GB2484735A - Blood typing method and instrument - Google Patents

Abstract

A method for crossmatching blood in which recipient serum is mixed with donor washed red blood cells and presence or absence of agglutination of red blood cells is recorded, wherein agglutination indicates an incompatible donor due to binding of antibodies in the recipient serum to the donor red blood cells, characterised in that the method comprises a step of incubating the mixed serum, donor cells and albumin at a preselected temperature greater than 37°C but not more than 50°C prior to assessment of agglutination. Preferably the temperature is between 39°C and 45°C. The method can be used for RhD (Du) testing in particular. Also claimed is a portable instrument for rapid blood typing or crossmatching of multiple samples using said method which comprises means for automatic control of sample incubation temperature and means to alert the operator upon incubation completion.

Description

BLOOD TYPING METHOD AND INSTRUMENT

The invention is in the field of medical laboratory technology, in particular, blood serology, blood bank and blood transfusion. The invention provides improved methods and instruments for blood grouping, RhD and weak D (Du) antigen typing, crossmatching, and has application for Direct Coombs tests for in vivo/auto antibodies.

Prior to conducting a blood transfusion, donor blood is tested for compatibility with the blood of the intended recipient in order to avoid triggering a dangerous immune reaction in the recipient. The compatibility testing of blood involves determining blood group (0, A, B, AB), RhD and weak RhD (DU) antigen type (positive or negative) and other non-ABO antigens, if present. Weak RhD (Du) testing to confirm RhD negative red cells and to detect weak RhD antigens is a major component of pre-blood transfusion compatibility testing which takes some time to complete.

A current crossmatching method is based on the principle of testing patient serum with donor washed red cells to determine the presence or absence of antibodies in the patient's serum that react with the donor's red cells. Binding of antibody to antigen is indicated by agglutination. Standard protocols for crossmatching and weak RhD testing are described in the

Comparative Example.

The problem with the standard methods is that they contain many manual steps, e.g. multiple washing steps, which make the tests labour intensive and slow to complete. For example, current techniques typically take 30-60 minutes to generate a result. The condition of a patient requiring emergency surgery may deteriorate significantly in this period. The total blood volume in the human body is about 5-6 litres; a person may die if he loses one third of his blood volume. A patient with severe bleeding may therefore lose a life-threatening volume of blood during the period required to perform a compatibility test for blood transfusion. At present, compatibility testing is performed in the laboratory; such testing cannot be done in the field because complex instruments and specialist technicians are needed in order complete the test in the least possible time -about half an hour.

Accordingly there is a need for methods and instruments for crossmatching of blood which give rapid, reliable results without the need for sophisticated laboratory facilities.

In order to enhance the agglutination of RhD and other antibodies it is desirable to reduce the distance between red cells in saline by decreasing the repulsion between red cells (zeta potential). Reducing the zeta potential makes it easier for sensitised red cells by lgG (warm) antibodies to form a lattice structure that bridges the sensitised red cells, which is referred to as agglutination, a visible clumping of red cells.

The inventors have achieved this objective by control of procedural steps and materials (samples and reagents). Parameters identified by the inventors for a rapid and sensitive test include the optimal ratio of red cell suspension (antigens on red cells) to antibody (antibodies in serum); pH (6.5-7.5) of test tube mixture; incubation time; Na and or ions in the 0.85% normal saline; and 22% bovine albumin (enhancer), which increases the dialectrical constant of the medium; and temperature.

Accordingly, the present invention provides a shorter method for crossmatching blood in which recipient serum is mixed with donor washed red blood cells and the presence or absence of agglutination of red blood cells is recorded, wherein agglutination indicates an incompatible donor due to binding of antibodies in the recipient serum to the donor red blood cells, characterised in that the method comprises a step of incubating the mixed serum, donor cells, and albumin at a pre-selected temperature greater than 37°0 but not more than 5000 prior to assessment of agglutination.

The operating temperature range of the instrument described herein is 38- 50°C. It is preferred that the incubation temperature is in the range 39-45°C.

Within this range the red cells are not ruptured but achieve maximum expansion. This expansion reduces the zeta potential (distance between red cells) and makes it easier for the corresponding antibodies to reach the antigens on the red cells' surface so that agglutination is achieved in a shorter time (within 3-5 minutes). For the standard manual procedure incubation is at 36-38°C for a minimum of 30 to 60 minutes. The method of the invention will also yield results with higher incubation temperatures of e.g. 46, 47, 48, 49 or even 50°C although, as the incubation temperature is increased, agglutination decreases and microscopic examination is required to detect the results of the test. However, at temperatures close to 50°C and higher lysis of red cells occurs.

Preferably the method includes a further step of adding antihuman globulin (Coombs reagent) to the mixed patient serum and donor cells after the incubation step and before assessment of agglutination.

Advantageously, agglutination may be apparent (i.e. observable) without the need for centrifugation.

A further advantage of the method of the invention is that it is not necessary to subject the mixed serum and donor cells to any washing steps between the incubation step and the addition of antihuman globulin.

The method of the invention is especially useful for RhD (Du) testing of blood.

The invention further provides a portable instrument for rapid blood typing or crossmatching of multiple samples which comprises a means for automatic control of sample incubation temperature and an indicator to alert the operator upon completion of selected incubation time.

The method and user-friendly instrument ("DR BOAUSA") of the invention can be used to produce fast and reliable crossmatching and RhD typing results outside the laboratory, e.g. on ambulances and/or emergency sites, to help save more accident victims and emergency patients.

The invention is described in the following example with reference to the figures, of which: Figure 1 is a photograph of a blood testing instrument according to the invention with the casing opened to expose its component parts: 1. Instrument base; 2. Isolating heating mental reaction chamber; 3. Testing tube; 4.

Cooling fan; 5. Testing tube cavity; 6. Heating source; 7. Thermal sensor switch and meter; 8. Bell alarm; 9. Operation and shut down light indicator; 10. Thermal sensor; 11. Electrical relay; 12. Transformer.

Figure 2 is a circuit diagram for an instrument according to the invention.

Features and advantages of the instrument described herein apply mutatis mutandis to the method of the invention, and vice versa.

The inventor has developed a portable electrical instrument ("DR BOAUSA") for performing the method of the invention and which can provide crossmatching and weak RhD antigen results within 3-5 minutes using the same basic materials used in the standard tube method.

The instrument consists of two major compartments mounted on a base (1). The first compartment contains the control mechanisms and a dry air cooling system. The second compartment contains the heating block comprising chamber (2) enclosing heating source (6) and having wells (5) to receive the sample tubes (3), and a second dry air cooling system (4). The whole system is electrically operated and includes a power supply control switch, indicator lamps (9) for power, start and end of incubation, an additional sound alarm (8) to indicate completion of test incubation time, an electronic thermostat regulator (7) to monitor and control heater temperature and dry air cooling system. The metal casing of the instrument may be finished with a coating of any colour. Multiple tests can be performed simultaneously: the maximum number of samples that can be accommodated at once is limited only by the number of wells provided in the heating block (six in the instrument shown in Figure 1).

The heating system is capable of smoothly and rapidly increasing the temperature of the test samples in the tubes from an initial temperature of 37°C to a temperature selected by the operator in the range 37°C to 50°C which provides maximum expansion of the red cells within the test medium (reagents and solutions) without causing rupture of the cells. This incubation temperature is selected to be optimal for enhanced antigen-antibody binding and decreasing the distance between the red cells (zeta potential) to achieve maximum red cell agglutination. Typically, the incubation temperature is attained within the first 21⁄4 minutes and maintained to the 5 minutes (incubation time) alarm. An incubation time of 5 minutes has been found to be sufficient for agglutination to be realised if antigen-antibody binding has occurred. Longer incubation times may be used if found necessary in particular circumstances. The thermostat mechanism activates the cooling fans as necessary to maintain the selected incubation temperature and not exceed it.

The instrument shown in Figure 1 is approximately 70cm wide and 25cm deep (front to back): the first compartment is approximately 25cm wide and 30cm high and the second compartment is approximately 45cm wide and 20cm high, with the casing closed. The instrument is therefore relatively compact and may be readily transported for use at any desired location. The overall size of the instrument may be reduced further by appropriate design and by selecting component parts of reduced size, making the instrument still more portable and easy to use in a wide variety of settings outside the laboratory.

The method and instrument of the invention can be used to perform blood grouping, RhD and weak RhD typing, and major crossmatch (detection of unexpected antibodies in the patient) and minor crossmatch (detection of unexpected antibodies in the donor) crossmatching. The invention is also expected to have utility for additional blood bank procedures such as Coombs' tests for the detection of unexpected antibodies.

The method and instrument of the invention provide enhanced sensitivity i.e. maximum detection of antigen-antibody binding. This includes agglutinating and potential non-agglutinating antibodies (antibodies that do not agglutinate red blood cells), which are detected after the addition of antihuman globulin (Coombs') reagent before centrifugation. This will ensure the detection of both in vitro (indirect Coombs') and in vivo (direct Coombs') alloantibodies. These antibodies include, among others, anti-rhesus (Rh) and non ABO and Rh antibodies.

The invention utilises the same reagents and washed red cell suspension used in the standard manual tube method, but is capable of producing reliable crossmatch results in 3-5 minutes compared with the 30-60 minutes, or longer, necessary for the standard tube method. The method steps for crossmatching using the instrument of the invention are set out in Table I (right column), alongside the steps of the standard routine test (left column). It can be seen that there is a 40% reduction in the number of method steps using the invention compared to the standard manual method. With the appropriate specific reagents these steps can also be used for e.g. RhD typing and detecting unexpected antibodies (other than ABO and RhD antibodies).

Table 1. Comparison of standard test procedure and procedure of the invention Steps for the performance of crossmatching & Rh-D (Du) _____ test ____________________________ _____ STANDARD Routine Test Invention Procedure _____ Sample/Patient Identification -Sample/Patient Identification - _____ validation validation ______ 2 Patient's Clot Separation -Serum Patient's Clot Separation -Serum 2 Labelling of donor cells washing Labelling of donor cells washing _____ tubes tubes ______ 4 Donor Cells-Washing I Donor Cells-Washing 1 4 Centrifugation Centrifugation 5 6 Donor Cells-Washing 2 Donor Cells-Washing 2 6 7 Centrifugation Centrifugation 7 8 Donor Cells-Washing 3 Donor Cells-Washing 3 8 9 Centrifugation Centrifugation 9 In case of emergency, the three washings can be replaced by the preparation of 2-5% dilution of Donor red ______ _____________________________________ cells. _______ Preparing 2-5% washed donor cells -Preparing 2-5% washed donor cells 10 ______ suspension -suspension ______ 11 Test-Tubes labelling Test-Tubes labelling 11 Mixing patient's serum to 2-5% 12 Mixing patient's serum to 2-5% washed/ diluted (2-5%) donor cells 12 washed/ diluted (2-5%) donor cells (or diluted donor red cells, in _____ ________________________________ emergency_cases) ______ 13 Immediate Spin (IS)-Centrifugation Incubation 38°C-50°C (selected 13 _____ ________________________________ optimal temperature) ______ Add Antihuman-globulin (Coombs') 14 Reading & recording. . 14 _____ _______________________________ reagent_and_mixing ______ Incubation 37° Centrifugation 15 16 37° -Centrifugation Microscopy -if indicated 16 17 Reading/recording Reading, Recording of results 17 18 Washing testing tube-1 _________________________________ ______ 19 Centrifugation _________________________________ ______ Washing testing tube-2 _________________________________ ______ 21 Centrifugation __________________________________ ______ 22 Washing testing tube-3 _________________________________ ______ 23 Centrifugation ________________________________ ______ 24 Add Antihumanglobulin (Coombs') _____ reagent -________________________________ ______ AHG Tube -Centrifugation ______________________________ ______ 26 Reading, Recording of results _______________________________ ______ 27 If negative, add Check Cells (CC) -________________________________ ______ 28 CC tube -Centrifugation -_________________________________ ______ 29 Reading, Recording of results -_______________________________ ______ Microscopy-if indicated IS = Immediate spin stage: The detection of antigen-antibody agglutination in the test tube that contains the patient's serum and the donor's red cells, immediately after the test tube is mixed and centrifuged (before the incubation stage).

AHG= AntiHuman Globulin stage: Where anti-human globulin antibodies (antibodies to human antibodies) are added to the washed (patient serum and donor cells) to detect the presence of any sensitized red cells (red cells with antibody/antibodies attached, but which cannot reach to other red cells to cause agglutination).

CC = Check Cells: These are sensitized 0 positive red blood cells which are used to check that the test -particularly the 3 washing steps and the addition of antihuman globulin reagent -has been carried out correctly.

In the standard procedure, the mixed patient serum and donor cells of step 12 are subjected to an immediate spin (IS) to test for agglutination caused by the presence of antibodies in the patient's serum that react with (i.e. bind to) the donor's red blood cells. If no agglutination is seen in the immediate spin stage or after incubation of the patient serum/donor cell mixture at 37°C, the sample is subjected to further treatment (AHG stage) to check that this is a true negative result and that no sensitized red cells are present. The cells are washed three times (to remove any free antibodies, if present) to yield red cells only and then antihuman globulin (AHG) is added.

If any sensitized red blood cells are present the antihuman globulin will cause them to agglutinate. Agglutination in the AHG stage indicates the presence of small lgG antibodies. Antibodies which cause agglutination upon incubation at 37°C (step 24 of standard procedure) may be termed warm antibodies, whereas antibodies which cause agglutination upon addition of antihuman globulin may be termed antihuman globulin antibodies.

The procedure of the invention is not concerned with the type of antibody detected (warm antibody or antihu man globulin antibody) -such classification can be done as part of the next process, which is the identification of the detected antibody/antibodies. The objective of the present invention is to produce reliable crossmatching and weak Rh-D (Du) typing results in an urgent/shorter time. This is achieved by focusing on whether or not agglutination is present without attempting to classify the type of antibody detected, which can be done later.

As noted above, in the AHG stage of the standard procedure the red cells are washed before addition of the AHG in order to reduce neutralisation of the AHG by excess antibodies in the serum. In the method of the invention the washing steps are dispensed with and any neutralisation of AHG by free antibodies is compensated for by addition of extra drop(s) of AHG compared to the standard procedure. The method of the invention therefore permits the omission of steps 13 to 23 of the standard procedure, which are replaced by the step of incubation at a selected optimal temperature above 37°C but not greater than 50°C.

Because of the built-in heater, it is not necessary to transfer the samples to a separate heating instrument (water bath or heating block). No washing steps are needed. Although centrifugation is recommended for best results, agglutination can most of the time be detected after the incubation and adding the last reagent. Elimination of the washing steps required with the manual method removes the possibility of loss of red cells and\or reagents during washing prior to the addition of the antihuman globulin (Coombs') reagent, and eliminates the possibility of having false negative results caused by pre-zone or post-zone effect.

The prototype instrument illustrated in Figure 1 can produce crossmatch results and confirm negative Rh (D) test in 3-5 minutes compared to the 30-45 minutes required by the standard manual method. To date, about 12,100 crossmatch and negative Rh (D) samples have been tested concurrently by "Dr BOAUSA" and the standard tube method and 100% accuracy results were achieved. These studies where conduced in Libya (at a government medical centre and two hospitals) and the United Arab Emirates (Sharjah Blood Transfusion And Research Services Center). The studies were supervised and coordinated by qualified pathologists and/or internationally certified clinical laboratory scientists.

Due to its compact size, the instrument of the invention is easy to install and requires little space. Its moderate power requirements can be supplied by mains electricity or a 12V battery. These features enable the instrument to be easily carried in a small bag and operated in minutes outside the laboratory in ambulances and mobile hospitals. The instrument is simple to operate which reduces the likelihood of technical and procedural errors. The simple design means that it is easy to maintain and repair. Operating costs are lower than existing crossmatching and RhD detection instruments/methods.

Most importantly, the instrument and method of the invention produces test results more quickly than the standard manual method adopted worldwide. The ability to perform crossmatching and the detection of weak RhD antigens in a shorter time will enable safer (compatible) blood transfusions and prevent transfusion reactions due emergency release of non-compatible blood and save more lives that would otherwise be lost because of the slower standard manual procedure.

Comparative Example

I. Current Crossmatchinq Technique This technique is based on the principle of testing patient serum with donor washed red cells for the detection of unexpected antibodies in the patient's serum against the donor's red cells.

The routine method for crossmatching test requires the following samples, reagents and instruments.

Samples: Patient's serum * 2%-5% suspension of Donor's blood Reacients: Albumin (22%) or Low Ionic Strength Saline Normal saline (0.85% NaCI), or Sensitized red cells "Check Cells" Coombs' (antihuman globulin) reagent Instruments: Incubator Centrifuge Microscope * In a properly labelled tube, donor's red cells are washed three tines with 0.85% saline and 2%-5% suspension is prepared for use.

Crossmatch Procedure: In a properly labelled glass test tube: a) add two drops of patient's serum, b) add one drop of 2% -5% suspension of washed (3 times) donor's red cells, c) mix gently and centrifuge at (3100-3400 RPM for 15 seconds), d) Gently re-suspend and record the presence and strength of the agglutination (0 (negative), 1+, 2+, 3, or 4+), if any, e) add two drops of 22% albumin, f) An Rh control test can (should be) be run in parallel as a control.

g) Mix gently and incubate at 37°C for 30-60 minutes, h) Centrifuge at (31 00-3400 RPM for 15 seconds), i) Gently re-suspend and record the presence and strength of the agglutination (0 (negative), 1+, 2+, 3, or 4+), if any, j) Wash three times with normal saline, k) Add two drops of antihuman globulin (Coombs' reagent) to the tube after removing the supernatant (saline), I) Mix gently and centrifuge at (31 00-3400 RPM for 15 seconds), m) Gently re-suspend and record the results for the presence and strength of the agglutination (0 (negative), I +, 2+, 3+, or 4+), n) if Positive, report positive for the crossmatch and identify the donor's blood as "Incompatible." o) if Negative (no agglutination), add 2 drops of sensitized red cells (check cells), and p) Mix gently and centrifuge at (3100-3400 RPM for 15 seconds).

q) Gently re-suspend and record the presence and strength of the agglutination. If positive, report negative for the crossmatch and identify the donor's blood as "Compatible." r) If Negative (no agglutination), an error in the procedure and repeat the test.

II. Current Technique for Detection of Weak RhD (Du) Antigens This test is used to confirm RhD negative red cells and to detect weak RhD antigens. It is a major component of pre-blood transfusion compatibility testing between the patient and the donor's blood which takes some time to complete.

The routine method for the detection of weak RhD antigens test requires the following reagents and instruments: Samples: Washed patient red cells: when testing for the patient's Rh type and washed donor red cells: when testing for the donor's RhD type.

Reagents: Monoclonal anti-D reagent Albumin (22%) or low Ionic Strength Saline Sensitized red cells "Check Cells" Coombs' (antihuman globulin) reagent Instruments: Incubator Centrifuge Microscope Weak RhD (Di Procedure: This is performed to check for Weak D antigen (Dc'), when RhD typing test is negative.

In a properly labelled glass tube: a) Add one drop of anti-RhD reagent and one drop of 2%-5% washed patient red cell suspension (prepared as described above), one drop of 22% albumin, and mix gently, b) An Rh control test should be run in parallel as a Control, as described in step#1, but with RhD control reagent instead of the anti-RhD reagent, c) Incubate both tubes (test & control) in a water bath at 37°C for 30-minutes (maximum), d) Mix gently and centrifuge at (3100-3400 RPM for 15 seconds), e) Gently re-suspend and record the results for the presence and strength of the agglutination (0= negative, 1+, 2+, 3+, or 4+ = positive), if any, f) Wash three times with normal saline, g) Re-suspend and add two drops of antihuman globulin (Coombs' reagent), h) Mix gently and centrifuge at (3100-3400 RPM for 15 seconds), i) Gently re-suspend and record the presence and strength of the agglutination (0= negative, 1+, 2+, 3-i-, or4+ = Positive), j) if Positive, report negative for RhD and positive for weak-RhD and the test is completed.

k) if Negative (no agglutination), add 2 drops of sensitized red cells (check cells), I) Mix gently and centrifuge at (3100-3400 RPM for 15 seconds), and m) Gently re-suspend and record the presence and strength of the agglutination. If positive, report RhD and weak RhD negative, and if negative, repeat the test.

Claims (8)

1. CLAIMS1. A method for crossmatching blood in which recipient serum is mixed with donor washed red blood cells and presence or absence of agglutination of red blood cells is recorded, wherein agglutination indicates an incompatible donor due to binding of antibodies in the recipient serum to the donor red blood cells, characterised in that the method comprises a step of incubating the mixed serum, donor cells and albumin at a preselected temperature greater than 37°C but not more than 50°C prior to assessment of agglutination.
2. 2. A method according to claim 1, in which the mixed serum, donor cells and albumin are incubated at a preselected temperature between 39°C and 45°C.
3. 3. A method according to claim 1 or claim 2, in which antihuman globulin is added to the mixed patient serum and donor cells after the incubation step and before assessment of agglutination.
4. 4. A method according to any of claims 1 to 3, in which any agglutination is apparent without the need for centrifugation.
5. 5. A method according to claim 3, in which the mixed serum and donor cells are not subjected to any washing steps between the incubation step and the addition of antihuman globulin.
6. 6. A method according to claim 1 for REiD (Du) testing of blood.
7. 7. A portable instrument for rapid blood typing or crossmatching of multiple samples which comprises a means for automatic control of sample incubation temperature and an indicator to alter operator upon completion of incubation time.
8. 8. A portable instrument for rapid blood typing or crossmatching of multiple samples substantially as hereinbefore described with reference to and as illustrated by the drawings.