Sep 10, 2020
Version 4
Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ) V.4
- Integrated Islet Distribution Program1
- 1Integrated Islet Distribution Program, City of Hope, Duarte, CA
- Integrated Islet Distribution Program
- Integrated Islet Distribution Program and Human Islet Phenotyping Program
Protocol Citation: Integrated Islet Distribution Program 2020. Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ). protocols.io https://dx.doi.org/10.17504/protocols.io.bk5vky66Version created by Integrated Islet Distribution Program
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 09, 2020
Last Modified: September 10, 2020
Protocol Integer ID: 41877
Keywords: Human Islets, Dithizone, Purity, Islet Equivalent, Actual Islet, Islet Quality Grade, Islet Index, Islets of Langerhans, human islets, islet equivalent
Abstract
This Standard Operating Procedure is adapted from the work of the 'National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities'' following the SOP cited in the document 'Purified Human Pancreatic Islet: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ) – Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium'
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.
Note
Notice: This SOP corrects an error which has been discovered on the conversion factor of islets to IEQ for the 101-150 range in Step 4.3 and on Attachment 1. The conversion factor should be 0.648 not 0.667. We apologize for the error, but the discrepancy in the conversion factor was difficult to trace historically, hence the importance for Rigor and Reproducibility provided by protocols.io. Documentation of this revision is found in Kin T. (2010) Islet Isolation for Clinical Transplantation.The Islets of Langerhans. Advances in Experimental Medicine and Biology. Volume 654.Dordrecht: Springer; p 683-710 referenced below.
Note
Integrated Islet Distribution Program (IIDP) (RRID:SCR_014387)
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.
DTZ staining is used as a lot release and as an in-process assay:
(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.
(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.
Guidelines
- Integrated Islet Distribution Program (IIDP) (RRID:SCR_014387): The IIDP is a contracted program commissioned and funded by the NIDDK to provide quality human islets to the diabetes research community to advance scientific discoveries and translational medicine. The IIDP consists of the NIDDK, the Project Officer (PO), the External Evaluation Committee (EEC) and the CC at City of Hope (COH). The IIDP CC integrates an interactive group of academic laboratories including the subcontracted IIDP centers.
- IIDP Coordinating Center (CC): Joyce Niland, Ph.D. is the Principal Investigator for the IIDP CC and leads staff from the Department of Research Information Sciences at COH to coordinate the activities of the IIDP and assist the participating centers and investigators in the distribution of human islets.
- Percent Purity: the percentage of islets compared to all tissue present in the islet preparation (islets, acinar and ductal cells) which is determined by visual inspection of a representative sample of the islet preparation. Islets are distinguished from non- islet tissue by using Dithizone (DTZ) to stain red the zinc granules in the beta cells.
- Actual Islets (AI) or Islet Particle Number (IPN): The actual number of islets or islet particles counted. For this SOP, the nomenclature of Actual Islets (AI) will be used.
- Islet Equivalent (IEQ) : An IEQ is defined as an islet with a diameter of 150 microns. The number of IEQ in
each size class is calculated by multiplying the number of islets by a conversion factor for each size.
- Islet Quality Grade: A qualitative designation given to the islet preparation after microscopic evaluation based on the parameters of shape, border, integrity, number of single cells, and overall islet diameter.
- Equations for Total Equivalent (Total IEQ) and Total Actual Islet(AI):
1. Total IEQ = Dilution Factor x
[(AI of diameter 50 – 100 μm x 0.167) +
(AI of diameter 101 – 150 μm x 0.648) +
(AI of diameter 151 – 200 μm x 1.685) +
(AI of diameter 201 – 250 μm x 3.500) +
(AI of diameter 251 – 300 μm x 6.315) +
(AI of diameter 301 – 350 μm x 10.352) +
(AI of diameter > 350 μm x 15.833)]
2. Total AI = Dilution Factor x ∑ AI of each diameter
- Islet Index (II): A quantitative designation given to the islet preparation after microscopic evaluation determined by dividing the IEQ by the AI. This designation determines the overall size distribution of the islets being shipped. If the majority of AI is around 150 μm (1-IEQ) then the II will equal 1.0. If the majority of AI is larger than 150 μm, then the II will be > 1.0. If the majority of the AI is smaller than 150 μm, then the II will be <1.0.
References:
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Materials
MATERIALS
Dithizone (Diphenylthiocarbazone)Millipore SigmaCatalog #D5130
Gibco DPBS without Calcium and MagnesiumFisher ScientificCatalog #14190136 or equivalent
Dimethyl sulfoxideSigma AldrichCatalog #D8779 or equivalent
Equipment
Gilson™ PIPETMAN Classic™ Pipets- F123601 or equivalent
NAME
Adjustable pipettor -P200
TYPE
Gilson
BRAND
F123601G
SKU
LINK
50 to 200μL, ±0.5, ±1μL
SPECIFICATIONS
Equipment
Fisherbrand™ Large-Orifice Pipet Tips, 1 to 200µL or equivalent
NAME
Genomic/Wide Orifice Pipet Tips
TYPE
Fisherbrand
BRAND
02-707-134
SKU
https://www.fishersci.com/shop/products/fisherbrand-large-orifice-pipet-tips-6/02707134?keyword=true
LINK
1 to 200μL
SPECIFICATIONS
Equipment
Fisherbrand™ Petri Dishes with Clear Lid or equivalent
NAME
Petri Dishes
TYPE
Fisherbrand
BRAND
FB0875713A
SKU
LINK
Round, Raised Ridge, 60mm, 15mm
SPECIFICATIONS
Equipment
Thermo Scientific™ Nalgene™ Rapid-Flow™ Sterile Disposable Bottle Top Filters with PES Membrane or equivalent
NAME
0.45 um Bottle Top filter
TYPE
Thermo Scientific
BRAND
09-740-62C
SKU
LINK
0.45μm nylon Filter
SPECIFICATIONS
Equipment
Invitrogen™ EVOS™ XL Core Imaging System or equivalent
NAME
Inverted Microscope
TYPE
Light Microscope
BRAND
AMEX1000
SKU
LINK
Equipment
Lab inverted light microscope
NAME
Eyepiece with Calibrated reticle, 1mm
BRAND
Microscope specific, lab SOP
SKU
Equipment
Bal Supply Cell Counter or equivalent
NAME
Cell Counter (Manual or Electronic)
TYPE
Bal Supply
BRAND
02-670-14
SKU
LINK
Equipment
Drummond™ Fixed-Volume Microdispensers or equivalent
NAME
Drummond 3000385
TYPE
Drummond™
BRAND
21176F
SKU
LINK
Volumetric Range 100/200UL with borosilicate glass bores
SPECIFICATIONS
Equipment
new equipment
NAME
Computer or calculator or equivalent
BRAND
NA
SKU
Computer software (e.g. Excel) with the mean and standard deviation functions
SPECIFICATIONS
Safety warnings
Dimethyl sulfoxide (DMSO) 
DMSO_MSDSAction.pdf
DMSO_MSDSAction.pdf - Hazard statement(s): Combustible liquid.
- Precautionary statement(s): Keep away from heat/sparks/open flames/hot surfaces. No
smoking. Wear protective gloves/ protective clothing/ eye protection/ face
protection.
- DMSO itself is not toxic but it can be a carrier of chemicals, viruses, etc. into the skin
Dithizone (DTZ)Dithizone_MSDSAction.pdf
Dithizone_MSDSAction.pdf - Hazard Statement(s): Causes skin irritation. Causes serious eye irritation.
- Precautionary statement(s): Wash skin thoroughly after handling. Wear protective gloves/ eye protection/ face protection.
Preparation of Working Dithizone
Preparation of Working Dithizone
Assemble all items described in the Materials section.
Prepare DTZ stain as described below. Observe all safety precautions when working with DMSO.
Dissolve 50 mg dithizone in 10 mL DMSO.
Add DPBS to bring the total volume to 50 mL .
Filter the combined solution using a 0.45 μm nylon filter.
Place solution in a 50 mL conical tube and label “Dithizone Stain or DTZ” with:
- Preparation Date and Time
- Expiration Date and Time (24 hours after preparation)
- Initials of person preparing solution
Preparation of Islets for Counting
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.
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.
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.
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
Islet Quantification
Identify AI, IEQ, and % purity.
Dithizone Stained Islets
Note
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.
- 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.
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.
Calculate the dilution factor as follows:
Example for a 100 µL sample from a 100 mL total volume =1000 dilution factor
Expected result
Dilution Factor = [(mL total volume / µL sample volume) X 1000]
Record and calculate the results using Excel Spreadsheet in Attachment 1.
Expected result
Example for a 100 µL sample from a 100 mL total volume
- 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.
Attachment 1-Islet Tabulation Counting Sheet for Culturing Islets 2020.xlsx Islet Quality Grade by Islet Ranking
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.
Attachment 2-Islet Ranking Guide.pdf Islet Purity
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
Photographic Documentation
Take digital images and upload (when applicable) for IIDP Islet Broadcast of the count sample.
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.
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
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.
Citations
Ricordi, C. Gray, D. W. Hering, B. J. Kaufman, D. B. Warnock, G. L. Kneteman, N. M. Lake, S. P. London, N. J. Socci, C. Alejandro, R. et al.,. Islet isolation assessment in man and large animals
https://doi.org/10.1007/BF02581331Ricordi, C. Pancreatic Islet Cell Transplantation
Kin T.. Islet Isolation for Clinical Transplantation
NIH CIT Consortium Chemistry Manufacturing Controls Monitoring Committee, NIH CIT Consortium. Purified Human Pancreatic Islet: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium
https://doi.org/pii:e1369Ricordi C, Goldstein JS, Balamurugan AN, Szot GL, Kin T, Liu C, Czarniecki CW, Barbaro B, Bridges ND, Cano J, Clarke WR, Eggerman TL, Hunsicker LG, Kaufman DB, Khan A, Lafontant DE, Linetsky E, Luo X, Markmann JF, Naji A, Korsgren O, Oberholzer J, Turgeon NA, Brandhorst D, Chen X, Friberg AS, Lei J, Wang LJ, Wilhelm JJ, Willits J, Zhang X, Hering BJ, Posselt AM, Stock PG, Shapiro AM, Chen X. National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities
http://www.ncbi.nlm.nih.gov/pubmed/27465220