Haemolytic Disease of the Newborn


Outline of the Undertaking

The selected cistron is KEL cistron, which plays a function in Haemolytic Disease of the Newborn ( HDN ) . The specific coding DNA that I will concentrate on is exon 6, which is known as K1. This normally affects newborns during gestation or from transfusions given to the female parent, therefore doing the disease HDN. I will magnify this cistron via PCR and noticing on its effectivity in placing polymorphisms. In add-on, I will besides be researching latest techniques used in placing KEL1 cistron in fetuss.

What is HDN?

HDN was one time a major cause of foetal loss and decease among newborns. HDN was foremost discovered in 1609 by a Gallic accoucheuse who delivered twins, one babe was swollen died merely after birth. The other babe developed icterus, which lead to decease several yearss subsequently. Many instances continued to develop for the undermentioned 300 old ages, taking to many deceases in neonates ( Dean, 2005 ) .

The principal cause was non identified until the 1950s, which was known to be the neonate ‘s ruddy blood cells ( RBCs ) being attacked by antibodies from the female parent. This occurs during the female parent ‘s gestation, while the babe is still in the uterus ( Dean, 2005 ) . This is due to the transportation of antibodies IgG1 and IgG3 sub-types across the placental barrier from the female parent to the foetus. These antibodies so cause devastation of foetal RBCs or erythroid primogenitors, which may go on after birth ( Fining et al, 2007 ) .

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Then in the sixtiess, trails were being conducted in the United States ( U.S ) and the United Kingdom ( U.K ) to develop curative schemes. They tested the usage of antibodies as a therapy, which could take antibodies that cause HDN from the female parent ‘s circulation. The trail proved to forestall HDN from happening by utilizing these curative antibodies during the gestation. By the 1970s, everyday antenatal attention was given to all female parents who were found to be at hazard of HDN and were given the preventive intervention. This has lead to a immense lessening in the prevalence of HDN, peculiarly decease ( Dean, 2005 ) .

How is HDN caused and different causes of HDN

In gestation, it is normal for the female parent ‘s antibodies to be transported across the placenta and enter the foetal circulation. This occurs because when the babe is born they merely have a crude immune system, hence, the presence of maternal antibodies guarantee they survive while their immune system is developing. However, the disadvantage of this protection is that by aiming foetal RBCs, maternal antibodies can besides do HDN ( Dean, 2005 ) .

A prevalent cause of HDN is mutual exclusiveness of the Rh blood group between the female parent and foetus. Hemolytic disease is largely triggered by the D antigen, nevertheless, other antigens such as degree Celsiuss, C, E and e, can besides do jobs ( Dean, 2005 ) .

The gestations that are at hazard of HDN are an Rh D-negative female parent who is transporting an Rh D-positive kid, in which the kid inherited the D antigen from the male parent. This causes the female parent to bring forth an immune response to the foetal D antigen by organizing IgG antibodies against it ( anti-D ) ( Dean, 2005 ) .

Incompatibility of the ABO blood group can besides do HDN. It occurs when a female parent with blood type O becomes pregnant with a foetus that has a different blood type, such as type A, B or AB. The female parent ‘s serum contains of course happening anti-A and anti-B of the IgG category and can hence traverse the placenta and haemolyse foetal RBCs. However, this is normally less terrible than Rh mutual exclusiveness ( Dean, 2005 ) .

Other causes of HDN include antibodies against antigens of the Kell blood group ( e.g. anti-K and anti-k ) , Kidd blood group ( e.g. anti-JKa and anti-Jkb ) , Duffy blood group ( e.g. anti-Fya ) , MNS and s blood group antibodies. However, these are less common compared to the others ( Dean, 2005 ) .

The Kell blood group

The Kell blood group system is complex and has many antigens which are extremely immunogenic. These antigens ( peculiarly KEL1 ) are 2nd to RhD as a cause of HDN at triping an immune reaction ( Li et al, 2008 ) .As mentioned antecedently, antibodies that mark Kell antigens can do HDN, every bit good as, transfusion reactions. Even though, HDN due to Kell immunisation is rare, instances cause terrible foetal anemia as maternal anti-Kell mark foetal RBC precursors, stamp downing the foetal production of RBCs ( Dean, 2005 ) .

The Kell blood group system was discovered in 1946 and was named after Mrs. Kelleher, a patient whose anti-Kell antibodies lead to HDN in her neonate. Since this determination, 25 Kell antigens have been established, which are expressed in different frequences in diverse populations ( as seen in Table 1 ) . However, the original K antigen is still of import in transfusion medical specialty and HDN ( Dean, 2005 ) . Furthermore, Kell is considered to be one of the major human RBC group ( Wagner et al, 2004 ) .

Table 1: The Kell antigens and the distribution among the population ( Redman et al, 1999 ) .

Low incidence

High incidence

KEL1 ( K )

KEL2 ( K )

KEL3 ( Kpa )

KEL4 ( Kpb )

KEL17 ( Wka )

KEL11 ( K11 )

KEL23 ( K24 )

KEL14 ( K14 )

KEL5 ( Ku )

KEL10 ( UIa )

KEL12 ( K12 )

KEL13 ( K13 )

KEL16 ( K16 )

KEL18 ( K18 )

KEL19 ( K19 )

KEL20 ( Km )

KEL22 ( K22 )


KEL25 ( VLAN )

KEL26 ( TOU )


Distribution of low incidence antigens


9 % in Caucasians, 2 % in Blacks, 12 % in Persian Hebrews


2 % in Whites


0.01 % in Caucasians, 20 % in Blacks


Less than 0.01 % in most populations, 2.6 % in Finns, 0.46 % in Nipponese


Approximately 0.3 % in most populations


Less than 0.01 % in most populations, 0.32 % in Nipponese


Less than 0.01 % in most populations


Less than 0.01 % in most populations


Merely found in 1 household

The antigens classified as high incidence occur in over 90 % of the population

The KEL cistron

KEL is inherited in an autosomal codominant mode and differs in different populations ( Lee, 1997 ) . KEL is located on chromosome 7q33 and consists of 19 coding DNAs in the scope of 63 and 288bp and crossing about 21.5Kb. The size of the noncoding DNAs range from 93bp to about 6kb. Exon 1 has an untranslated part dwelling of GATA-1 and Spl binding sites, with a individual codon for a possible induction methionine. The upstream part ( y176 ) contains a CACCC box and two other presumed GATA-1-binding sites. The nucleotide part, y176 to y1, placed in forepart of a newsman cistron, shows booster activity, nevertheless complete analysis has non been conducted. The KEL coding part, except exon 1 that novices methionine, resides in exon 2 to exon 19. Exon 2 is besides likely to originate methionine at nucleotide 178, amino acid residue 20. Neither two presumed interlingual rendition induction in KEL are authoritative Kozak sequences. The individual membrane crossing part of Kell protein is encoded in exon 3 and the zinc-binding catalytic site is in exon 16. The base sequences in coding DNAs 16 and 19 are the most homologous with the other members of the M13, Neprilysin and Zn endopeptidases household ( Redman et al, 1999 ) .

The Kell/XK composite, which shows amino acerb permutation in different Kell phenotypes. The transmembrane spheres of Kell and XK fractional monetary units are shown as cylinders. Cysteine residues are shown as ( C ) , but some cysteines in XK are non shown. The disulphide linkage between Kell Cys72 and XK Cys347 is illustrated. The amino acid alterations in Kell protein, ensuing from point mutants in KEL of different phenotypes are shown. The C-terminal sphere of Kell, which has the highest homology with other M13 Zn endopeptidases, is shaded, and the Zn binding, enzymatic active site ( HELLH ) is marked. N-glycosylation sites in Kell are designated “Y.” The hollow Y corresponds to the N-linked sugar that is non present in KEL1 protein ( Lee et al, 2000 ) .


KEL1 ( K ) is the strongest immunogenic Kell antigen and is the common cause of antibody production in mismatched blood transfusions, and maternal alloimmunization, which causes terrible anemia in newborns ( Lee et al, 2000 ) . The KEL1/KEL2 ( K/k ) DNA polymorphism is a individual base permutation ( C to T ) in exon 6 of the KEL cistron, doing exchange of threonine to methionine at residue 193 ( Li et al, 2008 ) . This amino acid permutation nothingnesss a compromised N-glycosylation site and the Kell protein in ruddy cells of KEL: 1, -2 phenotype has 4, instead than 5, N-linked oligosaccharides ( Lee, 1997 ) .

The KEL1 cistron is inherited in an autosomal codominant attack. If the pregnant adult female is KEL1-negative and has a spouse that is KEL1/KEL2 heterozygous, so the foetus has a 50 % opportunity of being KEL1-positive, puting it at hazard ( Lee, 1997 ) .

Anti-Kell and HDN

Anti-Kell is an of import cause of HDN. It seems to happen in female parents who have had several blood transfusions in the past, but besides arises in female parents who have been sensitized to the Kell antigen in old gestations ( Dean, 2005 ) .

Anti-K is prevailing in about 1 in 1000 pregnant adult females, and about 40 % of K+ babes of adult females with anti-K are affected with terrible anemia. K has a frequence of approximately 9 % in Caucasic but is much less in Africans ( 1.5 % ) and rare in Eastern Asian and in Native Americans. The k/K polymorphism consequences from a 698C & gt ; T single-nucleotide polymorphism ( SNP ) in KEL encoding a T193M permutation in the Kell glycoprotein. The pathogenesis of HDN caused by anti-K differs from anti-D. This is because it is much harder to foretell, as there is small association between anti-K titer and badness of the disease and anti-K HDN is linked with lower concentrations of amnionic fluid hematoidin than in anti-D HDN of matching badness and postpartum hyperbilirubinaemia is non present in babes with anemia caused by anti-K. Besides, there is decreased reticulocytosis and erythroblastosis in the anti-K disease. Fetal anemia in anti-K HDN is due to suppression of erythropoiesis. The Kell glycoprotein emerges on erythroid primogenitors, by macrophages in the foetal liver, before they develop into haemoglobinized erythroblasts ( Fining et al, 2007 ) . As RBC precursors contain no hemoglobin, the release of hematoidin is reduced during hemolysis, hence icterus is non as common, but terrible anemia may happen ( Dean, 2005 ) . This is supported by in vitro surveies which have shown that antibodies to Kell suppress growing of KEL1 primogenitor cells. The mechanism by which antibodies to Kell suppress erythropoiesis is unknown. However, surveies of primogenitor cells have besides demonstrated that Kell surface antigens are expressed in front of Rh, Landsteiner± Weiner, glycophorin A, set 3, Lutheran and Duffy. This suggests that antibodies to Kell may respond at an early phase of erythropoiesis where the primogenitor cells are foremost committed to erythroid distinction, stamp downing farther growing. As the endothelins are mitogenic and may hold an consequence on ripening of erythroid precursors, other possibilities are that the endothelins-3-converting enzyme activity of Kell may be involved in the procedure and antibodies to Kell may alter its capacity to procedure endothelins ( Redman et al, 1999 ) .

Management of HDN in adult females with KEL1 foetus

In order to forestall HDN, it is necessary for the sensing of the foetal KEL1 cistron ( Li et al, 2008. In pregnant adult females with anti-KEL1 and who have a spouse with KEL: 1, 2, anticipation of KEL1 phenotype from trials on foetal DNA allows accoucheurs to set direction of the gestation based on whether the foetus is at hazard of developing HDN. This Deoxyribonucleic acid can be acquired from amnionic fluid, chorionic villi, or maternal plasma, which requires the analysis of polymerization concatenation reaction ( PCR ) merchandises with the limitation enzyme Bsm1, PCR including an allelomorph specific primer, or allelomorphic favoritism by Taqman engineering ( Poole et al, 2006 ) .

Three methods have chiefly been used over the past decennary for anticipation of KEL1 phenotype or KEL1/2 genotype from genomic Deoxyribonucleic acid.

1. PCR with a KEL1 allele-specific contrary primer followed by agarose gel cataphoresis ( Poole et al, 2006 ) . This is performed utilizing an antisense K1-specific allele-specific PCR primer particular for exon 6 of the KEL cistron and a sense primer particular for exon 5. An internal control primer is besides included.
The PCR is performed with a concluding volume of 50µl incorporating the followers: 25ng each of Kell sense and anti-sense ASP-primers, 1ng each of Rh exon 5 internal control primers, 10mM tris-Cl pH 8.3, 500mM KCl, 3.0mM MgCl2, 1.25mM each dNTP and 1.25U Taq DNA polymerase. Then the undermentioned rhythms conditions are required: 5 min at 94°C followed by 30 rhythms at 94°C/1 min, 70°C/1 min and 72°C 1/min 30s, so in conclusion 72°C extension measure for 5 min ( Avent & A ; Martin, 1996 ) .

2. Direct sequencing of the part of KEL exon 6 including the KEL1/2 polymorphism. A 384-bp merchandise in amplified by PCR with the following primers: KellseqFor, GCTTCCTAGAGGAATCCAAG, and KellseqRev, TATCACACAGGTGTCCTCTTCC. The annealing temperature was 70°C for 1 rhythm and so reduced by 2°C for each of 8 rhythms, followed by 35 rhythms at 52°C. After cataphoresis of the amplified merchandise in a 1.5 % agarose gel, the set is cut out of the gel and the DNA removed. Sequencing reactions are conducted with a ready reaction kit and KellseqFor or KellseqRev primers for forward and contrary reactions, severally and an machine-controlled sequenator is used to transport out the sequencing ( Poole et al, 2006 ) .

3. Allelic favoritism by Taqman PCR. This involves PCR elaboration of the part of the KEL cistron including the KEL1/2 polymorphism and the elaboration of two Taqman fluorescent investigations, each transporting different reporter dyes and each specific enemy either the KEL1 or the KEL2 allelomorph. The sequences of the primers are the undermentioned: KELL_ADF, GGAGGCTGGCGCATCTC ; and KELL_ADR, GAGAGGCAGGATGAGGTCCA. The KEL1 particular and KEL2 specific investigations, which integrate the newsman dyes FAM and VIC, severally, are TAACCGAATGCTGAGACTTCTGATGAGTCAG and TAACCGAACGCTGAGACTTCTGATGAGCAG. Amplification happens in a 96-well home base. Incubation at 50°C for 2 proceedingss and so 95°C for 10 proceedingss is followed by 40 rhythms of 95°C for 15 seconds and 60°C for 1 minute. Fluorescence is so read by a sequence sensor. Relative end point fluorescence from the two newsman dyes us calculated and scored as homozygous for KEL1 or KEL2 or heterozygous for both ( Poole et al, 2006 ) .

PCR techniques for finding blood groups utilizing foetal DNA isolated from maternal plasma have allowed the application of non-invasive methods in a simple and dependable method ( AraUjo et al, 2005 ) . Furthermore, this is the method I will be utilizing in order to magnify K cistron and place any polymorphisms, hence, this will find how effectual the technique truly is.


Advent N.D. & A ; Martin P.G. ( 1996 ) . Kell typing by allele-specific PCR ( ASP ) . British Journal of Haematology, 93: 728-730.

AraUjo F. , Monteiro F. , Pereira C. , Duran J. , Nascimento H. , Lima L. , Cunha A. , Storry J. , Guimaraes J. ( 2005 ) . Prediction of the foetal Kell blood group reduces aggressive intercessions. Australian and New Zealand Journal of Obstetrics and Gynaecology, 45: 464-467.

Fining K. , Martin P. , Summers J. , & A ; Daniels G. ( 2007 ) . Fetal genotyping for the K ( Kell ) and Rh C, degree Celsius, and E blood groups on cell-free foetal Deoxyribonucleic acid in maternal plasma. Transfusion, 47: 2126-2133.

Lee S. ( 1997 ) . Molecular Footing of Kell Blood Group Phenotypes. Vox Sanguinis, 73: 1-11.

Lee S. , Russo D. & A ; Redman C. ( 2000 ) . Functional and Structural Aspects of the Kell Blood Group System, Transfusion Medicine Reviews, 14 ( 2 ) : 93-103.

Li Y. , Finning K. , Daniels G. , Hahn S. , Zhong X. & A ; Holzgreve W. ( 2008 ) . Noninvasive genotyping foetal Kell blood group ( KEL1 ) utilizing cell-free Deoxyribonucleic acid in maternal plasma by MALDI-TOF mass spectroscopy. Prenatal Diagnosis, 28: 203-208.

Poole J. , Nicole W. , Hustinx H. , Taleghani B.M. , Martin P. , Finning K. , Crew V.K. , Green C. , Bromilow I. & A ; Daniels G. ( 2006 ) . A KEL cistron encoding serine at place 193 of the Kell glycoprotein consequences in look of KEL1 antigen. Transfusion, 46: 1879-1885.

Redman C.M. , Russo D. & A ; Lee S. ( 1999 ) . Kell, Kx and the McLeod syndrome. Bailliere ‘s Clinical Haematology, 12 ( 4 ) : 621-635.

Wagner T. , Resch B. , Reiterer F. , Gassner C. & A ; Lanzer G. ( 2004 ) . Pancytopenia Due to Suppressed Hematopoiesis in a Case of Fatal Hemolytic Disease of the Newborn Associated With Anti-K Supported by Molecular K1 Typing. Journal of Pediatric Hematology/ Oncology, 26 ( 1 ) : 13-15.

Web sites

Dean L. ( 2005 ) . Blood Groups and Red Cell Antigens, [ online ] , NCBI, Last accessed 5th December 2009 at URL: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/bookshelf/br.fcgi? book=rbcantigen


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