Humidity stabilization during the preparation of biological samples for spectrometry

Abstract

The invention proposes preparing biological samples for spectrometry which contain cell structures and/or whole cells of human or animal origin (e.g. thin human and animal tissue sections) or prokaryotes (e.g. microorganisms), and which require constant relative humidity, in a temperature-controlled gas volume whose humidity is determined by a saturated substance solution, for example a suitable salt solution. The invention exploits a physico-chemical phenomenon called “deliquescence”, which manifests itself by keeping the relative humidity above the saturated substance solution constant with a high degree of precision when a specified temperature is maintained. Pure succinic acid exhibits deliquescence at approx. 99% relative humidity, for example. Since an enormous variety of deliquescent salts and other suitable substances are available, it is possible to find the suitable substance for almost any desired relative humidity, with adjustment of the temperature, where necessary.

Claims

1. A method for the preparation of a biological sample of human or animal origin for spectrometric analysis, comprising: providing the biological sample of human or animal origin; subjecting the biological sample to a preparation which renders it fit for a following spectrometric analysis, wherein the preparation takes place in a gas volume in which the relative humidity is kept constant by deliquescence; and transmitting the prepared biological sample to spectrometric analysis.

2. The method according to claim 1, wherein the biological sample is a thin tissue section and the spectrometric analysis is imaging mass spectrometry.

3. The method according to claim 2, wherein the imaging mass spectrometry works with ionization by matrix-assisted laser desorption in a time-of-flight mass spectrometer.

4. The method according to claim 2, wherein the preparation comprises chemical or enzymatic conversions of molecules in the thin tissue section, where a set level of humidity causes the tissue to swell to the desired extent, which is advantageous for the chemical or enzymatic reactions while largely maintaining the positional accuracy of the molecules.

5. The method according to claim 4, wherein the swelling of the tissue is assisted by a hygroscopic substance which is applied in a measured quantity.

6. The method according to claim 2, wherein the tissue is provided in chemically fixed form, for example fixed by formaldehyde.

7. The method according to claim 6, wherein the tissue is provided embedded in an organic solid material, such as paraffin.

8. The method according to claim 2, wherein the tissue is provided in frozen form.

9. The method according to claim 1, wherein macromolecules of the biological sample are broken into fragments by enzymatic digestion.

10. The method according to claim 1, wherein, during preparation, the temperature of the biological sample is briefly reduced to below the dew point to facilitate the controlled formation of a cohesive film of moisture on the surface of the sample.

11. The method according to claim 1, further comprising setting a high level of humidity just below the dew point.

12. The method according to claim 11, where the relative humidity is set to be equal to or higher than 95%.

13. The method according to claim 1, wherein the preparation of the biological sample takes place directly on the same sample support which subsequently serves as substrate for a mass spectrometric or infrared-spectrometric measurement.

14. The method according to claim 13, wherein the sample support takes the form of a flat steel plate, or flat ceramic plate/glass plate having a conductive coating.

15. The method according to claim 1, wherein the gas volume is supplied with a gas flow which has been brought to a specified relative humidity by interaction with a saturated solution of a deliquescent substance.

16. A preparation chamber to carry out a method according to claim 1, which comprises a gas-tight chamber, a holder for the sample support, a device for controlling the temperature of the chamber, and a dish for a saturated solution and a deliquescent substance to be dissolved.

17. The preparation chamber according to claim 16, further comprising a lock for the introduction and removal of the sample support.

18. The preparation chamber according to claim 16, further comprising a device for gently agitating the saturated solution and/or a device for gently agitating gas in the chamber.

19. A The method according to claim 1, wherein the biological sample contains cell structures and/or whole cells of human or animal origin.

20. A method for the preparation of a prokaryotic or fungi biological sample for a spectrometric analysis, comprising: providing the prokaryotic or fungi biological sample; subjecting the prokaryotic of fungi biological sample to a preparation which renders it fit for a following spectrometric analysis, wherein the preparation takes place in a gas volume in which the relative humidity is kept constant by deliquescence; and transmitting the prepared prokaryotic or fungi biological sample to spectrometric analysis.

21. The method according to claim 20, wherein the preparation of the prokaryotic or fungi biological sample is carried out on living microbes, which are incubated on a sample support plate in droplets of a nutrient liquid.

22. The method according to claim 21, wherein the droplets contain a measured quantity of an anti-microbial substance in order to allow for susceptibility testing.

23. The method according to claim 20, wherein the preparation of the prokaryotic or fungi biological sample is followed by infrared spectrometric analysis, whose aim is to determine a subspecies or variety of the sample.

24. The method according to claim 20, wherein the relative humidity is kept at about between 10% and 30%.

Description

BRIEF DESCRIPTION OF THE ILLUSTRATIONS

(1) FIG. 1 shows the image quality of mass spectrometric images of tissue sections as a function of the relative humidity during preparation of the tissue sections using enzymatic digestion. The best image quality is achieved at high relative humidity slightly below the dew point (95%<rH<dew point), particularly because the digestion times necessary in this case can be kept relatively short and therefore the radius of the lateral material transport, which degrades the image quality, can be kept small.

(2) FIG. 2 shows the relative humidities (DRH—deliquescence relative humidity) above saturated solutions for different salts as preferred deliquescent substances (taken from SaltWiki).

(3) FIG. 3 presents a flow diagram of a procedure for preparing an FFPE thin human or animal tissue section for imaging mass spectrometry.

(4) FIG. 4 shows a schematic representation of a thermally insulated chamber (16) including lid heating (14) and floor heating (15), whose floor is covered with crystalline salt (10) and a layer of water (11), for the preparation of a cell sample of human or animal origin or a prokaryotic sample (13) on a sample support (12), said sample requiring a specific humidity. The sample (13) can be a thin human or animal tissue section and the sample support (12) can be suitable for mass spectrometry (e.g. MALDI time-of-flight mass spectrometry).

(5) FIG. 5, panels A, B and C, illustrates the introduction of a sample support into an incubation chamber in which the humidity is stabilized by deliquescence using a lock.

DETAILED DESCRIPTION

(6) The invention is based on “deliquescence”, a relatively little known physico-chemical phenomenon. The deliquescence manifests itself here in that a gas volume above a saturated salt solution gives rise to a relative humidity that can be kept constant with high precision when a specified temperature is maintained. If the relative humidity in the gas volume increases, water condenses into the saturated solution, but the resulting higher degree of dilution is compensated by the dissolution of more salt. If the relative humidity in the gas volume decreases, water evaporates from the saturated solution, but the oversaturation thus created is reduced again by the recrystallization of the salt.

(7) Deliquescence is not the same as hygroscopicity. Hygroscopic materials reduce the humidity by removing water from the ambient air, but this water is bound by the materials, for example as water of crystallization or in pores. This process is therefore not easily reversible, and is thus susceptible to failure if it is necessary to set and maintain constant humidity conditions.

(8) Deliquescence is important in the building materials industry, which bears no relation to the field of the present disclosure, because it can cause moisture damage to buildings especially in freezing conditions. FIG. 2 depicts the deliquescence relative humidity (DRH) for several salts as a function of temperature.

(9) The invention now proposes that sample preparation which requires a constant relative humidity should be carried out in a temperature-controlled gas volume in which the humidity is maintained at a constant value by deliquescence, for example in a gas volume above a saturated salt solution. Since an enormous variety of salts and other suitable (water-soluble) substances are available, it is possible to find the suitable substance for almost any desired relative humidity, using specific temperature settings, where necessary.

(10) As can be seen from FIG. 1, high relative humidity just below the dew point is particularly important for the preparation of thin human and animal tissue sections including enzymatic digestion for imaging mass spectrometry. FIG. 3 shows a flow diagram of a possible procedure for preparing an FFPE thin human or animal tissue section that comprises the following steps. Step 300: Mount the section onto the specimen slide or sample support, e.g. a stainless steel plate; Step 302: Deparaffinize; Step 304: “Antigen retrieval”, basically a heat treatment or other energy treatment to partially break open formalin-induced cross-links in the section; Step 306: Spray on the enzyme (in a digest buffer, usually ammonium bicarbonate), e.g. trypsin; Step 308: Incubate in a humid atmosphere close to the dew point maintained by deliquescence; Step 310: Dry; Step 312: Spray on the matrix substance, e.g. sinapinic acid, α-cyano-4-hydroxycinnamic acid (α-CHCA), or 2,5-dihydroxybenzoic acid (DHB); and Step 314: Measure a distribution chart of analytes of interest, such as peptides and proteins, from the prepared section mass spectrometrically, e.g. using MALDI TOF.

(11) In the temperature range between 37° C. and 50° C., which is typically used for the digestion, a saturated solution of potassium sulfate (K.sub.2SO.sub.4), which maintains a humidity of around 98.0% at 37° C., and around 97.2% at 50° C., is suitable, for example (see curve 6 in FIG. 2). The times needed for a digestion leading to a quasi-optimal image are a few hours. At higher temperatures, which essentially entail higher (detrimental) diffusion rates for lateral molecular transport, shorter times are possible. The optimum temperatures and digestion times are preferably determined experimentally.

(12) FIG. 4 shows a rough schematic representation of a thermally insulated chamber (16) for the preparation of a cell sample of human or animal origin or a prokaryotic sample (13) on a sample support (12) which requires a specific humidity. The sample (13) can be a thin human or animal tissue section and the sample support (12) can be suitable for mass spectrometry (e.g. MALDI time-of-flight mass spectrometry). All holders for sample supports and chamber and all means of opening the chamber have been omitted for greater clarity. In this embodiment, two heating elements (14) and (15) heat the lid and the floor of the chamber. The floor is covered by crystalline or crystallized salt (10) and a saturated aqueous solution (11) of the salt. It does not matter if salt (10) protrudes from the solution. It is advantageous for the boundary surfaces between salt solution and air, and between solid salt to salt solution to be as large as possible. The aqueous solution should only form a comparatively thin layer so as to rapidly facilitate the compensation for evaporation or condensation by further dissolution or recrystallization of salt, without the salt molecules having to traverse long diffusion paths in the liquid. It is also possible to agitate the solution continuously (albeit gently), for example by stirring, preferably such that the formation of salt aerosol is avoided. For a similar reason, the gas in the chamber can also be carefully kept in motion, for example by a small fan (not shown).

(13) The deliquescence maintains the gas volume above the saturated solution at constant humidity, and practically no measurable control oscillations exist. To disturb the equilibrium of the relative humidity as little as possible when the sample support is introduced and removed, it is favorable for the sample support (12), which has been brought up to temperature, to be introduced into/removed from the chamber (16) through a suitably designed lock.

(14) FIG. 5, panels A, B and C, shows schematically the introduction of a sample support (204) carrying the sample, such as a human or animal tissue section or a microorganism culture in droplets of liquid nutrient, into an incubation chamber (200). The incubation chamber (200) is kept at constant humidity by means of deliquescence and shut off from the ambient atmosphere by lock chamber (202) and gates (206) and (208). For introducing the sample support (204) into the incubation chamber (200) from outside, gate (208) is opened while gate (206) remains closed, panel A. Once the sample support (204) has reached the lock chamber (202), gate (208) is closed and the conditions in the lock chamber (202) are harmonized with that in the incubation chamber (200) in order to be able to introduce it without affecting the conditions in the incubation chamber (200). Then gate (206) is opened in order to transfer the sample support (204) from the lock chamber (202) into the incubation chamber (200), panels B and C. The perturbation of the gas atmosphere in the incubation chamber (200) during the introducing (and in reverse sequence also during the taking out) of the sample support (204) can be kept small by the attached lock chamber (202) and gates (206, 208).

(15) If a lock is used for the introduction of the sample support, then the sample support carrying the sample is preferably first brought up to a temperature which is slightly above the temperature of the gas volume in order to prevent any condensation of water on the sample. This may take place in the lock, for example. If no lock is used, but instead a lid or a door to the chamber is opened, the humidity equilibrium is unavoidably disturbed initially. However, the disturbed humidity equilibrium can be used to bring the sample support, which is placed onto a solid holder at the target temperature, up to temperature before the humidity is restored again. For example, it is possible to equip the chamber with a lid or a door which can be controlled to close slowly. Condensation can also be avoided in this way.

(16) Instead of the thermally insulated chamber, a chamber (16) which contains no heating elements and no external insulation can simply be placed into an incubator (indicated by the outer, hatched wall in FIG. 4).

(17) With enzymatic digestion of the proteins of a thin tissue section, it is important that the thin tissue section is kept swollen as specified to allow the enzyme to penetrate into the thin section and act. The swelling is produced by the humidity but can also be improved by further measures. The application of a measured quantity of hygroscopic substances, for example glycerin, which is also familiar as a moisturizing agent in cosmetics, can increase the water content of the thin section. The hygroscopic substance can, for example, be sprayed on, highly diluted, together with the enzyme as a component of the enzyme buffer. The hygroscopic substance can, however, also be sprayed onto the thin section separately from the enzyme, in a rapidly evaporating solvent, for example acetone, in a carefully chosen dose.

(18) If the temperature of the sample support bearing the thin tissue section is reduced below the dew point, droplets do not form immediately, but instead a cohesive film of water forms on the surface of the already swollen tissue. This can be utilized to increase the moisture in the tissue. The procedure sometimes slightly reduces the temperature of the sample support for a short time, but it immediately returns to the original temperature again. This can be achieved by a heating element, such as a Peltier element, under the sample support plate, for example. This method practically pumps a small amount of moisture into the tissue. The optimal parameters are preferably explored experimentally.

(19) Other types of sample preparation for spectrometric analyses may also require stable humidity, however. If, for example, microbes have to be incubated for several hours in tiny droplets of a nutrient solution on the (flat) mass spectrometric or infrared spectrometric sample support, high humidity is necessary to ensure that the droplets do not dry up. On the other hand, uncontrolled condensation of water into the droplets should be avoided, too. In this case also, the deliquescence above a saturated solution of a suitable substance, e.g. potassium sulfate, proves to be advantageous and suitable. Incubation can serve to analyze the ability of the microbes to grow in the presence of specific anti-microbial substances such as antibiotics and antimycotics, while their resistance to these antimicrobial substances can be determined.

(20) In addition to this preferable, stationary method of stabilizing the humidity, it is also possible to conceive other methods using deliquescence, for example a dynamic method where a little water vapor is continuously fed into the gas volume, while the excess moisture is continuously absorbed by the saturated solution.

(21) A further, non-stationary method involves using deliquescence to bring a gas flow to a constant relative gas humidity and feeding this gas flow into a chamber in which the constant gas humidity is required for the preparation of biological samples. The gas flow can pass through a chamber containing a saturated solution, for example, as has been described above. The gas flow can, however, also be blown slowly, and in the form of small bubbles, through a gas washing bottle containing a saturated solution of a deliquescent substance, where a filter preferably cleans the gas flow of any entrained particles or droplets of solution. An impactor arrangement in the gas pipe can also be suitable to separate the humidified gas from any suspended particles of heavy substances.

(22) The patent specification DE 10 2013 022 016 B4 (equivalent to WO 2015/090727 A1), “Microbe identification by mass spectrometry and infrared spectrometry”, M. Kostrzewa, describes how IR spectrometry can be used for the identification of microbe subspecies or varieties. A method of exploring the subspecies level of Yersinia enterocolitica in a prevalence study using infrared spectrometry can be taken from I. Stamm et al., Journal of Clinical Microbiology, March 2013, Volume 51, Number 3, p. 887-893. It has meanwhile been found that it is necessary to keep the humidity constant when acquiring infrared spectra of samples containing tissues or microbes, but in this case at a set value specified for the method, preferably in the range between 10% and 30% relative humidity. Only when this humidity is precisely set, can the spectra be accurately reproduced and reliably compared with reference spectra which were acquired at the same relative humidity. If the humidity changes, the spectrum changes; if one returns to the specified humidity, the original IR spectrum returns also. In this case, a small air flow can be guided through a chamber which contains a saturated solution of a suitable substance having a large surface area, see for example curve 7 in FIG. 2 (magnesium chloride hexahydrate, MgCl.sub.2.Math.6H.sub.2O), and then the air flow can be fed at a set relative humidity to the sample in the infrared spectrometer. The sample here must be in a suitable vessel in order to seal it off from the ambient air. This type of identifying analysis of microbial subspecies and varieties is increasing in importance.

(23) The types of application and embodiments described here form only a fraction of the possible methods which are made available by the invention. With knowledge of this disclosure, the person skilled in the art will easily be able to develop further advantageous embodiments for preparation methods of biological samples which contain cell structures and/or whole cells of human and animal origin and/or prokaryotes for spectrometric measurement while maintaining a specific, stable humidity, and these shall be included in the scope of protection of the claims, including any equivalent implementations as the case may be.