Updates in processing of anterior fat pad aspirate for amyloid (with video and sketches)

INTRODUCTION

Fat pad aspiration has been a simple and minimally invasive method for the evaluation of systemic amyloidosis at a relatively lower cost with minimal morbidity.^[1-3] The procedure for performing anterior fat pad aspiration was reported previously in detail as a video article.^[1] In that article, the protocol suggested for processing of the aspirate for formalin-fixed paraffin- embedded FFPE and electron microscopy has shown some limitations. This article is to communicate the updates in processing of the fat pad aspirates so that the problems noted are resolved. This is critical for the best outcome with electron microscopic evaluation and preparation of FFPE for Congo red staining with polarizing microscopy with laser microdissection for mass spectrophotometric testing as indicated.^[1-3]

A commonly used method for the detection of amyloid in tissue sections is Congo red stain with the application of polarizing microscopy to detect apple green birefringence in the walls of small blood vessels in the fibrovascular tissue fragments in fat pad aspirates. Similarly, for electron microscopy, the requirement of at least 15 blood vessels may be compromised by the predominance of fat in the glutaraldehyde fixed aspirate. The abundance of the fatty component diluting the fibrovascular portion may lead to frequent suboptimal outcomes with inadequate sample quantity for electron microscopy and the FFPE material required for proteomic analysis with mass spectroscopy to confirm the amyloid subtype.^[3,4] This demands a proper approach to remove most of the adipocyte component and get more fibrovascular tissue with relative predominance of blood vessels.

DISCUSSION

The diagnosis of amyloidosis is based on tissue evaluation. It was dependent mostly on relatively invasive tissue biopsies such as from the kidney, liver, and/or heart with the potential for complications including hemorrhage.^[2] Some relatively less invasive approaches such as rectal, gingival, and bone marrow biopsies have been practiced as well.^[5-7] However, the introduction of abdominal fat pad aspiration was reported for the tissue diagnosis of systemic amyloidosis as a simple, minimally invasive, safe, and less expensive procedure.^[8] This procedure was refined further as a video article with the step- by-step procedure along with the suggested protocol of how to process the fat pad aspirate.^[1]

However, the fatty component in variable proportion may dilute and interfere with the evaluation of amyloid in the diagnostic fibrovascular component. This is especially problematic for the evaluation of microfragments with electron microscopy due to the need to get more than 15 blood vessels as recommended to be evaluated by electron microscopy.^[2] If the fatty component is not removed, the number of small vessels required for the evaluation of amyloid in their walls may not be sufficient in tiny electron microscopy sections of fibroadipose tissue fragments. Congo red staining for the evaluation of anterior fat pad aspirates for amyloid by light microscopy and polarizing microscopy is relatively less sensitive, especially when a representative number of blood vessels is scant in proportion to the fatty component.^[9] Similarly, FFPE sections required for laser microdissection in positive cases for mass spectrophotometry to subtype the amyloid for proper clinical management also need a greater number of vessels in fibrovascular tissue than the fatty component.^[3]

This technical update reports the modifications in processing of anterior fat pad aspirate by removing most of the adipocyte component for the evaluation of amyloidosis as reported previously.^[1] This is especially important in cases with early disease with scant amyloid. Based on our ongoing experience with the procedure,^[1] a standardized protocol for processing FNA of anterior fat pad for amyloid as demonstrated in the video 1 (please visit the article in HTML to watch the video at https://dx.doi.org/10.25259/Cytojournal_31_2020) with the commentary in reference to the video counter [Table 1] is presented in this methodology article.

Perform FNA of anterior fat pad with 18G needle as a mini- liposuction with aspiration of as many microfragments of fibroadipose tissue as possible with accumulation of at least 1.5 mL aspirate in the syringe as reported previously.^[1] The updated procedure explains how to remove the interference due to blood and fatty component with the help of a nylon mesh tissue bag [Figure 1]. This improved protocol to process the anterior fat pad aspirates for electron microscopy and paraffin embedding is demonstrated in the video with Figures 2-4.

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Figure 1:

Nylon mesh tissue bag.

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Figure 2:

Processing of anterior fat pad aspirate using nylon tissue bag to submit it for paraffin embedding after tissue processing.

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Figure 3:

Processing of anterior fat pad aspirate in nylon tissue bag for paraffin embedding after tissue processing.

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Figure 4:

Processing of anterior fat pad aspirate using nylon tissue bag to submit it for electron microscopy.

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First, remove the needle from the syringe nozzle. Transfer a tiny portion of the aspirate in the syringe on to the glass slide. Detach the fibroadipose tissue fragment from the nozzle of the syringe with the help of the tip of the used needle if required. Next, transfer about one-fourth of the fibroadipose tissue fragments in the aspirate to one nylon mesh tissue bag and remaining aspirate to the other nylon mesh tissue bag. Most of the contaminant fresh blood and fatty portion in the aspirate are squeezed out from both the nylon mesh bags by swiping with the index finger on an absorbent pad supported by a flat firm surface.

For the portion to be submitted for the preparation of FFPE, the nylon mesh bag with a larger amount of aspirate with squeezed out blood and fat is folded and transferred to the labeled container with 10% formalin fixative [Figures 2 and 3, Video 1 (please visit the article in HTML to watch the video at https://dx.doi.org/10.25259/Cytojournal_31_2020) at counter: From 0.44 to 1.19]. Achieving maximum proportion of fibrovascular tissue in the final FFPE of the cell block is important for a successful outcome with further evaluation for subtyping of amyloid in positive cases with mass spectrophotometry after laser capture microdissection^[3] and other evolving technologies.

To process for electron microscopy, the nylon mesh bag with smaller amount of aspirate is ripped open to expose the remaining fibrovascular tissue after squeezing out the fat and blood component. The fibrovascular tissue is swiped out with the needle used for the FNA procedure from the wall of the nylon mesh bag and transferred to the labeled container with glutaraldehyde fixative for electron microscopy. Close the cap and shake the tube to disperse the fibrovascular tissue fragments for optimum fixation in glutaraldehyde [Figure 4, Video 1 (please visit the article in HTML to watch the video at https://dx.doi.org/10.25259/Cytojournal_31_2020) at counter: From 1.20 to 2.19].

After this, the drop of the fibroadipose tissue on the glass slide is spread between the two glass slides to get direct smears. As the aspirate is rich in fat, the material does not dry in the time required for completing the processing to submit in 10% formalin for FFPE and in glutaraldehyde for electron microscopy. However, depending on the situation, the drop on the glass slide may be spread before starting the process for submitting in 10% formalin and in glutaraldehyde.

Both direct smears are air-dried. One of the smears is stained with DiffQuik for immediate onsite adequacy before the termination of the anterior fat pad aspiration procedure.

The other air-dried smears are rehydrated and post-fixed for Pap staining later in the Cytoprep Lab.^[10] Depending on the institutional choices and biases, the second direct smear may be wet fixed for Pap staining. However, the wet direct smears with fatty material may float and a predominant portion of the material on the glass slide may be lost with suboptimal smears made with scant tissue in addition to the usual problem of interference due blood contamination in wet fixed smears.^[10] The direct smears may also be used for the evaluation of amyloid in the smear with special stains, autofluorescence method, etc., as reported by some studies.^[11]