Metabonomics SOP

 

Safety Protocols

Everyone working in the NMR laboratory in ‘Statler Commissary’ is expected to act professionally and environmentally responsible. Our laboratory is committed to minimize the risk of personal and environmental hazards involved in working with high-field magnets and handling biological fluids. Thus, the protocols herein are established. These protocols are specifically tailored to our laboratory and subject to change if better procedures are identified in minimizing such risks.

Contact information

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Dr. Khalid Ahsan

• Chemistry Department Safety Officer
• ahsan@buffalo.edu
• 716-645-4115

Dr. Leonard Borzynski

• Manager, Biosafety Division
• Environment, Health & Safety (EH&S)
• lenb@buffalo.edu
• 716-829-3301; 716-829-2401 (sharps injury reporting)

University Police

• 716-645-2222

Mr. Ryan Sajdak

• PhD student
• rasajdak@buffalo.edu
• 716-474-1916

Ms. Victoire-Grace Karambizi

• MS student
• victoire@buffalo.edu
• 716-507-3880

Dr. Dinesh Sukumaran

• Director, NMR Center
• dks@buffalo.edu
• 716-725-1107

Dr. Thomas Szyperski

• Principal Investigator
• szypersk@buffalo.edu
• 716-472-7075

Szyperski Lab

• 716-645-4302, 716-645-4303, 716-645-4304, 716-645-4312

University at Buffalo Health Services

• 716-829-3316

Millard Fillmore Suburban Hospital

• 1540 Maple Road, Williamsville, NY 14221
• 716- 568-3600

STERICYCLE

• http://www.stericycle.com/medical-waste-disposal/customer-pickup-request.html
• 1-866-783-7422

NMR Safety Rules

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Cryogenic liquids

1. No person is allowed to use cryogenic liquids without training and permissio
2. Dr. Sukumaran should be notified first before any attempt to use cryogenic liquids
3. Protective glasses and thermal gloves should be used when working with cryogenic liquids
4. In the event of a magnet quench, all occupants must exit the NMR lab immediately
5. Any accident of exposure to cryogenic liquids must be reported to Dr. Sukumaran and Dr. Khalid Ahsan.
   

Magnetic materials

1. Persons with metallic implants (e.g. cardiac pacemakers, orthopedic pins, plates, arterial clips, etc.) must keep at least 10 feet distance from the magnets
2. Metal and magnetic objects (e.g spatulas, carts, keys, ATM cards, wristwatches, laptops, magnetic tapes, disks, etc.) must be left in the wet lab room or on the table outside the NMR room
   

Physical contact

1. Don’t lean on the magnet
2. Use the stairs provided for the spectrometers when loading the sample
   

Injury reporting

1. Report any injury to Dr. Sukumaran and Dr. Ashan

General Rules

1. In the event that the sample was broken inside the probe, immediately inform Dr. Sukumaran. Isopropyl alcohol must be used as a cleaner and disinfectant for the probe. Don’ t use bleach! Probe cleaning should only be performed with Dr. Sukumaran. If the sample is serum and if the probe/spectrometer needs fixing, inform the person who will fix the problem the potentially hazardous nature of the sample before he starts to work.
2. Use personal protective equipment (e.g. double purple nitrile gloves, lab coat, safety glasses).
3. Prepare 10% (v/v) bleach-water solution before handling serum samples. Keep the solution only for a week.
4. For NMR tube breakage, recover as much sample as possible from the tube, disinfect the broken tube by soaking it in freshly prepared bleach solution and disinfect the surface. Throw the disinfected glass fragments in the broken glass container.
   
5. In the event of blood serum spill, cover the spill with freshly prepared bleach solution and let it stand for at least an hour before wiping it off with paper towels. Redisinfect the surface with freshly prepared bleach solution. Throw the used paper towels in the biohazardous waste container.
6. If you cut or puncture yourself while handling potentially biohazardous biofluids, wash the ezposed area with soap and water and seek medical attention from Milliard Filmore Suburban Hospital or from Health Services as soon as possible (refer to Safety Protocols for NMR for injury logs and reporting).

Biosafety

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Safety training

Schedule for a discussion on the following should be made with Mr. Garcia (et. al.)

1. Exposure Control Plan
2. Metabonomics lab set up in Statler
3. Occupational Safety and Health Administration standards Regulations (Standards - 29 CFR) Bloodborne Pathogens
4. Questions regarding other aspects of biosafety can be directed to Mr. Leonard Borzynski of EH&S.

Training records

Write a paragraph on the WIKI regarding the date, location, resource person and bullet points of biosafety topics discussed

Vaccination

There are three shots that need to be legally administered to people who are front liners (have direct physical contact with blood and blood products):

1. Undergrad/Graduate students: Call University at Buffalo Health Services to schedule an appointment.
2. Post-docs: Make sure that you have an active health insurance. Search online for health providers that are affiliated with your current health insurance and schedule an appointment for vaccination. If you have a family doctor, he is also allowed to administer the shots, if he agrees.

Universal safety precautions and lab safety policies

1. Prepare 10% (v/v) bleach-water solution before handling serum samples. Keep the solution only for a week. Fill out the form that is posted beside the metabonomics hood for bleach solution preparation indicating the date and name of person who prepared it
2. Wash hands with hand soap and water before and after sample preparation
3. Wear double purple nitrile gloves whenever you are working with blood and other potentially infectious materials
4. Wear full body lab coats at the time
5. Wear safety glasses
6. Dispose used pipet tips, Eppendorf tubes and gloves that came in contact with serum and serum products in their designated biohazardous waste container. Contact STERICYCLE to schedule for pick up if these containers get filled. Note that the schedules should be made at least 2 days prior to pick up dates.
7. Disinfect contaminated safety glasses, lab coats and clothing that were contaminated with serum and serum products by soaking them in a freshly prepared 10% (v/v) bleach-water solution
8. Eating and drinking is strictly prohibited inside the NMR lab
9. In the event of blood serum spill, cover the spill with freshly prepared bleach solution and let it stand for at least an hour before wiping it off with paper towels. Redisinfect the surface with freshly prepared bleach solution. Throw the used paper towels in the biohardous waste container.
10. For NMR tube breakage, recover as much sample as possible from the tube, disinfect the broken tube by soaking it in freshly prepared bleach solution and disinfect the surface. Throw the disinfected glass fragments in the broken glass container
11. In the event that the serum sample was broken inside the probe, immediately inform Dr. Sukumaran. Isopropyl alcohol must be used as a cleaner and disinfectant for the probe. Don’ t use bleach. Probe cleaning should only be performed with Dr. Sukumaran. If the probe/spectrometer needs fixing, inform the person who will fix the problem the potentially hazardous nature of the sample before he starts to work
12. During data collection, post notes announcing that data acquisition for a potentially hazardous sample is on going
13. Wipe the NMR tube with Kimwipes before loading the sample to minimize transfer of dirt to the probe
14. If you generate garbage (e.g. used paper towels, Kimwipes, parafilms, or any clutter in general), throw them in regular waste bin
15. Clean your working area before leaving the lab

Injury logs and reporting

1. In case of fire or magnet quench, call the University police, Dr. Sukumaran and Dr. Szyperski.
2. If you cut or puncture yourself while handling potentially biohazardous biofluids, wash the ezposed area with soap and water and seek medical attention from Milliard Filmore Suburban Hospital or from Health Services as soon as possible.
3. Report the incident to Dr. Szyperski, Dr. Ashan and Dr. Sukumaran
4. You should also contact EH&S (sharps injury reporting)
5. Fill out an accident report form (http://hr.buffalo.edu/files/phatfile/Workers_Comp.pdf) and follow the procedures therein
6. Record the incident in ‘Sharp Injury Log book’

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NMR

Sample Storage and Preparation

Serum

Microflow Probe

Each serum specimen was thawed at room temperature inside a hood and prepared by combining 27 μL of serum and 3 μL of ‘lock solution’ (27 mM formate dissolved in D2O containing 0.9 % NaCl). The resulting solution was filtered through a barrier tip (catalogue # 87 001-866; VWR International, West Chester, PA, USA) into a 12 x 32 mm glass screw neck vial (Waters Corp., Milford, USA) by centrifugation. Additionally, 10 μL of the thawed serum was snap-frozen in liquid Nitrogen then stored at -80^oC until needed for MS analysis. The remaining thawed serum was snap-frozen and stored at -80^oC until needed for cryogenic probes data collection.

Cryoprobe

The serum was thawed at room temperature in a hood and prepared by combining 119 μL serum and 51 μL ‘lock solution’ (D2O containing 0.9 % NaCl) and transferred to a NMR thick-walled tube (inner diameter = 2.2 mm). The specimens were snapfrozen in the NMR tube after data collection and stored at -80^oC.

Data Acquisition

Profiling of serum

Microflow Probe

I. The proper functioning of the instrumentation is verified in five different steps:

1. Flow cell and path are cleaned and checked for proper functioning,
2. B0-field homogeneity is optimized by manual shimming,
3. the sample temperature (T = 25.0 ± 0.2 oC) is confirmed by recording a 1H spectrum for methanol,
4. the 1HDO signal line width (LW) and line shape detected for 99.96% D2O are assessed (acceptable values: at 50% of signal maximum< 1 Hz; at 10% < 3 Hz; at 0.55% < 10 Hz; at 0.11% < 20 Hz),
5. the S/N value of the anomeric 1H signal detected for a 10mM sucrose standard solution is measured (acceptable S/N ≥ 40).

II. The system performance for data collection for bio-fluid is evaluated in three steps:

1. A 1D 1H-NMR spectrum is recorded with 32 scans for a sample containing 27 mM of the internal standard formate dissolved in D2O containing 0.9% NaCl, after (i) the probe is tuned, and (ii) the 90o pulse width is determined, so that the LW and S/N of the formate 1H signal can be measured (acceptable values: S/N ≥ 150:1; LW at 50% of signal maximum < 1.2 Hz; at 10% < 5 Hz; at 0.55% < 15 Hz; at 0.11% < 30 Hz),
2. A sample of sterile-filtered fetal bovine serum (FBS) containing ‘lock solution’ (9:1 v/v, the ‘lock solution’ contains 27 mM of the internal standard formate dissolved in D2O containing 0.9% NaCl) is loaded in to the probe. The NMR probe is tuned, a 1D 1H-NMR spectrum without pre-saturation is recorded and the LW of the 1HDO signal is assessed (acceptable values: at 50% of signal maximum < 30 Hz; at 10% < 70 Hz), 3. for the FBS sample, the 90o 1H pulse width is determined and saturation parameters (saturation ‘carrier’ 1H frequency and saturation power) are optimized by recording standard 1D 1H NMR spectra with pre-saturation of the residual water line. The minimal saturation power is chosen which ensures that the intensity of the residual water line is lower than the intensity of the intensity of the internal standard.

III. The NMR data collection for biological specimens is pursued in eight steps:

1. The serum/plasma specimens are prepared as described in section 1.1.6.2.1,
2. The specimens are loaded in the auto sampler,
3. The NMR acquisition parameters obtained with the sterile FBS sample (see II. above) are used to set-up 1D ‘NOESY’ and 1D ‘CPMG’ 1H NMR data acquisitions,
4. The characteristics of the formate resonance line are assessed for the spectrum recorded for the first specimen (acceptable values: S/N ≥ 120:1 after 256 scans ; line width at 50% of signal maximum <1.5 Hz; at 10% < 5 Hz; at 0.55% < 15 Hz; at 0.11% < 30 Hz),
5. If the values obtained in step III.4 are acceptable, data collection continues for all remaining specimens, with D2O being used as the ‘push solvent’ and the probe being cleaned between recording of spectra with 2% Zymit™-H2O (Catalogue number Z-9701-12; International Products Corporation, Burlington, NJ), and rinsed with D2O,
6. After completion of data collection, proper spectrometer performance is confirmed by repeating step II.1 (see above),
7. The probe is cleaned using a solution of 1 M KOH in ethanol, formic acid/acetonitrile and 2% Zymit™-H2O followed by a D2O rinse,
8. The quality of all spectra is assessed by repeating step III.4 for each of the spectra

Cryogenic Probe

I. The proper functioning of the instrumentation is verified in four different steps:

1. The VT settings ( VT unit = 15 oC, VT air = 15, VT cooling air = 20 ) are verified and the temperature is measured accurately by recording a 1D 1H NMR spectrum for a 10 mM TmDOTP5- sample after which the chemical shift (relative to TSP) of the H6 is determined (acceptable value: -151.93±0.03 ppm) ,
2. B0-field homogeneity is optimized by manual shimming with a 1% CHCl3-acetone-d6 sample. Monitor the improvement of lineshape by overlaying the spectra (acceptable values: at 50% of signal maximum < 0.6 Hz; at 0.55% < 8.1 Hz; at 0.11% < 18.0 Hz). Note: Make sure that the probe is ‘detuned’,
3. The S/N value of the quartet around 1600 Hz detected for 0.1% ethyl benzene-CDCl3 and measured at 1200 – 4000 Hz spectral range is assessed (acceptable value: 1300:1),
4. The (S/N) value of the anomeric 1H signal comprising two scalar coupling fine structure components detected for a 2 mM sucrose standard solution is measured (acceptable S/N ≥ 140:1) within the 3000Hz – 5000 Hz spectral range. Additionally, the linewidth for each signal component and minimum between the two signal components are assessed (typical values: 1.5 Hz and < 20% from the baseline, respectively).

Operator Certification

It is important to assess not only the variability arising from the specimen collection, sample preparation, NMR data collection and processing but also to evaluate the variability arising from the operator pursuing the SOP. The operator-associated variability can be used to decide if one is ‘certified’ to pursue NMR-based metabonomics research, or if further training is required.

1. Read the Magn. Reson. Chem. 2009 paper (Sukumaran, DK; Garcia, E; Hua, J; Tabaczynski, W; Odunsi, O; Andrews, A; Szyperski, T. Standard operating procedure for metabonomics studies of blood serum and plasma samples using a 1H-NMR micro-flow probe. Magn. Reson. Chem. 2009, 47, S81-85) as an introduction to the certification procedure.
2. Perform the necessary checks to ensure proper functioning of the instrumentation (see SOP for 1H NMR data acquisition, Section I) and evaluate performance of the spectrometer for biofluids (see SOP for 1H NMR data acquisition, Section II.
3. Acquire 1D 1H NOESY and 1D 1H CPMG spectra for fetal bovine serum (FBS) (or any other test samples provided there is ample number of spectra (at least about 10 spectra) acquired by a certified operator on the same spectrometer and the test samples you want to use were prepared as one batch with those used by the certified operator) that were prepared in the same batch as those that were used by the certified operator. Also, make sure that you use the same type of NMR tube as the ones used by the certified operator. The trainee should collect spectra for at least 5 FBS samples per set up.
4. Perform spectral quality assessment (see SOP for 1H NMR data acquisition, Section IV.4) after data collection for each sample.
5. Process the 1D 1H NMR spectra (see SOP for data processing). Perform a PCA using SIMCA-P for your processed spectra and the spectra of a certified operator.
6. Repeat steps 2 – 5 at least 3X wherein someone else uses the spectrometer in between your set ups.
7. Certification is granted if all spectra from all set ups are indistinguishable from those acquired by the certified operator (i.e., your spectra should be scattered around the spectra of the certified operator within the 95 % ellipse of PCA score plots, see Figure 3.2.2 for example).
8. Bin (bin size = 0.005 ppm, unless other specification is asked for) your processed spectra together with the spectra acquired by the certified operator and send it to the statisticians (Prof. Chris Andrews and Mr. Su Qian, Department of Biostatistics, University at Buffalo) for additional analysis (if necessary) and for certification confirmation.