Histologic Analysis of the Lymphatic Collecting Vessels Used for Minimally Invasive Supermicrosurgical Lymphatico-venous Anastomoses in the Treatment of Lymphedema
Conditions: Lymphedema
Sponsor: Paolo Gennaro

OFFICIAL TITLE OF THE STUDY: LVA2017
NCT ID not yet assigned
DATE OF THE DOCUMENT: January 26th 2018

PATIENTS
patients of both sexes, age over 18 years, affected by secondary lymphedema of the upper or lower limb due to 
the treatment of breast, uterine, prostatic cancer or melanoma or by primary lymphedema.
SURGERY
- map the lymphatic system using indocyanine green and the venous system with Accuvein, 
- choose incision sites.
- premedication with Midazolam (0.02 mg/kg). Antibiotic prophylaxis, gastric protection and antiemetic drugs.  
- perform local anesthesia with a mixture of 2% lidocaine and epinephrine at a dilution of 1:200,000. Since the 
intervention requires immobility, deep sedation is recommended to increase patient’s comfort: Propofol (2 
mg/kg/h) and Remifentanil (0.015 mcg/kg/min) may be used while maintaining spontaneous breathing. Oxygen 
is administrated at 4l/min via nasal cannula or Venturi’s mask. Patients must be constantly monitored during the 
surgical procedure. 
- perform multiple (range 3 - 5) 2 cm linear skin incisions. Carry out microscopic dissection in order to identify 
lymphatic vessels and venules. Prepare lymphatic vessels and suture to veins using 11-0 or 12-0 sutures.
- postoperative analgesia with acetaminophen (1 g every 8 hours) for the first 24 hours.
Patients are dismissed the day after surgery and receive oral antibiotic therapy for 6 days. Stitches are removed 
two weeks after surgery.
Patients are evaluated at 2 weeks, 1, 3, 6, 9, 12, 18, 24 and 36 months after surgery.
Immunohistological analysis. 
- Fixation in 10% formalin and embedding in paraffin. Obtain 7μm thick serial sections.  
- Prior to immunohistochemical labelling, rehydrate sections via xylene and ethanol.
-  Perform  the  immunohistochemical  labelling  with  IHC  select  immunoperoxidase  detection  System  kit 
(Millipore) following the manufacturer’s instructions. As a primary antibody use a lymphatic marker (for 
instance the monoclonal antibody D2-40, DAKO). 
- reveal the reaction with Substrate Kit for Peroxidase (VECTOR).
- counterstain with Meyer’s hematoxylin and view under light microscope. 
Masson’s trichrome staining
- Use Trichrome stain Masson kit  following the manufacturer’s instructions to evidence smooth muscle cells in 
the wall of collecting vessels.