TITLE: Human Islet Quantification and Purity Assessment
AUTHORS: James Lyon, Aliya Spigelman, Jocelyn Manning Fox, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the Quantification and Purity of Assessment of human islets, as performed by the Alberta Diabetes Institute IsletCore. www.bcell.org/adi-isletcore</div></div>

[STEPS]
?. [Preparation and use of Dithizone stain in Human Islet Preparations]
Preparation of DMSO-dithizone (DTZ)Weigh out 0.2g of dithizone powder into a 50ml conical tube. Add 6mls of DMSO and mix until the powder is in solution. Bring the resulting dithizone solution to 40ml total volume with HBSS and mix. Transfer the dithizone solution to a 60cc syringe with a 0.45µm nylon filter. UseFor every ml of islet suspension add an equal amount of the prepared dithizone solution must be added to the sample. For visualization of staining, add another 2mls of HBSS to dilute the stain and reduce the background color.Alternatively: 50µl of islet suspension, 50µl dithizone, and 2000µl HBSS.UseFor every ml of islet suspension add an equal amount of the prepared dithizone solution must be added to the sample. For visualization of staining, add another 2mls of HBSS to dilute the stain and reduce the background color.Alternatively: 50µl of islet suspension, 50µl dithizone, and 2000µl HBSS.
?. [Islet Sampling]
Islet samples are prepared as described in Human Islet Sampling and Human Islet Isolation protocols.
?. [Human Islet quantification - Islet Equivalent (IEQ) counts]
Add ~1ml DTZ to sample and incubate until islets are visibly stained red. Add ~1ml HBSS to dilute staining background if necessary.
?. [Human Islet quantification - Islet Equivalent (IEQ) counts]
Place sample on stage and determine IEQ (single sample counted in duplicate) using the following steps.
?. [Human Islet quantification - Islet Equivalent (IEQ) counts]
Using the ocular with graticule (1 square = 100μm x 100μm), measure the diameter (or circular equivalent) of each particle in the sample and tabulate in the corresponding column. (Refer to the table in step 11).  Islet particles
?. [Human Islet quantification - Islet Equivalent (IEQ) counts]
Once the entire sample has been counted, calculate the sub-totals for each column and total of all columns and enter values into the table in step 11.  To facilitate easier counting, use a 9-channel benchtop cell counter (Bal Supply  - Catalogue number 808CI).
?. [Human Islet quantification - Islet Equivalent (IEQ) counts]
The multiplication factor (table, step 11) is determined by dividing the total volume by the sample volume. eg. 100mL / 50µL = 2000X (100/0.050=2000)
?. [Assessment of Islet purity]
Percent purity is recorded by estimating the ratio of islets to exocrine tissue. For example, if the area of islets is equal to the area of exocrine tissue, the purity would be 50%.
?. [Assessment of the percentage of trapped or mantled islets ]
Percent trapped is determined by estimating the ratio of trapped versus total islets
?. [Assessment of Islet Morphology ]
Visually assess the morphology of the islets and enter a score in the Islet Scoring Table. For example, if the islets are round and spherical enter a value of 2 for "shape".  If overall the islet borders are well-rounded enter a value of 2 under "border".  If the integrity of the islets are in-between (not fragmented and not solid/compact), enter a value of 1 under "integrity".  If there a few small dithizone-stained islets of less than 25µm in diameter, then mark a 1 under "Islets Islet scoring table  ABCDEFGHI1Shape (3D)BorderIntegrityislets &lt;25µmclumping&nbsp;&nbsp;
2Flat/planar - 0
irregular - 0
fragmented - 0
many - 0
many - 0
&nbsp;&nbsp;
3in between - 1
in between - 1
in between - 1
a few -1
a few -1
&nbsp;&nbsp;&nbsp;
4spherical - 2
well-rounded - 2
solid/compact - 2
almost none - 2
almost none - 2
Total scorePurity (%)Trapped (%)&nbsp;
5&nbsp;
&nbsp;
&nbsp;
&nbsp;
&nbsp;
&nbsp;&nbsp;
&nbsp;
&nbsp;
ABCDEFGHI1Shape (3D)BorderIntegrityislets &lt;25µmclumping&nbsp;&nbsp;
2Flat/planar - 0
irregular - 0
fragmented - 0
many - 0
many - 0
&nbsp;&nbsp;
3in between - 1
in between - 1
in between - 1
a few -1
a few -1
&nbsp;&nbsp;&nbsp;
4spherical - 2
well-rounded - 2
solid/compact - 2
almost none - 2
almost none - 2
Total scorePurity (%)Trapped (%)&nbsp;
5&nbsp;
&nbsp;
&nbsp;
&nbsp;
&nbsp;
&nbsp;&nbsp;
&nbsp;
&nbsp;
?. [Assessment of Islet Morphology ]
Islet Equivalent DeterminationDate(dd/mmm/yyyy)Suspension volume (mL)100Sample volume
  (ml)0.05Multiplication
  factor2000purityIEQ size range (µm)Conversion factor to correct to 150µmCount 1Count 2Mean CountCorrected Mean50-1000.1671a2a(1a+2a)/2Mean * 0.167100-1500.6481b2b(1b+2b)/2Mean * 0.648150-2001.6851c2c(1c+2c)/2Mean * 1.685200-2503.51d2d(1d+2d)/2Mean * 3.500250-3006.3151e2e(1e+2e)/2Mean * 6.315300-35010.3521f2f(1f+2f)/2Mean * 10.352350-40015.8331g2g(1g+2g)/2Mean * 15.833 IEQ in sampleTotal of corrected means = sum of above cellsTotal IEQTotal of corrected means * multiplication factor
Date(dd/mmm/yyyy)Suspension volume (mL)100Sample volume
  (ml)0.05Multiplication
  factor2000purityIEQ size range (µm)Conversion factor to correct to 150µmCount 1Count 2Mean CountCorrected Mean50-1000.1671a2a(1a+2a)/2Mean * 0.167100-1500.6481b2b(1b+2b)/2Mean * 0.648150-2001.6851c2c(1c+2c)/2Mean * 1.685200-2503.51d2d(1d+2d)/2Mean * 3.500250-3006.3151e2e(1e+2e)/2Mean * 6.315300-35010.3521f2f(1f+2f)/2Mean * 10.352350-40015.8331g2g(1g+2g)/2Mean * 15.833 IEQ in sampleTotal of corrected means = sum of above cellsTotal IEQTotal of corrected means * multiplication factor
?. [Assessment of Islet Morphology ]
Example of Islet Equivalent DeterminationExample isolationDate01-Jan-2000Suspension volume (mL)100Sample volume
  (ml)0.05Multiplication
  factor2000IEQ size range (µm)Conversion factor to correct to 150µmCount 1Count 2Mean CountCorrected Mean50-1000.167596461.510.271100-1500.64825232415.552150-2001.685565.59.268200-2503.5655.519.250250-3006.315121.59.473300-35010.352100.55.176350-40015.8330000IEQ in sample 68.989 Total IEQ137,977
Example isolationDate01-Jan-2000Suspension volume (mL)100Sample volume
  (ml)0.05Multiplication
  factor2000IEQ size range (µm)Conversion factor to correct to 150µmCount 1Count 2Mean CountCorrected Mean50-1000.167596461.510.271100-1500.64825232415.552150-2001.685565.59.268200-2503.5655.519.250250-3006.315121.59.473300-35010.352100.55.176350-40015.8330000IEQ in sample 68.989 Total IEQ137,977
?. [Determination of the number of Islet Particles (IP) and the Islet Particle Index (IPI)]
First, determine the total number of islet particles (IP). From the above table, total all the values in the "Mean Count" column and then multiply this value by the multiplication factor.    example (from above). - (61.5 + 24 + 5.5 + 5.5 + 1.5 + 0.5) * 2000 = 197,000 IPNext, calculate the islet particle Index by Dividing the number of Total Islet Equivalents by the number of Islet Particlesexample (from above) - 137,977/197,000 = 0.70 IPIAn IPI value of less than 1.0 means the islets tend towards the smaller end of the size distribution spectrum, whereas those with an IPI of greater than 1.0 tend towards the larger size range.