Haemolytic Disease of the Fetus and Newborn (HDFN)

Haemolytic unheal slightess of the Fetus and Newborn (HDFN)List and briefly describe three clinical bulls eyes for Haemolytic Disease of the Fetus and Newborn (HDFN)Haemolytic disease of the fetus and newborn (HDFN) is a r atomic number 18 disease that clears when agnate alloantibodies cross the placenta during motherhood and ca persona the destruction of fetal red blood cells (RBCs) (Delaney and Matthews, 2015 Haas et al., 2015). HDFN give the gate get out in fetal anaemia with progression to stark(a) morbidities, such as ascites, hydrops fetalis, heart failure, kernicterus, and death (Delaney and Matthews, 2015). The clinical presentation of HDFN is variable, in which at that place be some(prenominal) manifestations that may occur (Murray and Roberts, 2007). Three of the most prevailing clinical signs that al littles for neonatal paediatricians to suspect HDFN includes splenomegaly, oedema, and jaundice.HDFN is char recreateerised by the accele strided destruction of RBCs, which results in differing rates of haemolysis and fetal anaemia (Urbaniak and Greiss, 2000). The continuous and rapid do of haemolysis defecates extramedullary haematopoiesis, a result of erythropoiesis failing in the bone marrow, in the fetal liver and spleen (Dean, 2005). Organs, such as the liver and spleen, that are involved in the synthesis of RBCs increase the production to combat the rate of destruction and counteract the overall loss (Dean, 2005). The increased workload of the spleen results in its enlargement, termed splenomegaly (Bowman, 1997 Dunn, 1963). Oedema is an important clinical sign of HDFN that is to a fault associated with the microscope floor of haemolysis and anaemia (Delaney and Matthews, 2015). Oedema can occur collect to low takes of serum albumin by dint of a decrease in osmotic pressure (Dean, 2005). Moreover, as the body compensates for fetal anaemia, the fetus can set up a hyperdynamic circulation (Haas et al., 2015). This can result in hydrops fetalis, a severe and life-threatening condition in which in that respect is widespread oedema in the fetal and shinny and serous cavities (Haas et al., 2015).A nevertheless clinical sign of HDFN is jaundice. Jaunice may occur as a result of haemolysis, in which there is an increase in the direct of hematoidin within the body (Urbaniak and Greiss, 2000). passim gestation, bilirubin is removed via the maternal circulation by the placenta (Dean, 2005). at that placefore, a high level of haemolysis may be present with a low level of bilirubin (Murray and Roberts, 2007). However, aft(prenominal) stick out the haemolytic process continues. At this stage of development, the liver of the neonate is immature and uneffective to conjugate the excess bilirubin (Urbaniak and Greiss, 2000). The unconjugated bilirubin begins to build and accumulate in the blood of the neonate, causing the skin and whites of the eyes to turn yellow (Dean, 2005). Within 24 to 48 hours afterwards delivery, the level of bilirubin may increase substantially (Urbaniak and Greiss, 2000). Left untreated, this can lead to the development of kernicterus, a condition in which bilirubin deposits plant in the basal ganglia and brain stem nuclei (Haas et al., 2015).Describe the progression of HDFN, from sensitisation to fetal red cell destruction, in a D invalidating mother carrying a D confident(p) fetus that has non legitimate RhIg.Throughout motherliness, antibodies from the maternal circulation play a vital role in providing protection for neonates crossing the placenta from the maternal to the fetal circulation (Dean, 2005). This is essential for the fetus, as by delivery newborns have a relatively immature immune musical arrangement (Murray and Roberts, 2007). Although the straw man of maternal antibodies provides protection, the active transporting of antibodies across the placenta can result in HDFN (Dean, 2005).HDFN is triggered by a sensitisation event. This most oft en occurs during the first pregnancy however, it can also arise from a blood transfusion or organ transplant (Delaney and Matthews, 2015). During the course of pregnancy, the maternal and fetal circulations gradually salmagundi with each(prenominal) trimester (Delaney and Matthews, 2015). This results in maternal alloimmunisation, as the maternal circulation has been exposed to impertinent RBCs (Haas et al., 2015). Despite the relatively small amount of fetal blood that passes into the maternal circulation, only a small amount is needed for sensitisation to occur (Dean, 2005).This is typical for an RhD ostracize mother carrying an RhD supreme fetus. Sensitisation frequently transpires during the birth of the firstborn RhD positivistic child, where fetal maternal haemorrhage (FMH) is common (Delaney and Matthews, 2015). However, the danger of sensitisation increases in complicated and extensive comminutes (Dean, 2005). Sensitisation can also occur through earlierevents in pre gnancy, such as a prenatal lead, trauma, termination of pregnancy, chronic villus sampling, and miscarriage (Sebring and Polesky, 1990). The grea scrutiny risk of growth FMH is during the process of labour (Murray and Roberts, 2007). Subsequently, alloantibodies are most likely to form after delivery (Delaney and Matthews, 2015). After sensitisation of an RhD nix mother carrying an RhD positive fetus, the mothers serum will contain anti-D (Dean, 2005). Importantly, the maternal anti-D that is formed is of the IgM class and unable to effectively cross the placenta (Delaney and Matthews, 2015). As a result, HDFN is rare in first-born children and tall(a) to have any clinical consequence or significance (Dean, 2005). However, at once the maternal circulation has been exposed to the fetal circulation the maternal immune constitution has the latent to respond to foreign red cell antigens (Delaney and Matthews, 2015).Through extensive seek and cohort studies, it has been establish ed that HDFN is most likely to effect subsequent pregnancies (Dean, 2005). matriarchal alloantibodies of the immunoglobulin G1 and IgG class cause significant haemolysis, thus the most clinically significant forms of HDFN (Roberts, 2008). In the event that an RhD negative mother becomes fraught(p) for a second time, interaction with the RhD antigen stimulates the production of IgG type anti-D, which can be transported across the placenta into the fetal circulation (Delaney and Matthews, 2015). Once anti-D has entered the fetal circulation, it binds to the RhD antigens found on fetal RBCs and labels them to be destroyed (Delaney and Matthews, 2015). From here, the pathophysiology of the disease ensues, as illustrated in figure 4. apologize the action of RhIg in a D negative mother that has a D positive fetal leech.Rh immunoglobulin (RhIg) is routinely apply in clinical practice to prevent HDFN. RhIg is prepared from human plasma that has been immunised to the D antigen and functi ons by targeting RBCs that are positive for the D antigen (Brinc and Lazarus, 2009). The use of prophylactic anti-D remains the gold standard approach of antibody- arbitrate immunosuppression, having been utilize for several decades (Giancarlo et al., 2010). However, the mechanism of action of RhIg is not fully understood and there are three key hypotheses that have been proposed to explain its method of action.Antigen Clearance dead reckoningThe first hypothesis is the antigen dynamic headroom hypothesis and is considered the main mechanism of action. Here, IgG is understood to prevent an antibody response by increasing the rate of phagocytosis and the remotion of RBCs from circulation via the mononuclear phagocytic system, prior to recognition by the immune system (Brinc and Lazarus, 2009). IgG opsonised RBCs are believed to engage in the activation of IgG receptors (FcRs) on effector cells, stimulating phagocytosis. IgG is also suspected to increase the clearance of RBCs throu gh the stimulation of complement activation on the RBC surface. Anti-D does not activate complement and because it is believed that FcR-mediated phagocytosis is the mechanism by which anti-D is cleared (Brinc and Lazarus, 2009).FcRIIB mediated B-cell inhibition hypothesisThis mechanism is the most recently proposed and came about through the discovery of increased levels of transforming growth factor- and prostaglandin E2 in a number of pregnant women who were given RhIG. This mechanism proposes that RBCs and IgG form a complex in which a negative signal is delivered to inactivate antigen-specific B cells. However, mice models deficient in FcRIIBhave shown that the involvement of FcRIIB is not needed to induce antibody-mediated immune suppression. Furthermore, FcR-like molecules have been bump in both mice and humans. It is believed that the FCRLs mediate the B-cell inhibition, however, this has yet to be demonstrated (Brinc and Lazarus, 2009).Steric check HypothesisThe Steric h indrance hypothesis proposes IgG binds the antigen, preventing the B-cell receptor from recognising the corresponding epitopes. most anti-D epitopes are not blocked by RhIg. This allows free D epitopes to be honored after administration of RhIg. Monoclonal anti-D has been shown to prevent antibody responses by cover 10-15% of epitopes. This pathway has not been studied in detail and therefore association of the immunobiology is limited. However, it is believed that IgG binding of D epitopes allows a formation surrounded by RBCs and B cells, in which this prevents B-cell activation (Brinc and Lazarus, 2009).The established methodology in the UK for the Quantification of a D positive fetal bleed is via go cytometry with FITC-anti-D (FITC-BRAD3). Name and describe 3 alternative methods used oecumenic to detect fetal bleedsIn rise to power to the use of flow cytometry, several alternative screening methods are uncommitted to determine and quantify FMH (Kim and Makar, 2011). Three screening methods that are used worldwide include the rose window screen, Kleihauer-Betke acid elution attempt, and flow cytometry using anti-fetal hemoglobin antibodies.The rosette attempt is a screening method that is used to qualitatively detect fetal bleeds equal to or great than 10 mL and 0.2% of fetal cells present in the maternal circulation (Kim and Makar, 2011). The rosette test works by indirectly identifying the presence of D positive fetal RBCs in D negative mothers (Solomonia et al., 2012). To perform this test, a maternal blood sample is hive away, incubated with exogenous anti-D, and washed. D positive RBCs are added and are key as they act as an indicator. The sample is examined using a light microscope. In the presence of fetal D positive cells, the indicator RBCs form aggregates or rosettes some the coated fetal RBCs (Solomonia et al., 2012).A positive result is indicative of an FMH greater than 10 mL and requires quantification by Kleihauer-Betke acid elut ion test or flow cytometry to determine the dose of RhIg to administer (Kim and Makar, 2011). This test can encounter and resurrect fake-positive results. This largely occurs if the mother of fetus is weak D. Furthermore, in the presence of a direct antiglobulin test (DAT), the rosette test may produce a false-negative result. This can be attributed to crosslinking and agglutination of the mothers antibody coated cells (Kim and Makar, 2011).The Kleihauer-Betke acid elution test is a screening method that differentiates between fetal haemoglobin (HbF) RBCs and adult Hb (Bromilow and Duguid, 1997). The underlying notion of this test is fetal RBCs largely contain HbF and are resistant to acid elution, whereas in contrast, adult Hb is acid-sensitive (Kim and Makar, 2011). To perform this test, a maternal blood sample is taken to prepare a thin peripheral smear. The peripheral smear is dried, immersed in fixative, exposed to and incubated with an acid buffer, and varnished with eosin. Under a microscope, the test reveals fetal cells to be stained a dark pink-red colour, whilst adult red cells appear pale or as uncoloured ghost outlines (Kim and Makar, 2011). Under a microscope, the fetal cells are counted and report as a percentage of adult cells (Kim and Makar, 2011).Flow cytometry using anti-fetal haemoglobin antibodies is a variant of flow cytometry that detects RhD positive fetal cells (Kim and Makar, 2011). In this method, monoclonal antibody antibodies are directed against HbF (Davis, 2007). A maternal blood sample is collected and an RBC count is performed (Davis, 2007). Cells are then fixed and permeabilised with detergent to modify antibodies to enter the cellular membrane and bind HbF (Davis, 2007). A flow cytometer is used to analysed the antibody stained cells (Davis, 2007). This method uses positive and negative controls simultaneously to differentiation between fluorescence from fetal RBCs and non-specific background staining (Kim and Makar, 2011) . The positive control is also extremely important in setting out the parameters for gating a sample (Kim and Makar, 2011). little potato testKleihauer-Betke acid elution testFlow cytometry using anti-fetal haemoglobin antibodiesAdvantages commercialized kits Widley availableSimple to useFast InexpensiveAdvantagesNot helpless on presence of RhD antigenRequires only basic laboratory equipmentInexpensive butt be used to assess fetal welfare in RhD positive patientsAdvantagesQuantitativeAutomated good precisions, sensitivity, accuracy, and duplicabilityCost-effectiveLess labour intenseDisadvantagesOnly applicable to RhD negative mothers carrying RhD positive fetusqualitative only not quantitativeDisadvantagesLaborious to performAccuracy and precision limited due to variation in test characteristicsPoor reproducibilitySubjectiveDisadvantagesIf mother and fetus have the same RhD type or mother is RhD positive cannot be used to determine FMHFalse positives due to heritable persisten ce of fetal Hb, increased levels of Hb in pregnancy and certain disease statesa) A 2mL bleed is detected via acid elution test in a sample taken from a D negative mother. Quantification via anti-D flow cytometry results in a zero bleed. Explain 2 doable reasons for the opposing results.The differing results of the acid elution test and anti-D flow cytometry may be explained by the RhD status of the mother and fetus. If the mother is not RhD negative and is carrying an RhD positive fetus, this test would not reproduce the results of the acid elution test. Additionally, if the fetus has an RhD negative status, this would also cause the test to fail and detect a zero bleed. Furthermore, flow cytometry cannot accurately detect weak and partial D variants, resulting in a false negative result. Therefore, if the mother or fetus has either of these D variants, FMH would not be detected via flow cytometry. An alternative reason for the differing results between the two tests could be attr ibuted to haemoglobinopathies, in which the flow cytometer detects HbF. Lastly, there could be a slip ones mind in the tube, such that the antibody was not detected, causing an incorrect result.b) Suggest a suitable test alternative to those already conducted to investigate the sample further and explain your reasoning for the alternative test.The use of flow cytometry using anti-fetal haemoglobin antibodies would be a beneficial test to implement in regularize to clarify the results and detect if a bleed is present. This is important to ensure the admit and correct dose of RhIg is administered. Testing the sample using HbF flow cytometry would be useful as flow cytometry using anti-D failed to reproduce the results of the acid elution test.There is a possibility that the acid-elution test produced a false positive result. The acid-elution test is limited in that is has poor accuracy and is prone to variations. The false positive may arise as a result of adult hereditary persisten ce of HbF, which is known in 1 to 2% of the population. Furthermore, during pregnancy the level of HbF rises by 25%. Therefore, the use of HbF flow cytometry would be able to detect if this is what caused the result.The following bleeds were detected via anti-D flow cytometry in a D negative woman. Using the Mollison calculation work outThe bleed batch in mLThe total RhIg dose in each case to the nearest five hundred IUThe top up RhIg dose required in each case to the nearest viosterol IUThe Mollison calculation is used to calculate the volume of bleed. To do this, the background of the isotope matched control is subtracted from the number of events obtained in the D positive region, as recommended in the BCSH guidelines.The Mollison equation is as follows D (+) events D (-) events1800FMH = X -ml X 1.22 Total number of events 1The equation can be simplified to the followingFMH = % of D positive events x 18 x 1.220.81% x 18 x 1.22 = 17.79 mL18 ml bleed* = 18 x 125 = 2250 IU Ro unded to nearest d = 2 vitamin D IU2500 IU 500 IU = 2000 IU0.45 x 18 x 1.22 = 9.88 mL10 mL bleed = 10 x 125 = 1250 IU Rounded to nearest 500 = 1500 IU1500 IU 500 IU = 1000 IU0.091 x 18 x 1.22 = 1.99 mL2 mL bleed = 2 x 125 = 250 IU Rounded to nearest 500 = 500 IU500 IU 500 IU = 0 IUThis bleed is under 4 mL and therefore no top up is required as 500 IU is routinely administered for a bleed of up to 4 mL.1.09 x 18 x 1.22 = 23.94 mL24 mL bleed = 24 x 125 = 3000 IU Rounded to nearest 500 = 3500 IU3500 IU 500 IU = 3000 IU0.02 x 18 x 1.2 = 0.431 mL bleed = 1 x 125 = 125 IU Rounded to nearest 500 = 500 IU500 IU 500 IU = 0 IUThis bleed is under 4 mL and therefore no top up is required as 500 IU is routinely administered for a bleed of up to 4 mL.ReferencesBrinc, D. and Lazarus, A. (2009). Mechanisms of anti-D action in the legal community of hemolytic disease of the fetus and newborn. Hematology, online 2009(1), pp.185-191. accessible at http//asheducationbook.hematologylibrary.o rg/content/2009/1/185.long Accessed 6 Mar. 2017.de Haas, M., Thurik, F., Koelewijn, J. and van der Schoot, C. (2015). Haemolytic disease of the fetus and newborn. Vox Sanguinis, online 109(2), pp.99-113. functional at https//www.ncbi.nlm.nih.gov/pubmed/25899660 Accessed 6 Mar. 2017.Dean, L. (2005). Blood groups and red cell antigens. 1st ed. Bethesda, Md. NCBI.Delaney, M. and Matthews, D. (2015). haemolytic disease of the fetus and newborn managing the mother, fetus, and newborn. Hematology, online 2015(1), pp.146-151. Available at https//www.ncbi.nlm.nih.gov/pubmed/26637714 Accessed 6 Mar. 2017.Giancarlo maria Liumbruno, Angelo DAlessandro, Federica Rea, Vanessa Piccinini, Liviana Catalano, Gabriele Calizzani, Simonetta Pupella, Giuliano Grazzini (2010). Blood Transfus. 2010 Jan 8(1) 8-16. doi 10.2450/2009.0108-09Kim, Y. and Makar, R. (2012). Detection of fetomaternal hemorrhage. American Journal of Hematology, online 87(4), pp.417-423. Available at https//www.ncbi.nlm.nih.gov/pu bmed/22231030 Accessed 6 Mar. 2017.Murray, N. and Roberts, I. (2007). Haemolytic disease of the newborn. Archives of Disease in Childhood Fetal and Neonatal Edition, online 92(2), pp.F83-F88. Available at https//www.ncbi.nlm.nih.gov/pmc/articles/PMC2675453/ Accessed 6 Mar. 2017.Roberts, I. (2008). The ever-changing face of haemolytic disease of the newborn. Early Human Development, online 84(8), pp.515-523. Available at https//www.ncbi.nlm.nih.gov/pubmed/18621490 Accessed 6 Mar. 2017.Urbaniak, S. and Greiss, M. (2000). RhD haemolytic disease of the fetus and the newborn. Blood Reviews, online 14(1), pp.44-61. Available at https//www.ncbi.nlm.nih.gov/pubmed/10805260 Accessed 6 Mar. 2017.