ABORhCard. ABORhCard Package Insert ABO and Rh Blood Grouping Device

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1 ABORhCard Package Insert ABO and Rh Blood Grouping Device ABORhCard Intended Use The ABORhCard is a qualitative in vitro test that provides a simultaneous ABO and Rh determination of an individual s ABO blood group and Rh factor status. It is not for use for blood bank processing of blood products, for determining RHD status for the purpose of administration of Rh immunoglobulin, or for screening purposes prior to transfusion. The ABORhCard is intended for single use for individual blood group determination for educational and informational purposes. Explanation of the Test A person s ABO blood type either A, B, AB, or O is based upon the presence or absence of the A and B antigens on their red blood cells. The A blood type has only the A antigen, and the B blood type has only the B antigen. The AB blood type has both A and B antigens, while the O blood type has neither A nor B antigen. In routine blood grouping, a test for the D antigen, also known as the Rhesus factor or Rho[D] is also performed. If the D antigen is present, the person is Rh positive (Rh+). If the D antigen is not present, the person is Rhnegative (Rh ). Principle of the Procedure The Micronics ABORhCard utilizes microfluidics the ability to reduce sample and reagent volumes and to process fluids in small channels to provide an agglutination test result. This technology is centered on the generation and use of Laminar Fluid Diffusion Interfaces ( LFDIs ) in diffusion based reactive mixing. In the ABORhCard, the fresh whole blood sample stream and the hydrated antibody reagent stream are introduced into a common reaction channel and flow one on top of the other until they exit the channel. Agglutination results when particles, such as blood cells, are crosslinked or otherwise aggregated by formation of antigen:antibody affinity pairs at the interfacial boundary. The ABORhCard uses murine monoclonal antibodies, Anti A, Anti B, and Anti D (Rh) pre dispensed in channels in the device ( Card ), to determine the presence or absence of human blood group antigens A, B and D (Rh) on red cells collected in a capillary whole blood sample. A tube of rehydration buffer is included in the test kit packaging. The user dispenses the buffer into the Buffer well on the Card, from which the buffer flows directly into the channels and re hydrates the antibodies. The action of the buffer rehydration is visually confirmed by the user, who will see blue, red orange or green colors, respectively, in the A, B and D Verification windows. Once the user verifies that antibody re hydration has occurred, he/she then uses the alcohol wipe and lancet, packaged in the test kit, to first clean and then lance the finger from which a blood sample will be drawn. Using Micronics, Inc.

2 the 50 μl capillary pipette also included in the kit, a 50 μl sample of blood from this finger stick is then pipetted into the Card s Blood well. This sample flows into the three Verification windows, where a red color will be visually observed. The user presses the on Card finger Pump, then covers the Vent to draw antibody and blood together into the Card s three distinct channels, where agglutination or lack thereof is observed. The resultant ABO blood type and Rh factor status are indicated by the location of agglutination in the respective Blood Type windows. Agglutination of the sample s red cells indicates the presence of the relevant antigen, while no agglutination indicates its absence. Warnings and Precautions For In Vitro Diagnostic Use. Treat all biological specimens, including used Cards, as if capable of transmitting infectious agents. Guidelines for specimen handling are available from the U.S. Centers for Disease Control and Prevention and the Clinical and Laboratory Standards Institute 1. The ABORhCard Test Procedure should be followed closely. Perform the ABORhCard test rehydration using only the Buffer Tube supplied in the kit. Do not substitute water or other buffers for the supplied Buffer Tube. Re hydrated Anti A, Anti B and Anti D reagents should be visible in Verification windows within approximately 2 minutes. If reagents do not reach Verification windows within 2 minutes, discard Card and begin with new ABORhCard kit. Transfer of blood from capillary pipette to Blood well should occur as soon as possible after collection. Avoid expressing clotted blood into Blood well. Blood should be visible in Verification windows within approximately 2 minutes after addition to Blood well. If blood does not reach Verification windows within 2 minutes, discard Card and begin with new ABORhCard kit. Each single use ABORhCard is used to process one test. Do not reuse Cards or accessories provided in the ABORhCard kit. Do not use ABORhCard test kit beyond assigned expiration date on packaging label. Dispose of used ABORhCards according to established guidelines for hazardous material. Reagents and Materials Provided The ABORhCard Kit is packaged as 25 tests per box. Each box has 25 two-pouch components; one containing the Card and the other containing accessories for use with the Card. Card contents: Murine monoclonal Anti A, Clone Birma 1 (blue); pre printed into A channel Murine monoclonal Anti B, Clone LB 2 (orange); pre printed into B channel Murine monoclonal Anti D, Clone MS 201 (green); pre printed into D channel Each of the pre printed monoclonal solutions contains Sodium Azide 0.1% (w/v). ABORhCard Kit contents: Card (1) Quick Reference Guide (1) Buffer Tube (1) (Contains Phosphate Buffered Saline (PBS) ph 7.4 (1x) Liquid with green food coloring and Triton CF 87 Surfactant; Liquid volume: 0.5 ml) 50 μl Capillary Pipette (1) Lancet (1) Alcohol wipe (1) 2 Micronics, Inc.

3 Materials Required but not Provided Tape for use in sealing the Blood and Buffer wells prior to disposal of ABORhCard. Storage, Handling and Stability ABORhCard kits may be stored at room temperature. ABORhCard should be used within eight hours of opening the kit. ABORhCard should not be used beyond product expiration date on packaging. Specimen Collection The ABORhCard is designed for use with capillary fresh whole blood collected by a finger stick. Collection Procedure: 1. Using the tear notches, open the accessories pouch of the ABORhCard kit. 2. To ensure successful collection, inspect the test subject s hands. They should be warm with a pink red coloration, indicating adequate circulation. Select a finger tip that has little or no callus, preferably the ring or middle finger. Gently massage finger from base toward the tip to facilitate blood flow. 3. Open alcohol wipe packet and disinfect finger tip selected for collection. Allow to air dry. 4. Remove colored stem of lancet by twisting, then pulling on the stem. Lancet is now ready for use. 5. Place test subject s hand, palm upward, on a flat horizontal surface. 6. Place the lancet with the opening on the side of the selected finger facing the little finger, near the tip of the finger. 7. Push lancet down until a click indicates that the lancet has pierced the skin. 8. Using capillary pipette provided, collect 50 μl of fresh whole blood. Capillary pipette has a calibration line to indicate correct collection. 9. Follow Test Procedure (below) to complete the test. Test Procedure Use universal precautions when working with blood. Remove Card from packaging immediately before use Use of the card beyond 8 hours after opening the kit is not recommended. Place Card on a flat surface. [text facing up as shown below] 3

4 1. With Card on flat surface, twist off top of Buffer Tube and dispense drops of green buffer into well marked Buffer, taking care not to overflow out of the well. This is best achieved by directing the tube tip to the left, under the clear lip of the Buffer well. Wait until blue, red orange, and green colors appear in corresponding Verification windows, as shown at right. 2. While waiting for re hydration of the antibodies, clean and lance the finger from which a sample will be drawn. Using the capillary pipette, collect (fill to) 50μl of fresh whole blood. 3. When Verification is observed, dispense the 50μl fresh blood sample from the capillary pipette into the well marked Blood, taking care not to spill or overflow blood from the well. This is best achieved by directing the pipette tip downward, toward you, under the clear lip of the Blood well. 4. Wait until a red/purple color appears in all three Verification windows, as shown below. 5. Once mixed reagent and blood appear in the Verification windows, press down on the air Pump (P), with the index finger of the left hand and place the index finger of the right hand over Vent (V). 6. Release Pump (P), while still holding the Vent (V). Observe: Reagent/Blood mixture from the Verification windows will quickly flow to Blood Type (results) windows. 7. When the reagent/blood mixture first enters any of the Blood Type windows immediately release the Vent (V). 8. When flow into the Blood Type (results) windows has slowed or stopped, hold Card upright. 9. Read agglutination results through Blood Type (results) windows when the flow has stopped; this is typically within approximately 2 minutes. 10. Place tape over the Blood and Buffer wells. 11. Dispose of Card according to established guidelines for disposal of hazardous materials. Internal Controls Each Card has A, B, and D Verification windows. These windows: 1. Confirm the addition of buffer and its re hydration of Anti A, Anti B and Anti D reagents by the appearance of blue, red orange, and green color (reagent) in each window. 2. Confirm the addition of blood by the appearance of blood (red/purple) in each window. Results Once Test reactions are complete (see Test Procedure), examine the A, B, and D results in the Blood Type area of the Card. Agglutination, which is the visible clumping of red blood cells with a particular antibody, indicates the presence of the specific corresponding antigen on the red blood cells being tested. Absence of agglutination, observed as a smooth stream of diluted red blood cells (shown below as None ), with or without reagent stream color demarcation, indicates that the cells are negative for the specific corresponding antigen. 4 Micronics, Inc.

5 Examples of types of agglutination reactions seen with the ABORhCard: Strong Typical Weak None Agglutination results in Blood Type windows may be compared to the Quick Reference Guide simulations and as shown below to determine the presence (positive) or absence (negative) of agglutination and the resulting ABO and Rh type. AB A B O D(Rh)+ Blood types as determined by agglutination patterns: Blood Type (ABO & Rh) Blood Type (ABO & Rh) O Positive B Positive O Negative B Negative A Positive AB Positive A Negative AB Negative 5

6 Limitations of Procedure 1. The Anti D monoclonal antibody used in the ABORhCard does not recognize partial D category VI cells (D VI ). D VI cells occur in approximately 1 in 5000 Rh positive individuals The performance of the ABORhCard has been validated using the Test Procedure provided in this Package Insert. Modifications of these procedural steps, such as not observing reagent or sample in all Verification windows before proceeding, or pressing Pump (P) more than one time, or pressing Pump (P) after pressing Vent (V), could result in erroneous results. 3. The capillary pipette used for the collection of blood for this assay does not contain anticoagulants. It is possible that if the sample is not added to the ABORhCard in a timely manner, clotting or partial clotting will occur in the pipette. This will usually result in failure of blood to appear in all three Verification windows, invalidating the ABORhCard and requiring a retest with a new sample and ABORhCard. 4. The ABORhCard does not contain an isoagglutinin control. Therefore, a Blood Type result of AB Positive may be as a result of the presence of isoagglutinins in the blood sample and cannot be considered a valid test. 5. The performance of the ABORhCard has not been evaluated for use with anticoagulated blood samples. 6. The performance of the ABORhCard has not been established for use with lipemic blood samples. 7. The performance of the ABORhCard has been evaluated for use with hemolyzed blood samples and the blood group result is not affected by hemolyzed samples. Expected Values The common reaction patterns obtained in determination of ABO/Rh blood types in the U.S. are shown below: ABO/Rh Blood Type Frequency in the United States 3 ABO Group Rh + Rh O 37% 7% A 35% 6% B 9% 2% AB 3% 1% Specific Performance Characteristics A clinical study was conducted on a total of 1700 volunteers at three field trial sites selected to capture a diverse sample population, facility and environmental demographics. Testing with the ABORhCard was done with fresh capillary blood collected by finger stick. These results were then compared to sample results generated by each site using its test of record, and agreement/disagreement of ABO and Rh results was recorded. Only de identified samples that enabled routine laboratory testing using the licensed test of record for the trial site and repeat reference testing were included in the study. The study results are presented below: 6 Micronics, Inc.

7 Summary of ABORhCard Clinical Test Results # Samples/ Cards # ABO Discords ABO% Agreement # Rh Discords Rh% Agreement % Overall Card Agreement Total Results % % 97.12% 95% Confidence Intervals 97.30% % 98.15% % 96.25% % Discordant analysis Of the 1700 individual samples/cards tested, each was tested for determination of both ABO and Rh type, for a total of 3400 results recorded. Of these results, there were a total of 33 ABO type and 21 Rh type results that were discordant, for a total of 54 discordant results. The percent agreement of results (3346/3400) is 98.41%, with a 95% confidence interval of 97.95% to 98.79%. Five of the samples/cards tested were both ABO group discordant and Rh group discordant on the same card, resulting in a total of 49 discordant samples/cards, with an overall sample/card agreement (1651/1700) of 97.12%, with a 95% confidence interval of 96.25% to 97.86%, as reflected in the summary table above. Discordant analysis was performed by testing those individual samples that upon unblinding of the results did not agree between the ABORhCard result and the in house test of record result against a second ABORhCard and the in house reference method. Two of the three test sites used the Olympus PK 7200 as the in house test of record, and one used the Immucor Galileo. A retained EDTA anticoagulated vacutainer blood sample, stored at 2 8 C, was used for discordant analysis by testing on a second ABORhCard and with the in house referee method. The in house reference method used at all three sites was the tube test. The in house test of record and manual tube test reference method were 100% in agreement. Reproducibility Studies Reproducibility studies were performed using a blinded method at each of the three clinical trial sites. Product training was provided to all sites prior to reproducibility testing. A total of 27 users were trained. Three fourmember panels (EDTA anticoagulated vacutainer samples) of random ABO/Rh blood types were tested in duplicate on three separate days by each trained user, one panel each day. Results: The overall concordance rate of the proficiency sample test results with the correct blood type was 98.7%. Nine (9) of 690 proficiency sample tests were incorrectly typed on initial testing; all 9 were correctly typed upon blinded, repeat testing. References 1. Centers for Disease Control and Prevention. Biosafety in Microbiological and Biomedical Laboratories. Richmond, JY and McKinney RW (eds.) HHS Publication number (CDC) Complexities of RhD antigen and implications for NIPD, Daniels, Geoff, University of Bristol, Blood Type, Wikipedia. 7

8 Symbols Used in Packaging Labels Symbol Meaning For in vitro diagnostics use Kit lot number Catalog number Manufactured by Kit expiration date Consult instructions for use Do not reuse Micronics, Inc th Avenue NE, Redmond, WA Patent Protected. FDA 510(k) cleared All rights reserved.aborhcard is a registered trademark of Micronics, Inc. Micronics, Inc. ( E)

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