The Autoradiography Lab at QPS designs well-planned study protocols to address the needs of each client. The FAQs below address the most common practices.

1. What imaging hardware and software do you use for QWBA studies?
A: We utilize Leica CM3600 and CM3050 Microtomes, Molecular Dynamics Typhoon Phosphor Image Scanners, and three MCID Image Analysis Systems.

2. How many phosphor imaging plates do you have?
A: Currently, we have 30 Large format Fuji Imaging plates for 14C, and we maintain at least ten 20 x 40 cm Fuji TR plates for tritium. (Note: Tritium plates can be used only once, and the cost will be charged to the client.)

3. Where do you store your imaging plates during exposure?
A: In a lead exposure box. QPS has two large lead boxes, 2"-3" thick to reduce the effects of background radiation on autoradiographic image formation.

4. What is your typical exposure time?
A: 3-4 days for 14C, 125I and 7-14 days for 3H.

5. How many Cryomicrotomes do you run simultaneously?
A: Currently, one. However, a second one is coming in 2008.

6. What type and size tape do you use for whole body section collection?
A: Our standard tape is 3M, 810 (3"), but per client request, we can also order and use TESA, Instrumedics transfer tapes or WBA MAG Nakagawa tapes.

7. Do you use a sectioning tool to collect sections?
A: Typically, yes.

8. How do you verify section thickness consistency both within a section and throughout a study?
A: With Internal blood standards (IS) that involve holes drilled into the block and filled with whole blood containing known amounts of radioactivity. This can be tailored to the client's needs and is addressed in the protocol, but in general, analysis of the section thickness of every section will be performed by comparing the characteristics of the internal standards within sections and among different sections to determine if significant variations in section thickness exist. Density value/mm2 values of the IS must be within 15% of each other to be deemed acceptable.

9. How do you quantitate a tissue such as kidney that has heterogeneous distribution?
A: As per protocol, this typically includes: "renal cortex," "renal medulla," "renal papilla" or the entire cross section as "kidney."

10. What is your standard tissue list for quantitation?
A: The following is our default tissue list. However, the protocol drives our sampling and is determined/approved by the client as requested.

Adipose (brown and white), adrenal gland, blood, brain (cerebrum, cerebellum, medulla), bone, bone marrow, cecum and contents, epididymis, esophagus, eyeball (Uveal tract, aqueous humor, lens), Harderian gland, heart, kidney (cortex, medulla, papilla and entire section), large intestine and contents, liver, lung, lymph node submaxillary, pancreas, pituitary gland, prostate gland, salivary gland, seminal vesicles, skeletal muscle, skin, stomach (and contents), small intestine (and contents), spleen, spinal cord, trachea, thyroid, and urinary bladder (and contents).

11. How do you establish limits of detection?
A: The upper and lower limits of quantitation are determined by calibration curve regression analysis characteristics over the dynamic range (similar to that used for other bioanalytical techniques). Curves must be linear (correlation coefficient of > 0.9900 using a weighted linear regression) over the range.

12. What kind of standards do you use?
A: Radiolabeled blood standards made in-house. We use three different types of standards: a set for construction of the calibration curve (ten @ ~1-40,000 nCi/g), a set for checking the curve characteristics (four @ ~1-25,000 nCi/g), and internal standards (one @ ~ 1,000 nCi/g) for checking section thickness.

13. If you use commercially available standards, do you calibrate them in-house?
A: Not routinely, but if we use them, they will be calibrated / validated before use.

14 What acceptance criteria are used to determine if curve is usable or not?
A: Curves must be linear (correlation coefficient of > 0.9900 using a weighted linear regression) over the range.

15. When establishing calibration curves, what is the minimum number of standards used to construct curve?
A: Eight, but we routinely use 15 across the range of ~ 0.0008 -8.0 mCi/g tissue.

16. How do you prepare phosphor imaging plates?
A: Plates are cleaned with ethanol and "erased" for 10 minutes prior to exposure. All plates are tested for uniformity every year using a Raytest Imaging Plate uniformity standard. Tritium plates are purchased new for each study and used once only.

17. How many QWBA personnel do you currently have?
A: Eight dedicated to autoradiography.

Q: What is your current capacity to conduct QWBA studies?
A: We anticipate the ability to conduct >50 QWBA studies annually.

18. Do you routinely calculate dosimetry?
A: Yes, we use both the "MIRD" and/or "Marinelli" equations as accepted by the FDA ("equation 1" as described in: Dain, Collins and Robinson (1994). Pharmaceutical Research, 11(6):925-928).

19. What program are you using to calculate dosimetry?
A: Tissue pharmacokinetic parameters are determined in WinNonLin, and the predicted human exposure is determined using either of the equations cited above.

20. Do you currently exsanguinate your animals prior to freezing?
A: This is protocol driven by the sponsor, but typically, animals will not be exsanguinated. We would obtain 1-2 mL of blood for plasma and blood radioactivity LSS determinations if requested.

21. How do you currently euthanize your QWBA animals?
A: Deep anesthesia with isoflurane and death by snap-freezing, but this can also be altered to suit sponsor needs.

22. Have you validated your computer image analysis system and how?
A: The complete system validation was completed at the end of 2002. The study design employed an IQ/OQ/PQ approach in a "Black Box" testing design. Tests were designed to measure User Specifications, System Specifications and Functions, and stress tests in an integrated system (hardware and software).

23. Do you have the capability to conduct infusion studies in rodents?
A: Yes, we have performed rodent infusions up to 24 h using a variety of formulations, and we have performed numerous direct brain infusions in rats over a 7 day period.

Related Information:

Tissue Distribution and Autoradiography

QWBA Tissue Distribution and Quantitative Whole Body Autoradiography