WO2009118551A1

WO2009118551A1 - Ultrasound method for analysis of blood for blood typing, antibody detection and flow cytometry

Info

Publication number: WO2009118551A1
Application number: PCT/GB2009/050276
Authority: WO
Inventors: Damian Joseph Peter Bond; Larisa Alexandrovna Kuznetsova
Original assignee: Prokyma Technologies Limited; Heyns, Ine, Lutgart, Peter; Steenhoudt, Oda, Fracine, Lieven, Ghislena
Priority date: 2008-03-25
Filing date: 2009-03-24
Publication date: 2009-10-01

Abstract

An ultrasound method for analysis of blood for blood typing, antibody detection or flow cytometry is described in which a blood, serum or plasma sample is introduced with one or more reactants into a chamber which is associated with means providing a standing wave to form an initial aggregate or agglutinate of particles at a pressure node of the standing wave. The product of the blood sample and reactant is subjected to a fluid flow by providing for a fluid flow through the conduit. The behaviour of the aggregate or agglutinate in the fluid flow, when the rate of flow or acoustic pressure is changed identifies a characteristic of the blood, serum or plasma sample, or its content. This characteristic can be identified by monitoring or observing the fluid flow downstream of the pressure node at different flow rates and/or acoustic pressures in the standing wave. Various means to achieve this are described, including fluorescent labelling of a reactant.

Description

ULTRASOUND METHOD FOR ANALYSIS OF BLOOD FOR BLOOD TYPING,

ANTIBODY DETECTION AND FLOW CYTOMETRY

[0001] This invention relates to methods and apparatus utilising an ultrasound standing wave for distinguishing the product of agglutination of a particle from the product of aggregation of a particle.

[0002] An ultrasound or acoustic standing wave field is capable of localising particles within a liquid at either the pressure nodes or antinodes of the field. Localisation is dependent upon a number of different factors including the relative densities and compressibility of the particles and the fluid.

[0003] An acoustic standing wave field is produced by the superimposition of two waves of the same frequency travelling in opposite directions either generated from two different sources, or from one source reflected from a solid boundary. Such fields are characterized by regions of zero local pressure (acoustic pressure nodes) with spatial periodicity of half a wavelength, between which areas of maximum pressure (acoustic pressure antinodes) occur.

[0004] Ultrasound is sound with a frequency over 20,000 Hz. It has long been established that acoustic radiation force generated in an ultrasound standing wave resonator can bring evenly distributed particles/cells in aqueous suspension to the local pressure node or anti-node planes. The radiation force arises because any discontinuity in the propagating phase, for example a particle, cell, droplet or bubble, acquires a position- dependent acoustic potential energy by virtue of being in the sound field. Suspended particles tend therefore to move towards and concentrate at positions of minimum acoustic potential energy. The lateral components of the radiation force, which are about two orders of magnitude smaller than the axial, act within the planes and concentrate cells/particles in a monolayer. This phenomenon has successfully been used to separate particles from a suspension, and particularly separation of blood cells from There are more than 20 genetically determined blood group systems.

[0007] The traditional method of ABO blood typing involves antibodies against the specific glycophorin. The antibodies can bind to antigens on more than one blood cell, cross- linking to form a complex of cells. Multiple cross links lead to agglutinates forming, which can be measured by a number of means. The Rhesus positive and Rhesus negative blood factors can be determined in a similar way.

[0008] Blood typing is critical as antibodies in donor blood may be incompatible with a patient's blood sample (or vice versa), whereby the antibodies may attach to antigens on the foreign blood cell surface stimulating the immune system to attack the blood cells as foreign particles and stimulating a haemolytic reaction. This can be fatal. The most common antigens are A, B and D (for rhesus). There are a further 26 antigens that are clinically significant. Blood is therefore characterised when it is obtained from a donor or a patient to determine its ABOD classification and also to see whether it contains any antigens or antibodies of concern. Patient blood is also checked before a transfusion is administered to ensure that the donor sample is compatible. Finally every donor sample transfused is cross-matched with patient blood to ensure that no adverse reaction can occur.

[0009] Agglutination is a general term for particles cross linking in the presence of a cross linking agent or target analyte. Agglutination is usually mediated by antibodies and antigens, wherein the particles are typically polystyrene spheres. Agglutination may also be mediated by other agents providing for cross linking of particles to form a complex such as biotin-avidin. Haemagglutination is a sub-set of agglutination whereby the particle is a red blood cell.

[0010] Blood grouping determination is based upon an agglutination reaction, whereby a blood sample is mixed with a cross-linking agent specific for the glycophorin. Monoclonal IgM antibodies are typically used as cross linking agents for the major blood groups, such as A, B and D. The IgM binds to antigens on more than one blood cell, cross-linking to form a complex, leading to haemagglutination of the red blood cells. The product of heamagglutination can be measured by a number of means.

[0011] For determining incompatibility of a patient/donor cross match, a test to determine whether the patient serum contains antibodies that could bind to antigens on the donor red blood cell (or vice versa) is used. This intermediate and generic method is termed the Coombs method or Coombs test, whereby patient and donor samples are mixed and treated with anti-human globulin reagent, known as Coombs reagent. This reagent comprises anti-human-IgG and anti-human complement C3 (C3b+C3d) antibodies, as well as anti-human IgM and anti-human IgA antibodies, which can bind to human antibodies in blood. If Coombs reagent binds to these human immunoglobulins that have attached to antigens on the red blood cell, agglutination will occur. The Coombs method also comprises a washing step, prior to addition of the reagent, to remove non- bound antibodies. This prevents non-bound antibodies in the patient serum from binding to the reagent, which may lead to a false result.

[0012] In the Coombs method non-bound antibodies are interferents, and the requirement to remove these interferents has led to the design of a test device for blood grouping, which is achieved by a system that essentially uses a gel to separate populations of differing sizes, before performing the Coombs reaction. Disadvantages of the method include a requirement for a centrifugation step. Such an approach is described in US patent 5460940.

[0013] It is the aim of this invention to overcome the disadvantages of the previously known blood group test systems in one simple system.

[0014] Accordingly the present invention provides a method of blood typing or red blood cell antibody detection comprising the steps

[0015] of;

• introducing a sample containing red blood cells and one or more reactants capable of providing for haemagglutination into a chamber associated with means providing a standing wave such that an aggregate or agglutinate is formed at a pressure node of the standing wave; D

• subjecting the aggregate or agglutinate and reactant to a fluid flow by providing for the fluid flow through the chamber; and D

• monitoring or observing the behaviour of the aggregated or agglutinated product at different fluid flow rates or acoustic pressures in the standing wave, whereby the occurrence of positive haemagglutination can be distinguished from negative or weak positive haemagglutination and weak positive haemagglutination can be distinguished from negative haemagglutination or aggregation. D

[0016] The reactants may be introduced into the chamber with the sample or separately.

[0017] One aspect of the invention, which is particularly useful when the Coombs test is to be carried out, comprises the steps of introducing the sample containing red blood cells as a suspension into an ultrasound resonator, initiating the ultrasound to cause red blood cells to collect in a standing wave, establishing a flow of fluid, through the chamber to wash away interferents such as non-bound antibodies, introducing a reactant into the fluid flow, increasing the velocity of the flow or reducing the acoustic pressure in the standing wave, monitoring or observing the behaviour of material collected in the standing wave as an indication of the blood cell type. In the example of the Coombs test, the red blood cells would have been pre-incubated with human serum to allow any antibodies to attach to the surface of the cell.

[0018] As an alternative to the previous paragraph, particularly when the ABO test is to be carried out, the reactants can be introduced into the chamber with the blood cells or in the initial fluid flow, without the need for a preliminary washing step.

[0019] In this invention the aggregate or agglutinate may be characterised by monitoring or observing the fluid flow downstream of the pressure node at different flow rates and / or acoustic pressures in the standing wave.

[0020] In another aspect of the invention a method of analysing particles comprises introducing a sample and one or more reactants into a conduit associated with means providing a standing wave to form an initial aggregate or agglutinate of particles at a pressure node of the standing wave, subjecting the product of the sample and reactant to a fluid flow by providing for a fluid flow through the conduit, and monitoring or observing the fluid flow downstream of the pressure node at different flow rates or acoustic pressures to characterise the reaction on the aggregate or agglutinate.

[0021] The method of the previous paragraph is particularly useful for analysing particles in blood, serum or plasma.

[0022] In serology a reactant may be an antibody or antigen carried on particles inert to the reaction. In this approach a further reactant may be an antibody capable of binding to antibodies attached to the inert particle.

[0023] In this invention typical examples of "reactants" could be one or more monoclonal and/or polyclonal antibodies, naturally occurring antibodies in the blood sample, antigens attached to a solid phase, such as a polystyrene particle and Coombs reagent. The "reactants" react with "targets" in the sample. Typical examples of "targets" could be cell surface antigens, such as the glycophorins specifying the blood group, or CD4 or other cell surface antigens used in disease diagnosis, or antibodies in blood, serum or plasma for reverse blood typing or serology detection of infectious disease, or antibodies in blood, serum or plasma that can bind to an antigen on a red blood cell in the Coombs test. The sensitivity of the invention is determined by the ability to measure the interaction of the "reactant" with the "target" and this can be prevented or reduced by the presence of "interferents". In some cases, such as ABO or reverse blood typing, the reaction is not affected by interferents to any noticeable degree, but as previously mentioned in the Coombs test, the "interferents" are predominately an excess of antibodies that have not bound to the blood cells. In other examples, "interferents" can comprise a wide range of materials that diminish the avidity of antibody binding, or blocks or quenches light passing through the liquid. In this invention, the detection is improved by removing the presence of "interferents" by establishing a flow of fluid prior or during the action of the "reactants".

[0024] The red blood cells and serum can be mixed preincubated at a temperature that is about that of a subject species from which the blood cells or serum are derived. When the subject species is human the preincubation is at about 37°C for about 15 minutes. [0025] By setting the initial rate of fluid flow to low levels and/or the acoustic pressure in the standing wave to a high level, interferents such as unbound antibodies are washed from the chamber without single cells in an aggregate or agglutinate being washed away. In an apparatus used by the inventors, a flow rate to achieve this was less than lmm sec ¹, the voltage applied to the ultrasound transducer used in this invention to get the same effect through acoustic pressure is between 25 - 50V applied to a 1.5 MHz ultrasound resonator generator. In one aspect of the invention the red blood cell sample and reactants are premixed before entry to the chamber associated with means providing a standing wave.

[0026] In this invention the occurrence of negative haemagglutination is identified by individual blood cells being swept from the aggregate or standing wave. In the inventors' apparatus this occurred at a flow rate of less than about 4.5 mm sec ¹, alternatively variations of the acoustic pressure will achieve a similar effect.

[0027] The occurrence of weak positive haemagglutination is identified by dislocation of the agglutinate in increasing flow rates, or at lower powers. Clumps of blood cells leave the standing wave as the flow rate increases.

[0028] A strong positive haemagglutinate resist cells being washed away as single cells or small clumps from the main agglutinate, which is swept from the standing wave substantially as a whole at high flow rate, in the inventors' apparatus this occurred typically at 10mm sec¹ or more.

[0029] To aid viewing one of the reactants can have a fluorescent label. Normally this will be attached to at least one antibody.

[0030] Ideally aggregates should form at a single node, however, the lateral reflections of acoustic waves of the side walls of the chamber can lead to a complicated pattern of pressure nodes throughout the chamber. Small changes in frequency or in factors that can affect pressure, such as viscosity of the sample or temperature can shift cells to a different node. The frequency used can also determine where the main node is positioned in the chamber and this may be off centre. Once cells have aggregated into the main node they tend to be held firmly. The blood typing assay performs more re- producibly if all the cells are in a single node held at the centre of the chamber. Frequency sweeping across a predetermined range of frequencies moves cells around the chamber and encourages formation of nodes that coalesce into the central node at the end of the sweep. The appropriate range for frequency sweeping varies with each chamber, but the range 1.412 to 1.438 MHz is an example in one chamber investigated for this invention.

[0031] The method can be performed by introducing the reagents separately into the standing wave or by premixing the sample containing red blood cells and reactants prior to insertion into the standing wave. [0032] The premixing can also involve Coombs reagent or it can be inserted into the fluid flow. Washing of the blood sample and reactants is advisable before introducing the Coombs reagent, although in this system, that is not absolutely necessary, which is an unexpected finding.

[0033] By varying the flow rates through the standing wave, different characteristics of aggregates and haemagglutinates gathered at the standing wave can be identified. If a washing stage is used, the flow rate should be set sufficiently low to achieve washing but without other materials leaving the standing wave. As the flow rate increases aggregates, collections of materials trapped by the standing wave but which have not reacted, start to leave the standing wave (this is known as negative haemagglutination). At slightly higher flow rates, materials associated with weak positive haemagglutination leave the materials as small clumps of cells. As the flow rate increases further, negative and weak positive haemagglutinates will leave the standing wave altogether, and eventually at still higher flow rates any strong positive haemagglutinate will leave as a single mass. In this way, the method can be calibrated for the different outcomes of a blood typing test. Similar effects are achieved by progressively reducing the acoustic pressure in the standing wave.

[0034] The rate of change of flow rate can have an impact upon the interpretation of the result. In strong agglutination reactions, a single flow rate selected above the point at which the negative loses cells, but below the positive can be selected. In a negative reaction, cells leave the aggregate, in a positive no cells leave. In another embodiment, the flow rate can be increased in steps. This is often used for example, with a manually controlled peristaltic pump. The step changes cause a pulse and a surge of pressure, which can dislodge some cells before the flow settles down. Whilst not affecting the basic difference between positive and negative reactions, these lost cells could cause problems in interpretation by an optical system. A mechanism that smoothed the changes in flow rate is preferred to maximise the ability to control the reaction and discriminate between negative and weak positive samples and computer control of the pumps is ideal for this application.

[0035] The application lends itself to automated detection using an instrument. In the simplest example, cells will wash off the aggregate in a negative sample, whilst no cells leave a positive sample. A simple optical sensor measuring the pattern of light scatter can be employed to measure the difference. In a weak positive sample, the presence of clumps can also be measured as the light scatter will be different from a sample containing single cells. Such techniques are widely described and employed in instruments based upon measurement of turbiditry or nephelometry to measure agglutination reactions.

[0036] Observation or calibration can be enhanced if at least one of the reactants has a flu- orescent label. This could be used to label agglutination complexes or even single cells.

[0037] This is particularly useful for identifying the behaviour as a result of a weak positive haemagglutinate being formed in the standing wave and for calibration of results.

[0038] The applicant has additionally found that acoustic streaming may occur between the node and anti-node planes and/or within the planes in the standing wave. This is particularly effective in aiding movement of fluid and soluble material through that standing wave, and introducing reactant to the particle or aggregate and removing non- bound material from the particle or aggregate facilitating washing and mixing.

[0039] Efficient mixing of cells and reactants is important to maximise the sensitivity of the assay. In particular the Coombs test uses cells mixed in human serum, which has a higher viscosity than buffer and can have an impact on mixing. Several strategies have been employed to improve this including introducing the sample and antibodies from 2 separate input tubes to ensure turbulence and mixing.

[0040] This invention can be applied to a number of presentations of immunoassay technology. Most immunoassay tests detect antibodies (serology), or antigens in a sandwich or competition format. Serology tests detect the presence of specific antibodies in a blood sample, and are typically used to measure IgG or IgM antibodies, to determine the progression of a disease course, but can also detect other classes of antibody such as IgA, IgD or IgE.

[0041] Other examples use fluorescent labelled antibodies to measure sub-populations of cells in a blood sample. For example the application has demonstrated that improved fluorescent staining of CD4 and CD8 from a whole blood sample can be achieved. In this example, a blood sample is reacted with fluorescently labelled antibodies and then the red blood cells are selectively lysed using a commercial product such as Beckman Coulter Optilyse C. The ultrasound standing wave aggregates the leukocytes and the red cell debris is washed away. The washed leukocytes can be examined using fluorescent microscopes or in a flow cytometer showing sharper imaging and therefore discrimination of labelled cells compared to the unwashed sample. Usually, agglutination would be avoided in these cases as the instruments are designed to detect single cells, however it could also be applied to agglutinated clumps of cells exhibiting better contrast and resolution due to more light emission due to the concentration of fluorophores in the agglutinate, combined with removal of interferents in the background that would block or quench light.

[0042] The previous paragraph along with example 6 below describes a method for labelling leukocytes in a blood sample. A similar approach could be used for a serology assay, using serum or plasma and adding inert particles such as polystyrene particles with antigens attached on the surface. Circulating antibodies specific for the antigens would attach and a secondary labelled antibody could be flowed through as a reactant to attach for detection by fluorescent or other means known in the art of immunoassay detection.

[0043] The following examples of use of the invention are given for illustrative purposes. They are not intended, in any way, to be limiting.

[0044] The wash solution, or rinsing fluid, used for the blood grouping experiments was

0.01 M Phosphate buffered saline (Sigma, UK) prepared using deionised water. Other wash solutions that could be used for blood grouping include (i) Dulbecco's Phosphate Buffered Saline (PBS), obtained from Biological Industries, Beit Ha'emek, Israel; (ii) a solution made from PBS diluted 1:1 in water with 4% (w/v) poly ethylene glycol (PEG) 15000-20000MW (Fluka) and 0.3% (w/v) dextran sulfate sodium salt (Amersham Biosciences); (iii) a solution of PBS with 0.001 - 0.01% (w/v) poly- oxyethylene-10-tridecyl ether (Sigma).

[0045] Red blood cell suspensions and reactants used for the blood grouping experiments were obtained from DiaMed and Immucor Gamma. The suspensions used were:

• Ai, A₂, B and O human origin red blood cells in DiaMed buffered suspension, containing trimethoprim and sulfamethoxazole as preservatives - the Al, A2 and B Cells are rhesus negative whereas the O cells are rhesus positive; D

• DiaClon anti-A, DiaClon anti-B or DiaClon anti-D from DiaMed (IgM-type murine monoclonal antibodies). Preservative < 0,1% sodium azide; D

• "Coombs-control IgG" human origin red blood cells sensitised with IgG, in DiaMed buffered suspension, containing trimethoprim and sulfamethoxazole as preservatives; D

• DiaCell human origin red blood cells I, P and III in DiaMed buffered suspension, containing trimethoprim and sulfamethoxazole as preservatives; D Three types of Plasma -anti-D/C N" 10, anti-D/C N" 19 and anti-C N^25; D

• DiaClon Coombs reagent from DiaMed, preservative < 0,1% sodium azide D

• Anti-human Globulin, Anti-human IgG (Murine monoclonal), anti-human C3d (Immucor Gamma), preservative <0.1% sodium azide. D

• Capture-R^® , Positive and negative Control Serum (Immucor Gamma), Preservative 0.1% Sodium Azide. D

[0046] DiaMed Anti-D Reference Reagent contains a blend of monoclonal anti-D IgGl (cell line ESD-I) and monoclonal anti-D IgG3 (cell line LHM59/19), suspended in human AB serum. Lyophilised. Preservative < 0,1% NaN3.

[0047] The required concentration of red blood cells was obtained by diluting the initial suspensions with PBS or ID-Diluent 2 (DiaMed). The final red blood cell concentration introduced into the ultrasound chamber was 0.3% unless otherwise stated.

[0048] DiaClon anti-A, anti-B and anti-D antibodies (DiaMed) were diluted with PBS or ID- Diluent 2 to the concentrations required (normally 20-fold dilution unless otherwise stated).

[0049] The Coombs reagent comprised i) DiaClon Coombs reagent (DiaMed), which is a polyspecific anti-human-globulin (AHG) reagent and contains rabbit anti-human IgG, and monoclonal anti-human C3d, cell line C 139-9; DiaMed), including < 0.1 % sodium azide as preservative; ii) Anti-human Globulin, Anti-human-IgG (Murine monoclonal) and anti-human-C3d (Immucor Gamma) 0.1% sodium azide as preservative.

[0050] DiaClon anti-human serum antibodies (DiaMed) and Anti-human-IgG (Murine monoclonal), anti-human-C3d (Immucor Gamma) were diluted with PBS or ID- Diluent 2 (DiaMed) to the concentrations required.

[0051] The ultrasonic apparatus used for blood typing comprises a disk piezoelectric transducer, a quartz glass reflector, a spacer layer for a sample solution, and a coupling stainless steel layer separating the transducer from the spacer layer. The apparatus has been described in L. A. Kuznetsova et al, Langmuir2007, 23, 3009 - 3016. The spacer layer was filled with a red blood cell suspension by a syringe. The inlet to the spacer layer was then reconnected to a KDSlOO syringe pump (KD Scientific Inc., Ma, USA) which pumped PBS, antibody suspension or Coombs reagent through the chamber. Cell movement was monitored with an Olympus BX41M epi-fluorescent microscope or a standard PAL CCD JVC video camera (Victor Company, Japan) with a TV zoom lens. The camera was connected via a 0.5 microscope adaptor and the images were recorded onto a standard video tape.

[0052] Other experiments used a Gilson MiniPuls 3 peristaltic pump (Anachem, Luton, UK) in place of the syringe pump and BMZ GL Trinoc Long Arm ST4 microscope (Brunei Microscopes, Chippenham, UK) to monitor cell movement. The video was captured on computer hard drive via a PCI Eurosys card.

[0053] A preliminary voltage/frequency scan established the optimal frequency. A suspension of PBS and human red blood cells was pumped into the spacer layer by syringe. The scan was performed by sweeping the frequency in small increments in a range near the transducer's nominal resonant frequency (1.5 MHz) and identifying the frequency at a minimal voltage. The established resonant frequency was maintained manually during the initial blood grouping experiments. Later work showed that the aggregate formation can be automated using programmed sweeps of frequencies and voltages using the control and wave generator developed for the inventors by D4 Technologies Limited.

[0054] The acoustic pressure amplitude at the chosen frequency was estimated experimentally from the balance of the axial direct radiation force and gravitational force acting on a particle in suspension as described in L. A. Kuznetsova et al, Langmuir 2007, 23, 3009 - 3016. Acoustic pressure amplitude P₀ at the threshold voltage of 1.3 V was 39 kPa, which allows its estimation at experimental conditions from P₀ vs V linear dependence.

[0055] For each experiment a fresh portion of erythrocyte suspension was pumped into the chamber. Upon application of a standing wave the erythrocytes are driven to the pressure node plane by the acoustic radiation force and therein form aggregates in the pressure node. The difference between aggregates and agglutinates was clearly observed upon application of a hydrodynamic flow. After each experiment the spacer layer was washed with detergent and rinsed with isopropanol followed by PBS. The stability of an ultrasonically formed aggregate in a flow depends on the acoustic pressure amplitude, which is linearly proportional to the voltage across the transducer. A voltage of 30 V was chosen for the experiments unless specified otherwise.

[0056] Example 1 - Negative-Control Haemagglutination Experiments

[0057] Negative control experiments comprised the interaction of A group cells with anti-B antibodies, B group cells with anti-A antibodies, Ai, A₂ or B group cells and anti-D antibodies, and O red blood cells with both anti-A and anti-B antibodies.

[0058] Equal volumes of group Ai and A₂ red blood cell suspensions and anti-B antibodies were pre-mixed, pumped into the spacer layer and exposed to ultrasound. A large aggregate was seen growing in the centre of the spacer layer. Major contributors to that growth were several lines of single cells and small clumps of cells. At the periphery of the chamber were smaller aggregates, some of which eventually linked with the central aggregate leading to reorganization of the main aggregate. The aggregates of approx. 2 mm in diameter formed within one minute of the exposure to ultrasound. The flow of wash solution started immediately after that at a rate 1 mm sec ¹. It led to slow but continuous single cell detachment from the edges of the main aggregate, clearly seen at x2 to x5 microscope magnification, and rapid disintegration of smaller aggregates scattered around the chamber. As the flow rate increased, so increased disintegration of the central aggregate. It was found that at between about 4.5 mm sec ¹ and 5mm sec ¹ the aggregate fully dissociated within about 2 min.

[0059] A modified experimental procedure involved pumping a suspension containing A group cells into the spacer layer, initiating the ultrasound and forming a central cell aggregate. After that anti-B antibodies were pumped into the chamber at a flow rate of 1 mm sec ¹. for 2 min. As the flow rate increased the pattern of aggregate dissociation was the same as described above. No agglutination was observed.

[0060] Premixed suspensions of equal volumes of Ai, A₂ or B group cells and anti-D antibodies exposed to ultrasound led to cell aggregation in the pressure node plane. Aggregates disintegrated and were washed away at about 4.5mm sec ¹. The same result was obtained when pre-formed cell aggregates were washed in a flow of anti-D antibody at a slow flow of lmm sec ¹ and then subjected to a higher flow rate of about 4.5mm sec ¹.

[0061] Exposing a suspension of premixed O group red blood cells and anti-A and anti-B antibodies to ultrasound led to cell aggregation. Dissociation was as described above.

[0062] Example 2 - Positive- Control Haemagglutination Experiment

[0063] Positive control experiments comprised the interaction of A group red blood cells with anti-A antibodies, B group red blood cells with anti-B antibodies, and O group red blood cells with anti-D antibodies.

[0064] Premixing equal volumes of group A₁ and A₂ red blood cell suspensions with anti-A antibodies or a group B red blood cell suspension with anti-B antibodies, and exposing to ultrasound resulted in production of agglutinates at the pressure node of the standing wave within one minute of exposure. The pattern of formation is quite different from that of aggregation as described in Example 1. Instead of single cells being attracted by the radiation force to form a central aggregate, small and medium sized agglutinates are attracted by the radiation force to form a central agglutinate. One large central and several small peripheral agglutinates were formed within one min of exposure to ultrasound. The flow started immediately after that. The central agglutinate showed no sign of disintegration at low and medium flow rates and remained intact, although its position shifted slightly in the direction of the flow, whereas the smaller agglutinates were swept from the field by the flow. In some cases the smaller agglutinates were swept past the main agglutinate and if contact occurred became attached to the main agglutinate. The main agglutinate remained intact until the flow was increased to between 10mm seσ¹ and 13.75mm seσ¹ whereupon the agglutinate was washed away as a whole, i.e. the detachment of single cells as seen in Example 1 for aggregates did not occur with agglutinates.

[0065] Exposure of premixed suspensions of equal volumes of O group (rhesus positive) red blood cells with anti-D antibodies to ultrasound led to agglutinate formation at the centre of the chamber, which showed no sign of disintegration until it was swept away as a whole at a flow rate of 10mm sec ¹. Introduction of anti-D antibodies to O group red blood cells aggregated at the pressure node of a standing wave also had the same effect. In this case, agglutination is indicative of a positive Rhesus blood group.

[0066] The experiments in Example 1 and Example 2 were performed at a transducer voltage of 30 V. It was noted that at lower voltages the difference between the flow rate at which an aggregate product was swept from the standing wave and the flow rate at which an agglutinate product was swept from the standing wave was less, and thus distinguishing a positive result from a negative result would be more difficult to achieve. At voltages higher than 50 V cavitation air bubbles often interfered with the process. [0067] Examples 3A to 3E - Coombs Method

[0068] The Coombs method actually encompasses two different tests, the direct Coombs test and the indirect Coombs test. The direct Coombs test is used to detect antibodies or complement system factors that have bound to red blood cells surface antigens in vivo, whereas the indirect Coombs test is used to detect low concentrations of antibodies present in a patient's or donor's plasma or serum prior to a blood transfusion. The two tests are based on the concept that anti-human antibodies, produced by immunized non-human species, will bind to human antibodies, commonly IgG or IgM. Animal anti-human antibodies will also bind to human antibodies that may be fixed onto the surface of red blood cells, and in the appropriate test tube conditions such red blood cells may agglutinate.

[0069] Example 3A Direct Coombs test - Positive Control

[0070] A suspension of 0.4% Coombs-control IgG pre-loaded cells (i.e. red blood cells with IgG antibodies already attached for use as a quality control test) was flowed into the chamber and exposed to ultrasound. Cells aggregated in the pressure node at the centre of the chamber. Flowing a 10-fold dilution of Coombs-serum through the chamber at a flow rate of lmm sec¹ led to cell agglutination. The agglutinates were washed away as a whole at a flow rate of 10mm sec ¹.

[0071] Agglutination also occurred when a premixed suspension of equal volumes of 0.8% of Coombs-control IgG pre-loaded cells and 10-fold dilution of Coombs-serum (giving a final cell concentration of 0.4 %) were exposed to the ultrasound field. The agglutinates were again washed away intact at flow rate of 10mm sec ¹.

[0072] Example 3B Direct Coombs test - Negative Control

[0073] Exposing a suspension of premixed equal volumes of 0.6% group A₁ A_2, B, or O red blood cells with 10-fold dilution of Coombs-serum (giving a final cell concentration of 0.3%) to ultrasound led to cell aggregation in the node plane. No agglutination occurred, and the aggregates were washed at a flow rate of about 4.5 mm sec ¹.

[0074] Example 3C Coombs reagent titration

[0075] Titration was performed to determine the minimum concentration of Coombs reagent to result in cell agglutination. It was found that when the initial Coombs reagent concentration was diluted 10- and 100-fold, strong cell agglutination occurred. Agglutinates showed stability and were only swept from the ultrasound field as a whole at a flow rate of 10mm seσ¹ to 13.75 mm sec ¹. At 1000-fold dilution partial cell agglutination was observed, with small cell clusters being washed away from a central agglutinate/aggregate at a medium flow rate. At 10,000- and 100,000-fold dilution cells began to be washed from the central agglutinate/aggregate at a slow flow rate.

[0076] Example 3D Indirect Coombs test - Negative Control

[0077] A pre-mixed suspension of equal volumes of DiaCell III and Plasma anti-D/C Nr 10 was incubated for 15 minutes at room temperature and exposed to ultrasound. A red blood cell aggregate was formed at the centre of the chamber. The aggregate was washed in a PBS flow at a rate of lmm sec ¹ for 2 min to remove unbound antibody molecules which were present in the plasma. One of the problems encountered with the Coombs method is that of 'neutralisation' of the Coombs reagent by antibody molecules present in plasma. The present method allows the red blood cells to be washed, thus avoiding this problem. A 10-fold dilution of Coombs reagent was pumped through the chamber at lmm sec ¹. As the flow rate increased to 1.5mm seσ¹ the aggregate started to lose cells and at about 4.5 mm sec ¹ rapid aggregate dissociation occurred. This indicated that the plasma contained no corresponding antibodies. Therefore the Coombs test was negative.

[0078] Example 3E Indirect Coombs test - Positive Control

[0079] A pre-mixed suspension of equal volumes of DiaCell I and Plasma anti-D/C Nr 10 was exposed to the procedures used for the negative control. Following washing of the aggregate, introduction of a 10-fold dilution of Coombs reagent resulted in agglutination. The agglutinate was swept away as a whole at 10mm sec-1.

[0080] Example 4 Weak Agglutination

[0081] A weak agglutination reaction was simulated using DiaCell control cells and anti-D reference reagent. Anti-D antibodies will bind to DiaCell II cells but not to DiaCell III. Addition of Coombs reagent will agglutinate type II cells but not type III. At a dilution of 1/32 anti-D limited antibody has bound and consequently a weak agglutinate is formed.

[0082] A 1:5 dilution of Coombs serum was prepared using PBS

[0083] 75μl of 1/32 Anti-D is mixed with 75 μL of DiaCell cells (II or III as required per test) in an Eppendorf tube and incubated at 37°C for 15 minutes. The sample was loaded into ultrasound chamber at a flow rate of 5.0 rpm on the Gilson Miniplus 3 peristaltic pump. A programmed frequency and voltage sweep formed a central aggregate of cells. The voltage of the transducer was set to 50V holding power.

[0084] The diluted Coombs serum was flowed through the chamber for 5 mins at a flow rate of 0.5 rpm on the Gilson Miniplus 3 peristaltic pump. The voltage was reduced to 20V for the assay.

[0085] A fluid flow of PBS was started through the chamber starting at a flow rate of 0.5 rpm on the Gilson Miniplus 3 peristaltic pump increasing the flow rate by 0.5 rpm on the Gilson Miniplus 3 peristaltic pump every 30 seconds until the readout was 10 rpm, increasing by 1.0 rpm thereafter

[0086] The appearance of a tail of cells or clumps washing away from the aggregate/agglutinate was recorded on a video, as well as the time at which the aggregate/agglutinate is completely washed from the chamber. [0087] In the case of a negative antigen- antibody reaction, single cells started leaving the aggregate at a flow rate of 1.5-1.6 mm sec ¹. As the velocity further increased the rate of an aggregate disintegration also increased. In the case of weak agglutination, small clumps of approximately 20-50 cells started to leave the agglutinated product at a PBS flow rate of 2.4-2.6 mm sec ¹. As the velocity increased larger cell clumps start to become detached.

[0088] In addition the time taken for disintegration of the aggregate/agglutinate was quicker for the negative than the weak positive by several minutes. Typically a time difference of 5 minutes can be observed between the disintegration of a negative aggregate and a weak positive agglutinate based on a 1/32 anti-D reaction.

[0089] Example 5 Use of fluorescent reagents in an agglutination system

[0090] The method of the present application can also be performed using fluorescent reagents such as a fluorescently labelled antibody specific for blood cells. The behaviour of the aggregated or agglutinated product in the fluid flow, such as the pattern or process of loss of material from the standing wave, could therefore be more easily observed and/or characterised through fluorescence, and in particular through the intensity of the fluorescent signal or the arrangement or organisation of fluorescence over the surface of the product. The example below describes a method of fluorescently staining the cells before introducing them into the flow chamber and performing the reactions described in examples 1 to 4 above.

[0091] Fluorescence Based Indirect Assay

[0092] A pre-mixed suspension of equal volumes of DiaCell I and Plasma anti-D/C Nr 10 was incubated for 15 minutes at room temperature. IgG antibodies in the plasma bound to antigens on the surface of the DiaCell I erythrocytes. The suspension was adjusted to a concentration of 3-4 % red blood cell solution (100 ml), suspended in phosphate buffered saline (PBS; 900 ml), mixed and then washed by centrifugation for 5 min at 1000 rev/min. The supernatant was removed and the pellet reacted with a 1:10 dilution of Coombs reagent (1 ml), washed 3x with PBS, centrifuged and the supernatant removed. To the pellet, a 1/6 dilution of a mixture of FITC labelled anti-rabbit antibodies diluted in PBS containing lmg/ml BSA and 0.9 % sodium azide, or alternatively Q-dots anti-rabbit antibodies, is added. The solution is then incubated for 1 hr at room temperature in the dark. The pellet is washed 3x with PBS, and then a suspension of the pellet analysed in the method of the present application. The cells were removed from the chamber and examined with a fluorescent microscope.

[0093] Example 6 Use of fluorescent agents in a non-agglutination system.

[0094] Equipment

[0095] 2 channel signal generator (obtained from D4 Technologies, Southampton UK); Oscilloscope - Hameg HM 1008, Combiscope, Gilson Miniplus 3 peristaltic pump; Ul- trasound chamber PK/CH/0014; Tubing - Anachem 116-0549-090P PVC pump tubes LD. 1.02mm; RF leads RS cat no 284-3792; T-Piece - RS cat no 295-8030; SMA plug to BNC jack connector - RS Cat no 242-102; RS232 serial lead; USB serial connector; Whole blood, drawn from venipuncture (9.00 am, 6th Mar 09) in Sarstedt blood collection tube with EDTA; Optilyse C lysis solution (Al 1895), Beckman Coulter, Lot 14, Exp 02/07/2010; IoTest Dual stain CD4-FITC, CD8-PE (PN-IM0747U), Beckman Coulter, Lot 53, Exp 21/01/2011; 0.01M PBS pH7.4; Epics Altra flow cytometer (Beckman Coulter)

[0096] Investigation of the ratio of different types of T-CeIl is significant in monitoring progression of an immune response. A common test using a flow cytometer is the measurement of CD4 and CD8 glycoproteins on T-CeIIs. In a disease such as HIV / AIDS, the ratio of CD4 and CD8 cells can help diagnose the progression of the disease and its response to therapy. Without prior treatment, CD4 and CD8 cells are difficult to detect in whole blood as they are outnumbered by other blood cells, particularly red blood cells. In a typical blood sample, each mL of whole blood will contains 5xlO⁹ red blood cells but only 8xlO⁵CD4 cells and 5xl0⁵CD8 cells. CD4 / CD8 cells therefore only represent 0.03% of all cells in the blood sample. A preliminary treatment step is therefore employed to Iy se the red cells, leaving the white cells intact.

[0097] The first step is to label the cells with fluorescently labelled antibodies. 100 μL of whole blood (less than 24 hours after drawing from veniculture with anti-coagulent, such as EDTA) was mixed with 20 μL of IoTest Dual stain CD4-FITC, CD8-PE ((PN-IM0747U), Beckman Coulter, Lot 53, Exp 21/01/2011) and incubated in the dark for 10 minutes at room temperature (18-25°C).

[0098] The next step is to selectively lyse the red blood cells. 500 μL of Optilyse C lysis solution ((Al 1895), Beckman Coulter, Lot 14, Exp 02/07/2010), is added to the blood sample and incubated in the dark for 10 minutes at room temperature. At the end of 10 minutes, the lysis buffer is neutralised by addition of 500 μL of PBS buffer and incubated for a further 5 minutes. An aliquot of the sample is retained for examination under fluorescent microscopy and with flow cytometry.

[0099] lOOμl of the Optilyse treated sample is flowed into the empty ultrasound chamber.

[0100] A programmed algorithm sweep is applied, which sets the voltage at the Transducer to 50V, applies a frequency of 1.468 MHz for 5 mins, then changes to 1.492 MHz decreasing in steps of 0.02 MHz every 2 seconds until it reaches 1.468 MHz. These conditions were found optimal in the chamber used to form an aggregate of leukocytes in a short time period.

[0101] 2 Mins after starting the algorithm, the peristaltic pump (Gilson Miniplus 3) was started to flow PBS through the chamber for 3 minutes at a flow rate of 0.5 as recorded on the pump display. [0102] At the end of the 3 mins, the pump was stopped, whilst the ultrasound was still active, but before the frequency step changes. The purpose of the step change is to move the aggregate around the chamber enhancing the washing. The ultrasound was stopped and the cells removed from the chamber and collected in a clean Eppendorf tube for examination by fluorescent microscopy and flow cytometry.

[0103] The sample that had only been treated by Optilyse C showed clear staining under microscopy. The sample that has undergone ultrasound processing in addition provided a more intense fluorescent signal as well as larger looking cells. The presence of red cell debris and haem in the original sample appeared to quench the light absorption / emission through the sample and inclusion of a washing step enhanced the resolution of the cells, improving detection. These observations were confirmed by tests on an Epics Altra flow cytometer, which showed the ultrasound washed sample recorded a higher incidence of fluorescent cells in both red and green gates compared to the unwashed sample.

[0104] Variations on the basic approaches described above are possible. Some are exemplified below.

[0105] Branched inlet tubes could be used to allow a better mixing of different components prior to entering the chamber.

[0106] The indirect Coombs test is designed to detect antibodies in the patient's serum that can bind to donor blood cells and cause hemolysis. We refer to this in the earlier description when we describe the Coombs test, although we refer to antibodies in the patients blood, test cells from the proposed donor blood can be mixed with patient serum. In this example the test procedure is best done by incubating of serum from the patient with a sample of blood cells. This is incubated at 37°C (ie about normal human body temperature) for 15 mins to allow any antibodies to find and bind to the antigens on the blood cell. The test cells are quality control cells with known antigens on them to test the patient serum.

Similarly it is also possible to bring together patient blood cells and test monoclonal and or polyclonal antibodies.

[0107] For veterinary purposes the incubation step would be carried out at the body temperature of the relevant subject.

[0108] Multiple branched inlet tubes can be used to control the sequence of mixing and reaction steps, e.g. for an indirect Coombs assay, a tube connected to a reservoir of washing buffer could have three branched inlet tubes arranged in sequence with number 1 closest to the ultrasound chamber and number 3 the furthest away (upstream and closest to the washing buffer). Whilst tube number 3 and the washing reservoir tube are sealed, tube numbers 1 and 2 can add test cells and patient serum respectively into the main tube where they mix and are held at 37°C for a 15 minute incubation. Tubes numbers 1 and 2 are closed and the washing reservoir tube is opened and the cells flowed into the ultrasound chamber, where they aggregate and are washed as described earlier. The washing reservoir tube is closed and tube number 3 is opened to flow through Coombs reagent. Tube number 3 is closed, the washing reservoir tube opened and the cell aggregate/agglutinate washed to determine positive or negative reaction as previously described.

[0109] An alternative to the use of the branched tubes could be micromixing chambers.

These chambers may also be used to pre-incubate the cells/serum mixture at body temperature.

[0110] Instead of using the washing flow rate and time needed to completely disintegrate the agglutinate/aggregate as the assay end point, one could adapt the washing procedure (using a rather high flow rate from the start or applying a very steep flow rate increase) and use the visual appearance of the cells (agglutinated cell clumps versus stream of individual or grouped cells) in the outlet tube as the assay end point.

[0111] In case of using visual inspection as the end point, the duration of the washing step might even be further reduced by rapidly increasing flow rate and then reducing the voltage applied to the ultrasound generator by applying very low voltage (amplitude) values to the ultrasound generator significantly lowering the holding power of the pressure nodes to speed up the movement of either the agglutinated cell clumps or the stream of individual or grouped cells into the fluid flow. The ultrasonic generator may ultimately be turned off altogether.

[0112] Addition of agglutination enhancers may lead to increased assay sensitivity.

Enhancers are reagents used to enhance the strength of agglutination reactions. Liss, Polyethylene Glycol and bovine albumin are typical. They provide a dehydrating effect and concentrate the water soluble elements into a smaller volume ensuring the reactants are more likely to meet.

[0113] The outlet pipe from the flow chamber can also be designed to introduce shearing, either by sharp edges or by constriction to a narrower channel exiting into a wider channel, which will disperse cells and exaggerate the difference between single cells which will spread through the larger volume and cells attached to each other, which will not spread so far.

[0114] As used herein, a particle is particularly intended to mean a bacterial cell, blood cell, blood platelet, cell fragment, spore, plasmid or virus, but also includes synthetic particles which may or may not be modified, or coated, with one or more different chemical or biological moieties or synthetic derivatives thereof. Examples of synthetic particles include, but are not limited to, polymers, such as latex and polystyrene, composites such as gold coated polystyrene, particles with a paramagnetic core and glass/silica beads that may or may not be coated with proteins, capture moieties, recognition elements, ligands, amplification moieties or other chemical or biological agents. It should be emphasised that where flow rates and voltages are given in this specification, they relate to flow rates and voltages used by the inventors on their apparatus. The appropriate flow rates and voltages may vary depending on the exact configuration and parameters of the ultrasound system in use, and appropriate calibration will be required. This step is within normal experimental skills.

Claims

[0001] A method of blood typing or red blood cell antibody detection comprising the steps of: introducing a sample containing red blood cells and with the sample or separately one or more reactants capable of providing for haemagglutination into a chamber which is associated with means providing a standing wave such that an aggregate or agglutinate is formed at a pressure node of the standing wave; subjecting the aggregate or agglutinate and reactant to a fluid flow by providing for the fluid flow through the chamber; and monitoring or observing the behaviour of the aggregate or agglutinate at different fluid flow rates or acoustic pressures in the standing wave, whereby the occurrence of positive haemagglutination can be distinguished from negative or weak positive haemagglutination and weak positive haemagglutination can be distinguished from negative haemagglutination or aggregation.

[0002] A method according to claim 1 comprising: introducing the sample containing red blood cells as a suspension into an ultrasound resonator; initiating the ultrasound to cause red blood cells to collect in a standing wave; establishing a flow of fluid through the chamber; introducing a reactant into the fluid flow; increasing the velocity of the flow, or reducing or removing the acoustic pressure; monitoring or observing the behaviour of aggregate or agglutinate collected at the standing wave as an indication of the blood cell type.

[0003] A method according to claim 1 comprising: introducing a sample containing red blood cells and at least one reactant as a suspension into an ultrasound resonator; initiating the ultrasound to cause red blood cells to collect in a standing wave; establishing a fluid flow through the chamber; increasing the flow velocity, or reducing or removing the acoustic pressure; monitoring or observing the behaviour of aggregate or agglutinate collected at the standing wave as an indication of the blood cell type.

[0004] A method according to anyone of claims 1 to 3 in which the behaviour of the aggregate or agglutinate collected at the standing wave is monitored by monitoring or observing the fluid flow downstream of the pressure node at different flow rates and /or acoustic pressures in the standing wave to characterise the aggregate or agglutinate.

[0005] A method of analysing particles in a sample comprising: introducing the sample and one or more reactants into a conduit associated with means providing a standing wave to form an initial aggregate or agglutinate of particles at a pressure node of the standing wave; subjecting the aggregate or agglutinate to a fluid flow by providing for a fluid flow through the conduit; and monitoring or observing the fluid downstream of the pressure node at different flow rates and/or acoustic pressures in the standing wave to characterise the reaction on the aggregate or agglutinate.

[0006] A method of analysing particles according to claim 5 including the step of increasing the flow velocity, or reducing or removing the acoustic pressure.

[0007] A method of analysing particles in a sample according to claim 5 or claim 6 in which the sample comprises blood, serum or plasma.

[0008] A method according to any one of Claims 1 to 7 in which Coombs reagent is introduced into the fluid flow after the formation of an initial aggregate to form an agglutinate in the case of positive reaction.

[0009] A method according to any one of claims 1 to 8 in which the initial rate of fluid flow is set at a low levels allowing unbound antibodies or other soluble materials to be washed from the chamber but below that at which single cells in an aggregate or agglutinate are washed away.

[0010] The method of any of preceding claim in which the occurrence of negative haemagglutination is identified by individual blood cells being swept from the aggregate or standing wave.

[0011] The method of any of claims 1 to 10 in which the occurrence of weak positive haemagglutination is identified by dislocation of the agglutinate and in which clumps of blood cells leave the standing wave.

[0012] The method of any of claims 1 to 11 in which a strong positive haemagglutinate is swept from the standing wave substantially as a whole.

[0013] A method of analysing particles in a blood, serum or plasma sample for serology according to claim 7 in which a reactant is an antibody or antigen carried on particles inert to the reaction.

[0014] A method of analysing particles in a blood, serum or plasma sample for serology according to claim 13 in which a reactant is an antibody capable of causing agglutination by binding to antibodies attached directly or indirectly to the inert particle.

[0015] A method of analysing particles in a blood, serum or plasma sample for serology according to claim 13 in which a reactant is an antibody capable of attaching to antibodies or antigens attached directly or indirectly to the inert particle.

[0016] A method of analysing cells such as leukocytes or platelets in a blood, serum or plasma sample according to claim 14 or 15 in which a reactant is an antigen or antibody carried on or attached to the cells.

[0017] A method according to any of Claims 1 to 16 in which at least one of the reactants has a fluorescent label.

[0018] A method according to Claim 17 in which the fluorescent label is attached to at least one antibody. A method according to Claim 17 or 18 in which the fluorescent label is used to observe the appearance of aggregates or agglutinates formed in the standing wave or the materials leaving the standing wave in the fluid flow.