Claims:
1. An apparatus (100) for horizontal extraction of a thin component layer of a buffy coat from an anticoagulated centrifuged blood sample without being contaminated comprises:
a modified centrifuge tube (300) having transparent side walls (341) provided with a polymeric section (343);
a handle (101) extended at a back of the apparatus (100), wherein the handle (101) is adapted for 3 axis of movement with respect to a vertical cylindrical barrel (102);
a vertical cylindrical barrel (102) to support the modified centrifuge tube (300);
a holder (103) adapted to hold an extraction device (111) by means of a locking assembly rigidly connected to a viewfinder and aligner module (104), and slidably moving the extraction device vertically and horizontally towards as well as away from the modified centrifuge tube (300);
an extraction device (111) adapted to extract the thin component layer;
a driving knob (105) located at a bottom of the apparatus (100) and attached to a linear mechanism (400) to adjust a position of the holder (103); and
a viewfinder and aligner module (104) to which the holder (103) is rigidly connected,

wherein the buffy coat is horizontally and accurately extracted from the anticoagulated centrifuged blood sample by (i) adjusting the holder (103) to a level of buffy coat layer by means of the driving knob assembly (105), (ii) focusing at the buffy coat through the viewfinder and alignment module (105), and (iii) piercing a needle of the extraction device (111) through the polymeric section (343) of the modified centrifuge tube (300).

2. The apparatus as claimed in Claim 1, wherein the holder (103) comprises a locking flange (201) to grip the extraction device (111); a knob (202) for axial traversing of the extraction device (111); and the horizontal cylindrical barrel assembly to hold the extraction device (111).

3. The apparatus as claimed in Claim 2, wherein the horizontal cylindrical barrel assembly comprises of an internal horizontal cylindrical barrel (203) to hold the extraction device (111); and an external horizontal cylindrical barrel (204) for axially traversing of the internal horizontal cylindrical barrel (203).

4. The apparatus as claimed in Claim 1, wherein the locking assembly comprises a rotatable locking knob (107) having a slitted cylindrical end on one side and threaded end on the other side.

5. The apparatus as claimed in Claim 1, wherein the linear mechanism (400) comprises of a shaft (401) to connect the rotary knob (105) to a driving gear (402); a driven gear (403) connecting a lead screw (404) to convert rotary motion to linear motion.

6. The apparatus as claimed in Claim 1, wherein the viewfinder and alignment module (104) comprises of a removable lens assembly (109) with magnification lens selected from a group comprising fresnel lens, spherical lens, cylindrical lens or similar lens thereof.

7. The apparatus as claimed in Claim 6, wherein the magnification lens is provided with a polymer film having markings.

8. The apparatus as claimed in Claim 7, wherein the markings (108) on the magnification lens are selected from gridlines, crosshair, and such.

9. The apparatus as claimed in Claim 1, wherein the polymeric section (343) is provided with a self-resealing polymeric material.

10. The apparatus as claimed in Claim 1, wherein the modified centrifuge tube (300) is of a shape selected from as a curved bottom test tube, an hourglass with a narrow central region, a zig-zag sidewall, and such.
, Description:FIELD OF INVENTION:
[001] The present invention generally relates to separation and selective horizontal extraction of component layers or fractions from a multicomponent suspension. The present invention particularly relates to a system and a method of extraction of buffy coat layer from blood samples.

BACKGROUND & PRIOR ARTS:
[002] Centrifugation is a technique employed for separation of a substance of different shape, size, density and/or viscosity from the solution by the application of centrifugal force. When the solution is subjected to the rotation around a central axis, the denser substance is pushed outwards at the periphery while less dense substance rotates around the central axis.

[003] Biologists and chemists often use this technique applications such as purification of two miscible liquids, alcohol clarification, purification of mammalian cells, making of products such as skimmed milk by removing fat from milk, preparation of platelets rich plasma for transfusion, analysis of body fluids such as urine, blood, bone marrow aspirates etc., purification of proteins and extraction of lipoproteins, cells, organelles, RNA, DNA, etc. This technique is also useful in nuclear reactor for fuel enrichment.

[004] The centrifugation method is routinely used for testing of blood samples and extraction of buffy coat, peripheral blood mononuclear cells (PBMCs), RBCs, plasma separation etc., for various laboratory analysis and procedures. The buffy coat is a thin layer of blood sample separating plasma at the top and granulocytes-RBCs at the bottom, when the anticoagulated blood sample is subjected to differential centrifugation. The buffy coat occupies less than 1 percent of the total volume of anticoagulated blood. The buffy coat contains white blood cells (WBCs) and platelets, and is widely studied for various laboratory purposes.

[005] Extraction of the really thin buffy coat layer sandwiched between the plasma and the RBCs is commonly carried out by arresting the centrifuged tube on a stand and slowly aspirating with a pipette inserted through the plasma layer on top. This process is tedious, time consuming and requires highly skilled technicians to overcome challenges including poor visibility of the buffy coat layer through the tube while pipetting, reduced stability and control while extracting the buffy coat layer resulting in mixing of various centrifuged fractions and difficulty in selective extraction of buffy coat layer without any RBC contamination. Removal of the top plasma layer or the bottom RBC layer are suggested methods in the art to overcome these issues. However, lack of skill or stability can result in mixing of the buffy coat with other centrifuged fractions requiring more than one centrifugation step for achieving maximum yield and purity of the buffy coat layer.

[006] The extraction of any ultrathin liquid is a cumbersome task for which an expert technician is needed. For extraction of buffy coat, the centrifuged container is held at a neutral position on a stand. A pipette is inserted from the top into the liquid component and the buffy coat is slowly extracted. Several problems are associated with this method. From the top, the buffy layer is not visible. Therefore, locating buffy layer is a cumbersome job. Contamination of the buffy layer is another major problem associated with this method. If not extracted properly and gently, there is always a probability of recirculating platelets back into plasma, thereby contaminating the sample.011405
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[007] Some patent documents have been listed here which speak of extraction of liquid sample, or more particularly blood sample, by piercing the needle from the top of the container into the buffycoat through plasma region without contamination of the sample. EP2940119 relates to a buffy coat extraction kit used for extracting buffy coat from a blood collection for the purpose of autologous blood or PRP (Platelet Rich Plasma) cell regeneration treatment and skin regeneration treatment.

[008] EP1014088 relates to a device and method for separating heavier and lighter fractions of a fluid sample by means of centrifuge device. Referring to figures and description, the device defines a well area 60 having a thin diaphragm or a self sealing septum for directing and receiving the point of a needle to be vertically inserted into and through the stopper. The self-sealing septum material allows penetration by a piercing element such as a needle and then reseals when the piercing element is withdrawn.

[009] US4055501 relates to fluid collection devices and more particularly to blood collection devices having means for partitioning the lighter and heavier phases of blood. Referring to Figures and specification, fluid collection device 10 includes a closure or a stopper 16 which extends into the open end in sealing engagement with the side walls of the tube. The stopper 16 is vertically pierceable by a needle and self-sealing, and may be formed of a suitable elastomer, such as butyl rubber.

[010] US4663032 relates to an apparatus for the decanting, successively, of an upper blood-plasma layer and an intermediate buffy-coat layer through a flexible tube opening into the top of a flexible bag containing blood which has been centrifuged to form these two layers above the erythrocyte or red blood cell concentrate. Referring to Figure 2, a clamping element 8, provided between the upper and lower press plates 3 and 2, is a horizontally movable horizontal plate and can be displaced in the direction 23 to clamp the bag and define two compartments one above and one below the clamping site C. The clamping element 8 is controlled by a sensing device for the blood plasma/buffy-coat interface. Further, the sensing device being provided heightwise in the region of the upper press plate 3 in the form of a horizontally oriented row of infrared light emitters 11 and detectors 12. It may be noted that the liquid is pushed out of the bag by moving the plates to extract the layers of the liquid from the top of the bag.

[011] KR101140551 discloses a Buffy coat extraction kit which is used to extract the buffy coat in the blood, consisting of the kit body, stopper, and the piston, the blood is injected into the buffy coat extraction kit. The buffy coat extraction kit is mounted in a centrifuge to separate blood into layers, and when the buffy coat layer is formed out of the buffy coat portion, the piston is rotated to rotate the buffy coat layer into the buffy coat portion. The buffy coat is extracted with the needle tip of the syringe reaching the buffy coat layer. However, since the buffy coat layer is extracted by inserting a needle from the top of the kit through a plasma layer, the extracted buffy coat may also contain some amount of plasma. This makes the method inaccurate in extracting the buffy coat layer and hence inefficient.

[012] However, these documents pertain to extraction of liquid layer from the top. Hence there is a critical need for a method of locating and extraction of thin component layer or fractions, such as like the buffy coat layer from a centrifuged blood sample, with greater stability, visibility, accuracy, purity, consistency and reproducibility.

SUMMARY OF INVENTION:
[013] Accordingly, the present invention describes an apparatus for horizontal extraction of desired component layer, as well as fraction of interest from a multicomponent system.

[014] The apparatus comprises of a modified centrifuge tube having transparent side walls provided with a polymeric section; a vertical cylindrical barrel to support the modified centrifuge tube; a handle extended at a back of the apparatus; a holder adapted to hold an extraction device by means of a locking assembly rigidly connected to a viewfinder and aligner module and slidably move horizontally & vertically as well as towards as well as away from the modified centrifuge tube; a driving knob located at a bottom of the apparatus and attached to a linear mechanism to adjust a position of the holder; and a viewfinder and aligner module to which the holder is rigidly connected.

[015] The holder comprises a horizontal cylindrical barrel assembly and a locking assembly. The horizontal cylindrical barrel assembly comprises an internal horizontal cylindrical barrel and an external horizontal cylindrical barrel. The internal horizontal cylindrical barrel has a provision for the extraction device to fit in.

[016] The viewfinder and alignment module includes a removable lens assembly having a magnification lens. In an embodiment, the lens is selected from a group comprising fresnel lens, spherical lens, cylindrical lens or similar lens thereof. Further, the lens has a polymer film, which is provided with markings in the form selected from gridlines, crosshair, and such.

[017] The polymeric section in the modified centrifuge tube is provided with a self-resealing polymeric material.

[018] In an embodiment, the multi-component liquid may be a blood sample from which the desired thin buffy-coat is intended to be extracted.

[019] The present invention further describes a method of extraction of a desired component layer from a multicomponent liquid sample, which comprises (i) adjusting the holder to a level of desired component layer by means of the driving knob, (ii) focusing at the desired component layer through the viewfinder and alignment module, and (iii) piercing a needle of the extraction device through the polymeric section of the modified centrifuge tube.

BRIEF DESCRIPTION OF DRAWINGS:
[020] Following figures represent various embodiments, including the preferred embodiment of the invention:
Figure 1 illustrates a schematic representation of an apparatus (100) of the present invention.
Figure 2 illustrates a sectional view of a holder (103).
Figure 3(a) – Figure 3(e) illustrate various designs of the modified centrifuge tube (300).
Figure 4 illustrates mechanism of driving knob (105)

DETAILED DESCRIPTION OF INVENTION:
[021] Accordingly, the present invention discloses an apparatus and a method for horizontal extraction of desired component, and desired specific quantity from a multicomponent suspension which overcomes the drawbacks of the aforementioned separation practices dependent on the centrifugation method. More particularly, the invention discloses an apparatus and a method for horizontal extraction of a buffy coat layer from the centrifuged blood sample.

[022] The present specification describes main embodiment and various optional embodiments, which may be apprehended by the figures appended at the end of the specification. However, the figures do not restrict the scope of the invention.

[023] Figure 1 illustrates an apparatus (100) for horizontal extraction of a desired component from a multicomponent liquid. The apparatus (100) comprises a modified centrifuge tube (300) having transparent side walls (341) provided with a polymeric section (343); a handle (101) extended at a back of the apparatus (100); a vertical cylindrical barrel (102) to hold the modified centrifuge tube; a holder (103) adapted to hold an extraction device (111) by means of a locking assembly rigidly connected to a viewfinder and aligner module (104), and slidably moving vertically as well as towards as well as away from the modified centrifuge tube (300); a driving knob (105) located at a bottom of the apparatus (100) and attached to a linear mechanism (400) to adjust a position of the holder (103); and a viewfinder and aligner module (104) to which the holder (103) is rigidly connected to the said extraction apparatus. The apparatus is further provided with a tube entry barrel (106) to stabilize and hold the modified centrifuge tube (300) in position while the extraction of liquid component is in process.

[024] The handle (101) is ergonomically designed and coated with soft material to gain a firm grip for the user. In a preferred embodiment, the said soft material may be rubber. In another embodiment, the soft material may be TPU or silicone and such. In one of the embodiments, the handle (101) has a freedom of 3 axis movement with respect to the vertical cylindrical barrel (102), which ensures that the vertical barrel is always inline to the gravity irrespective of the handle (101) orientation. The free 3 axis movement is similar to a mechanical a gyroscope instrument having 2-3 gimbals providing pivoted support, which are used in camera stabilizer which ensures that the camera is always aligned to the object of interest irrespective of hand rotations and movements. Such a 3-axis movement mechanism with gimbal provided pivoted support could resemble a Steadicam or instruments having passive gyroscopic effects. The free 3 axis mechanism is packaged between the handle (101) and vertical cylindrical barrel (102) so that the thin component layer and the modified centrifuge tube (300) is always vertical and parallel to the direction of gravity.

[025] The viewfinder and alignment module (104) comprises of a removable lens assembly (109) with a magnification lens, wherein the removable lens assembly (109) is affixed to the holder (103). The lens is selected from a group comprising Fresnel lens, spherical lens, cylindrical lens or similar lens thereof. The said lens in the lens assembly can be removed for cleaning and replacing as needed. The lens preferably has a magnifying power of 20x for a clear view of the desired thin component to be extracted from the modified centrifuge tube (300). The lens further includes a film of polymer with markings (108). The viewfinder and alignment module (104) is positioned in the device such that it has a lot of open space for light to enter easily for better illumination of the sample.

[026] Figure 2 describes the holder (103) that firmly holds the extraction device (111) in place such that the extraction device (111) may be accurately pierced into the modified centrifuge tube (300) for the purpose of extracting the desired component as well as desired specific quantity from the multiple components suspended in the liquid. In a preferred embodiment, the desired component is a buffycoat which is intended to be extracted from the centrifuged blood sample. The holder (103) is rigidly connected to the viewfinder and aligner module (104). The holder (103) is provided with a locking flange (201) to grip the extraction device (111); a knob (202) for axial traversing of the extraction device (111); and the horizontal cylindrical barrel assembly to hold the extraction device (111). The horizontal cylindrical barrel assembly is provided with an internal horizontal cylindrical barrel (203) to hold the extraction device (111) and an external horizontal cylindrical barrel (204) for axially traversing the internal horizontal cylindrical barrel (203).

[027] The locking assembly of the holder (103) includes a rotatable locking knob (107) having a slitted cylindrical end on one side and a threaded end on the other side. At the time when the rotatable locking knob is rotated, the locking flange (201) grips the extraction device (111).

[028] The extraction device (111) is a system which extracts the desired component using a pressure driven flow. In the preferred embodiment, the extraction device (111) is a syringe type device which allows extraction of desired component in a specific quantity through manual plunger movement. In another embodiment, the extraction device (111) is a micro-pipette with adjustable quantity knob, through which user can define the desired quantity before starting the extraction process. This ensures the preciseness of the desired specific quantity of the desired component. In another embodiment, the extraction device (111) is a self-extracting device where desired negative pressure is existing in the chamber and once activated, it takes the desired quantity based on the negative pressure. The activation of the extraction device can be done using knob (202).

[029] In a preferred embodiment, the holder (103) has a manufacturing tolerance of less than 300 µm such that the tip of the needle attached to the syringe in the holder does not wobble during operation. In the preferred embodiment, the manufacturing tolerance of the internal horizontal cylindrical barrel (203) is approximately 200 µm. To prevent additional wobbling of the needle while sliding and piercing the needle entry point, an additional manufacturing tolerance of preferably 50 µm is set for the sliding fit between the locking assembly and the horizontal cylindrical barrel assembly in the holder (103). The holder (103) further has a lubricant coating in the internal horizontal cylindrical barrel (203) as well as the external horizontal cylindrical barrel (204) to enable easy and smooth sliding.

[030] As illustrated in Figure 3, the modified centrifuge tubes (300) may be of different shapes. Figures 3(a) to Figure 3(e) demonstrate shapes ranging from tube with curved bottom, tube having zig-zag shape, tube having hourglass shape, tube with thin diameter and increased height. The said shapes assist in better viewing of the component to be extracted, either by creating an acute angle of the tube at the said component or by reducing the diameter of said tube corresponding to the said component and the length of the fraction is increased for a given volume of the said component.

[031] The modified centrifuge tube (300) is provided with a self-sealing polymeric portion (343) at the central portion of the tube. The said polymeric section of the tube is connected to the rest of the tube material with fusion welding such that there are no leakage while handling and using the said tube for centrifugation, extraction, sample collection, transportation, storage etc.

[032] The component desired to be extracted is denoted by 342(b) in Figure 3(a), 319(b) in Figure 3(b), 331(b) in Figure 3(d), and 322(b) in Figure 3(c) as well as in Figure 3(e).As illustrated in Figure 4, the driving knob (105) is joined to the linear mechanism (400) to translate rotational motion into linear motion. The linear mechanism (400) comprises of a shaft (401) to connect the rotary knob (105) to a driving gear (402); a driven gear (403) engaged with the driving gear (402) & connecting a lead screw (404) to convert rotary motion to linear motion. In another embodiment, the driven gear (403) is connected to the pin slot mechanism.

[033] The method of operation of the said apparatus (100), which is exemplified for buffy coat extraction from the centrifuged blood sample, is as follows:
The whole blood is collected in the modified centrifuge tube (300). The modified centrifuge tube (300) is then centrifuged at the desired speed in the centrifuge such that different components of blood are separated in the modified centrifuge tube (300). The apparatus (100) is held vertically and the modified centrifuge tube (300), with blood components separated, is placed in the vertical cylindrical barrel (102) through the tube entry barrel (106). For the purpose of piercing, the apparatus is held firm at the handle (101) by the operator, and the holder (103) is adjusted by activating the driving knob (105). While observing through the lens assembly (109) in the viewfinder module (104), the driving knob (105) is activated until the crosshair (108) is aligned to the buffy-coat layer.
[034] The extraction device (111), having a pierceable element, is inserted through locking assembly of the holder (103). The extraction device (111) is inserted into the locking assembly until it touches the modified centrifuge tube (300) at the polymeric section (343). The marking (110) defines the entry point of the pierceable element of extraction device (111). The user locks the extraction device (111) using the locking knob (107). Subsequently, the user holds the axial traversing knob (202) and rotates in a direction to move the extraction device (111) towards the modified centrifuge tube (300). In the said movement of extraction device (111), the internal horizontal cylindrical barrel (203) and the external horizontal cylindrical barrel (204) slide relative to each other, thereby achieving perfect linear motion. As the extraction device (111) moves, it pierces the polymeric section (343), as exemplified in Figure 3(a), of the modified centrifuge tube (300) to reach the desired location of the thin component layer (342(b)) for the purpose of extraction. While extraction, the thin layer (342(b)) is monitored by the user from the viewfinder module (104), such that once the thin layer (342(b)) is completely extracted, the user deactivates the extraction device (111). The axial traversing knob (202) is then rotated in the opposite direction to move the extraction device (111) away from the modified centrifuge tube (300). As the rotations of the axial traversing knob (202) are completed, the extraction device is completely taken outside the modified centrifuge tube (300). The self-resealing polymer of the polymeric section (343) ensures that the liquid does not come out of the modified centrifuge tube (300) after the extraction device (111) is taken outside the modified centrifuge tube (300). The extraction device (111) is unlocked by rotating the locking knob (107), and the user takes the extraction device (111) containing the thin layer substance (342(b)) outside the holder (103). The thin layer substance (343(b)) can be extracted to the desired container for the desired test.

[035] The apparatus described in the present specification is advantageous for easy, user friendly, and cost-effective extraction of the buffycoat from the centrifuged blood sample. The apparatus as well as the method described above is particularly advantageous for it aids in horizontal extraction of the buffycoat from the coagulated sample. It may be noted that the apparatus described above and illustrated in figures includes preferred embodiment, and is not limited to the description of the present specification.