Required from Customer

• Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
• Molecular mass (exact mass) of the test compound and its salt form
• MSDS and handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

• Percent of hemolysis in each sample
• Rate of hemolysis, if applicable

Substrate

• Test compound dissolved in DMSO applied to a suspension of red blood cells in isotonic buffer or plasma. The final dosing concentration is typically 10 uM or the clinical C_max

Assay System

• A suspension of red blood cells from a species specified by the customer
• The positive control: red blood cells treated with 0.1% SDS (sodium dodecyl sulfate) to produce 100% hemolysis
• The negative control: red blood cells treated with DMSO

Assay Conditions

• Prepare three separate test tubes (N=3) containing red blood cells dosed with the test compound dissolved in DMSO
• Incubated at 37°C in a water bath with mild agitation
• Sample each replicate at 0, 5, 15, 30, 60, 120 and 180 minutes
• Centrifuge each sample
• Measure the absorbance of the supernatant from each sample using a UV/Vis spectrometer
• Calculate percent hemolysis at each sampling time point

Assay Dynamic Range

• Hemolysis can be determined from between 10 and 100% with a typical precision (N=3) of 10%

Assay QC

• Positive and negative controls run with each compound

Notes

1. This assay can also be run in plasma rather than buffer if the customer wants to include the effects of protein binding. The concentration of SDS in the positive control will have to be adjusted accordingly to achieve 100% hydrolysis of the positive control.
2. The percent hemolysis is calculated using the following formula:
   Percent Hemolysis = 100 x [(Absorbance of sample – Absorbance of negative control) ÷ (Absorbance of positive control – Absorbance of negative control)]

Options

1. The customer may select:
   • either the basic, standard or custom report format
   • the species of blood donor plasma or isotonic buffer
2. The customer can request:
   • additional replicates
   • the use of a control buffer in which the stability of the test compound is measured
   • the concentration of the test compound