How Histotechnology Plays a Role in Lung Transplant Technology Advancement

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Lung transplantation has always been a challenge in the medical field as there is always issue in shortages of suitable donor lung for patients who are waiting for years with their deteriorating conditions with lung diseases. Even with patients who are fortunate to find the right donor lung, often there are complication with primary graft dysfunction and long-term chronic lung allograft dysfunction (CLAD).

Ranked as the 2nd top hospital in the World, The Toronto General Hospital (TGH) at University Health Network (UHN) is a leading institute in lung transplant technology and research. In 2008, the Ex-vivo Lung Perfusion (EVLP) system was invented at TGH to advance the technology and improve the chance of success in lung transplant. In recent years, TGH has been conducting extensive research in expending the scope of application on the EVLP system. There are also extensive researches conduct at TGH investigating the post-transplant allograft rejection on fibrosis and immune cells responses to the grafted lung tissue. In this blog article, we will look at how Histotechnology play a role on evaluation and validating findings on all these studies related to lung transplant technology research.

What is EVLP?

EVLP is a perfusion machine in which donor lungs are sustained outside the body, mimicking the conditions of the living body. The system allows functional assessment (eg., lung oxygenation capacity, ventilatory mechanics, degree of lung edema) of marginal quality donor lungs, which would normally not be considered for transplantation. EVLP can also potentially lead to improvement of lungs before transplantation, including removal of blood clots, high dose antimicrobial therapy against donor pathogens, and gene editing.

Benefits of EVLP

Expanding donor pool: allow the use of lungs that would normally be discarded due to marginal quality
Improve lung function: edema or inflammation
Reduce risk of complication: transplant rejection
More time for lung function assessment
Logistical benefits: donors from distance, better resource allocation

How does Histotechnology play a role in EVLP system improvements?

H&E staining

Lung tissue biopsy samples from the donor lung are stained for H&E to evaluate the structural integrity and injury level during and after perfusion. This assessment will determine whether the marginal donor lungs are suitable for transplantation. Main focus of the H&E evaluation is looking for any indication of alveolar damage, edema and inflammation.

TUNEL: Assessment for cell damages results in apoptosis

TUNEL staining

This is a test in complement to the H&E staining to assess tissue damage during prolong perfusion, which would lead to cell death on the donor lung tissue. The assay uses Terminal deoxynucleotidyl transferase dUTP nick end-labeling to tag on fragmented DNA resulted from apoptosis due to cell damage.

Immunohistochemistry

Specific protein markers expressed on surface of cells can be labelled with immunohistochemistry to assess immune responses and any blood vessel damages as a result from the EVLP treatment to the donor lungs.

CD31/PECAM-1: Assessment of tissue integrity and evaluate any vascular damage during
CD20: Rituximab is a drug delivered during EVLP to the donor lung to target B cells, preventing donor-transmitted EBV to the recipient. The effectiveness of the treatment on B-cell depletion in lymph node and donor lung can be assess with IHC results.
CD68 and CD11b: These are markers for macrophages to evaluate inflammation in the EVLP treated lungs and the immune cells response to the reconditioning efforts.
CD3: To assess T-cell and NK cells immunomodulation effects to pre and post EVLP treatment.

Post-Transplant Complication

Allograft rejection after lung transplant is common, but it can be managed by immunosuppression medication and close monitoring care. There might be other acute post-transplant complication such as hypoxemia injury during lung reperfusion or pneumonia infections. Acute cellular rejection is mostly resolved by treating patient with immunosuppression. However, if acute graft dysfunction persists, there will be higher risk in leading to Chronic Lung Allograft Dysfunction (CLAD) that often lead to mortality in lung transplant patient after 5 years.

What are the histological tests used in diagnosis and related studies on post-transplant lung allograft dysfunction?

H&E and Immunohistochemistry on Acute Cellular Rejection diagnosis

T-lymphocyte mediated response on the allograft lung is the main cause of Acute cellular rejection (ACD). Pathologist will look at the alveolar septum, intra-alveolar lumen and the subpleural, interlobular airways for cell infiltration in these regions of the lung. An H&E staining will provide the cellular morphology information in grading the severity of ACD. Immunohistochemistry can be used to label and differentiate types of the cell infiltrating, including lymphocytes, macrophages and eosinophils. Antibody markers such as CD3 and CD68 are often use for the IHC staining.

Precision-Cut Lung Slices (PCLS) on Ischemia-Reperfusion Injury intervention testing

Ischemia-reperfusion injury (IR) often occur as complication when there is an interruption of blood flow and reperfusion of the allograft lung, causing inflammation, pulmonary edema and hypoxia. Precision-Cut Lung Slices (PCLS) is a technique that is used by researcher to model the pathophysiology of IRI process ex vivo in a live cell cultured environment. The PCLS procedure involves sectioning live lung tissue 50-100um in thickness using vibratome and the slices are placed in culture media to be tested on different condition and drug treatment. This technique allows real-time imaging of the live tissue responses to testing. The resulted thin slices are often processed into FFPE blocks after live treatment, sectioned into the routine histology 4-5um thickness for H&E and IHC testing. While the PCLS section are very thin slices, there is not much leeway for trimming when performing paraffin sectioning. All points on the PCLS slice has to be carefully pressed down flat during paraffin embedding to ensure getting full face of the tissue on the section.

Elastin Masson trichrome: Combine Verhoeff-elastin and Masson trichrome to assess chronic damage in lung

Elastin Masson trichrome staining on Chronic Lung Allograft Dysfunction (CLAD)

Chronic Lung Allograft Dysfunction is featured by fibrous scarring of the bronchioles, leading to bronchiolitis and lumen occlusion. There are 2 main types of CLAD: Brochiolitis Obliterans Syndrome (BOS) and Restrictive Allograft Syndrome (RAS), both causing fibrosis in the bronchioles. A combination of elastin stain (Verhoeff) and Masson trichrome is used to assess the loss of elastic fibres in airway and vessels and fibrosis in the submucosal regions of the bronchioles. There might also be vascular changes with pulmonary vessels thickening, the Elastin Masson trichrome can highlight these arteriosclerotic changes. In this stain, the Verfoeff stain is first performed to highlight the elastin fibers around airway and blood vessels in black with ferric-chloride iodine hematoxylin. Then followed up by Mason trichrome staining to highlight the fibrous regions of the bronchioles with fast green. Staining collagen fibres in fast green provides better contrast over the Aniline blue in traditional Masson trichrome protocol.

The Latest Assay and Histological Tests used in Lung Transplant Research

In recent years, there has been increase interest in tagging multiple markers on tissue sections to provide high throughput of data in short period of time. There has been interest in some lung studies looking on Imaging Mass Cytometry (IMC) that combines the principle of immunohistochemistry coupled with mass spectrometry to produce a highly multiplexed image for protein detection on tissue section. Spatial-omics is also another technology that lung researches are interested where it combines imaging and sequencing, enabling high-throughput in mapping out the microenvironment of immune cells and drug treatment progression on the tissue samples. A lot of studies would consider building Tissue Micro-Array (TMA) for these multiplex assay to make the assay more efficient when multiple tissue blocks are combined on 1 slide and the assay is run on the same time.

Histotechnology continues to evolve in adapting to new technologies in meeting the needs on lung transplant research studies, helping to save lives and improving care for patients.

Written by: Napoleon Law, BSc, HTL(ASCP)^CM

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