CA2365178A1

CA2365178A1 - Method and apparatus for blood typing analysis

Info

Publication number: CA2365178A1
Application number: CA002365178A
Authority: CA
Inventors: Fariborz Dehghan
Original assignee: Royce Technologies LLC USA
Current assignee: Royce Technologies LLC USA
Priority date: 2001-12-14
Filing date: 2001-12-14
Publication date: 2003-06-14

Abstract

A method for blood typing analysis including the steps of providing a suitable macro-array or micro-array substrate having a plurality of known sites each of which have been seeded with a reactant. The identity of the reactant at each of the sites being known. The sites are exposed to a serum or plasma solution to be tested containing unknown antibodies or unknown antigens. A reaction at one of the sites enables the identity of the unknown antibodies or antigens to be determined by using the known identity of the reactant at the reactive site.

Description

TITLE OF THE INVENTION:
Method And Apparatus For Blood Typing Analysis FIELD OF THE INVENTION
The present invention relates to a method and apparatus for blood typing analysis with particular application to blood bank testing.
BACKGROUND OF THE INVENTION
Before any blood transfusion takes place, it is prudent medical practise to test the blood of the donor to ensure that it is compatible with the blood of the patient. The presence of incompatible antibodies or antigens results in agglutination reactions within the blood which places the patient at risk.
In addition, the blood must under go pathogen screening for blood transmissible pathogenic factors, such as HIV, HAV, HBV, HCV, herpsvirus, syphilis, and parasites.
Traditional test tube testing tends to be a somewhat subjective art. The test involves shaking of the test tube along with a visual examination for an agglutination reaction.
Variations in results often arise between technicians due to different degrees of shaking and different interpretations of the visual results. The task is made more difficult by the fact that the agglutinations being examined are not stable due to re-suspension of red blood cells. It is also made more difficult when anti-bodies are present in such low concentration that agglutination does not immediately occur.
There are also conditions that lead to false agglutination results. Rouleaux formation is caused by an excess of high molecular portein in the serum. This is seen in patients with conditions that lead to abnormal globulin levels. The presence and appearance of Rouleaux obscures agglutination.
Agglutination is also unreliable in blood samples that are lipemic, icteric or contain weak alloantibodies.
In view of the shortcomings of traditional test tube

2 testing, a number of alternative technologies have been developed, namely: gel testing, solid-phase immunoassays, and affinity column chromatography. The gel test uses the principle of controlled centrifugation of red blood cells through a gel containing appropriate reagents. The agglutinants are trapped by gel particles. Solid-phase immunoassays affix target antigens or antibodies to the bottom of a micro titer well. In positive reactions, indicator cells adhere to a layer of IgG coated red blood cell wall to form a second immobilized layer. In negative reactions, the indicator cells will not attach to the cell wall, falling to the bottom ~of the well to form tightly agglutinated red blood cell buttons. Affinity column chromatography relies upon the ability of protein A (staphylococcus aurous) and protein G
(group G staphylococcus) to bind the Fc portion of IgG
molecules. Immuno-reactive columns are prepared by covalently linking these two proteins to a bead gel matrix. In positive reactions, the red blood cells coated with IgG adhere to - immuno-reactive gel to produce a band of red blood cells atop the column. In negative reactions, all red blood cells pass through the immuno-reactive gel and settle to the bottom.
The major advantages that these technologies provide over routine tube testing are standardization, stability, decreased sample volumes, and enhanced sensitivity and specificity. Each of the gel, solid phase immunoassays, and affinity column chromatography technologies require the use of special centrifuges and multiple samples.
SUMMARY OF THE INVENTION
What is required is a method and apparatus for blood typing analysis that provides desired standardization, stability, decreased sample volumes, enhanced sensitivity and specificity without requiring a special centrifuge.
According to one aspect of the present invention there is provided a method for blood typing analysis. A first step

3 involves providing a suitable macro-array or micro-array substrate having a plurality of known sites each of which have been seeded with a reactant. The identity of the reactant at each of the sites being known. A second step involves exposing the sites to one of a serum or plasma solution to be tested containing unknown antibodies or antigens. A third step involves examining for evidence of a reaction at one of the sites and using the known identity of the reactant at the reactive site to determine the identity of one of the unknown antibodies or antigens.
It is envisaged that sites will be assigned an alpha-numeric identifier and that each alpha-numeric identifier will be associated with a different one of the known blood testing reactants. As will be hereinafter further described, the blood testing reactants will include known antibodies when the purpose of the analysis is to detect unknown antigens. The blood testing reactants will include known antigens when the purpose of the analysis is to detect unknown antibodies. The blood testing reactants may consist of either known antibodies, antigens or DNA when the purpose of the analysis is pathogen screening. For the most rapid and thorough analysis, it is preferred that all of the tests be combined into one by using as blood testing reactants a combination of known antibodies, known antigens and DNA. This enables a single test to detect unknown antibodies, unknown antigens and pathogen screening.
According to another aspect of the present invention there is provided an apparatus for blood typing analysis which includes a suitable macro-array or micro-array substrate having a plurality of known sites seeded with blood testing reactants .
An identity of the blood testing reactant located at each of the sites being known.
The method and apparatus, as described above, enables more extensive blood testing more rapidly with smaller quantities of blood, as will hereinafter be further described.

4 BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings, the drawings are for the purpose of illustration only and are not intended to in any way limit the scope of the invention to the particular embodiment or embodiments shown, wherein:
FIGURE 1 is a diagrammatic representation of blood typing analysis to detect the presence of unknown antibodies in accordance with the teachings of the present method, using non fluorescent checkers to improve visibility.
FIGURE 2 is a diagrammatic representation of blood typing analysis to detect the presence of unknown antigens in accordance with the teachings of the present method, using non fluorescent checkers to improve visibility.
FIGURE 3 is a diagrammatic representation of blood typing analysis to detect the presence of unknown antibodies in accordance with the teachings of the present method, using fluorescent checkers to improve visibility.
FIGURE 4 is a diagrammatic representation of blood typing analysis to detect the presence of unknown antigens in accordance with the teachings of the present method, using fluorescent checkers to improve visibility.
FIGURE 5 is a perspective view of a first embodiment of an apparatus for blood typing analysis constructed in accordance with the teachings of the present invention.
FIGURE 6 is a perspective view of a second embodiment of an apparatus for blood typing analysis constructed in accordance with the teachings of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The preferred method and apparatus for blood typing analysis will now be described with reference to FIGURES 1 through 6.
Referring to FIGURE 5, there is illustrated an apparatus for blood typing analysis, generally indicated by reference numeral '11. Apparatus 11 consists of a suitable array substrate 30. The array on the substrate can be a macro-array or a micro-array. This substrate may be a microscope glass

5 slide or a slide of glass, plastic, metal, ceramic or any other form of substrate considered suitable by one skilled in the art. Although a rectangular substrate has been illustrated, the substrate can be in any imaginable shape. Substrate 30 has a plurality of sites 31, the location of each site being known.
Sites 31 are seeded with blood testing reactants 40, 42, 44, etc. The identity of each blood testing reactant located at each of sites 31 is known. In FIGURE 5, each blood testing reactant has been assigned a different reference numeral to reflect their known identity. It is envisaged that sites 31 will be assigned an alpha-numeric identifier and that each alpha-numeric identifier will be associated with a different one of the known blood testing reactants. As will be hereinafter further described, the blood testing reactants will include known antibodies when the purpose of the analysis is to detect unknown antigens. The blood testing reactants will include known antigens when the purpose of the analysis is to detect unknown antibodies. The blood testing reactants may consist of either known antibodies, antigens or DNA when the purpose of the analysis is pathogen screening. For the most rapid and thorough analysis, it is preferred that all of the tests be combined into one by using as blood testing reactants a combination of known antibodies, known antigens and DNA.
This enables a single test to detect unknown antibodies, unknown antigens and pathogen screening.
The method for blood sample analysis involves a first step of providing apparatus 11, as described above, having known sites 31 with known blood testing reactants. A second step involves exposing sites 31 to a serum or plasma solution to be tested containing unknown antibodies or unknown antigens. A
third step involves examining apparatus 11 for evidence of a reaction at one of sites 31 and using the known identity of the

6 blood testing reactant at the reactive site to determine the identity unknown antibodies or unknown antigens.
Antibody Screening Referring to FIGURE 1, bodies of antibody 20, against red blood cells (RBC) are attached to the surface of substrate 30.
The art of attachment of protein and antibodies are well known to persons skilled in the art and will, therefore, not be further described. In order to screen the different antibodies, one has to array different antigens in a known location on the substrate. The layer 20 is an anti-human red blood cell, antibodies made of goat. It could also be made of proteins such as protein G and protein A, which specifically attach to the Fc portion of antibodies. Or, organic molecules that act as a glue for attachment of red blood cells. Therefore, the Fab portion remains free for binding to antigens. Red blood cells fractionate 12a and 12b are attached to substrate 30 via antibodies 20 in FIGURES 1 and 3. In order to screen for different antibodies in serum/plasma, one has to array different fractions of red blood cells that posses a known antigen in a known location (50,52). This will give substrate the ability to have arrays of red blood cells, 12a and 12b, 25 with different antigens in known locations 50,52. By passing the serum/plasma over the substrate 30, any compatible antibody 18, which recognizes the antigen in fractionated red blood cell 12b, will be bound to an antigen. Since the antibody 18 does not recognize antigens from 12a, it therefore does not bind to 30 it. As will hereinafter be further described, reactive sites can be made more visible using either non-fluorescent checkers as illustrated in FIGURE 1, or fluorescent checkers as illustrated in FIGURE 3.
Referring to FIGURE l, after the attachment of unknown antibody 18 from serum/plasma to the known antigen 12b, there is a bridge made by the addition of monoclonal antibody 16 to

7 a non-fluorescent checker cell 14 to form a complex 22a.
Checker cell 14 could be red blood cells of animal origin, such as sheep, rabbit or human, sensitized to human globulin IgG.
Complex 22a with monoclonal antibody 16 could also be replaced by human RBC sensitized with anti-human IgG via its Fc portion of antibody leaving the Fab portion free for binding to human antibodies captured by cell antigen from the surface of the substrate. The role of checker cells 14 could also be performed by micro-sphere beads made from material such as polystyrene which can easily accommodate attachment of antibodies to its surface.
If there is no antibody 18 serum/plasma, the bridge will not be formed and the whole complex is not formed, as is the case in 12a. A red appearance of the complex is an indication of positive results. Since the antigen and the location are known, the antibody will be determined.
Referring to FIGURE 3, After antibody 18 is attached to red blood cells, fraction 12b, a fluorescent-tagged anti-human globulin (AHG), 24 are passed on the complex. This will bind to antibody 18 and shows positive results after reading with a chip reader (available from such manufacturers as Virtek located in Hamilton, Canada). Tagged particle 24 could be an enzyme or gold particles or coloured micro-sphere beads from molecular probe. As described above, checker cell 14 could also be changed with others such as micro-sphere beads of different colour or red blood cells from different animals, such as sheep, rabbit or human.
Antigen Screening FIGURES 2 and 4 are diagrammatic illustrations of an antigen screening on apparatus 11. FIGURE 2 is non-fluorescent and FIGURE 4 is a fluorescent version in which the captured red blood cell 26 has taken up some fluorescent dye. FIGURE 2 shows the known array of antibodies 40, 42, 44 at different known locations on the surface of substrate 30. Upon passing

8 of red blood cells 10 from human, if there is a match, it will be formed at the site when antibodies 40 is located. The antigen from red blood cell 10 will be determined by knowing the identity of antibody 40 at the reactive site. Referring to FIGURE 4, it will be easier using a chip reader or fluorescent detector since red blood cell 26 has taken on some fluorescent dye.
Advantages The method, as described above, provides advantages with respect to cost, efficiency, and sensitivity. In addition, it provides for some degree of automation, while being less prone to human error. This form of micro analysis completely eliminates any need to centrifuge the blood solution. A vast number of sites can be placed on bimolecular chips enabling all testing to be done simultaneously for antigens, antibodies and pathogen screening. The amount of blood required for the test is minute, less than one milli-litre of liquid is sufficient to cover the entire surface of the bimolecular chip. This makes the testing ideal for extreme situations in which the patient has already lost large amounts of blood and time is critical. The test provides a blood typing analysis in a fraction of the time which was formerly required.
fluorescent vs. non-fluorescent Some molecules are naturally fluorescent or autofluorescent. For this reason, chemiluminscent technology using non-fluorescent techniques is increasingly being used as an alternative to fluorescent technology.
Variations:
Referring to FIGURE 6, in this embodiment the substrate takes the form of a disc, generally indicated by reference numeral 100. Positioned on disc 100 are a plurality of known

9 sites 102 seeded with blood testing reactants 104. As described above, the identity of the particular blood testing reactant 104 located at each of sites 102 is known. This embodiment of the apparatus is preferred for a number of reasons:
- the mechanical parts used for compact disc (CD) players can be used as a base technology from which scanners can be constructed. CD players are already mass produced and, as such, is comparatively inexpensive. It is easier to miniaturize the instrument as less mechanical parts are needed;
- scanning merely requires rotation in a manner similar to a CD. There is no requirement for X,Y movement to scan the entire disc. Rotation is faster than X, Y movement scanning;
- a disc has more room for binding molecules;
- it is easier to spot the discs with primary molecules as rotation of the disc results in positioning for spotting.
This enables spotting instruments to be built at less expense;
- liquid washing and draining can be accomplished through the rotational motion. With the thickness of the liquid being adjusted by the speed of rotation.
In this patent document, the word "comprising" is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. A reference to an element by the indefinite article "a" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
It will be apparent to one skilled in the art that modifications may be made to the illustrated embodiment without departing from the spirit and scope of the invention as hereinafter defined in the Claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for blood typing analysis, comprising the steps of:
providing a suitable array substrate having a plurality of known sites each of which have been seeded with a reactant, the identity of the reactant at each of the sites being known;
exposing the sites to one of a serum or plasma solution to be tested containing unknown antibodies or antigens; and examining for evidence of a reaction at one of the sites and using the known identity of the reactant at the reactive site to determine the identity of one of the unknown antibodies or antigens.

2. The method as defined in Claim 1, the array being a macro-array.

3. The method as defined in Claim 1, the array being a micro-array.

4. The method as defined in Claim 1, at least some of the sites using as blood testing reactants known antibodies for the purpose of detecting unknown antigens.

5. The method as defined in Claim 1, at least some of the sites using as blood testing reactants known antigens for the purpose of detecting unknown antibodies.

6. The method as defined in Claim 1, at least some of the sites using as blood testing reactants least one of an antibody, an antigen and DNA for the purpose of pathogen screening.

7. The method as defined in Claim 1, the sites using as blood testing reactants a combination of known antibodies, known antigens and known DNA, for the purpose of detecting antibodies, antigens and pathogen screening, all in a single test.

8. A method for blood typing analysis, comprising the steps of providing a bimolecular imaging chip having a plurality of known sites which have been seeded with an ordered micro-array of blood testing reactants, there being only one blood testing reactant at each of the sites with the identity of the blood testing reactant at each of the sites being known;
exposing the sites of the bimolecular imaging chip to a blood solution to be tested; and examining the sites on the bimolecular imaging chip for evidence of a reactive site at which a reaction has occurred between the blood solution and one of the blood testing reactants and determining which of the blood testing reactants has reacted by correlating the reactive site with the known identity of the blood testing reactant at each of the sites.

9. A method for blood typing analysis, comprising the steps of:
providing a bimolecular imaging chip having a plurality of known sites which have been seeded with an ordered micro-array of blood testing reactants, there being only one blood testing reactant at each of the sites, each of the sites having a unique identifier which identifies the blood testing reactant seeded at that site;
exposing the sites of the bimolecular imaging chip to a blood solution to be tested;
examining the sites on the bimolecular imaging chip for evidence of a reactive site at which a reaction has occurred between the blood solution and one of the blood testing reactants; and determining which of the blood testing reactants has reacted with reference to the site identifier for the reactive site.

10. The method as defined in Claim 8 or 9, the blood testing reactants being at least one of antigens, antibodies or DNA.

11. The method as defined in Claim 8 or 9, the blood testing reactants being adapted for pathogen screening.

12. The method as defined in Claim 8 or 9, examining including the use of fluorescent or non-flourescent checkers.

13. An apparatus for blood typing analysis, comprising:
a suitable array substrate having a plurality of known sites seeded with blood testing reactants, an identity of the blood testing reactant located at each of the sites being known.

14. The apparatus as defined in Claim 13, wherein the array is a macro-array.

15. The apparatus as defined in Claim 13, wherein the array is a micro-array.

16. The apparatus as defined in Claim 13, wherein at least some of the sites are using as blood testing reactants known antibodies for the purpose of detecting antigens.

17. The apparatus as defined in Claim 13, wherein at least some of the sites are using as blood testing reactants known antigens for the purpose of detecting antibodies.

18. The apparatus as defined in Claim 13, wherein at least some of the sites are using as blood testing reactants least one of an antibody, an antigen and DNA for the purpose of pathogen screening.

19. The apparatus as defined in Claim 13, wherein the sites using as blood testing reactants a combination of known antibodies, known antigens and known DNA, for the purpose of detecting antibodies, antigens and pathogen screening, all in a single test.

20. The apparatus as defined in Claim 13, wherein the substrate is in the form of a rotatable disc.

21. An apparatus for blood typing analysis, comprising:
a bimolecular imaging chip having a plurality of known sites seeded with an ordered micro-array of known blood testing reactants there being only one blood testing reactant at each of the sites, each of the sites having a unique identifier which identifies he blood testing reactant seeded at that site, thereby permitting rapid identification of a reactive site on the bimolecular imaging chip by reference to the site identifier for the reactive site.

22. The apparatus as defined in Claim 21, wherein the blood testing reactants include at least one of antigens, antibodies or DNA.

23. The apparatus as defined in Claim 21, wherein the blood testing reactants being adapted to perform pathogen screening.

24. The apparatus as defined in Claim 21, wherein the chip is in the form of a rotatable disc.