When is abo reverse grouping indicated

In some scenarios a donor may express blood type O by forward typing and a different blood type by reverse typing when the donor has received un-crossmatched blood type O transfusions which can convolute the forward typing result. For example, if a donor receives massive transfusions of blood type O packed red blood cells PRBCs , then blood type forward-typing indicates blood type O with reverse-typing indicating blood type A, it is likely the blood type O blood transfusions have affected the forward typing by reflecting the blood type of the transfused PRBCs.

In such a scenario, the safest course of action is to conclude the donor is blood type A. Concluding that the donor is blood type O in error would potentially expose transplant candidates to organs that are incompatible for transplant. By concluding the donor is blood type A in this scenario subtyping in this scenario would not be an option then all candidates matched to the donor would be ABO type A or AB or Platelet Transfusion Refractory PTRs listed as accepting organs of incompatible blood type as allowed by policy.

It is best in such scenarios to consult with blood banking physicians and scientist experts to review the entirety of the circumstances, donor medical history, transfusion history and blood type results to ensure the safest course is followed when the final determination of donor blood type is made. If there is doubt about the conclusions of donor blood typing, extreme caution should be exercised to avoid the possibility of exposing candidates to such risk. Conflicting blood typing results are certainly the more concerning scenarios OPOs may face.

In the event the donor blood typing by one lab or blood draw time is conclusive but conflicting with the conclusive results of another lab result or result on a donor blood sample drawn at a different time, the OPO should review of donor transfusion history and review of all forward and reverse blood type results obtained to determine the source of the conflict.

The reliability of the blood sample source must also be called into question in such a scenario. OPTN policy requires that blood type be determined using two blood samples drawn at separate times. The purpose of this requirement is to confirm blood type determination and ensure that samples have been drawn from the correct patient to prevent conflict that may have occurred due to possible sample labeling error.

Some OPOs have employed policies to re-draw donor blood samples after an interval of time has passed and have the samples re-tested for blood type. While this may resolve some conflicts it may not always be a reliable means since no criteria is known for determination of when a donor would revert to their natural blood type.

Re-testing may result in further conflict or such a practice may result in blood type results that are no longer in conflict and enable more confidence in the original result. The utilization of alternative new testing methods for determination of blood type DNA-based determination of blood type as described above could be an adjunct in efforts to resolve conflicting, discrepant or indeterminate blood type results.

As a last resort, when donor blood typing results remain in conflict and unable to be resolved, the safest course of action is to consider the donor to be blood type AB to ensure that only AB blood type candidates, as universally ABO compatible recipients, would be considered to receive the organs from that donor.

This does however carry the consequence that urgently ill candidates in need of a lifesaving transplant may be excluded from consideration of the organs in such a scenario. When the ABO gene was cloned in , it was found that the genes for A the B glycotransferase enzymes differ by four single nucleotide polymorphisms SNPs in exon 7, designated according to the cDNA sequence as c.

Recently [ 9 ], a modern strategy was explored where elastic scattering of laser radiation is followed by digital imaging for determining human BGs. This hybrid acousto-optical approach has demonstrated high resolving power in monitoring sedimentation of RBCs and their agglutinates [ 10 ].

Polymerized chain reaction with sequence-specific priming PCR-SSP has also been put forward by researchers for molecular genotyping [ 11 ] of human BGs. Some cutting edge technologies aim at the determination of rare or weak alleles of BG; however, in both classical and advance techniques, there is a compromise between sensitivity, time of analysis and ultimate cost of that particular test. Furthermore, in some techniques, highly-trained personnel are required for interpreting blood typing analysis reports.

Therefore, it is difficult to prefer a single testing method that offers sensitive and speedy results at a relatively low cost. Miniaturized chemical sensors [ 12 ] are receiving increasing attention for their sensitive and selective response, rapid results and with the feasibility of in-field measurements [ 13 ]. These sensors can perform efficiently in complex mixtures [ 14 ] and, therefore, have found numerous applications in many fields, e.

In the design of biochemical sensors, the interfacial part could range from natural antibodies to synthetic receptor materials [ 18 ]. Although natural antibodies are greatly selective and specific in their binding, they suffer from regeneration problems, storage stability, are highly expensive and their derivation is not straightforward. In this scenario, synthetic antibodies crafted by molecular imprinting [ 19 , 20 , 21 , 22 ] offer comparable sensitivity and selectivity as that of natural competitors, are easy to synthesize and can be reused for several analyses with adequate stability.

These features make synthetic antibodies exceedingly suitable candidates in modern biosensor [ 23 ] design and especially for blood typing. In this article, we present a concise overview of some selected strategies in blood typing extending from classical methods to state-of-the-art modern biosensor devices equipped with synthetic antibodies as generated by molecular imprinting. Additionally, some latest developments in this area will also be highlighted briefly.

The proposed methods will be discussed in view of their sensitivity, specificity, analysis time, cost and their applicability for in-field applications. In routine clinical analysis, there is a wide range of established procedures and practices for blood typing, where nearly all of them deal with the formation of agglutinates.

Even though some of these classical methods are not highly sensitive, nonetheless, they still hold importance in ABO grouping tests. There is a wide range of blood typing techniques [ 6 ], which differ from each other in terms of sensitivity, reagents and equipment required, the time of operation and throughput analysis. Herein, we describe some general approaches of blood grouping along with their inbuilt advantages and drawbacks. The slide test is relatively the least sensitive method among others for BG determination, but due to its prompt results, it is very much valuable in emergency cases.

In this method, a glass slide or white porcelain support is divided into three parts, as for each part, a drop of donor or recipient blood is mixed with anti-A, anti-B and anti-D separately. The agglutination or blood clumping pattern can be visually observed from which the ABO and rhesus D RhD type of blood can be determined. The test completes in 5—10 min and is inexpensive, which requires only a small volume of blood typing reagents.

However, it is an insensitive method and only useful in preliminary BG matching for getting an early result. The test cannot be conducted for weakly or rarely reactive antigens from which the results are difficult to interpret, and additionally, a low titer of anti-A or anti-B could lead to false positive or false negative results. Although the slide test [ 6 ] is useful for outdoor blood typing, it is not reliable enough for completely safe transfusion.

In comparison to the slide test, the tube test is more sensitive and reliable; therefore, it can be used conveniently for blood transfusion. In this method, both forward cell , as well as reverse serum grouping is carried out. The forward grouping suggests the presence or absence of A and B antigens in RBCs, whereas reverse grouping indicates the presence or absences of anti-A and anti-B in serum.

In forward grouping, blood cells are placed in two test tubes along with saline as a diluent media, and then one drop of each anti-A and anti-B is added separately in these samples.

These tubes are subjected to centrifugation for few minutes, and then, the resultant matrix is gently shaken for observing agglutination. For precise blood grouping, the two tubes can be categorized according to the extent of blood clumping. The purpose of centrifugation is to ensure enhanced chemical interactions, particularly for weaker antibodies to react, thus leading to agglutination.

Some potentiators could also be added to promote the agglutination; moreover, the long incubation of tubes also favors these reactions without drying of the test samples.

In a similar fashion, reverse grouping can be performed, as here, the blood serum is treated against RBC reagent groups of A1 and B, and the subsequent agglutination pattern is monitored.

The grading of agglutinates in both forward and reverse grouping is useful in comparing the difference in the strength of hemolysis reactions. In general, the tube method is much more sensitive than the slide test and requires a low volume of reagents, and some unexpected antigens can also be detected; therefore, it is a better option for safer transfusions. However, in infants, reverse grouping is somewhat difficult to perform, since they produce insufficient amounts of antibodies to be determined.

Among classical methods, microplate technology is a further step towards more sensitive and fast blood typing analysis with the feasibility of automation. In this technique, both antibodies in blood plasma and antigens on RBCs can be determined. Following centrifugation and incubation, the subsequent agglutination can be examined by an automatic read out device. The microplate technique was first introduced in early s; however, since then, considerable developments have been made in the design to improve the performance.

The foremost advantage of microplate technology is its fast response, low reagent volumes and high throughput analysis. Apart from microplates, gel cards or strips can also be used for blood grouping in modern immunoassay machines. Column agglutination technology or gel centrifugation is a relatively modern approach that has gained substantial interest in ABO blood grouping, as it intends to establish a standard procedure for quantifying cell agglutination.

Here, the column is made of small microtubes that contains gel matrix to trap agglutinates. Blood serum or cells are mixed with anti-A, anti-B and anti-D reagents in microtubes under controlled incubation and centrifugation. The gel particles trap the agglutinates, whereas non-agglutinated blood cells are allowed to pass through the column. The analysis time can be reduced by using glass beads in place of gel material, since in this way, faster centrifugation speeds can be achieved, which leads to rapid results.

This technology is sensitive, straightforward and relatively easy to operate for less trained personnel. Synthetic receptors [ 24 , 25 ] crafted via molecular imprinting have shown considerable impact in designing advance biosensors. The utmost importance of molecular imprinted polymers MIPs is due to their substantial bio-recognition competency and their high chemical stability.

The synthesis of these biomimetic materials [ 26 ] is straightforward and relatively easy, which altogether results in its low cost. They can be stored at ambient conditions for several months, keeping the same recognition characteristics, whereas the lifetime of natural antibodies is limited, and they need to be stored under controlled environments.

These features add value to molecular imprinted receptors and, therefore, are the main driving forces in switching from natural antibodies to synthetic polymers. This clearly indicates the cost benefit analysis in using MIPs in contrast to natural receptors. Realizing the potential advantages of MIPs, they have been utilized in a variety of fields, such as chiral separations [ 28 ], immunoassays [ 29 ], drug delivery systems [ 30 ], chemical biosensors [ 31 ] and many others.

In the context of blood typing, MIPs can be designed according to the target shape, as the templating process [ 32 ] results in analogous cavities for analyte binding. Conventional bulk imprinting [ 33 ] deals with the smaller size targets, whereas for large bio-macromolecules [ 34 ], relatively modern techniques, like surface [ 35 ] or epitope imprinting [ 36 ], are preferred. Although in the bulk method, the number of interaction sites is larger, the diffusion pathways are long, which leads to prolonged response time; additionally, the complete release of target species is somewhat difficult, which ultimately reduces the regeneration of receptor sites.

Contrarily, surface or epitope imprinting strategies offers shorter diffusion corridors for analytes with complete reversibility, which leads to prompt response and improved reusability. In surface imprinting, the soft lithographic technique [ 37 ] can be applied to structure the polymeric layer material, especially for sensitive and fragile bio-species.

Moreover, soft lithographic imprinting [ 38 , 39 ] of the target analyte can be done directly on the transducer device surface, which results in appropriate integration of the sensor layer with the device. A suitable densely-packed template stamp is pressed on an already spin-coated pre-polymer surface and allowed to cure under mild temperature and humidity for some time.

This initiates the oligomer chains to self-organize [ 40 ] around the template and fixes their positions. After removing or washing the template cells, replicate cavities are formed in accordance to the shape of target species. The washing is done with mild solvents that eradicate only the template molecules and do not disturb the polymer structure.

The imprinted structures are capable of recognizing a target form their shape, as well as chemical interactions.

A schematic representation of the soft lithographic surface imprinting methodology is illustrated in Figure 1. This technique can be applied to imprint erythrocytes on a pre-polymer surface. The designing of stamp generation is relatively different, as the erythrocytes are a soft cell species and require special purification and pretreatment steps to discard added substances.

The basic principle of the erythrocyte stamp was derived from the osmotic effect, i. First of all, the erythrocyte concentrate is washed three to four times with isotonic NaCl, i. This solution is centrifuged at rpm for 10 min, thus recollecting blood cells and removing the supernatant. After each washing step, the process is repeated in the same way to obtain the desired purity of RBCs. A suitable volume of resultant erythrocytes, i. The use of glass instead of PDMS could lead to tight adhesion of blood cells, which makes the complete removal of the template difficult.

The erythrocyte stamp is pressed over an already spin-coated pre-polymer surface and allowed to cure overnight at a suitable temperature. After polymer curing, the templates can be removed by washing with hot water.

Now, this surface contains the imprints of RBCs of exactly the same shape, and it can be viewed by atomic force microscopy AFM. One such image is displayed in Figure 2 , where the polyurethane surface has the impression of the RBCs.

The soft lithographic imprinting [ 41 , 42 ] procedure is interesting and promising in order to transfer the structural details of bio-species onto a synthetic polymer interface. The imprinted polymer surface can reversibly reincorporate the template cells.

It is interesting to note that the shape and size of all of the blood groups is the same; nonetheless, synthetic antibodies derived from MIPs can differentiate blood groups on the basis of the difference in their cell surface.

In view of sensor measurement for blood typing, the beneficial aspect is that imprinting can be made on a polymer surface that is already coated on the transducer device. Therefore, no problems exist concerning polymer transducer integration [ 43 ], and thus, sensor measurements can be made straightaway after template removal.

The purpose of using silicone polymer is to avoid any damage to the shape of RBCs, as they are flexible in nature, and using a rigid substrate, like glass, as a support material is not useful.

Furthermore, glass stamps could lead to tight adhesion of template cells on the polymer surface, which is sometimes difficult to wash out. As the erythrocytes are a fragile species and need extra care during the stamping process, a modified version [ 44 ] of cell surface imprinting can be adopted.

In this way, blood cells become more rigid and robust, which can then undertake the imprinting process, as shown in Figure 3. Furthermore, the imprinting density is also enhanced, since there are more interaction sites compared to the previous imprinting method. This is also beneficial in improving sensitivity. After modification, cells are washed and diluted accordingly to develop a monolayer on the support. This stamp is coated with PDMS and placed in a vacuum for one hour in order to remove air bubbles.

Polymerization is completed at room temperature after a certain time, and by sonication, the stamp can be removed. Casting prepolymers on these patterned materials results in the generation of plastic or artificial cells, which are more robust than natural blood cells and, therefore, can be more useful for structuring polymer surfaces.

The artificial cell stamp can be called the master imprinting stamp. This brings the possibility of crafting synthetic cells of the same shape as that of native cells. AFM image of the erythrocyte imprinted polyvinylpyrrolidone surface; adhered erythrocytes are shown, whereas white circles indicate the cavities formed after washing away erythrocytes; adapted from [ 44 ].

The imprinting of blood cells can be directly made without using any stamping method. In such a practice, a pre-polymer mixture is first coated on the transducer surface, and after, packed cells are spread or deposited over this pre-polymer layer by spin coating. Now, the polymer can be hardened thermally or by a photochemical reaction under UV light, i. At the start, the polymer layer is relatively less viscous, and cells are easily deposited without using any support material, and during the course of the reaction, oligomer chains are self-organized around the cells.

The important point in this methodology is that biological cells, i. Therefore, considering this point, rapid polymerization is favored immediately after cell sedimentation.

Imprinted cells can be washed from the polymer surface simply by rinsing with water. Biomimetic surfaces [ 45 ] as designed by soft lithographic imprinting are highly useful for selectively recognizing erythrocytes. For developing a typical sensor, surface imprinted layers are combined with a suitable transducer, such as acoustic or mass-sensitive devices.

The foremost advantage of using acoustic devices [ 46 , 47 ] is that they are universal transducers, because mass is the fundamental property of any analyte that can be determined. Moreover, they are remarkably sensitive, which makes them highly valuable while detecting low analyte concentrations.

Quartz crystal microbalance QCM and surface acoustic wave SAW resonators are typical examples of mass-sensitive devices and are widely used for developing chemical sensors. The integration of the layer material with the transducer is straightforward, as it was previously mentioned that the pre-polymer matrix is spin coated on QCM before erythrocyte imprinting.

Figure 4 represents the relative sensor responses of four different polyurethane layers for the ABO BG system [ 48 ]. The sensor response of each layer for all four BGs is summarized in this graph. As one can see, each polyurethane layer exhibits the highest sensor signal for the BG that was used as the template during the surface imprinting of that polymer layer. For example, the polyurethane layer imprinted with BG A shows the maximum response for this BG during measurements, whereas the sensor response is relatively less for other BGs, i.

For the other three polyurethane layers, a similar trend was observed. It is interesting to note that all of these BGs have nearly the same size, i. Therefore, the imprinted polymer surface recognizes them by the difference in their surface groups, rather than by simple shape.

The imprinted polyurethane films precisely incorporate target analyte cells by chemical interactions between erythrocyte surface antigens and polymer functional groups, thus providing chemical fitting. The haemovigilance services did not report any adverse transfusion reactions. Discrepancy: I carefully repeated the test procedure with new cell test preparation to eliminate any technical nature.

Donors rarely present discrepancies for the biological qualification selection. An irregular antibody agglutinated B cells in room temperature. With a negative auto-test, presence of cold allo antibodies theory is accepted. Normally the detection of irregular antibodies needs to be performed.

In this case it was impossible for lack of screening and identification reagent red blood cells. Fortunately, the blood was not transfused. ABO group percentage distribution Table 1 did not reflect population percentage distribution, 9 the transfusion center needs influenced them, those needs responded to repeated transfusion for known patients with AB and B group in hematology service.

After day5, hemolysis increases to be microscopic then macroscopic and the antigenic activity decreases. Laboratory made test cells were used for 4days, I predicted 05days. In developing countries with low incomes, reverse grouping with laboratory made test cell for ABO group identification, is an interesting economic alternative with a goal to ensure blood components and blood products and optimize patient outcomes.

If weak or discrepant reactions cannot be resolved, or if an ABO subgroup incompatible with transfusion is identified, the donor will be deferred. As required by the Canadian Standards Association CSA Z 4 , hospital blood banks must perform forward typing confirmation of red blood cell units received by Canadian Blood Services in order to allow for release of units via electronic cross match.

In these cases, it is important to contact Canadian Blood Services distribution to ascertain whether a subgroup was identified and investigated by Canadian Blood Services donor testing during routine typing procedures. A Canadian Blood Services technical specialist can view donor history and confirm whether a subgroup was identified. In cases when a weak reaction is unexpectedly observed during confirmation testing i. A hospital customer feedback form and return of the unit to Canadian Blood Services may be required and discussion with a hospital transfusion medicine physician is recommended.

Figure 1. Algorithm for hospital blood banks for follow up of potential ABO subgroup donor units. For an introduction to immunohematology and the foundations of blood bank compatibility testing, visit LearnSerology. The curriculum consists of six modules and includes an interactive module for completing an antibody investigation panel. Vox Sang ; Daniels G. Human Blood Groups, 3rd Edition.

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