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EP-4377666-B1 - APPARATUS AND METHOD FOR PREPARATION OF A BIOLOGICAL SAMPLE FOR ANALYTICAL OR DIAGNOSTIC PURPOSES

EP4377666B1EP 4377666 B1EP4377666 B1EP 4377666B1EP-4377666-B1

Inventors

  • Franchi, Gian Martino
  • CARBONE, ROBERTA
  • SCARINCI, Daniele Timothy
  • LECCARDI, Matteo Bruno
  • STEPIEN, Grazyna Elzbieta
  • BERTINI, Claudia

Dates

Publication Date
20260513
Application Date
20220728

Claims (15)

  1. An apparatus (1) for the automated processing of a sample of body fluid, preferably of whole blood, for clinical analysis, in particular of liquid biopsy, which apparatus (1) comprises: ▪ a box-like body (10) comprising an internal chamber (20) and an opening (12) for accessing said internal chamber (20), ▪ first housing means (31) configured to house said sample in said internal chamber (20), ▪ second housing means (33) configured to house reagents in said internal chamber (20), ▪ third housing means (32) configured to house a planar support suitable for receiving cells of said sample, ▪ fourth housing means (31p) configured to house a container apt to contain the liquid fraction of said sample, ▪ means for collecting and dispensing the sample and/or the reagents and/or intermediate preparations of the sample into said internal chamber (20), ▪ centrifugation means (40) operatively associated to said collecting and dispensing means, ▪ a control unit configured to control said collecting and dispensing means and said centrifugation means (40) according to an automated protocol, characterized in that the overall configuration of the apparatus being such that said automated protocol allows to obtain from the fluid sample entering said internal chamber (20) two or more informative components of the fluid sample and a waste component, said two or more informative components comprising a first component, a second component and/or a third component of the fluid sample separate and distinct from each other, wherein said first component includes cells of the sample immobilized on said planar support, wherein said second component includes suspension sample cells and wherein said third component includes the sample liquid fraction contained in said container, in particular a test tube.
  2. The apparatus (1) according to claim 1, wherein said collecting and dispensing means is further configured to stabilize said first component by collecting and dispensing a fixing solution.
  3. The apparatus (1) according to claim 1 or 2, wherein said collecting and dispensing means is further configured to stabilize said third component by collecting and dispensing a fixing solution.
  4. The apparatus (1) according to anyone of the preceding claims, operatively associated to a device configured to count the number of cells, in particular of white blood cells, present in the fluid sample.
  5. The apparatus (1) according to anyone of the preceding claims, wherein said dispensing means is configured to dispense a single uniform layer of cells in a substantially not overlapped way on the planar support.
  6. The apparatus (1) according to anyone of the preceding claims, wherein said collecting and dispensing means comprises reading means configured to identify the body fluid sample, the reagents, the first component, the second component and/or the third informative component.
  7. The apparatus (1) according to anyone of the preceding claims, wherein said collecting and dispensing means is configured to work with replaceable tips, chargeable in fifth housing means (34).
  8. The apparatus (1) according to anyone of the preceding claims and further comprising a filtering system (50) configured to confine and filter the vapours generated by the reagents in said internal chamber (20).
  9. A system comprising an apparatus (1) for the automated processing of a sample of body fluid, preferably of whole blood, for clinical analysis, in particular of liquid biopsy, according to any one of claims 1 to 8, and at least a planar support intended to receive the first component and comprising a surface for immobilizing cellular elements, the surface being treated with nano-materials.
  10. A method for the automated preparation of cells immobilized on a planar support, of suspension cells and of the liquid fraction starting from a body fluid sample, through an apparatus according to any one of claims 1 to 8, which method comprises the steps of: a) providing and introducing the fluid sample in first housing means present in the internal chamber (20) of said apparatus (1), b) collecting the sample from said first housing means (31) and dispensing it in centrifugation means (40), c) centrifuging the sample to obtain a component comprising cells and a component comprising the liquid fraction, d) collecting the component comprising the liquid fraction from the centrifugation means (40) and dispensing it in a container, in particular a test tube, housed by additional housing means (31p) present in the internal chamber (20), e) collecting the component comprising cells from the centrifugation means (40) and, alternatively or in combination, dispensing it - on a planar support housed by additional housing means (32) present in the internal chamber (20), - in an additional container, in particular a test tube, housed by said additional housing means (31p) f) immobilizing said cells on said planar support.
  11. The method according to the preceding claim, further comprising a step of providing reagent elements, in particular a fixer solution, in third housing means (33) of said internal chamber (20) and a step g) of administering the fixing solution to said first component and/or to said third component to obtain a stabilization thereof.
  12. The method according to claim 10 or 11, further comprising a step of counting the cells present in the fluid sample to obtain a reference value and of dispensing a number of cells on said planar support depending upon said reference value.
  13. The method according to any one of claims 10 to 12 wherein the body fluid sample is a sample of whole blood and said third component comprises plasma.
  14. The method according to the preceding claim wherein the centrifugation step c) allows to obtain two initial components, in the form of an intermediate preparation comprising cells and a component comprising plasma, wherein downstream of said step d) il method further provides of d') collecting reagent elements and dispensing it in said intermediate preparation, d") centrifuging the intermediate preparation to obtain an intermediate component substantially comprising white blood cells, and wherein the step e) comprises the fact of collecting the white blood cells from the centrifugation means (40) and dispensing on said planar support one single uniform layer of substantially not overlapped white blood cells.
  15. The method according to any one of claims 11 to 14, wherein the reagent elements comprise Phosphate Buffer Saline, Red Blood Lysis Buffer, solution containing formaldehyde.

Description

Technical field of the invention The present invention relates to the field of the apparatuses and methods for preparation of biological samples for subsequent analyses or for diagnostic purposes. In particular, the present invention relates to an apparatus allowing the automated and standardized processing of a body fluid sample, preferably for analysis or diagnosis based upon the liquid biopsy, near the point for collecting the starting sample and configured so as to provide informative and stable components of said sample, particularly suitable to analysis of biological contents correlated to multiple conditions, thereamong tumour or degenerative or infective pathologies, or pathologies which can be detected in prenatal context. Background In the clinical contexts it is known that the preanalytical phase represents a crucial moment within the activities for analysing the biological samples, since it comprises different procedure passages in which the incidence of different variables may affect the quality of the sample itself, the results of the subsequent analyses and their usability. The suitability and acceptability of the sample are fundamental components of the preanalytical phase thereon the variability of the results of the laboratory investigations are to be parameterized. The preanalytical variables are several and include biological, physiological events and technical modes related to the collection, preparation, preservation and transportation of the sample which, often, determine uncertainties, errors and, sometimes, an unwished expense item by the laboratory for the management thereof. The above-mentioned variables may affect in an important way the result of the laboratory tests by adding an additional uncertainty factor with respect to the obtainable results or they may cause responses not corresponding to the real biological situation of the patient, in particular in the diagnoses based upon the so-called liquid biopsy. The liquid biopsy relates to that field of diagnostics, mainly oncology diagnostics, aimed at identifying and/or characterizing biological components through the analysis of samples of body fluids, such as for example the peripheral blood, and in particular of the elements correlated to the pathology, for example with the tumour, released in the blood circulation. The analyses of liquid biopsy can be aimed at identifying and characterizing circulating tumour cells (CTC) or foetal cells, in case of prenatal diagnosis, or of other cell-free elements (for example nucleic acids, proteins, exosomes) which are extracted from the (mainly acellular) liquid component of the sample after having eliminated the cellular component; the suspension cellular component can be used for extremely informative genetic analyses to characterize the mutational state of genome DNA and to compare it with the mutational features identified in the fraction of the cell-free DNA present in the liquid component. In the suspension cellular fraction genetic mutations can be found which have no pathological meaning and then their identification is fundamental not to run into false positives: this phenomenon, which has frequency increasing with age of individual, is known as CHIP (Clonal Haematopoiesis of Indeterminate Potential) and represents one of the major sources of wrong diagnosis. The suspension cellular component can further be used for proteomics and transcriptomics analysis of the immune cells. The three components, however, have sample preparation needs and analysis modes different from each other which limit the possibility of obtaining clinical information from all of them without having to recourse to multiple collections. In order to maintain the integrity and composition of the sample, the known apparatuses and methods used in the sample preparation preanalytical activities generally face problems due to causes very different from each other, for example at the preservation temperature, the possible presence of preservatives, mechanical/physical stresses due to centrifugation, filtration, freezing, transportation and type of used test tubes/containers. As from the very moment of the collection, the body fluid starts to deteriorate and several degradation phenomena may be observed. The cells modify their morphology and disintegrate, by releasing the genetic material contained inside thereof, which mixes with the one circulating in the liquid component. The not cellular material present in the liquid component (exosomes, proteins, vesicles, free nucleic acids), for example in plasma in case of blood collection, starts to damage, with consequent information loss, and then it requires the use of preservatives to stop or at least to slow down the process. However, these substances can interfere with the cellular biology, for example by changing the morphology thereof or by interfering with the protein expression. For example, some types of preservative are contraindicated in case one wishes to p