GB2461076A - Devices and methods for the separation of blood serum from a blood sample - Google Patents

Abstract

A blood sample from a patient may be taken into a pre-evacuated sample chamber 21 via a double ended needle (27). After centrifugation one separated component may be transferred to a second pre-evacuated sample chamber. Subsequently, the serum 12 may be transferred into a third pre-evacuated sample chamber 20. Transfer between chambers may be executed via double ended needle 27. Also disclosed are: a needle carrier (37); a serum preparation and extraction kit comprising an agitation module (e.g. centrifuge), an evacuated sample tube with end cap seal and a double ended transfer needle (where the centrifuge device may be may be a hand powered spinner with push down drive actuation (40), a handheld elongate sleeve (61) or a rotary rattle, clapper or clacker (50)). The devices and methods may allow blood serum separation to be performed in the absence of other specialised equipment (e.g. away from a laboratory setting).

Description

Serum Separation and Extraction This invention relates to separation and extraction techniques, methodology and apparatus and is particularly, but not exclusively concerned with separation and extraction of serum from a fresh' -ie recently taken -blood sample, including separation and extraction without the benefit of laboratory facilities. Sampling and sample transfer techniques are also addressed. The techniques and apparatus described may admit of wider use.

Term mo logy The terms separation' and extraction' are used herein respectively to identify the mutual physical distancing or isolation of constituent parts and the transfer relocation or removal of a target isolated component for independent assessment or treatment, such as analysis or testing.

Serum' and plasma' have related meanings or significance and in less rigourous usage or according to context may be used interchangeably. Thus generally plasma designates the liquid component of blood, in which the blood cells are suspended and makes up about 55% of total blood volume. It is mostly (some 90% by volume) water, and contains dissolved proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the main medium for excretory product transportation), as well as blood cells.

Serum or Plasma can be prepared by spinning a confined blood sample in a centrifuge until the red blood cells fall to the bottom of the confinement chamber. Separation reflects the strength of centrifugal action. The plasma is then poured or drawn oil the top. Serum reflects plasma without fibrinogen or the other clotting factors. Serum is generally an amber coloured protein rich liquid (including white cells) of which plasma is a colourless component fluid part. The terms plasma and serum are used informally on occasion somewhat

interchangeably within this disclosure.

The terms sample' and sampling' are used herein to embrace taking a fresh sample from a patient and transfer of a sample, in particular while still fresh but after some preparatory separation into component parts.

Serum Extraction Essentially, separation must preface extraction and isolation of the serum.

Separation into Constituents In order to carry out certain diagnostic tests upon a patient blood sample, such as preparatory to therapeutic treatment, it is necessary firstly to separate red blood cells from the serum solution carrier medium', in which they are normally suspended.

Urgency There is some urgency to this separation, as over the course of some 4-6 hours the red blood cells will die -once the glucose nutrient' in the sample is used up' or consumed for nutrition. When cells die, their membranes break down and cell contents are released, a process known as haemolysis, or disruption of red blood cell membranes, causing release of haemoglobin -contaminating the serum composition, and invalidating any subsequent test.

Similarly the concentration of certain solutes which may be subject to testing differs between the serum solution and cell contents, so if haemolysis occurs the outcome of other diagnostic tests would be voided. Thus, for certain biochemical tests, it is a priority to separate serum and blood cells within a critical circa 4 hour time window.

Gravity and Accelerative g' Loading Blood samples will naturally settle over time under their own weight countered by buoyancy, with (heavier I denser) red blood cells sinking to the bottom and the (lighter I less dense) serum floating on the top -but not within the required time window before the onset of deterioration or contamination.

Separation into primary constituents can be hastened by accelerative loading, such as by centrifuging the blood sample. The centrifuge action applies an accelerative or temporarily enhanced apparent or effective weight or so-called g' loading arising by centrifugal or centripetal action of rotation which increases the effective weight and load upon a body mass.

Aside from centrifuges, mechanical or biochemical devices are also known such as floating gates, beads or gels (per Gelss) which separate the red blood cells and blood plasma and/or keep them separated.

(Blood) Sampling Typically, patients might be required to attend a specialist sampling department designated phlebotomy (a title reflecting surgical vein opening to withdraw blood or introduce fluid) department, where a blood sample can be taken and processed immediately with laboratory equipment. While still fresh, the sample taken would be placed into an automated high power centrifuge and processed within an optimum time frame.

Sample Taking -Prefacing Statement of Invention

Although a conventional (piston-in-cylinder I positive displacement) syringe might be employed for the initial sample taking, according to one aspect of the invention, the Applicant envisages an alternative evacuated chamber and double-ended (transfer) needle for sample-taking and a corresponding arrangement for transfer and extraction for isolation of a separated out serum.

Thus a homogenous fresh' blood sample would pass from one pre-evacuated chamber, centrifuged to separate out principal constituents respectively of red blood cells and serum, then the isolated serum component transferred into another pre-evacuated isolation chamber ready for testing.

The degree of pre-evacuation is just sufficient to effect transfer of a modest sample quantity and can be determined empirically so that when the sample is transferred pressure equilibrium with the outside is restored.

Vacuum Tube Prior Art

Vacuum sample tubes are known per se and reflect what might be termed a passive' loading or fill facility reliant upon the pressure differential between outside and within the tube, rather than, say, a separate syringe. An example is the so-called VACUTAINER (Trade Mark).

An initial low or negative pressure, as opposed to a positive or atmospheric outside ambient presssure, engenders an inherent tendency to draw in contents until the pressure differential is consumed or exhausted.

Also known, such as per US 5,181,523 Wendelborn is a blood sampler with pre-evacuated sample chamber and double-ended transfer needle.

Double-Ended (Sampling and Transfer) Needle A double-ended needle is envisaged for selective sampling and transfer, with a facility to puncture and penetrate a barrier membrane, whether it be, say, a closure cap seal (of a sample chamber) or a patient skin tissue.

Different (double-ended) needles can be employed respectively for sample-taking and sample component transfer, to obviate cross-contamination between samples. That is to eliminate residue of a homogenous initial sample from transfer of a separated out serum component.

Dual Separate Needles Deployed Back-to-Back In some aspects of the invention, the Applicant envisages a two-part or back-to-back single sharp or point ended needle, with an abutment base for mounting contact with another corresponding single ended needle, so a pair of corresponding separate needles are co-operatively disposed.

Needle Holder I Carrier A dedicated needle holder or carrier is envisaged with an external profile and cross-section contrived for ease of needle capture, manual grip and manipulation (eg orientation and positioning) -for both vein insertion and between sample chamber transfer.

Remote Blood Collection In practice, blood samples may have to be taken remotely, even away from a hospital site. A sophisticated high-power centrifuge, as used in a phlebotomy department, is too expensive and unwieldy a facility to be of practical use at a remote in the field' collection point. Rather, samples must be despatched by courier to a phlebotomy lab within a critical four hour time window, incurring significant costs.

Local Extraction A physical proximity and short time window correspondence arises. Aspects of the present invention envisage local' separation and extraction of serum from blood into a separate sample tube. Local' means at or close to the point of collection, and without expensive centrifuge equipment -as a both convenient and time-saving measure. Once serum is separated -ie there is no longer a transition interface between serum and red cells, and even more so once serum is extracted into isolation, the risk of contamination and degradation is alleviated, so the urgency to test the sample is alleviated. The serum can be tested at the point of collection, or the sample can be stored and taken to the phlebotomy lab at a convenient point -say as part of a weekly collection.

Statement(s) of Invention

Separation + Extraction Method A method of serum separation and extraction directly and promptly upon taking a blood sample comprising the steps of loading a sample chamber, such as from a syringe or other transfer device* used to take a blood sample; agitating the sample, such as by spinning in a centrifuge, to effect separation into red blood cell and serum (concentration) regions; inserting a transfer tube into the sample chamber, with one end immersed within a serum concentrate region and the opposite end within a pre-evacuated isolation chamber, using the relative pressure differential between chambers to transfer only serum to the isolation chamber.

Syringe plunger (positive-pressure) displacement and/or optional pre-evacuation of the sample chamber may be employed for original sample taking from a patient vein and/or to load or transfer to a sample chamber.

Separation and Extraction Modules The Applicant envisages a simple, but effective, low-cost, manually-driven, portable centrifuge separator module, used in conjunction with a simple, robust separation and isolation facility, such as a pre-evacuated sample draw-up and transfer module.

Manual Centrifuge Module A manually driven centrifuge separator configured as a rotary rattle or clapper format with a radial arm carrying a sample chamber, mounted upon a handle as a rotational axis, to swing around an operator.

Drive Action A manual centrifuge drive could feature a push I press-down or reciprocal pump action linear to rotary actuator drive with automatic release, as with spiral twist coupling of toy spinner tops. A spiral drive rotary whisk mechanism could be adopted. Alternatives would be a tethered restraint or tied catapult style rotation.

Embodiments There now follows a description of some particular embodiments of the invention, by way of example only, with reference to the accompanying diagrammatic and schematic drawings, in which Figures 1A to 1 B show transfer of a live' blood sample from a syringe 10 to a sampler 20 which may be pre-evacuated; more specifically...

Figure 1A shows a syringe filled with a patient blood sample, juxtaposed to a pre-evacuated sealed sample tube; Figure 1 B shows insertion of the syringe needle through a resilient end cap seal of the sample tube, and transfer of syringe contents into the tube.

Figures 2A to 2B show centrifuging of the sample tube of Figure 1 B; more specifically...

Figure 2A shows the sample tube of Figure 1 B, containing a live blood sample, being subjected to a centrifugal spin action; Figure 2B shows the sample tube of Figure 2A after centrifuge, the blood sample has separated into respective concentration regions of two primary components, namely red blood cells and serum.

Figures 3A to 3B show extraction and isolation of the serum layer from the sample tube of Figure 2B with a transfer syringe; more specifically Figure 3A shows a sterile syringe needle, inserted through the resilient end cap seal 22 of the sample tube 21 of Figure 2B into the serum layer 12. The syringe plunger 26 is pulled out, drawing out the serum into the syringe barrel chamber 24 up to the inboard end of the syringe needle 28; Figure 3B shows the syringe of Figure 3A containing serum, juxtaposed with the sample tube 21 of Figure 3A, with the majority of the serum layer 12 having been transferred.

Figures 4A to 4D show serum extraction and isolation (from an original blood sample) with a bespoke transfer tube configured as a double-ended needle 27 and a pre-evacuated sampler 20 with a tubular chamber body 32; more specifically Figure 4A shows a sample tube 21 containing a separated blood sample with separated red blood cell and serum layers 11, 12 juxtaposed with an elongate hollow transfer tube with opposed needle ends 27; Figure 4B shows insertion of one needle 27 end into the sample tube 21, to a point between the top and bottom of the serum layer 12. The opposite outboard' needle 27 end is juxtaposed with a pre-evacuated sterile sample isolation chamber 32; Figure 4C shows the outboard needle 27 end of Figure 4B pierced through a resilient end cap seal 22 of the sample chamber 21 of Figure 4C, and serum layer 12 in transit between sample and isolation chambers; Figure 4D shows a the needle 27 of Figure 4C withdrawn from the sample chamber leaving predominantly red blood cells 11 along with residual serum 12; and separate isolation sample chamber 32 with isolated a pure serum sample 12; Figures 5A to 5D show a push-button manually-operated centrifuge; more specifically...

Figure 5A shows a manually -operated centrifuge 40 with a base platform 42 surmounted by rotatable centre turntable 44, with a sample tube 21 end capture throat fitment 43 and top drive plunger actuator 41; juxtaposed with a sample tube 21 containing a live blood sample; Figure 5B shows the sample tube 21 fitted to the centrifuge centre portion 43, 44. Downward force applied to the drive plunger 41 by an operator results in rotation of the turntable 44 and temporarily conjoined sample tube 21; Figure 5C shows spring return action of the drive plunger 41, ready for the next cycle of downward force applied by an operator (not shown); Figure 5D shows the centrifuge of Figure 5A, at a standstill following rotation, with the entrained sample tube 21 still in situ, but with its contents separated out internally; Figures 6A to 6C show a hand-operated centrifuge configured in the format of a rotary rattle, clapper or clacker 50; more specifically Figure 6A shows a centrifuge rattle 50 comprising a rotary radial arm 52 mounted upon a handle 51, with a cut-out well compartment 53 configured to receive and locate a sample chamber 21; juxtaposed with a sample tube 21 containing a live (initially homogenous) blood sample; a demountable restraint (not shown) might be used to constrain the sample tube 21 in place; Figure 6B shows rotary action of the centrifuge 50 of Figure 6A, with the sample tube 21 inset within outward end compartment 53 in a rotating arm 52; Figure 6C shows the centrifuge of Figure 6B, upon removal of the sample tube 21 with its contents separated out.

Figures 7A to 7C show an elongated restraint sheath 61 and agitator grip with an wide opening, snug-fit end for sample chamber restraint upon agitation, such as in particular being flung around in a catapult centrifuge manner; by an operator hand 42 and arm movement 30 of an operator more specifically....

Figure 7A shows an agitator or centrifuge restraint sheath 61 with an inboard end access opening or port for insertion and withdrawal of a sample tube 21; Figure 7B shows the sample tube 21 at the outward end of the restraint sheath 61 of Figure 7A being swung in circular or orbital motion by hand; Figure 7C shows removal of the sample tube 21 from the restraint sheath 61, the sample contents having been separated out by centrifugal agitation; Figures 8A through 8C show a variant of the sample transfer of Figures 4A through 4D using carrier for a double-ended transfer needle; more specifically Figure 8A shows a needle carrier 24 juxtaposed with a centrifuge sample chamber 21; Figure 8B shows one end of a transfer needle 27 inserted into an end seal cap 22 of a centrifuge sample chamber 21; with the carrier 24 being used for safe manual handling support and manipulation with reduced risk of inadvertent skin puncture and thus flesh wound infection; and juxtaposition of an evacuated sample isolation chamber 21; Figure 8C shows insertion of an evacuated sample isolation chamber 21 in the outboard end of the transfer needle 27; Figures 9A through 9C depict a sequence of blood sample taking from a subject patient arm vein 71 using a double-ended needle 72 mounted in a carrier tube 24 configured to accommodate a pre-evacuated sample tube 21; More specifically Figure 9A shows a double-ended needle 72 with one outboard end juxtaposed with a patient arm 71 and an opposite end disposed within a carrier 24 configured as a hollow open ended tube 24 with a top aperture to receive a sample tube bounded by an edge rim; Figure 9B shows the needle 72 having punctured the skin and penetrated the arm into a vein, and a pre-evacuated sample chamber configured as a closed-ended tube 21 with an end cap seal juxtaposed with the access port aperture of the needle holder 24; Figure 9C shows the sample chamber 21 disposed within the carrier 24 with the projecting end of the needle 72 having punctured an end cap seal and entered the sample tube 72 to draw up a blood sample 10 from a vein in the patient arm 71; Figures 1 OA through 1 OC show in 2-D side elevation a follow-on sample separation and transfer sequence from the initial patient sample taking steps of Figures 9Athrough 90; More specifically Figure 1 OA shows the initial sample chamber 21 after centrifuge agitation, to separate the homogenous blood sample into concentrate regions of serum 12 and red blood cells 11; juxtaposed with another double-ended needle 27' mounted in a carrier tube 24'; Figure lOB shows the needle 27' mounted in carrier 24' embedded though and end cap seal 22 of the sample chamber 21 with its lower end penetrating a serum concentrate region 12; Figure 1OC shows another pre-evacuated sample chamber 21" mounted upon the needle 27" within the carrier 24' with the upper needle 27' end penetrating an end cap seal and a portion of serum 12 transferred from the upper end of the sample chamber 21'; Figures hA through 110 show 2-D summaries respectively of Figures 9A, 9B and 90; the sample chamber 21 and carrier 24 configurations admit of considerable variation as does the transfer needle 72; onward transfer post separation of a sample into component parts can be effected in a similar manner with corresponding but separate apparatus, as reflected in Figures 10A through bC, to avoid cross-contamination between the original homogenous blood sample 10 and the separated out serum component 11; Figures 1 2A through 1 2G explore detail of a transfer needle and dedicated carrier; thus more specifically Figure 1 2A shows an assembly of double-ended (ie with a sharp or point at each end) transfer needle and carrier mounting; Figure 12B shows a cartridge case of opposed end shrouds for a double-ended needle; Figure 12C shows unpacking of the sample case of Figure 12B with one shroud removed to expose an associated needle end; Figure 12D shows both end shrouds removed to expose both needle ends; Figure 12E shows a double-ended needle with an mid-riff handling disc; Figure 1 2F shows a split needle with a common end joint at the base of a carrier tube; Figure 12G shows the split needle of Figure 12F fully assembled ready for use; Referring to the drawings Figures 1 A through 1 C reflect aspects of a more traditional sample taking approach with a conventional hypodermic (ie with a needle end or sharp penetrating beneath the skin) syringe; whereas Figures 9Athrough 8C reflect an alternative sample taking technique and apparatus of the invention using pre-evacuated sample tubes and a double-ended transfer needle.

In Figures lAthrough 1C, a patient blood sample lOis taken from a vein using a needle 26 of a standard syringe 23 with (finger I thumb-operable) plunger piston 25 within a cylindrical barrel chamber, in accordance with standard procedures.

Needle 26 is then inserted through an end cap seal 22 of tubular sample chamber 21 and most, if not all, of the blood sample 10 taken is transferred.

The sample tube 21 is subjected to centrifugal agitation action until the entire transferred blood sample separates into two distinct concentration regions, respectively red blood cells 11 and serum 12.

Figures 2A and 2B reflect sample tube agitation such as by centrifuge to separate out principal component parts by weight or density -in this case In Figures 3A and 3B a transfer syringe 23' (distinct from the original sample taking syringe) fitted with an elongate transfer needle 28 is inserted into a centrifuge sample tube 21; the needle tip is positioned mid-way or between 50-60% immersion within the top serum layer 12 and the syringe plunger 25' is withdrawn, drawing the serum 12 into the syringe chamber 24'.

Thus, overall, Figures lAthrough 3B reflect a dual use of conventional syringes respectively for pro-active' sample taking and transfer in distinct successive steps; whereas a preferred passive' alternative places reliance upon a pre-evacuated sampler modules cartridges or capsules or sample chambers for the sample transfer and optionally also initial sample chamber.

An alternative transfer apparatus and methodology for a separated out sample after centrifugal agitation is reflected in Figures 4Athrough 4D; employing an elongate double-ended needle 27 with opposite ends inserted respectively into a separated blood sample 11, 12 to a point above the transition of red blood cells 11 and serum 12.

The opposite needle end is used to pierce an (eg resilient) end seal of a pre-evacuated sample tube 20, so a serum layer 12 above the needle end is drawn through into the evacuated chamber of a sample tube 20.

After disposal of the transfer needle 25 two sample tubes remain, the original 21 containing red blood cells and a remainder of serum and a second sample tube 21 containing serum only.

In practice the needle 27 would be mounted within a tube holder 29, for ease of handling and safety as depicted in Figures 8Athrough 8C.

Figures 5A through 5D, 6A through 6C and 7A through 7C reflect methods of agitating or centrifuging the sample to engender serum separation Figures 9A through 9C and 11 A through 11 C reflect sample taking without a syringe, but rather with a pre-evacuated sample chamber; However the initial sample is taken -transfer of separated-out serum is desirably effected with another pre-evacuated sample chamber and double-ended needle arrangement of Figures 4Athrough 4D, or with the carrier variant of Figures 8A through 8C and Figures lOAthrough 100.

Components whole blood sample 11 red blood cells 12 serum sample tube -pre-evacutated 21 sample tube -no longer contains a vacuum 22 sample tube rubber seal /valve cap 23 syringe 24 syringe barrel syringe plunger 26 syringe needle 27 elongate double-ended needle 28 elongate syringe needle 29 tube holder centrifuge action 31 fluid flow 32 a vacuum needle end 36 needle end 37 needle carrier 38 needle mid-riff handling disc 39 split needle with joint push action manual centrifuge 41 plunger 42 base 43 sample tube fitment 44 rotating portion football clacker style centrifuge 51 handle 52 rotating portion 53 sample tube cut out portion hand held centrifuge 61 sock I elongated sleeve/sheath 62 hand 71 arm