What is the principle behind frozen aliquotting?

To collect a frozen aliquot, a hollow stainless steel bit with a serrated tip is used to drill into a sample. Once coring is initiated, the probe spins at a pre-defined speed and is lowered into the sample. As the drill bit bores into the sample, the push rod inside the drill bit is forced up and the hollow bit fills with frozen sample. When coring is complete, the push rod is forced down to expel the frozen core into a destination vial.

What sample types have been cored?

The CXT 353 and CXT 750 Frozen Sample Aliquotters are designed to core a wide range of frozen samples. Sample types cored most often include serum, plasma, whole blood, urine, raw feces, and stabilized feces. The CXT 353 is routinely used to core a wide range of tissue types, including liver, heart muscle, pancreas, and thyroid tissue. The CXT 353 has also been used to core bone (femur). Other sample types that have been cored include breast milk, sputum, saliva, CSF, and buffered solutions, such as PBS.

How are samples and aliquots maintained at -80°C?

The CXT 353 benchtop aliquotter is cooled via liquid nitrogen (LN2) and can maintain temperatures between -80°C and -150°C. Sample and vial fixtures extend into the LN2 tank, maintaining uncored samples and frozen cores at temperature. The CXT 750 Automated Frozen Sample Aliquotter is maintained at -80°C via two mechanical chillers. Sample and destination fixtures are in direct contact with the frozen deck.

What is the typical volume of a frozen aliquot (core)?

Core volume will depend on the vial height, fill volume, and diameter/length of probe used. When using a 2 mL cryovial filled to 1.8 mL with a standard 3mm probe, the volume of a single core typically ranges from 100 µL to 120 µL, depending upon the vial shape. Round-bottom vials enable slightly longer cores than vials with a conical bottom.

How many frozen aliquots (cores) can be extracted from a single vial?

The maximum number of aliquots will depend upon the diameter of the vial. Most standard 1.8 mL to 2.0 mL cryovials can accomodate four to five frozen aliquots. (Four aliquots for applications requiring the greatest core volume consistency). Fewer frozen aliquots can be extracted from narrower cryovials. If using the CXT 353 with a large diameter sample container, a larger number of aliquots can be extracted from each sample.

What is pre-analytical sample QC?

It is generally acknowledged that poor sample handling can affect sample quality and downstream analytical results. Pre-analytical sample QC involves testing a sample to assess general sample quality and identify samples that may have been subjected to poor handling, including long time intervals on the bench or repeated freeze-thaw cycles. This assessment can be used to determine whether or not to maintain or utilize a set of banked samples, to audit sample collection sites, or to determine if particular samples should be omitted from a clinical trial study.

Though standard tests to routinely check for pre-analytical events have not yet been established; clinical chemistry markers(1), mass spec profiling, metabolomics, and even RNA quality have been used to identify suspect samples. With frozen aliquotting, pre-analytical sample QC can be performed without thawing the sample, preserving sample integrity. (1) H.J. Kang, et al. (2013). Biopres. Biobank. 11(2):94-100. Doi:10.1089/bio.2012.005

How can I target specific tissue sections?

With the CXT 353, a laser guide and movable stage enable targeting of specific areas within a tissue sample. Standard probes collect samples 3 mm in diameter, while 1.5 mm probes can collect samples that are just 1.5 mm in diameter.

How long does it take the instruments to cool to temperature?

It takes approximately 1.5 to 2 hours for the CXT 750 Automated Frozen Sample Aliquotter to reach -80°C, depending upon environmental temperature. The CXT 353 reaches temperature (below -80°C) in 15 to 30 minutes.

How frequently do probes need to be changed?

The probes for the CXT 353 instrument are single-use. Probes for the CXT 750 instrument should be discarded after 100 to 200 cores (after every 100 cores if core volume consistency is important for downstream applications).

How does sample homogeneity affect frozen aliquotting?

Scientists at CryoXtract have recently completed several studies looking at the distribution of small and large molecules in frozen biofluid samples. In studies conducted to date comparing center and perimeter cores, horizontal gradients were not observed. Top (50 µL), Mid (100 µL) and full (150 µL) cores were compared to sample remainders and un-cored controls to assess vertical gradients. For the snap freeze condition, vertical gradients were minimal and cores correlated well with liquid samples. Increasing vertical gradients were seen at higher freezing temperatures and lower freezing rates, but drug concentrations from cores were very reproducible and differences between top/mid cores and full cores were very consistent, indicating that frozen aliquots could be integrated into the standard workflow for a validated assay. This will be of particular use for analysis of labile drug compounds.

How large are the instruments?

The CXT 353 Frozen Sample Aliquotter is a bench-top instrument. It is 25.5 in. (71.7 cm) deep, 21.1 in. (53.7 cm) wide, and 28.2 in. (64.8 cm) high. The CXT 750 Automated Frozen Sample Aliquotter is a floor-standing instrument. It is 34.5 in. (84 cm) deep, 52 in. (134 cm) wide, and 71 in. (180 cm) high.

Are there specific facility requirements?

The CXT 353 Frozen Sample Aliquotter requires liquid nitrogen (LN2) along with proper ventilation (minimum of six air changes per hour). A standard electrical outlet can be used for power (90-260V 50/60Hz 15A circuit). For optimal performance laboratory temperature should be 50 - 80°F (10 - 27°C) and relative humidity (non-condensing) should be less than 30%. Higher humidity can cause frost build-up.

The CXT 750 Automated Frozen Sample Aliquotter requires a steady flow of clean, dry air (200 LPM continuous flow). If air supply is insufficient or inconsistent, a compressor (Kaeser Airtower 4C or 5C, or equivanent) is recommended for a single CXT 750 installation. A larger compressor may be required if shared by multiple instruments. The CXT 750 requires two outlets for power (120VAC, 60Hz @ 2 x 20A circuits or 220VAC, 50Hz @ 2 x 16A circuits). The recommended laboratory temperature is 60 - 80°F (16 - 27°C). Ambient temperature below 71.6°F (22°C) is recommended for optimal low-temperature operation. Relative humidity (non-condensing) should be less than 30%. Compressed air requirements will vary based on the relative humidity in the laboratory.







Methodology for Assessing Protein Biomarker Distribution in Frozen Biological Fluids Using Frozen Aliquotting Technology

Researchers at CryoXtract have developed a frozen aliquotting technique for the assessment of protein distribution profiles in a frozen matrix. This technique was used to assess distribution of p-selectin in serum and plasma samples SNAP frozen in LN2 vapor, frozen at -80°C, and frozen at -20°C. Frozen cores collected from the perimeter of frozen samples were compared to center cores to assess lateral distribution. 50µL, 100µL, and full-length cores (~150µL) were compared to sample remainders and un-cored controls to assess vertical distribution. For all conditions, no lateral gradients were observed. SNAP frozen cores correlated well with the un-cored control at all vertical depths. When freezing at -80°C, soluble p-selectin began to settle into the bottom two-thirds of the cryovial. At -20°C, a greater degree of settling was observed. While individual biomarkers should be evaluated for distribution upon freezing and analyte stability, SNAP freezing is strongly recommended in conjunction with frozen aliquotting to preserve sample and analyte integrity. Read more >