Field of Invention:
The present invention relates to a novel diagnostic device for differential detection of blood groups namely A, B and D. The device utilizes the antigens present on the cell surface of erythrocytes (Red Blood Cells, RBC) present in human whole blood as markers and does not require any other special markers.

Background of Invention:
The human ABO blood groups was a concept that was put forth by a biologist Karl Landsteiner in 1901. Landsteiner found that there are substances in the blood; antigens and antibodies, that induce clumping of red blood cells when red cells of one type are added to those of a second type. He recognized three groups – A, B and O – based on their reactions to each other. A fourth group, AB was identified a year later by another research team. Red cells of the A group clump with donor blood group (cells) of the B group; those of the B group clump blood group (cells) of the A group; those of the AB group clump with those of the A or the B group because AB cells contain both A and B group antigens; and those of the O group do not generally clump with any group, because they do not contain either A or B antigens. The application of this knowledge of the ABO system in the life saving practice of blood transfusion is of enormous importance, since mistakes can have fatal consequences.

The discovery of the Rh system by Landsteiner and Alexander Weiner in 1940 was made because they tested human red cells with antisera developed in rabbits and guinea pigs by immunization of the animals with the red cells of the rhesus monkey, Macaca mulatta.

The knowledge and testing for the presence of blood group antigens is a very important requisite in the field of health care, the testing of blood groups is of immense importance in blood transfusions during accidents, surgeries and therapeutic transfusions especially in cases involving hemolytic diseases. Also blood grouping is important during pregnancy, in cases when the fetus conceived is of a positive blood group while the mother is of a negative blood group, which may result in a condition called Erythroblastosis fetalis or Hemolytic Disease of the Newborn (HDN) can occur in which there is mass destruction of fetal red blood cells which in turn, results in jaundice and can even cause neonatal death.

Ever since the discovery of the blood grouping system, there have been various attempts to make the detection of specific blood group antigens more effective in order to avoid or completely eliminate transfusion reactions or the accompanying mishaps.

Prior Arts:
Amongst the arts that has been most widely employed; is the use of antisera. Antisera is sera that have antibodies to the antigen/s present on blood cells. The antisera can be for the identification of either of the three, that is either anti – A, anti – B and anti – Rh, which agglutinate blood samples containing A, B and Rh antigens respectively.
These tests can be done either as slide tests which would require two drops of blood and 2 drops of antisera or alternatively can be done as tube tests, which will require proportionate quantities of blood sample and antisera.

However it is noteworthy to mention that either of these tests have to be performed under proper lab conditions, infrastructure and standard settings. These kinds of tests though are highly efficient are relatively tedious and require the individuals performing the test to have a basic training in the test procedure, which would also require him/her to understand the principle that underlies the test.

Another limitation of this art is that the reagents used are heat labile and therefore have to be stored at temperatures ranging from 2 to 8 deg. C., which may not be convenient in rural and field conditions therefore affecting the efficacy of the test or alternatively rendering the test useless.

Also, during the storage of the antisera, it should be free from any sort of contamination with any matter, which will in turn influence the outcome of the test, thus making the storage of these a more critical parameter that has to be undertaken.

WO/1982/002484: (EN) The device comprises a photographic group, a screen and a liquid crystal display device to visualize the name and the first name of a person of whom the blood group is to be determined. A arm, carrying capillaries, allows to effect automatically the blood takings. A Printer unit provides, in form of graphs, the results of the tests effected and analyzed automatically by the apparatus. The photographic unit takes a simultaneous photography of the person, of his/her description card and of the graphs illustrating the results of the tests.

The limitation of the art is that it is highly sophisticated and a man lacking the necessary knowledge and or of ordinary prudence will not be able to operate the device. The kit being so will have to be used under standard laboratory conditions, proper infrastructure and will have to conducted by persons trained in the art. All these conditions may not always be available in field and rural settings. The time that the test will require to be performed is not mentioned in the patent, which is a key factor in rapid In – vitro diagnostics.

WO/1987/001461: (EN) A device for determining a blood group comprises at least one closed chamber, means for introducing into said chamber a blood sample to be tested in order to contact the sample with a test serum and means enabling to observe the reaction of the test-serum with the blood.

The patentee in his claims has failed to mention the time that the test will require to be performed and whether or not the test can be performed under field conditions and in rural settings. The conditions of storage are also not mentioned in the patent, which may affect the performance of the test and thus the results.

WO/2005/005986: (EN) The invention relates to a device for the simultaneous qualitative or quantitative determination of several analytes in a liquid sample, comprising a membrane with a charging zone, for the application of the liquid sample, at least two indicator zones which can interact with the analyte(s) and at least one absorption region, which accepts the fluid after passing through the indicator zones, whereby the indicator zones lie between the charging zone and an absorption region, characterized in that the flow directions (flow tracks) are essentially parallel from the application zone through each indicator zone to an absorption region and at least two different flow tracks are present. The invention further relates to a method for the determination of several analytes or derivatives thereof in a liquid sample, comprising: application of the sample to the charging zone of a membrane of the device, whereby said sample is present in sufficient amounts to permit the sample fluid to flow in the direction of the absorption region through the indicator zones and to permit the analytes or derivatives thereof in the liquid sample to form a complex in the indicator zone.

Here the patentee has not described the storage conditions of the device and nor is there a mention of the time which the test requires to be conducted, therefore does not explain whether the test is a rapid test or not. The amount of sample that is used in the test is also not given,

WO/2005/005991: (EN) The invention relates to a device for simultaneously, qualitatively or quantitatively identifying a number with: a charging zone for applying the liquid sample; at least two indicator zones, which can interact with the analyte(s), and at least one absorption area, which absorbs the liquid after passing the indicator zones, whereby the indicator zones are located between the charging zone and an absorption area. The invention is characterized in that the flowing directions from the charging zone through the respective indicator zones to an absorption area (flow paths) are essentially parallel, and at least two different flow paths exist. The invention also relates to a method for identifying a number of analytes or the derivatives thereof in a liquid sample, consisting in the application of the sample to the charging zone of a membrane of the device according to one of cited claims 1 to 8, whereby this sample is present in an amount sufficient for causing the sample liquid to flow through the indicator zones toward the absorption area and for causing the analytes or the derivatives thereof in the sample liquid to form a complex in the indicator zones.

Limitations of the art include the absence of the details of the time factor that the test requires to be performed, which is a critical factor in all diagnostic tests. Also the invention makes use of a conjugate and or indicator particles, which are affixed in the form of a conjugate pad to the device, therefore increasing the cost of the production of the device and thus the rate at which the device is availed. The storage of the device is not mentioned, therefore, it is not clear whether or not the adequate storage of the device will be possible in underdeveloped settings.

Brief description with figures:
Figure 1 is a diagrammatic plan view of the device constructed as per the invention. It shows the test device with three colored lines, each at regions marked ''A'', ''Rh'' and ''C''.

Figures 2-8 are diagrammatic plan views of the device constructed as per the invention. They show the results of the tests conducted as per Example 2.
Statement of Invention:
A device for differential detection of blood groups, said device comprising a receptacle and a lid, the substrate comprising a sample receiving zone, a buffer receiving zone, a detection zone comprising a control band coated with a control particle and plurality of indicator bands coated with indicator particles specific to blood groups A, B and ‘Rh’; the sample, buffer and detection zones being arranged in any order and wherein in use the sample received is diluted by the buffer received and flows over the indicator bands and the control band, and generates a color signal.

Detailed description of the invention:
The present invention relates to a novel device for differential detection of blood groups. The device of the invention enables easy visual detection and typing of the blood group and can be performed by any skilled or unskilled person. The device enables sensitive and qualitative immunoassay for detection of blood groups.

The device comprises a receptacle and a lid, the substrate comprising:
- a sample receiving zone,
- a buffer receiving zone,
- a detection zone comprising a control band coated with a control particle and plurality of indicator bands coated with indicator particles specific to blood groups A, B and ‘Rh’;
- the sample, buffer and detection zones being arranged in any order and wherein in use the sample received is diluted by the buffer received and flows over the indicator bands and the control band, and generates a color signal.

In particular, the device is a two-part device, wherein the receptacle includes a chromatographic strip for detecting the presence of A, B and Rh antigens present in Red Blood Cells. The pore size of the chromatographic strip which may be made of nitrocellulose material may be in the region of 0.1 to 0.6 µm. The strip is operatively attached to the receptacle. The lid is configured to fit onto the receptacle. The lid may include an aperture for receiving sample, receiving buffer and a region with inscriptions of various blood groups, i.e. A, B and Rh. Preferably the device may be made of polystyrene and or High Impact Polystyrene (HIPS) and or Acrylonitrite Butadiene Styrene (ABS).

The chromatographic strip on the receptacle comprises various zones for receiving the sample and buffer and a detection zone. The detection zone of the strip includes bands labeled as ‘A’, ‘B’ and ‘Rh’. The bands are coated with monoclonal or polyclonal antibodies raised against A, B and Rh antigens. The amount of antibody coated may be in the region of 3 to 10µl. The invention utilizes the ability of Red Blood Cells which contain A, B and Rh antigens to bind with the polyclonal antibodies coated onto the chromatographic strip. The polyclonal or monoclonal antibodies may be preferably mouse or goat anti-human antibodies. The strip may include a control band coated with a lectin or glycophorin A protein. The lectin may be obtained from any plant source such as phaseolus vulgaris. The antibodies and proteins used for coating may be diluted with 10 – 25 mM phosphate buffer or phosphate buffer saline (PBS).

To facilitate easy detection of the blood groups, a sample pad is positioned just below the aperture of the sample receiving zone and a sink pad is positioned just below the aperture of the buffer receiving zone and the detection zone follows thereafter. As stated earlier, the letters ''A'', ''B'', ''C'' and ''Rh'' are inscribed on the lid part top component where ''A'', ''B'' and ''Rh'' represents the anti A, anti B and anti Rh polyclonal and or monoclonal mouse antihuman specific antibody coated regions, and the letter ''C'' represents the control line.

In operation, the test sample is introduced into the sample receiving aperture alongwith the sample running buffer, which is introduced through the aperture for receiving buffer. The sample and the buffer flow laterally on the absorbent pad and upon reaching the detection zone, it contacts the antibody coated on the nitrocellulose strip. If the target antigen is present on the cell surface within the sample, a few cells from the sample get immobilized to that region; while other cells flow through with the sample running buffer to the subsequent immobilized antibodies. In each of the subsequent regions, respective antigens; if present on the Red Blood Cells are captured by the specific capture antibody, which can be either polyclonal and or monoclonal mouse anti-human antibodies.

The Red blood cells themselves being colored, form a dark red colored band on the particular region on which they are immobilized by the specific complementary antibody.

To serve as a procedural control, an additional control line C has been immobilized at a distance from the three test lines on the membrane. It is desirable that the distance between the control region and the bands should be at least 4.0 mm, for the reason that blood sample has a tendency to flow rapidly beyond a point and if a specific distance is not maintained then detection of the color signal generated would be difficult and further, it would be very difficult to find out whether the device is working at all. It is also desirable that the distance between the bands labeled A, B and Rh should be at least 2.5 mm.

The invention also provides a kit for differential detection of blood group. The kit includes the device as described above and desiccant, a loop, sample running buffer and fixing solution. All these components are provided along with the kit together with an instruction manual so that the user finds it easy to operate the device and the kit.

The buffer may comprise 10 – 15 mM Phosphate buffered saline (PBS) or 10 – 15 mM Phosphate Buffer and 10 – 15mM NaCl solution + 2 - 5% of a surfactant either brij or Tween – 20 or Tween 80 (surfactant), The pH of the buffer may be in the region of 7.2 + 0.4. The fixing solution comprises 10–15 mM Phosphate Buffered Saline (PBS) or 10–15 mM Phosphate buffer and 10–15 mM NaCl solution + 2-5% of a surfactant containing either brij or Tween–20 or Tween– 80 + 0.5–1.0% glutaraldehyde or 0.5 – 1.0% formaldehyde. The pH of the fixing solution may be in the region of 7.2 + 0.4.

The device and kit of the present invention can be used by a man of ordinary prudence and the device is so designed that the results are evident within a short period of about 10 minutes. The device can be stored at temperatures of up to 30 deg. C. enabling use of the device even in conditions where refrigeration or low temperature maintenance of the device is not feasible.

The invention is now illustrated with the following examples and drawings which are for illustrative purposes only and not meant to restrict the scope of the invention in any manner.

Example 1: Construction of the device
Plastic housing with a receptacle and a lid was manufactured. The lid contained two apertures and inscriptions labeled as A, B and Rh. A nitrocellulose strip coated with polyclonal antibodies raised against A, B and Rh antigens was assembled within the plastic housing. Care was taken to ensure that distance of 2.5 mm was maintained between the bands and distance of 12 mm was maintained between the last band and the control band. The device was tightly sealed for further use. The device as constructed is shown in figure 1.

Example 2: Detection of blood group
With the help of the loop provided, approximately 5µl of whole blood was added via aperture on the lid into the device. Six drops of the sample running buffer was added using a dropper into the buffer receiving cavity. The reaction between the sample and the buffer as well as bands coated on the strip was allowed to take place. The results were visible within 2-5 minutes; however, the results were finally read and readings were taken after 10 minutes. In order to save the results for a longer period, 10 - 25µl of the fixative solution was added after reading the results.

Results:
Dark coloured band was found at the spot labeled B, which means that the sample contained blood of B group.

Interpretation of results:
Appearance of three colored lines, each at regions marked ''A'', ''Rh'' and ''C'' - Interpretation: blood group; A+.

Appearance of two colored lines, each at regions marked ''A'' and ''C'' - Interpretation: blood group; A-.

Appearance of three colored lines, each at regions marked ''B'', ''Rh'' and ''C''. - Interpretation: blood group; B+.

Appearance of two colored lines, each at regions marked ''B'' and ''C''. - Interpretation: blood group; B-.

Appearance of two colored lines, each at regions marked ''Rh'' and ''C''. - Interpretation: blood group; O+.

Appearance of one colored line, at region marked ''C''. - Interpretation: blood group; O-.

Appearance of four lines colored, at regions marked ''A'', ''B'', ''Rh'' and ''C''. - Interpretation: blood group; AB+

Appearance of three colored lines, each at regions marked ''A'', ''B'' and ''C''. - Interpretation: blood group; AB-.

Invalid test: In any case, if the control band does not appear the test is to be considered invalid.

The results as set out above are shown in figures 1-8.

Similarly, tests were conducted with 10 other samples obtained from different individuals. The results were read and 4 belong to A+ type blood group, 3 of O+ and 3 were of AB+ group.

Example 3: Sensitivity and specificity
The sensitivity of the device was compared with the sensitivity of the test currently used, i.e. detection of blood type using anti-sera. The results are set out in the table below.

Assay Sensitivity
No. of samples tested / No. giving correlating results %age sensitivity of the device
Anti-sera 85 / 85 100
Device of the invention 85 / 85 100

From the results above, it is clear that the device has a sensitivity of more than 99%, which is considered highly accurate in blood group typing.

Advantages of the invention:
The device in the present invention is designed as a self performing test kit for the analyses of the presence of blood group antigens in on erythrocytes in human whole blood. The device in the present invention is a major improvement in the field of In – vitro diagnostics, it is designed in such a manner that the red blood cells that serve as the analyte for the test, themselves also serve as the indicator molecule for monitoring the test. This is a very significant improvement, which is for the first time available in a device for the detection of antigens present on the Red Blood cells, which are red in color by itself, and are employed as the marker entities. Thereby substantially reducing manufacturing costs. The device mentioned in the present invention is self operative on addition of blood sample and sample running buffer, and the simplicity by which the test can be performed ensures that a man of ordinary knowledge can operate the device with confidence and ease. The device is designed to be robust and portable, having a storage temperature ranging from 2 to 30 deg. C, thus making it an appropriate choice for field conditions. The operation of the device is such that it requires minimum infrastructure and training. Therefore the device can not only cater to the requirements of sophisticated labs but also be used with the same ease in rural and underdeveloped settings.

This Rapid lateral flow device is a visual, sensitive and accurate test for analyzing the presence of blood group antigens in human blood. The device has a minimum sample requirement, of 2 to 5 µl, for which a finger – prick sample should suffice, thus making the device even more user friendly. The device is a rapid diagnostic test device for the qualitative detection of blood group antigens. The proper and accurate determination of the blood group is possible within 2 to 5 minutes.

The novel invention also has an added advantage from the other available blood grouping devices, in that it is provided with a fixing solution, that fixes the reaction, which can be kept for a long period, of more than 3 months for future referral.

The present invention has the following advantages:

a) The advantage of the present invention is to provide a rapid, self performing, sensitive, and visual test, with minimum sample requirement, and with no sample treatment required prior to the test.

b) The Marker molecule that is used in the test is the analyte itself in that the red blood cells themselves serve as a signal and a visual indicator for viewing the test, therefore decreasing the production cost of the device since there is no need of additional requirement of signal molecules and or gold conjugates and or biotinylated peptides for the detection of antigens. This principle of using the analyte itself as the marker is a novelty in the field.

c) The device in the present invention is a self performing kit, which on addition of the sample and the sample running buffer; the results can be visualized within 5 to 10 minutes from the addition of both the test components, therefore making the test immensely easy to perform and can be done by any individual having minimum or no expertise in the area.

d) The sample quantity that is required in this novel invention, is also very less, 2 to 5 µl even a finger – prick blood sample is useful in the effective detection of blood group antigens present on the cell surface of the RBCs in human whole blood.

e) The test requires minimum infrastructure and therefore can be used in field conditions. The buffer is also provided in dropper bottles therefore there is no need of specialized equipment.

f) The device has a convenient storage ranging from 2 to 30 deg. C. thus making it robust.

g) The device is made up of a low weight material which has a high tensile strength like polystyrene and or High Impact Polystyrene (HIPS) and or Acrylonitrite Butadiene Styrene (ABS) and therefore many devices can be carried easily and transported with minimum or no self imposed damage.

h) The kit is inclusive of a fixative solution and hence the reaction can be fixed with the addition of a few drops of the fixative solution and thus the test can be preserved for more than 3 months for future reference and documentation.

i) The device in the present invention aims to bring about precision, efficacy and ease of operation.

j) Two devices used simultaneously can aid in the direct testing of patient / donor compatibility during transfusions.

WE CLAIM:

1. A device for differential detection of blood groups, said device comprising:
a receptacle and a lid, the substrate comprising
i. a sample receiving zone,
ii. a buffer receiving zone,
iii. a detection zone comprising a control band coated with a control particle and plurality of indicator bands coated with indicator particles specific to blood groups A, B and ‘Rh’;
iv. the sample, buffer and detection zones being arranged in any order and wherein in use the sample received is diluted by the buffer received and flows over the indicator bands and the control band, and generates a color signal.

2. A device as claimed in claim 1, wherein an absorption pad made of nitrocellulose is situated at the sample receiving zone.

3. A device as claimed in claims 1 and 2, wherein a sink pad made of nitrocellulose is situated at the buffer receiving zone.

4. A device as claimed in claims 1 to 3, wherein the sample receiving zone, the buffer receiving zone and the detection zone are collinearly located on the substrate.

5. A device as claimed in claims 1 to 4, wherein the substrate is a membrane made of a nitrocellulose material having a pore size in the range 0.10 to 0.60 µm.

6. A device as claimed in claims 1 to 5, wherein the indicator particle is a polyclonal or monoclonal antibody raised against ‘A’, ‘B’ and ‘Rh’ antigens.

7. A device as claimed in claim 6, wherein the control particle comprises glycophorin A or lectin obtained from a plant source such as Phaseolus vulgaris.

8. A device as claimed in claim 1, wherein the buffer comprises 10 – 15 mM Phosphate buffered saline (PBS) or 10 – 15 mM Phosphate Buffer and 10 – 15mM NaCl solution + 2 - 5% of a surfactant either brij or Tween – 20 or Tween 80 (surfactant), pH 7.2 + 0.4, and preferably the buffer is a phosphate buffered saline (PBS) or phosphate buffer.

9. A device as claimed in any of the preceding claims 1 to 8, wherein the amount of indicator particle coated is between 3 to 10µl.

10. A device as claimed any of the preceding claims 1 to 9, wherein the receptacle and the lid portion is made of a material selected from the group comprising Polystyrene, High Impact Polystyrene (HIPS) and or Acrylonitrite Butadiene Styrene (ABS).

11. A kit for detection of antigens present on human red blood cells, said kit comprising:
a. a device as claimed in any of the preceding claims 1 to 10;
b. a desiccant;
c. a loop;
d. sample running buffer; and
e. fixing solution.

12. A kit as claimed in claim 11, wherein the sample running buffer comprising 10 – 15 mM Phosphate buffered saline (PBS) or 10 – 15 mM Phosphate Buffer and 10 – 15mM NaCl solution + 2 - 5% of a surfactant either brij or Tween – 20 or Tween 80 (surfactant), pH 7.2 + 0.4.

13. A kit as claimed in claims 11 and 12, wherein fixing solution comprises 10–15 mM Phosphate Buffered Saline (PBS) or 10–15 mM Phosphate buffer and 10–15 mM NaCl solution + 2-5% of a surfactant containing either brij or Tween–20 or Tween– 80 + 0.5–1.0% glutaraldehyde or 0.5 – 1.0% formaldehyde, pH 7.2 + 0.4.

14. A method for differential detection of blood groups, said method comprising the steps of:
(a) introducing 2 - 5µl of human whole blood into the sample receiving zone;
(b) adding 2 to 7µl of buffer into the buffer receiving zone; and
(c) allowing the sample running buffer to flow along with blood sample to the detection zone; and
(d) reading the colour signal generated, wherein presence of a dark coloured band is indicative of the type of blood group.

15. A device for detection of antigens present on human red blood cells substantially as herein described with reference to the accompanying drawings.