The Quantitation of Anti-D by Flow Cytometry


It has been determined that the major causative factor for the haemolytic disease of the newborn is the maternal immunization against D and incompatibility. Medical research has developed a medication that can help to prevent this complication. In the D– incompatibility pregnancy cases, governments and health policies in many countries recommend that these pregnancies be monitored to determine the levels of the maternal anti-D. This is in most cases done by the indirect anti-globulin test which is then complemented by a process of quantitation. This is a very important process that includes automated antibody detecting techniques. Flow cytometry effectively determines the level of the anti-D serum and is also helpful in assessing IgG and the presence of IgM anti-D in the samples. The results are then compared to indirect anti-globulin test titre and the results from the automated antibody process and quantitation. Flow cytometry is sensitive enough and very precise method of determining the anti-D level and show very low inter-assay and intra-assay variability. It is also simple, fast and reliable hence it is the most suited process for quantitation of Anti-D antibodies during suspected D-incompatible pregnancies.


As medical practice or profession advances through better care is a necessity. To ensure better service production and evidence-based care, there is a lot of research being conducted and analytical processes devised accurate diagnoses. Early accurate diagnosis means that the service providers will be able to give preventative medication rather than wait to respond to the damage already done. In maternal care, the quantitation of the Rhesus antibodies has become a clinically important measure in determining the risk of a newborn developing haemolytic disease. Flow Cytometry is a technique developed for the process of quantitation of these Rhesus antibodies.

Anti-D (Rho) Immunoglobulin

When a woman gets pregnant, the child inherits blood group genes from both parents but by the virtue of the newborn having stayed in the womb of the mother, there is often a possibility of haemorrhage (Christenson 1). This is often very common when a Rhesus D (RhD) negative mother carries or gives birth to a foetus that is RhD positive. The first pregnancy is likely to survive but the successive pregnancies if the foetuses are Rh-positive again, there will be problems and the mother may even miscarriage. This is because the mother develops antibodies against the Rhesus D positive foetuses which are deemed by the body to be foreign to it (Christenson 1, p. 203). This easily results in Rhesus haemolytic disease of the newborn.

Research in medicine had found that when the Rhesus D –ve mother is given an anti-D medication for prophylaxis just immediately after birth (when haemorrhage takes place), the delivery of the Rhesus positive baby will not cause the body of the mother to get sensitized against Rhesus factor. Essentially, the mother will not develop antibodies against the Rhesus D +ve child. This medication strategy has been a very good solution to problems of infant deaths by this cause across the world (Christenson 1, p. 203). Many mothers have benefitted from it. Considering that, the dose of the anti-D that is administered in such a case is related to the foetal-maternal bleeding, it is, therefore, significant to carry out quantitation of the haemorrhage. The quantitation of the Rh antibodies thus helps predict the possibility of the disease occurring in the newborns.

The anti-D immune globulin G contains very high levels of the Rhesus-antigen antibodies (Morrison 2, p. 372). This is often found in the human plasma and can effectively prevent active alloimmunization. Administering anti-D and following it with an antibody test gives a positive response. This is a low titre and the antibodies are not strongly reactive.

The anti-D crosses the placenta and binds to the red blood cells of the foetus and in such cases, they do not cause anaemia, haemolysis or jaundice (Morrison 2, p. 372). Even studies of mothers who gave birth to Rh +ve babies conducted on mothers who were on the antepartum medication of anti-D immune globulin showed the positive test to the anti-globulin but the haemoglobin levels and bilirubin levels were normal just as compared to the Rh-negative babies. This makes it important for quantitation of the anti-D antibodies so that Rh –ve mothers giving birth to Rh +ve babies can take anti-D IgG for prophylaxis (Morrison 2, p. 372).

Sensitization takes place in 12% to 16% of the mothers who do not get these postpartum medications compared to 1.6% to 1.9% of mothers who get postpartum prophylaxis medication.

Why the Quantitation

As already mentioned above, the reason for quantization is to determine the risk for RhD haemolytic disease. This condition is an innate process where the mother gets naturally immunized against the D antigen. Consequently, there is a natural transfer of foetal red blood cells. It is usually not possible to detect Anti-D at 5 to 15 weeks after sensitization (Lee, et al., 3). This is a very slow response compared to the usual immune response when the body detects foreign antigens in the body faster, like when the body detects micro-organisms. The significance of this process in Europe and the United States is big. This is because there is 1 to 1.5% of at-risk D –ve mothers experience sensitization regardless of whether RhD immune globulin was administered for prophylaxis or not.

The RhD immune globulin (RhD Ig) usually administered is comprised of a concentrated solution made up of IgG anti-D generated from human plasma. From this solution, a one-millilitre dose vial is sufficient enough to counter the immunizing impact of 15 millilitres of Rh +ve red blood cells (Lee, et al., 3). This strategy was developed to cut the natural tendency of immunization in Rh -ve mothers happening when sensitized against the D antigen and therefore preventing haemolytic disease of the newborn which is caused by the anti-D reaction.

Process of Flow Cytometry

It is recommended that future non-invasive foetal testing for the RhD gene will be done routinely for the mothers. The process of sorting the maternal blood to test foetal cells by use of flow-Cytometry has had success in the identification of the RhD +ve foetus (Johnson 4). Recent developments have seen the free foetal DNA in the maternal plasma getting used for detection of the RhD sequences in the case of RhD +ve foetus. Many nations in Europe are now employing this method as a diagnostic test (Johnson 4).

Flow Cytometry makes use of the theory of light scattering, light bending and emission of the fluorochrome molecules. The process goes through a number of actions which then produce a particular multi-parameter data from the particles and cells that measure 0.5 um to 40 um in diameter. The cells generated from the above process are subsequently hydro-dynamically concentrated through a sheath of Phosphate Buffered Saline. When they come out, they then intercept an optically focused light. The light source commonly used is a laser. The interception causes scattering of light and fluorochromes which are also excited at the same time. Energy photons are released in a definite range of spectra distinctive to the different fluorochromes (Johnson 4). Flow Cytometry is different from spectrophotometry in that it measures absorption per specific cell of particle rather than per bulky sample.

The emitted light from the cells and the particles pass through an optical laser which converts them into electrical pulses. A series of processes take place through optical devices and filters. The electrical pulses from the light are detected by Photomultiplier Tubes (PMTs). After going through the PMTs, the light is processed by a number of linear and log amplifications. After amplification, the signals are processed and converted from analogue to digital hence allowing the plotting of graphical scales (Johnson 4). The profile produced as output data is stored in digital forms as files. The FCS 2.0 or 3.0 standards are often used in storage and data presentation. One sample of data can be stored as a histogram file.

Effectiveness of the Anti-D test

There are a number of studies that proved anti-D prophylaxis is efficient in the prevention of the problems that come as a result of Rh incompatibility. The administration of Anti-Antibodies needs to be done within 72 hours following a foetal-maternal haemorrhage (Davis 5). Studies show that a dose of 100 µg (500 IU) antibody can sufficiently protect a person as it is able to block antibody response to 5 ml foetal RBC (Davis 5). This is what emphasizes the need to quantify the size of the foetal-maternal haemorrhage accurately. This way, practitioners can be able to correctly calculate the dose of anti-D antibody that needs to be administered to manage the condition and hence effectively decreasing the rate of developing complications or the disorder (Navenot 6).

Over the years, the Kleihauer-Betke acid-elution Technique (KBT) has been used as a standard for detecting the foetal RBC’s; however, the procedure has not been an accurate one. Besides, it has been a very time-consuming process. In recent times, however, the advancement of flow Cytometry has increasingly been used in the foetal RBC detection in the mother’s peripheral blood (Navenot 6). The test is done based on the antigen-antibody concept and differences. This has often produced a clear pattern of the mother’s red blood cells and the foetus RBCs. The different antigens are then examined since there is a possibility that the mother and the foetus share similar profiles or have differences that could spark health problems. The commonly used method for detecting the foetal cells has been the use of antigens especially the anti-D polyclonal and monoclonal reagents (Béliard 7).

The foetal Red Blood Cells can be precisely identified and quantified by the use of intracellular detection of the foetal haemoglobin (HbF). This is a new process for doing the red blood cells isolation (Geifman-Holtzman 8). Flow Cytometry has two processes of detecting the FMH; they include the use of anti-HbF or the use of anti-D antibody from the blood of the mother. The accuracy of these tests needs justification. Therefore researchers have often used artificial models to ascertain and develop standards (Geifman-Holtzman 8).

Research by Lloyd-Evans (8), concerning quantitation of the Anti-D antibodies, showed that the detection limit for the foetal cells was 0.02% for 200,000 events and 0.03% 100,000 events. This means that the method was very sensitive. The flow Cytometry protocol for the assessment of the anti-D which allow quantification of the D+ve foetal cells from the mother’s blood also gave high sensitivity. Its sensitivity was higher than that of the antibody to HbF which was only determined to be at 0.10%. clinically, this method should be sensitive enough to detect the very minimal foetal blood volume that would be more than the dose of the anti-D normally administered to prevent the production of the antibodies (or immunization), Finning et al., (10). The standard dose ranges from 100 to 300 mg as determined by the medical regulation of the specific country in question. This helps prevent immunization expected to take place when 10 or 30 ml of foetal blood is mixed. KBT and flow Cytometry are satisfactory as far as this requirement is concerned.

Flow Cytometry has been proved by research to be more precise and has a high rate of reproducibility compared to the KBT (Gautier et al 11). Two reasons make flow Cytometry a better process;

The first is that, with the method, it is possible to counter a very large number of cells in a very short span of time. Therefore, the process gives a much more accurate quantification of the rare occurrences like the presence of foetal cells in the mother serum.

Second, the flow Cytometry process is easier or simpler to conduct. The process is also more objective than the KBT regardless of attempts to standardize it. The KBT process has the problem f having the blood film preparation, its staining and precise identification are greatly reliant on the experience of the person conducting it (Moise 12).

These reasons have made many countries in Europe and Australia recommend the use of flow Cytometry rather than the KBT whenever necessary to ensure accurate quantification of the Anti-D. However, health institutions still retain the KBT for screening positive samples (Maayan-Metzger et al., 13). Certainly, it is the fact that flow Cytometry is more accurate and has a linear profile that allows it to be effectively adopted for doses of anti-D which have to be administered based on the level of the FMH (Crowther & Middleton 14). This is where 100 mg of anti-D are to be administered for 0.20% of foetal cells that a patient has.

Because of the significance of the Rh D problems, the United Kingdom has a policy for pregnant women and children’s health which requires that all the Rh D negative mothers giving birth to Rh D positive children should be injected with the anti-D immunoglobulin at 500 IU (Nordvall M, et al 15). This is sufficient enough to block the effects of up to 4 ml of foetal red blood cells.

In clinical practice, the Kleihauer screening has become a common test that is carried out in all cases of birth to detect 1% of the mothers who have bled more than 4 ml, Abassali et al., (16). In such instances, the Kleinhauer test is done for quantification of the haemorrhage and determination of the additional doses of anti-D immunoglobulin. Conditions with large bleeding are to be monitored for the disappearance of the foetal haemoglobin (HbF) cells by use of this test as well as test for the anti-D presence in the mother’s serum Abassali et al., (16). The expression of RhD antigen detects the presence of foetal cells in the mother.


Studies show that when patients are treated with ant-D immunoglobulin based on the estimates that were derived from the Flow Cytometry, there was the likelihood that significantly low doses of anti-D immunoglobulin would be required, especially when the free anti-D was detected within 48 hours. Sometimes the test can fail to detect the anti-D immunoglobulin, in such a case, the test has to be redone again after a span of 24 hours because there is a possibility that at 48 hours it was too early for the absorption to have begun or elicited a reaction in the body. Basically, flow Cytometry could be of great help for the accurate quantitation and management of the problems of haemolytic disease of the newborn. It is also helpful in circumstances where the maternal blood has its haemoglobin F cells, therefore, rendering the Kleihauer technique irrelevant. Even though the Kleihauer test is deemed appropriate for screening the foetal-maternal haemorrhage, scientific investigations done to assess the role of flow cytometry in the detection of the anti-D are also reasonable.


Whenever a non-sensitized mother who is Rh-negative gives birth to a Rh-positive child, she should be given an anti-D Ig of 300 µg either Intra-Muscularly or Intravenously. This has to be done 72 hours after delivery. Based on the foetal-maternal haemorrhage, which goes beyond 15 ml, there will be a need for additional Anti-D Ig. An alternative Anti-D Ig 120 µg could be administered in 72 hours and more anti-D Ig administered following more FMH of beyond 6 ml of foetal RBCs.

In case the Anti-D was not administered in 72 hours following delivery or after evidence of other incidences that are sensitizing, anti-D should be administered as soon as the need is identified for up to 28 days.

There is however a challenge of lack of evidence concerning the inclusion and exclusion of the routine testing of the postpartum FHM. This is because there is yet to be conducted a serious and validated cost-benefit analysis of the mothers at risk. It is recommended that anti-D Ig 300 µg be administered routinely to every Rhesus negative non-sensitized mother at 28 weeks of pregnancy when the foetal blood type is Rhesus positive or unknown.

References List

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Johnson PR, et al., Flow Cytometry in Diagnosis and Management of Large Fetomaternal Haemorrhage. J Clin Pathol 1995; 48:1005-8.

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Lloyd-Evans P et al., Use Of A Directly Conjugated Monoclonal Anti-D (BRAD-3) For Quantification Of Fetomaternal Haemorrhage By Flow Cytometry. Transfusion 1996; 36: 432-7.

Finning KM, Martin PG, Soothill PW, & Avent ND. Prediction of Foetal D Status from Maternal Plasma: Introduction of a New Non-invasive Foetal RHD Genotyping Service. Transfusion 2002; 42:1079.

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Abassali, P., et al.,., (2008), The Assessment Of Feto-Maternal Haemorrhage In An Artificial Model Using Anti-D And Anti-Foetal Haemoglobin Antibody By Flow Cytometry, Iranian Biomedical Journal 12 (1): 43-48.