# US 12,181,387 B2 · System and method for sample preservation

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- Number: US 12,181,387 B2
- Document ID: US12181387B2
- Status: Granted
- Kind: B2
- Application number: 18/120,887
- Filed: 2023-03-13
- Published: 2023-07-06
- Granted: 2024-12-31
- Earliest claimed: 2016-12-22
- Assignees: Ventana Medical Systems, Inc.
- Applicants: Ventana Medical Systems, Inc.
- Inventors: Daniel Bauer; David Chafin; Michael Otter
- Web page: https://patentgrep.com/patent/US12181387B2

## CPC

- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- G01N 1/31: Apparatus therefor
- G01N 29/024: by measuring propagation velocity or propagation time of acoustic waves
- G01N 29/07: by measuring propagation velocity or propagation time of acoustic waves
- G01N 29/4418: {with a model, e.g. best-fit, regression analysis}
- G01N 29/4427: {with stored values, e.g. threshold values}
- G01N 29/4472: {Mathematical theories or simulation}
- G01N 2001/305: {Fixative compositions}
- G01N 2291/011: Velocity or travel time
- G01N 2291/023: Solids
- G01N 2291/02475: Tissue characterisation
- G01N 2291/02809: Concentration of a compound, e.g. measured by a surface mass change
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method is disclosed that permits calculation of reagent concentrations (in SI units) over time and space within a tissue sample as the sample is immersed in the reagent and the reagent diffuses into the tissue sample. The disclosed method has yielded the surprising result that once a formaldehyde concentration at all points within a tissue sample exceeds about 90 mM during a cold step of a cold+hot fixation protocol, the hot step of the fixation protocol can be commenced to provide reliable detection of molecular targets and preservation of tissue morphology in downstream analyses.

## Claims

### Claim 1

A method for fixing a tissue sample, comprising:
immersing the tissue sample in a cold formalin solution until a point at the center of the tissue sample is determined to have a formaldehyde concentration of at least 90 mM, wherein the immersing of the tissue sample in the cold formalin solution is performed under conditions sufficient to diffuse the formaldehyde to the center point of the tissue sample; and
contacting the tissue sample with a hot formalin solution once formaldehyde concentration at the point at the center of the tissue sample is determined to be above 90 mM for a time sufficient to form cross-links, thereby fixing the tissue sample;
wherein the cold formalin solution has a temperature of between about 0° C. and about 15° C., and
wherein the hot formalin solution has a temperature of between about 20° C. and about 55° C.

### Claim 2 (depends on 1)

The method of claim 1, wherein the immersing in the cold formalin is conducted until the formaldehyde concentration at the point at the center of the tissue sample is determined to have a formaldehyde concentration of at least 100 mM; and wherein the contacting of the tissue with the hot formulation solution is performed after the concentration of formaldehyde at the point at the center of the tissue sample is determined to be above 100 mM.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cold formalin solution has a temperature of between about 2° C. and about 15° C.

### Claim 4 (depends on 1)

The method of claim 1, wherein the contacting of the tissue sample with the hot formalin solution comprises raising the temperature of the cold formalin solution while the cold formalin solution remains in contact with the tissue sample.

### Claim 5 (depends on 1)

The method of claim 1, wherein the formaldehyde concentration is determined by a method selected from time-of-flight (“TOF”), radio-label tracing, mid-infra-red (“IR”) spectroscopy, and magnetic resonance.