**4.7 Challenges associated with the provision of antigen negative red cells to patients with clinically significant red cell antibodies**

Evidenced based best practice in management of patients including pregnant women requires that patients are transfused with red cells lacking the group specific antigens to which the recipient alloantibody is specific [136]. This aim of this implementation is to prevent immune mediated destruction of the donor red cells containing the offending antigen [137, 138]. For example, pregnant women who have alloantibody Kell should be given K-negative donor units. The provision of antigen negative blood units for these patients is a special challenge particularly in Nigeria and many other countries in Africa [139]. Although these countries have national blood transfusion services, majority of them are often not fit for purpose. Majority of them test blood donors only for their ABO and Rhesus antigen status [139]. Routine testing of blood donors for other clinically significant red sell antigens are not routinely done. This has a significant implication on haemolytic transfusion reaction and HDFN. In the alloimmuned pregnant women who have low molecular weight immune IgG antibodies, IAT crossmatching should ideally be carried out using donor red cells suspended in low ionic strength saline preferably using highly sensitive column agglutination technique instead of the conventional less sensitive tube methods. Many of these technologies are often not available in most settings in Nigeria and other African countries. Transfusion of antigen positive donor red cells to patient with the group specific antibodies like it potentially happens in Nigeria and some other African countries have significant negative consequences [140]. These antibodies can cause clinically significant haemolytic transfusion reactions, difficulty in cross-matching

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*Distribution of Clinically Relevant Blood Group Antigens among Nigerians…*

**4.8 Lack of facilities for feto-maternal haemorrhage testing**

blood and getting compatible blood in future, cause decrease in RBC survival and thus negate the aim of red cell transfusion of managing anaemia, improving the quality of life and improving the oxygen carrying capacity of the recipient's blood [141]. Also, there is also the effect of managing donor red cell clearance and the product of red cell breakdown due to haemolysis which often results in multiple organ failure, electrolyte perturbations, coagulopathy and in some severe cases, death [142]. Nigerians and citizens of other African countries deserve the best quality transfusion service like their counterparts in the West. It is the responsibility of African government to work smartly and effectively by avoiding waste and eradicating corruption to ensure that citizens get the best quality health care

The accurate detection and quantification of foetal red blood cells (RBCs) in the maternal circulation are necessary for the prevention of Rhesus D alloimmunization among D-negative women because of FMH. It is critical to the administration of adequate amount of anti-D prophylaxis are necessary for the prevention of Rhesus D alloimmunization. As a result of D incompatibility between mother (Rhesus D negative and foetus (D-positive), foetal red cells can enter into maternal circulation and sensitise the mother to produce anti-D alloantibody. These anti-D alloantibodies (small molecular weight IgG antibodies) can pass through the placenta barrier in subsequent D positive pregnancies cause HDFN (haemolysis, foetal anaemia, hydrops foetalis, kernicterus or even death). FMH testing helps obstetrician to determine that a potential sensitising event has taken place and facilitate the administration of adequate amount of prophylactic anti-D (125 IU per 1 ml bleed of foetal red cells into maternal circulation). The widespread use of FMH testing in the evidenced-based provision of adequate immunoprophylaxis with anti-D immunoglobulin has resulted in a significant reduction in the incidence of anti-D related HDFN mortality. Evidence-based best practice implementation of FMH test and provision of immunoprophylaxis with anti-D immunoglobulin in England and Wales has brought about a significant reduction in the number of infants affected with HDFN from affected in 1.2 per 1000 births in 1970 to 0.02 per 1000 births by 1989. There are several methods available for FMH testing (Quantitative and qualitative). Three qualitative methods (micro Du, rosette test, and PEG Du) and two quantitative methods (acid elution and Flow Cytometry) for assessing FMH were evaluated with particular attention given to PEG Du and FC. Of the qualitative techniques, the micro Du test was the least sensitive with 20% false-negative results occurring at 0.5% foetal cells. The PEG Du test was only slightly more sensitive and offered no clinical advantage. The rosette test was the most sensitive, consistently detecting foetal cells at concentrations of 0.25% or greater. Flow Cytometry and acid elution showed similar results, with good correlation obtained between measured and expected quantities of foetal cells (r = 0.99 and 0.96, respectively). One of 26 postpartum samples was positive by all screening techniques; acid elution and FC detected 0.3% concentrations of fetal cells and 0.17% [143, 144]. The rosette screen is a highly sensitive in qualitatively detecting 10 mL of foetal whole blood in the maternal circulation. As the screen is reliant on the presence of the D antigen to distinguish foetal from maternal cells, it cannot be used to detect FMH in D-positive mothers or in D-negative mothers carrying a D-negative fetus. The Kleihauer-Betke acid-elution test, the most widely used confirmatory test for quantifying FMH, relies on the principle that foetal RBCs contain mostly foetal haemoglobin (HbF), which is resistant to acid-elution whereas adult haemoglobin is acid-sensitive. Although the Kleihauer-Betke test is inexpensive

*DOI: http://dx.doi.org/10.5772/intechopen.90372*

they deserve.

*Distribution of Clinically Relevant Blood Group Antigens among Nigerians… DOI: http://dx.doi.org/10.5772/intechopen.90372*

blood and getting compatible blood in future, cause decrease in RBC survival and thus negate the aim of red cell transfusion of managing anaemia, improving the quality of life and improving the oxygen carrying capacity of the recipient's blood [141]. Also, there is also the effect of managing donor red cell clearance and the product of red cell breakdown due to haemolysis which often results in multiple organ failure, electrolyte perturbations, coagulopathy and in some severe cases, death [142]. Nigerians and citizens of other African countries deserve the best quality transfusion service like their counterparts in the West. It is the responsibility of African government to work smartly and effectively by avoiding waste and eradicating corruption to ensure that citizens get the best quality health care they deserve.

#### **4.8 Lack of facilities for feto-maternal haemorrhage testing**

The accurate detection and quantification of foetal red blood cells (RBCs) in the maternal circulation are necessary for the prevention of Rhesus D alloimmunization among D-negative women because of FMH. It is critical to the administration of adequate amount of anti-D prophylaxis are necessary for the prevention of Rhesus D alloimmunization. As a result of D incompatibility between mother (Rhesus D negative and foetus (D-positive), foetal red cells can enter into maternal circulation and sensitise the mother to produce anti-D alloantibody. These anti-D alloantibodies (small molecular weight IgG antibodies) can pass through the placenta barrier in subsequent D positive pregnancies cause HDFN (haemolysis, foetal anaemia, hydrops foetalis, kernicterus or even death). FMH testing helps obstetrician to determine that a potential sensitising event has taken place and facilitate the administration of adequate amount of prophylactic anti-D (125 IU per 1 ml bleed of foetal red cells into maternal circulation). The widespread use of FMH testing in the evidenced-based provision of adequate immunoprophylaxis with anti-D immunoglobulin has resulted in a significant reduction in the incidence of anti-D related HDFN mortality. Evidence-based best practice implementation of FMH test and provision of immunoprophylaxis with anti-D immunoglobulin in England and Wales has brought about a significant reduction in the number of infants affected with HDFN from affected in 1.2 per 1000 births in 1970 to 0.02 per 1000 births by 1989. There are several methods available for FMH testing (Quantitative and qualitative). Three qualitative methods (micro Du, rosette test, and PEG Du) and two quantitative methods (acid elution and Flow Cytometry) for assessing FMH were evaluated with particular attention given to PEG Du and FC. Of the qualitative techniques, the micro Du test was the least sensitive with 20% false-negative results occurring at 0.5% foetal cells. The PEG Du test was only slightly more sensitive and offered no clinical advantage. The rosette test was the most sensitive, consistently detecting foetal cells at concentrations of 0.25% or greater. Flow Cytometry and acid elution showed similar results, with good correlation obtained between measured and expected quantities of foetal cells (r = 0.99 and 0.96, respectively). One of 26 postpartum samples was positive by all screening techniques; acid elution and FC detected 0.3% concentrations of fetal cells and 0.17% [143, 144]. The rosette screen is a highly sensitive in qualitatively detecting 10 mL of foetal whole blood in the maternal circulation. As the screen is reliant on the presence of the D antigen to distinguish foetal from maternal cells, it cannot be used to detect FMH in D-positive mothers or in D-negative mothers carrying a D-negative fetus. The Kleihauer-Betke acid-elution test, the most widely used confirmatory test for quantifying FMH, relies on the principle that foetal RBCs contain mostly foetal haemoglobin (HbF), which is resistant to acid-elution whereas adult haemoglobin is acid-sensitive. Although the Kleihauer-Betke test is inexpensive

and requires no special equipment, it lacks standardisation and precision, and may not be accurate in conditions with elevated F-cells. Flow cytometry is a promising alternative, although its use is limited by equipment and staffing costs. The two well-established confirmatory tests are the Kleihauer-Betke acid-elution assay and flow cytometry. The rosette screen is a highly sensitive FDA approved method to qualitatively detect 10 mL or more of foetal whole blood, or 0.2% foetal cells (volume/volume) in the maternal circulation [143]. The principle is based upon the formation of microscopic foetal D+ aggregates upon incubation of foetal cells with enzyme0-treated group O D-positive indicator RBCs and reagent anti-D serum. The foetal cells must be D-positive and the maternal cells D-negative for the test to be valid. In summary the maternal blood sample is first incubated with anti-D and then washed. The indicator D-positive RBCs are added and the sample is examined under a light microscope. In the presence of anti-D coated foetal D-positive cells, the indicator cells will form aggregates (or rosettes) around the foetal cells. The main limitation of this method is that falsely positive result may occur is the mum has a variant of the D antigen and falsely negative if the foetus or neonate is weak D. Also, mothers that have a positive direct antiglobulin test (DAT) or an autoantibody can produce a false positive result due to the agglutination of the mother's antibody coated red cells.

A positive rosette test is indicative of FMH > 10 mL. In a negative rosette test administration of is 300 μg (1500 IU) of Rh Ig is sufficient to prevent immunisation in 99% of patients. If the rosette test is positive quantification of the FMH by either the Kleihauer-Betke acid elution test or flow cytometry is indicated to determine the optimal dose of prophylactic anti-D immunoglobulin to be administered. The Kleihauer-Betke acid-elution test is an inexpensive method and requires no special equipment and thus can possibly be implemented in developing country like Nigeria and other resource-limited settings. The method is based on the principle that foetal RBCs containing foetal haemoglobin (HbF) to resist acid elution whereas adult haemoglobin does not. When a peripheral smear prepared from a EDTA anticoagulated maternal blood sample is exposed to an acid buffer such as Shepherds stain, haemoglobin in the cytoplasm of maternal red cells are eluted while HbF in the foetal red cells resist acid elution. Subsequent counterstaining with haematoxylin results in red cell containing foetal haemoglobin staining pink while the maternal adult haemoglobin containing red cells appear as ghost. **Figure 1** shows a positive Kleihauer-Betke test. To calculate the number of foetal cells in maternal circulation using Kleihauer-Betke test, get a stained thin film on a microscope and replace one of the objectives of the microscope with a graticule (Miller square) (**Figure 2**). Focus the microscope making sure the graticule is on the top left-hand corner. Select the ×40 lens and count at least 50 fields of the area of the film where the red cells are just touching each other. For each field count all the foetal cells in the large and small square. Count all the maternal cells in the small square. Count fields with at least 25–30 maternal cells. To calculate the total number of maternal cells, add the total number of maternal cells together and multiply by 9. Also add the total number of foetal cells from the 50 fields. The calculation of FMH is done using Mollison's rule.

Mollison's rule = 2400 × number of foetal cells ÷ number of maternal cells For example, if the number of foetal red cells is 20 and the maternal cells is

2000, the FMH = number of maternal cells = 2000 × 9 = 18,000

25 × 2400/18,000 = 3.3 mls bleed.

Calculation of dose of anti-D. Anti-D administered is calculated by giving 125 IU per ml of FMH. For a 3.3 ml bleed you will have to administer 3.3 × 125 IU = 413 IU of anti-D.

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*Distribution of Clinically Relevant Blood Group Antigens among Nigerians…*

The Kleihauer-Betke method is a labour intensive and subjective method. The accuracy and precision of this method is hampered by lack of standardisation which can potentially lead to slight variations in result (thickness of blood smear, pipetting skill of analyst, pH variations in the buffer used, inter analyst variations). Over- and underestimation of FMH using the Kleihauer-Betke test has been reported [145, 146]. The disadvantages of this method include; laborious to perform, lacks standardisation, is imprecise and may not be accurate in conditions associated with elevated haemoglobin F containing red cells. However, standardisation of the Kleihauer-Betke test can make the result potentially comparable to result from flow cytometry [147]. Haemoglobin F containing red cells are increased in haemoglobinopathies including sickle cell disease and β-thalassemia and hereditary persistence of foetal haemoglobin (HPFH). In 25% of pregnant women, HbF tend to be increased and can cause false positive results [148, 149]. In pregnant women with conditions such as sickle cell disease, thalassaemia and HPFH an alternative method for FMH testing such as flow cytometry should be employed. Despite its limitations, a good correlation has been reported between the Kleihauer-Betke test

Flow cytometry involves using a flow cytometer using monoclonal antibodies directed against. Flow cytometric determination of FMH is superior to Kleihauer-Betke test for a number of reasons; cytometric methods can accurately distinguish adult foetal haemoglobin containing red cells from foetal RBCs, flow cytometers rapidly analyses a significantly higher number of cells (≥50,000), thus improving

and flow cytometry for both small and large FMH [150, 151].

*DOI: http://dx.doi.org/10.5772/intechopen.90372*

**Figure 1.**

**Figure 2.**

*Diagram of Miller square (graticule).*

*Positive Kleihauer-Betke test.*

*Distribution of Clinically Relevant Blood Group Antigens among Nigerians… DOI: http://dx.doi.org/10.5772/intechopen.90372*

**Figure 1.** *Positive Kleihauer-Betke test.*

**Figure 2.** *Diagram of Miller square (graticule).*

The Kleihauer-Betke method is a labour intensive and subjective method. The accuracy and precision of this method is hampered by lack of standardisation which can potentially lead to slight variations in result (thickness of blood smear, pipetting skill of analyst, pH variations in the buffer used, inter analyst variations). Over- and underestimation of FMH using the Kleihauer-Betke test has been reported [145, 146]. The disadvantages of this method include; laborious to perform, lacks standardisation, is imprecise and may not be accurate in conditions associated with elevated haemoglobin F containing red cells. However, standardisation of the Kleihauer-Betke test can make the result potentially comparable to result from flow cytometry [147]. Haemoglobin F containing red cells are increased in haemoglobinopathies including sickle cell disease and β-thalassemia and hereditary persistence of foetal haemoglobin (HPFH). In 25% of pregnant women, HbF tend to be increased and can cause false positive results [148, 149]. In pregnant women with conditions such as sickle cell disease, thalassaemia and HPFH an alternative method for FMH testing such as flow cytometry should be employed. Despite its limitations, a good correlation has been reported between the Kleihauer-Betke test and flow cytometry for both small and large FMH [150, 151].

Flow cytometry involves using a flow cytometer using monoclonal antibodies directed against. Flow cytometric determination of FMH is superior to Kleihauer-Betke test for a number of reasons; cytometric methods can accurately distinguish adult foetal haemoglobin containing red cells from foetal RBCs, flow cytometers rapidly analyses a significantly higher number of cells (≥50,000), thus improving

its quantitative accuracy and flow cytometry is automated non-subjective and has greater reproducibility. The only disadvantage is that it requires trained staff to perform test coupled with the fact that it is it is expensive and may not be affordable in low-resource settings.
