Name: performing and processing fna of anterior fat pad for amyloid
Uploaded: 2010-10-30T17:00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

We thank Ms. Bonnie Phetteplace, RN for assistance during video graphing the FNAB procedure demonstration.

Introduction
Systemic amyloidosis is a highly variable disease that refers to extracellular deposition of various proteins that are collectively referred to as amyloid. Deposition of these amyloid fibrils with beta pleated configuration leads to various clinical presentations depending on the organs involved. The most clinically relevant manifestations of systemic amyloidosis are noted when there is involvement of critical organs such as the heart, liver, and/or kidney. Historically, these involved organs would be biopsied to demonstrate the presence of amyloid. These moderately invasive procedures carried a significant risk including hemorrhage. Fat pad aspiration has since been shown to provide a reliable and noninvasive method for the detection of amyloid in systemic amyloidosis 1. This procedure is essentially comparable to the liposuction of the subcutaneous fat in the anterior abdominal wall under local anesthesia to retrieve fibroadipose tissue for evaluating scant amyloid deposits in small blood vessel walls 2.
The most frequently utilized method for amyloid identification in tissue remains the characteristic apple-green birefringence pattern seen when Congo red stained sections are visualized under a polarized light microscope 3. When fat pad aspiration is performed, Congo red stains can be done on either direct smeared slides or cell block preparations of the aspirated adipose tissue. However, patients in early stages of amyloidosis have scant amyloid deposits, which greatly reduces the sensitivity of Congo red stained cell block sections 4,5. Ultrastructural evaluation of fat pad aspirates by electron microscopy has better reproducibility and improved sensitivity 4. Therefore, it is recommended to submit all fat pad aspirates for both preparation of a cell block and for performing electron microscopy 2.
Fat pad aspiration is a relatively low cost and noninvasive method for obtaining tissue to diagnose systemic amyloidosis. This article describes fat pad aspiration procedure along with details about sample processing to submit specimen for both Congo red staining and ultrastructural evaluation by electron microscopy. In this video, we demonstrate this reproducible and simple procedure to retrieve optimal diagnostic material.

1. Performing the FNA of Anterior Fat Pad
A. Anesthetization of the local area (Figure 1 &amp; 2)
<ol>
	<li>Using alcohol swabs or the skin cleansing agent preferred by a particular institution, clean the skin on the lower quadrant area of the abdomen lateral to the midline and below the umbilicus (Figure 1).</li>
	<li>Mark a rhomboid shaped area about 2 x 2 inches as shown in Figure 1 with a marking pen.</li>
	<li>Aspirate approximately 10 mL of 1% lidocaine with an 18G needle. Attach a 25G 1 &frac12; inch needle to the syringe. Remove any trapped air by tapping the upright syringe while pressing the plunger until liquid is dispensed and no air bubbles are present.</li>
	<li>Anesthetize along the borders of the rhomboid area already marked as shown in Figure 2. Start by inserting the 25G needle just under the skin at point A and carefully push the needle subcutaneously to point X. Pull back on the syringe plunger to ensure that you are not within a vessel. Then slowly push the plunger to infiltrate up to 2.5 mL of lidocaine (about &frac14; of the lidocaine in 10 mL syringe) while withdrawing the needle to point A without letting the needle come out of the skin at point A (Figure 2a3). With the needle still under the skin change the direction towards point Y (Figure 2a4) and push the needle subcutaneously to point Y (Figure 2b1). As before, confirm that the needle is not in a vessel and dispense approximately 2.5 mL of lidocaine while slowly withdrawing the needle through point A (Figure 2b3 through 2b4). Repeat similar steps starting from point B and dispensing lidocaine subcutaneous from points B to X and B to Y (Figure 2c1 through 2d4). Prevent bleeding from the pricks A and B by firm application of sterile gauge piece.</li>
	<li>You may now check that the area is anesthetized by lightly touching the skin within the anesthetized rhomboid by tip/corner of cotton gauze piece, by comparing with adjacent unanesthetized skin.</li>
</ol>
B. Performing fat pad aspiration (Figure 3)
(Application of local anesthesia prior to performance of fat pad aspiration may be bypassed, depending on the regional and individual preferences. Properly performed FNAB procedure for anterior fat pad aspiration can be completed in one prick. However, depending on the pain threshold of individual patient, maneuvering of 18G needle back and forth in to the subcutaneous fat tissue is relatively distressing. The anesthetization method described here achieves the effect in only two pricks with 25G needle and averts the pain with improved tolerance to the procedure.)
<ol>
	<li>Assemble 18G 1 &frac12; inch needle onto a 10 mL syringe. Mount the syringe-needle assembly in the syringe grip ("FNAB grip/gun") for proper application and release of vacuum.</li>
	<li>Insert the tip of the needle into the subcutaneous fat within the cleansed and anesthetized rhomboid area (Figure 3a).</li>
	<li>Fully retract the plunger of the syringe with the needle inserted in the subcutaneous tissue to generate vacuum (Figure 3b).</li>
	<li>Maintain the vacuum and maneuver the needle back and forth in various directions in to the subcutaneous fat (Figure 3c through 3i). Each stroke should be as long as possible with the length of the needle selected without letting the needle come out of the skin. Maximum sampling is achieved by changing direction with each stroke (Figure 3i). It is important to keep the direction of the needle tangential to the serosa and parallel to the skin surface to avoid puncturing of the peritoneal cavity.</li>
	<li>The fibroadipose tissue accumulates in the syringe. Once enough fibroadipose tissue fragments (up to 1 mL of fragments rich blood mixed specimen) are retrieved, release the vacuum completely and remove the needle (Figure 3k).</li>
	<li>Have the patient or the assistant apply firm pressure on the area of procedure with a pad of gauze to prevent extravasation of blood.</li>
</ol>
2. Specimen Processing
<ol>
	<li>Place at least 5-6 fragments of fibroadipose tissue in glutaraldehyde solution for electron microscopy (Figure 4B).</li>
	<li>Depending on the institutional protocol, a few smears of the fibroadipose tissue fragments may be prepared by spreading them between two slides (Figure 4A).</li>
	<li>The remaining material is allowed to clot in the syringe (this may take 5-7 minutes, depending on the clotting time) (Figure 4C).</li>
	<li>Aspirate 10% formalin in the syringe so that the clotted fibroadipose tissue material is dislodged from the wall of the syringe and free floating (Figure 4C2). Remove the plunger from the syringe (Figure 4C3) and transfer the clotted fibroadipose tissue into the 10% formalin container from the open end of the syringe opposite the nozzle end (Figure 4C4).</li>
	<li>Label the containers appropriately and submit the glutaraldehyde for electron microscopy and the formalin for H &amp; E section and Congo red stain for evaluation under polarizing microscopy. If smears are prepared, they may be processed according to the protocol of the individual laboratory.</li>
</ol>
<img src="/files/ftp_upload/1747/1747fig1.jpg" alt="Figure 1" /> Figure 1. Area to be anesthetized for FNAB of anterior fat pad.
<img src="/files/ftp_upload/1747/1747fig2.jpg" alt="Figure 2" /> Figure 2. Anesthetization of the local area.
<img src="/files/ftp_upload/1747/1747fig3.jpg" alt="Figure 3" /> Figure 3. Performing FNAB of anterior fat pad.
<img src="/files/ftp_upload/1747/1747fig4.jpg" alt="Figure 4" /> Figure 4. Processing of anterior fat pad aspirate to be submitted to laboratory for detecting amyloid deposits.
3. Representative Results
<img src="/files/ftp_upload/1747/1747fig5.jpg" alt="Figure 5" /> Figure 5. Amyloid in the wall of small blood vessels in fibroadipose tissue fragments. Fibroadipose tissue was formalin-processed, paraffin embedded, stained with Congo red, and examined by polarizing light microscopy. Amyloid in the wall of small blood vessels shows apple green birefringence (white arrow).
<img src="/files/ftp_upload/1747/1747fig6.jpg" alt="Figure 6" /> Figure 6. Tissue lacking amyloidosis. The apple green birefringence is absent in tissues of a different patient without amyloidosis. Blue birefringence (white arrow) of collagen fibers, are usually present in almost all specimens.
<img src="/files/ftp_upload/1747/1747fig7.jpg" alt="Figure 7" /> Figure 7. Fibrils consistent with amyloid in the blood vessel wall. Electron micrograph of straight, non-branching, randomly-scattered 8-10 nm diameter fibrils formed by amyloid in the blood vessel wall.

<ol>
	<li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+diagnosis+of+systemic+amyloidosis.">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li><li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates:+Is+Congo+red+sufficient?.">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?.</a> Amyloid. , .</li><li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Amyloidosis.">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li><li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis:+How+can+the+clinical+role+of+the+test+be+improved?.">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?.</a> Mod Pathol. 20, 1A-380A (2007).</li><li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+typing+of+systemic+amyloidosis:+The+role+of+abdominal+fat+pad+fine+needle+aspiration.">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li><li>Hazenberg, I. I., Rijswijk, C. V. a. n., H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice.">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li><li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis:+specificity,+positive+predictive+value,+and+diagnostic+pitfalls.">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li><li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+Amyloidosis.">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li><li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence.">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li><li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+amyloid+in+fat+pad+aspiration:+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy.">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li><li>DeMay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+Needle+Aspiration+Biopsy.">Fine Needle Aspiration Biopsy.</a> The Art &#38; Science of Cytopathology. , 465-46 (1996).</li><li>Arbustini, E., Vrga, L., Concardi, M., Pallandini, G., Obici, L., Merlini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+and+Immuno-electron+Microscopy+of+Abdominal+Fat+Identifies+and+Characterizes+Amyloid+Fibrils+in+Suspected+Cardiac+Amyloidosis.">Electron and Immuno-electron Microscopy of Abdominal Fat Identifies and Characterizes Amyloid Fibrils in Suspected Cardiac Amyloidosis.</a> Amyloid. 9, 108-114 (2002).</li></ol>

No conflicts of interest.

Diagnosis of amyloidosis is usually achieved with a tissue biopsy of the affected organs such as the kidney, liver, and/or heart. This approach has high diagnostic yield, however, is invasive and may be associated with complications including hemorrhage 2. Rectal, gingival, and bone marrow biopsies were preferred for diagnosis as relatively less invasive approach in 1960s 6,7,8. Abdominal fat pad aspiration was reported in 1973 as a safe, minimally invasive, simple, and less expensive procedure for the tissue diagnosis of systemic amyloidosis 1. However, the details of the procedure and approach to processing of the retrieved specimen is not coherently reported. This video and the article describe the procedure step by step.
The approach for detecting amyloid in fat pad aspiration, similarly, is also not well standardized with a few studies comparing and evaluating various approaches 1,5,6,7,8. Evaluation of anterior fat pad aspirates with Congo red staining is less sensitive with lower interobserver reproducibility especially in early cases of amyloidosis with scant amyloid deposits 5. Immunohistochemistry performed on formalin fixed paraffin-embedded cell block sections and Congo red fluorescence performed on cytology smears have been reported to improve the diagnostic sensitivity 9,10. Electron microscopy improves the detection and identification of scant amyloid fibrils in small blood vessel walls in the fibroadipose tissue in fat pad aspirates 4, 5. Based on this information, this article describes processing of the specimen to prepare cell blocks (for evaluation of Congo red stained cell block sections under polarizing microscope for diagnostic apple green birefringence and also for immunohistochemistry as indicated) and for electron microscopy. However, depending on the approach selected for evaluation of the specimen for amyloid, it may be subjected to cytology smear preparation, cell block preparation, and processing for electron microscopy. Some laboratories perform the tests on the smears prepared from the aspirated fibroadipose tissue fragments and stained with Congo red to study the fluorescence 10.
Generally in a late amyloidosis, the light microscopy with Congo red polarizing microscopy may be sufficient; however, in early stages of the disease Congo red staining with polarizing microscopy on cell block sections is less sensitive and reliable for fat pad aspirates 4,5,11. Other special stains including Thioflavin T may be used with similar limitations. Other approaches to detect amyloid include evaluation of the aspirate smears for amyloid with Congo red polarizing microscopy and fluorescence 10. In our experience this method is less reproducible. Evaluation of the blood vessels in fat pad for amyloid by electron microscopy overcomes these limitations 4,11. Further characterization of amyloid has been reported by immunoelectron microscopy 12; however, these methods may not be available widely in a non-tertiary care setting.
Needles used for performing aspiration procedures may be categorized according to their gauge sizes into fine (21-25G), intermediate (18-20G), and large (eg- 14G) 11. Anterior fat pad aspirations have been reported to be performed by using variable gauges ranging from intermediate (18 to 20G) to fine (21 to 22G) 2,10. Most of the mass lesions are aspirated with finer gauge needles (such as 25G) to obtain good cytology smears along with additional passes with wider gauge needle (such as 18G) to retrieve additional sample for cell blocks.
During anterior fat pad aspiration, the cohesive fibroadipose tissue does not aspirate well with finer needles. Cell block preparation and electron microscopy are important for evaluation of small blood vessel walls in fibroadipose tissue fragments in the fat aspirates. Wider gauge needles (such as 18G) are recommended to yield diagnostic adequate material 2,10. As the procedure is comparable to the conventional FNAB, fat pad aspiration is referred to as FNAB even though wider gauge needles may be used for performing it 5.

A correction was made to <a href="http://www.jove.com/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

Alcohol swabs, gauze pads, marking pen, 10 mL syringes x2, 1% lidocaine (local anesthetic- If lidocaine is used alone, it causes an initial burning sensation after instillation. This can be prevented by using a 1:1 mixture of 1% lidocaine and 1% sodium-bi-carbonate), 18 gauge(G) 1 &frac12; inch needles x2 (for performing FNAB), 25G 1 &frac12; inch needle (for injecting local anesthetic), FNA syringe holder ("gun"), bandage, vial of glutaraldehyde (for electron microscopy), and biopsy container of 10% formalin.

performing and processing fna of anterior fat pad for amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

performing-and-processing-fna-of-anterior-fat-pad-for-amyloid

Research

JoVE Journal

Medicine

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although several studies have investigated the role of VC as a direct hepatotoxicant, the concept that VC modifies sensitivity of the liver to other factors, such as nonalcoholic fatty liver disease (NAFLD) caused by high-fat diet (HFD) is novel. This protocol describes an exposure paradigm to evaluate the effects of chronic, low-level exposure to VC. Mice are acclimated to low-fat or high-fat diet one week prior to the beginning of the inhalation exposure and remain on these diets throughout the experiment. Mice are exposed to VC (sub-OSHA level: &#60;1 ppm) or room air in inhalation chambers for 6 hours/day, 5 days/week, for up to 12 weeks. Animals are monitored weekly for body weight gain and food consumption. This model of VC exposure causes no overt liver injury with VC inhalation alone. However, the combination of VC and HFD significantly enhances liver disease. A technical advantage of this co-exposure model is the whole-body exposure, without restraint. Moreover, the conditions more closely resemble a very common human situation of a combined exposure to VC with underlying nonalcoholic fatty liver disease and therefore support the novel hypothesis that VC is an environmental risk factor for the development of liver damage as a complication of obesity (i.e., NAFLD). This work challenges the paradigm that the current exposure limits of VC (occupational and environmental) are safe. The use of this model can shed new light and concern on the risks of VC exposure. This model of toxicant-induced liver injury can be used for other volatile organic compounds and to study other interactions that may impact the liver and other organ systems.

The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although ...