closest staining technique to manual staining.


Figure 1. Open, personalized slice staining technique with reagents automatically added to the top of the tissue via a liquid dispenser. This principle has been applied to many staining systems.
           

Liquid Overlay Technology

This technique acts on the entire slice by the flow of liquid, thereby enabling the reagent to cover/deposit to cover the fluid. This technique allows for mixing of the reagents on the sections while temperature control to avoid evaporation of the reagents, but must be washed away before the slides are dyed and sealed.


Figure 2. Liquid overlay technology, reagent covering or depositing to cover fluid
Tubular Gap Staining

This technique holds the reagent in two planar units by capillary force, either as two sections with tissue, or as slices and covers. A narrow, fixed space is required between the two planes to maintain capillary forces on the entire section.


Figure 3. Tubular gap dyeing technique, where a very small space between two planes creates capillary forces, allowing reagents to be stored between two planes
           

Dynamic Gap Staining

The latest dynamic gap dyeing technique uses the principle of capillary force, which moves forward-up-down-backward in a 16-second cycle during incubation and cleaning. This ensures that the reagents are evenly distributed across the entire surface of the slice, effectively mixing the reagents, and ensuring uniform reaction conditions in the incubation zone of the reagents and effective and controlled cleaning conditions in the wash step. The application of the dyed lid, combined with the high moisture retention of the dyeing chamber, avoids the evaporation of the reagents, so the dyeing can ensure the uniformity and reproducibility of the dyeing results under temperature control conditions.


Figure 4. Dynamic gap dyeing technique with the dyed lid moving forward-upward-downward-backward in a 16-second cycle
The staining process of most automated immunohistochemical staining systems is continuous. Once the slices are uploaded, all staining processes are processed according to the procedure, from uploading the slices to the end of the staining, and the entire step is done in the same staining module.

In recent years, with the continuous optimization of automated immunohistochemical staining machines, dyeing machines with the advantages of continuous uploading and parallel processing have received increasing attention. In the different modules of the instrument, different dyeing procedures can be processed simultaneously, some modules are used for dewaxing and repair of the tissue, and some modules are used for tissue dyeing. The advantages of this parallel processing principle are:

Each module can greatly optimize and process specific staining steps

Highly flexible for program adjustments for new upload slices

Save time by processing multiple steps simultaneously

Continuous uploading enables priority handling of emergency case sheets and reduces the time required for the first batch of slices, while continuously optimizing dyeing conditions and increasing workflow flexibility. The dyeing time of a dyeing machine with a continuous upload function relies on the "idle" time of the instrument, so new slices and new reagent uploads do not affect the slicing process being processed.


           

Ruijin Hospital introduced the automated immunohistochemical staining system - immunohistochemistry and in situ hybridization system Omnis, launched by Agilent Dako in 2015, six months ago. Omnis uses state-of-the-art dyeing technology, Dynamic Gap Staining, which greatly reduces the uneven staining and edge effects of stained sections in the department. The dyeing system is a continuous upload and parallel processing mode with high sample throughput. In addition, the reagent storage unit of this system has a temperature control system that can be maintained at 18 ° C, which can avoid the influence of changes in ambient temperature on reagent titer during long-term dyeing and overnight dyeing, and the temperature can be achieved throughout the dyeing process (32 °C) and humidity (70%-80%) are controllable, thus ensuring the stability of dyeing conditions and providing objective guarantee for quality control. The Omnis system has 60 temperature-controlled reagents, which can hold up to 53 primary antibodies at the same time. These multi-species reagents basically guarantee the primary resistance items that the department needs daily. At the same time, all the reagents and waste liquids used in the Omnis instrument have safety and prompt functions, and each liquid can have multiple reagent barrels. The liquid can be added, replaced and cleaned while the machine is running. The liquid exchange design is highly user-friendly and daily operation. Simple.

At present, the series of antibodies in the breast cancer and lymphoma packages of Ruijin Hospital are stained on Omnis. The following are the results of staining in two cases [1].

1. Male, 17 years old, with enlarged left inguinal lymph nodes. Pathological diagnosis: (left inguinal lymph node) T lymphocytic lymphoma


                 
2. Female, 68 years old, found a right breast mass for half a month, right breast puncture specimen. Pathological diagnosis is considered invasive ductal carcinoma.


                   
As of June 2017, the Agilent Dako Omnis system has been operating normally for six months at Ruijin Hospital. Some teachers commented: "The system can perform immunohistochemical staining and fast FISH detection at the same time. The two systems work in parallel, which is very convenient for the flexible selection of the department. At the same time, the system can realize continuous uploading and parallel processing, and can complete the first two and a half hours. Batch dyeing, a new batch of dyeing can be completed every 50 minutes, and can be processed overnight, which greatly improves the efficiency of our department, and obtains the ideal dyeing results and doctors' praise."

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