TITLE: Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ)
AUTHORS: Integrated Islet Distribution Program

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This Standard Operating Procedure is adapted from the work of the '</span><span style = "font-weight:bold;font-style:italic;">National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities'' </span><span style = "font-weight:bold;">following the SOP cited in the document </span><span style = "font-weight:bold;font-style:italic;">'Purified Human Pancreatic Islet: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ) – </span><span style = "font-style:italic;">Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium'</span><span></span></div><div class = "text-block">This SOP defines the assay method for quantitative and qualitative determination in the identification of  human isolated islet preparations, which include endocrine and exocrine tissue, for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP).  This protocol is written to assist the participating islet isolation centers and investigators who are part of this program.</div><div class = "text-block">Dithizone (diphenyl thiocarbazone; DTZ) is an organic chemical that chelates the zinc in the insulin granules present in the beta cells of the pancreatic islets. The islet cells are stained red while the acinar cells remain unstained.</div><div class = "text-block">DTZ staining is used as a lot release and as an in-process assay:</div><div class = "text-block">    (i)Lot release testing: DTZ staining is used to identify islets and to determine the quantity and quality of the final islet product. Islet quantity is expressed as the number of islet equivalents (IEQ), which is calculated based on the number and diameter of the islets present in the preparation, mathematically corrected for islet volume.</div><div class = "text-block">    (ii)In-process testing: DTZ staining is used to identify islets and to assess the effectiveness of the digestion, isolation and purification processes. The quality of the preparations is expressed as percent islet purity, and percent trapped islets. Islet quantity (IEQ) is also assessed.</div></div>

[STEPS]
?. [Preparation of Working Dithizone]
Assemble all items described in the Materials section.
?. [Preparation of Working Dithizone]
Prepare DTZ stain as described below. Observe all safety precautions when working with DMSO.
?. [Preparation of Working Dithizone]
Dissolve  dithizone in  DMSO.
50 mg
10 mL
?. [Preparation of Working Dithizone]
Add DPBS to bring the total volume to  .
50 mL
?. [Preparation of Working Dithizone]
Filter the combined solution using a 0.45 μm nylon filter.
?. [Preparation of Working Dithizone]
Place solution in a 50 mL conical tube and label “Dithizone Stain or DTZ” with:Preparation Date and TimeExpiration Date and Time (24 hours after preparation)Initials of person preparing solutionExpiration Date and Time (24 hours after preparation)Initials of person preparing solution
?. [Preparation of Islets for Counting]
Samples for islet quantitation and purity should be taken for islet broadcast either after the isolation or post culture. Sampling must be repeated prior to packaging for shipment in order to ensure that samples after culture are representative of what is being distributed to the investigators.
?. [Preparation of Islets for Counting]
Mix the final islets suspension very gently but thoroughly by inverting the islet prep in a conical 2-3 times before quickly taking a sample. (Do not swirl.) As islets settle rapidly, care must be taken to ensure a representative sample is taken from the middle of the suspension.
?. [Preparation of Islets for Counting]
Take replicate 100-200 µL sample volumes from 100 mL final prep using a widebore tip pipettor.  Two duplicate counts should be performed by two separate technicians or 2 separate counts by one technician.
?. [Preparation of Islets for Counting]
Add 3 drops (30 μL) of the DTZ solution to the islets sample and allow staining for 1 – 2 minutes at room temperature. Cover the bottom of the counting dish with DPBS to approximately ½ the height of the dish. Count the islets under the microscope following the steps below.
?. [Islet Quantification]
Identify AI, IEQ, and % purity. Islets are counted by size of the Actual Islets (AI), tabulated, and mathematically calculated to determine the Total Islet Equivalents (IEQ). Examine the islets sample (stained islets will appear red) using the 10x eyepiece and the 4x objective to give a total magnification of 40x. Using the grid lines on the counting dish as a guide, methodically scroll through the dish from side to side, and top to bottom, examining each islet. Count islets within the perimeter of the grid’s squares, including only islets touching the top and right lines (not the bottom and left lines), to avoid counting the same islet twice.Examine the islets sample (stained islets will appear red) using the 10x eyepiece and the 4x objective to give a total magnification of 40x. Using the grid lines on the counting dish as a guide, methodically scroll through the dish from side to side, and top to bottom, examining each islet. Count islets within the perimeter of the grid’s squares, including only islets touching the top and right lines (not the bottom and left lines), to avoid counting the same islet twice.Using the grid lines on the counting dish as a guide, methodically scroll through the dish from side to side, and top to bottom, examining each islet. Count islets within the perimeter of the grid’s squares, including only islets touching the top and right lines (not the bottom and left lines), to avoid counting the same islet twice.
Dithizone (diphenyl thiocarbazone; DTZ) is an organic chemical that chelates the zinc in the insulin granules present in the beta cells of the pancreatic islets. The islet cells are stained red while the acinar cells remain unstained.
?. [Islet Quantification]
There are different types of reticles that can be used but all should be certified in the eyepiece to the correct specification by the microscope company representative.  Once calibrated, the eyepiece of the microscope is used to determine the size of each islet. Using the table below as a guide, regardless of which type of reticle is used, count (tally) the number of Actual Islets (AI) in each diameter group using the manual or electronic cell counter.  Do not count islets smaller than 50 μm because their contribution is not significant.
?. [Islet Quantification]
Calculate the dilution factor as follows:            Example for a 100 µL sample from a 100 mL total volume =1000 dilution factor
Dilution Factor = [(mL total volume / µL sample volume) X 1000]
?. [Islet Quantification]
Record and calculate the results using Excel Spreadsheet in Attachment 1.    Calculate the Total Actual Islets (∑ AI), and the Total Islet Equivalents (∑ IEQ) by inputting the data from the tally, in step 4.1, into the Excel spreadsheet in Attachment 1 for Most Pure Shipment and Least Pure Shipment if applicable.  Calculate the total number of IEQ present by entering the total Actual Islets (AI) in each range which will be converted using the conversion factor, to IEQ per each range.  This conversion normalizes the AI to 150 μm sized islet. Both the AI and IEQ will be totaled and multiplied by the dilution Factor for a Final AI count and Final IEQ Count of the preparation.The Islet Index (II), a calculation made by dividing the IEQ by the AI, is calculated automatically in Attachment 1.  This designation determines the overall size distribution of the islets being shipped. Repeat process, as needed, for a Least Pure Shipment.Calculate the total number of IEQ present by entering the total Actual Islets (AI) in each range which will be converted using the conversion factor, to IEQ per each range.  This conversion normalizes the AI to 150 μm sized islet. Both the AI and IEQ will be totaled and multiplied by the dilution Factor for a Final AI count and Final IEQ Count of the preparation.The Islet Index (II), a calculation made by dividing the IEQ by the AI, is calculated automatically in Attachment 1.  This designation determines the overall size distribution of the islets being shipped. Repeat process, as needed, for a Least Pure Shipment.Both the AI and IEQ will be totaled and multiplied by the dilution Factor for a Final AI count and Final IEQ Count of the preparation.The Islet Index (II), a calculation made by dividing the IEQ by the AI, is calculated automatically in Attachment 1.  This designation determines the overall size distribution of the islets being shipped. Repeat process, as needed, for a Least Pure Shipment.The Islet Index (II), a calculation made by dividing the IEQ by the AI, is calculated automatically in Attachment 1.  This designation determines the overall size distribution of the islets being shipped. Repeat process, as needed, for a Least Pure Shipment.
?. [Islet Quality Grade by Islet Ranking]
Determine the Islet Quality Grade based on the chart below and Islet Ranking Guide (Attachment 2). Record and calculate the results on Attachment 1 for Most Pure Shipment and Least Pure Shipment, if applicable, and on broadcast website.
?. [Islet Purity]
Calculate the percent purity of the islets to the nearest 5% by estimating the portion of dithizone (red stained islets)  to all tissue (islets, acinar, ductal cells). Record the purity of each sample on Attachment 1.  The average will be calculated automatically.
?. [Photographic Documentation]
Take digital images and upload (when applicable) for IIDP Islet Broadcast of the count sample.
?. [Photographic Documentation]
Gently swirl the count sample in order to center the islets in the dish. This will give a representative photographic image of the purity and size distribution of the islet preparation. If possible, take a well focused digital photo, using the center’s SOP for it's specific microscope and camera set-up, so the final image size is a 10x-40x magnification. Label and save image with RRID#, date, time when photo was taken (prior to broadcast), and magnification.
?. [Photographic Documentation]
With the use of the digital imaging techniques on the IIDP website, upload images and match the background grid lines per the online instructions in the broadcast section (when applicable).
?. [Subsequent Counting, Grading, Purity, and Viability]
Repeat all counting, grading, purity, and viability estimates and digital image uploads for islets prior to packaging for distribution if more than 6 hours post original broadcast evaluations. Update broadcast records under the “Confirm Islet Information” section of the broadcast and repeat these procedures for rebroadcasts and shipments with every subsequent 24 hour rebroadcast/shipment.