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The molecular basis ofblood cell alloantigens 273

Short-read sequencing Long-read sequencing

between SNPs/variants

https://t.me/med1917

Whole gene HLA amplicons

Amplicons are fragmented No fragmentation

SNP/varient No coverage

• Variable depth of sequencing coverage across the gene

• No coverage in some regions due to missing sequencing reads

• Short reads unable to phase across long conserved regions

Figure19.3 Schematic diagram showing the differences between sequencing short- reads and long- reads.

• Even depth of sequencing coverage across the gene

• Long reads span entire amplified gene region

• Fully phased unambiguous HLA typing results

development of DNA microarrays for genotyping blood cell antigen systems. Although genotyping yields less informa-

substitution in the gene encoding the relevant glycoprotein,

with the exception of HPAtion than sequencing systems, the data analysis can be faster and requires less computer processing than sequencing.

HPA allele frequencies

Typically, microarray genotyping techniques rely on DNA hybridization of a DNA probe specific for previously known genotype designed to hybridize at the target sequence. Microarrays for genotyping Human Erythrocyte Antigens (HEA), Human Leucocyte Antigens (HLA), and Human

The allele frequencies vary between populations (Table19.3). For example, GPIIIa- Pro33 (HPA- 1b) is extremely rare or absent in East Asia, while the opposite is the case for the GPIIIa- Gln143 form (HPA- 4b).

Platelet Antigens (HPA) using microarray technology using 50,000 probes to genotype one individual for the blood cell antigens systems have been described and have the potential for replacing serological typing techniques commonly used in blood centers for routine phenotyping.

The molecular basis of platelet- specific antigens or HPA

The HPA alloantigens are expressed on the surface of platelets but are not truly platelet- specific since some are also found on the surface of other blood cell types (Table19.1). To date, 41 HPAs have been defined by immune sera, which are grouped into 35 platelet- specific alloantigen systems. The molecular basis of these have been determined and are all located on the surface of certain platelet membrane glycoproteins. Twelve of the most clinically relevant HPAs are grouped into six biallelic systems. Of the 35 HPA systems, 27 are located on the integrin heterodimer αIIbβ3 or glycoprotein (GP)IIb/IIIa (Table19.3 and Figure19.4). Of the remaining eight, three are on GPIb/IX/V, four on the integrin α2β1 or GPIa/IIa, and one on CD109. The difference between the two alleles in each bi- allelic HLA system is a single- nucleotide

Molecular detection methods

Amplification of genomic DNA by PCR- SSP and SBT can be used to type donors and patients, even when the latter are thrombocytopenic. NGS methods have also been developed for HPA typing, which has enabled the detection of novel variants that result in the formation of alternative glycoprotein structures that are exposed on the platelet surface. Thus, this technique could help to identify and confirm the presence of novel alloantigens.

throughput donor HPA typing can also be per-

Highformed using the 5′- nuclease TaqMan® assay. Two TaqMan HPA- specific probes are covalently linked with a 5′ reporter dye VIC or FAM and a 3′ quencher dye. When the probe is intact, the quencher suppresses the fluorescence of thereporter dyes. In the PCR reaction, the TaqMan® probe hybridizes to the target sequence and during extension, the 5′ exonuclease activity of Taq polymerase releases the reporter from the probe and fluorescence is emitted for detection. Taq exonuclease activity only occurs when the probe is fully hybridized with no mismatches present. The fluorescent signal for the two reporter dyes is measured at

14b (Table19.2).

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274 Molecular Hematology

https://t.me/med1917

Table19.2 Platelet- specific alloantigen systems

System Antigen

1 HPA- 1a

HPA-

HPA- 1b

HPA- 2 HPA- 2a

HPA- 2b

HPA- 3 HPA- 3a

HPA- 3b

HPA- 4 HPA- 4a

HPA- 4b

HPA- 5 HPA- 5a

HPA- 5b HPA- 6b Caa, Tu HPA- 7b Mo HPA-

8b Sr HPA- 9b Max HPA- 10b La HPA- 11b Gro HPA- 12b Iy HPA- 13b Sit HPA- 14b Oe

HPA- 15 HPA- 15a

HPA- 15b HPA- 16b Duv HPA- 17b Va HPA- 18b Cab HPA- 19b Sta GPIIIa 487A > C Lys > Gln137 HPA- 20b Kno GPIIb 1947C > T Thr > Met619 HPA- 21b Nos GPIIIa 1960G > A Glu > Lys628 HPA- 22b Sey GPIIb 584A > C Lys > Thr164 HPA- 23b Hug GPIIIa 1942C > T Arg > Trp622 HPA- 24b Cab2 HPA- 25b Swi HPA- 26b Sec HPA- 27b Cab HPA- 28b War GPIIb 2311G > T Val > Leu740 HPA- 29b Kha HPA- 30b Lab HPA- 31b Cab4 HPA- 32b Dom HPA- 33b Bl HPA- 34b Bzh HPA- 35b Efs

Alternative names Glycoprotein

, Pl

Zwb, Pl

Koa, Sib Baka, Lek Bak Yukb, Pen Yuka, Pen Brb, Zav

Bra, Zava, Hc

Gov

3a+

GPIIIa 176T

GPIbα 482C

GPIIb 2621T

GPIIIa 506G

GPIa 1600G

GPIIIa 1544G > A Arg > Gln489 GPIIIa 1297C > G Pro > Ala407 GPIIIa 1984T > C Arg > Cys636 GPIIb 2602G > A Val > Met837 GPIIIa 263G > A Arg > Glu62 GPIIIa 1976G > A Arg > His633 GPIbβ 119G > A Gly > Glu15 GPIa 2483C > T Thr > Met799 GPIIIa 1109- 1911delAAG Lys611del CD109 2108C

GPIIIa 497C > T Thr > Ile140 GPIIb/IIIa 662C > T Thr > Met195 GPIa 2235G > T Gln > His716

GPIIb 1508G > A Ser > Asn472 GPIa 3347C > T Thr > Met1087 GPIIIa 1818G > T Lys > Asn580 GPIIb 2614C > A Leu > Met841

GPIIIa 98C > T Thr > Met7 GPIIb 2511G > C Gln > His806 GPIX 368C > T Pro > Leu368 GPIIIa 521A > G Asp>Ser148 GPIIIa 1373A > G Asp>Gly432 GPIIIa 349C > T Arg > Trp91 GPIIIa 1514A > G Arg > His479

Nucleotide change

176C

482C

2621G

506A

1600A

2108A

Amino acid change (mature protein)

Leu33 Pro33 Thr145 Met145 Ile843 Ser843 Arg143 Gln143 Glu505 Lys505

Ser682 Tyr682

The superscript letters were the previous nomenclature (alternative names) for the serological definition of human platelet antigens.

the end point of the PCR reaction and the ratio of signals is used to determine the genotype.

HPA alloimmunization and clinical implications

Alloimmunization against HPA is associated with three clinical syndromes: post- transfusion purpura (PTP), platelet refractoriness, and neonatal/fetal alloimmune thrombocytopenia (NAITP).

本书版权归John Wiley & Sons Inc.所有

The two most clinically relevant HPA antigens are HPA- 1a and HPA- 5b on the β3 and α2 integrins, namely those on platelet glycoproteins GPIIIa and GPIa, respectively. In cases of platelet refractoriness as a result of HPA alloimmunization, HPA- 1b, - 5b, and - 2b antibodies are commonly found. In NAITP cases observed in European Caucasoid populations, approximately 75% of cases are due to HPA- 1a

The molecular basis ofblood cell alloantigens 275

GPIa

GPIIa

GPVI

GPIIb

GPIIIa

CD109

GPIbα

GPV

HPA-18w TQ716H

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HPA-5 E505K

HPA-13w T799M

α2

2 3

βTD

β2

HPA-10w R62Q

HPA-20w T619M

HPA-9w V837M

HPA-3 18435

αIIB

β3

2 3

βTD

HPA-19w K137Q

HPA-16w T140l HPA-17w T195M

HPA-4 R143Q

HPA-7w P407A

HPA-1 L33P

HPA-6w R489Q

HPA-8w R636C

HPA-11w R633H

HPA-14w Del K611

HPA-21w E628K

HPA-15 S703Y

GPIbβ

HPA-12w G15Q

GPIX

HPA-2 T145M

Talin

Figure19.4 Schematic presentation of the platelet glycoproteins (Adapted from Practical Transfusion Medicine Third Edition, 2009, Wiley- Blackwell by G. Lucas). Schematic presentation of the platelet glycoprotein from which the HPAs can be found. The amino acid substitutions arising from allelic variation of the GPIIb and GPIIIa genes are depicted by black dots and the name of the HPA system is noted. Amino acids are given as three- letter acronyms.

Table19.3 Table showing the% HPA allele frequencies observed inindividuals ofEuropean andEast Asian ethnicities

Antigen

HPAHPA- 1b HPA- 2a HPA- 2b HPA- 3a HPA- 3b HPA- 4a HPA- 4b HPA- 5a HPA- 5b HPA- 15a HPA- 15b

European Caucasoid n=1266

86.9

13.1

91.9

8.1

61.1

38.9

100

90.5

9.5

49.5

50.5

East Asian n=1170

99.1

0.9

93.5

6.5

55.1

44.9

99.7

0.3

96.9

3.1

46.7

53.3

Data from the 1000 Genomes Project (30× coverage).

antibodies, 15% are caused by HPA- 5b antibodies, and 4% are a result of HPA- 15b antibodies.

Alloantibodies against other HPA alloantigens are observed less frequently in pregnancy but do occur such as the case for HPA- 4b alloantibodies being more prevalent in East Asian populations.

Post- transfusion purpura

PTP is defined as thrombocytopenia arising 5–12days following transfusion of cellular blood components (red cells or platelets) associated with the presence in the patient of antibodies directed against the HPA systems with HPA- 1a antibodies being the most frequent. There is some evidence of autoreactive antibodies destroying the patient’s own platelets during the acute phase of PTP in addition to the alloreactive HPA- 1a antibodies.

Alloantibodies against other HPA alloantigens have been observed in hemato- oncological patients and other patients on long- term prophylactic platelet transfusion, albeit at a low frequency.

Platelet refractoriness

Prophylactic platelet transfusions are essential for preventing bleeding during intensive chemotherapy or other myeloablative therapies. Poor increments following the infusion of an adult dose of donor platelets (>25 × 109/L) can be caused by a variety of factors. Non- immune factors, such as splenomegaly, bleeding, sepsis, fever, and certain drugs (e.g. amphotericin), can compromise the beneficial effect of donor platelet infusions. In 10–20% of patients, the problem of poor increments is further compounded by antibodymediated destruction of donor platelets. Despite this, the

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276 Molecular Hematology

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clinical definition of refractoriness remains much disputed; clinically, the picture of an increased frequency of platelet transfusions to maintain satisfactory platelet counts and effective hemostasis requires further laboratory investigations for HLA class I and for HPA alloantibodies, platelet autoantibodies or high- titer anti- A or anti- B antibodies.

The ability to type donors and patients for HLA class I and HPA by molecular techniques has resulted in more accurate matching of donors with patients, with improved posttransfusion increments. The algorithm currently used for the management of the alloimmunized patient with poor increments is shown in Figure19.5. Traditionally, HLA matching for the provision of compatible platelets for patients who have become immunologically refractory to random platelet transfusion has been based on the serological definition of these antigens. A new approach based on the identification of conformational epitopes present in each HLA allele has been described. According to this strategy, each HLA antigen is converted into a string of potentially immunogenic epitopes, which are represented by amino acid triplets (called eplets) on exposed parts of the HLA chains accessible to alloantibodies. These eplets consist of amino acid residues that are located within small clusters (with diameters of about 0.3–0.35 nm) around a non- self- residue and are formed by amino acids in linear sequences and from discontinuous regions of the sequence brought together by the folding of the molecule. This is therefore considered to be a more accurate representation of the epitope than that derived from the triplet HLA Matchmaker model developed by Duquesnoy. Consequently, it is possible to determine the number of eplets which are either shared or different between the donor and the recipient. The algorithm also performs intra- and inter- locus comparisons of polymorphic eplets in amino acid sequence positions in order to determine the spectrum of non- shared eplets between HLA antigens of the donor and the patient.

Neonatal alloimmune thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAITP) was first described by van Loghem in 1959 and was initially thought to be a rare disorder. Prospective screening studies in pregnant European women have shown that 1in 1100neonates have severe thrombocytopenia (<50 × 109/L) due to maternal anti- HPA- 1a, confirming the notion that the most frequent cause of severe thrombocytopenia in the term newborn is maternal alloantibodies against a fetal HPA alloantigen.

This serious clinical condition is caused by the destruction of fetal/neonatal platelets by maternal HPA alloantibodies of the IgG class. Cerebral bleeds in the perinatal period are the most concerning complication, which either occur in utero or

during delivery. In cases of severe thrombocytopenia

× 109/L), there remains a small but definite risk of this

(<25 serious complication in the first days of life, warranting treatment. For proper clinical management, the cause of severe thrombocytopenia in an otherwise healthy term neonate should be determined with urgency and prompt correction of a count less than 25 × 109/L by platelet transfusion is of utmost importance. This should precede the outcome of platelet antibody investigation, which can be a time- consuming process.

Incidence of and HPA immunogenicity

Alloantibodies against the HPA- 1a alloantigen occur in 1in 365 pregnancies and cause severe thrombocytopenia, with a neonatal platelet count of less than 50 × 109/L in 1in 1100 term neonates.

Maternal IgG alloantibodies against a fetal HPA alloantigen can cross the placenta and bind to the fetal platelets, thus reducing platelet survival. The HPA- 1 and HPA- 5 systems are the two most clinically relevant in the majority of cases caused by antibodies against HPA- 1a (75%) and HPA- 5b (10–15%).

Why the immunogenicity of HPA- 1a is magnitudes higher than that of its antithetical antigen HPA- 1b was initially not well understood. In the early 1980s, it was discovered that the formation of anti- HPA- 1a in pregnancy was positively associated with the HLA haplotype A*01, B*08, DRB1*03. Further studies revealed that nearly all antibody formers were positive for the *01 : 01 allele of the DRB3 gene (DRB3*01 : 01 or DR52a). A prospective study in 25,000 pregnant women showed that this class II marker has an odds ratio of 140, which makes it one of the most reliable HLA associations reported to date, with negative predictive power equal to that of HLA- B*27 in ankylosing spondylitis. A difference in the efficiency of presentation of the GPIIIa- Leu33 (HPA- 1a)derived oligopeptide between DRB3*01 : 01- positive and

- negative antigen- presenting cells to CD4+ T cells is the most likely explanation of this restriction in alloimmunization. The frequency of the HLA DRB3*01 33%, and this marker therefore has a high negative predictive value but a low positive one for anti- HPA- 1a formation. The allele frequencies vary between populations; for example, HPA- 1b is extremely rare or absent in East Asia, while the opposite is the case for the HPA- 4b.

: 01 allele in Caucasians is

Characteristics

NAITP presents in the otherwise healthy newborn as petechiae or ecchymoses or is found coincidentally by a whole blood count. Severe cases can present neurological symptoms due to cerebral bleeds or with hydrops fetalis or cerebral cysts. NAITP can affect the first pregnancy and has a 10% risk of severe intracranial hemorrhage. Diagnosis is based on the detection of HPA alloantibodies in the maternal

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Patients likely to receive multiple platelet transfusion

Investigation of immune refractoriness to platelet transfusion

https://t.me/med1917

Assess transfusion response

Ab positive

provide HLA

selected platelets

Good response to

HLA selected

platelets

Continue

transfusing HLA

selected platelets

Test for HLA

antibodies at

regular intervals

Poor response to random donor platelets on two or more occasions

HLA type

and test for

HLA specic

antibodies

HLA antibody

test result

Poor response to

HLA selected

platelets

Provide ABO

compatible, “A”

grade matches if

possible

Test for HPA

specic antibodies

Ab negative

Factors associated with non-

immune platelet destruction

Absent Present

Consider trial of

HLA selected

platelets

Poor response Good response

Treat cause consider further platelet transfusion based on clinical status of the patient e.g. increase dose of platelets or discontinue prophylactic transfusions

Figure19.5 Platelet transfusions in alloimmunized patients. An algorithm outlining the decision process for the management of alloimmunized patients refractory for random donor platelets. After confirmation of refractoriness for random donor platelets, patients are screened for HLA class I alloantibodies and, if positive, HLA class I- matched platelets are transfused. In 20–30% of patients, increments with HLA class I- matched platelets are poor and screening for HPA antibodies should follow. Also, the possible presence of potent anti- A or anti- B should be excluded since platelets do carry ABO blood group antigens. If there are no detectable HLA class I antibodies, a trial of HLA- matched platelets and screening for HPA antibodies should be considered (right arm of algorithm).

本书版权归John Wiley & Sons Inc.所有

Hpa antibody

test result

Positive Negative

Provide HLA and

HPA selected

platelets

Consider

1. Non immune consumption

2. ABO antibodies

Continue

transfusing HLA

selected platelets

278 Molecular Hematology

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serum combined with a parental HPA incompatibility, as determined by molecular typing methods such as PCR- SSP, Taqman assay, or sequencing methods such as SBT or NGS.

Treatment

Although guidelines for the treatment of NAITP can vary between different countries, most agree that a neonatal platelet count of between 30 and 25 × 109/L should be corrected immediately before the confirmation of NAITP by diagnostic tests. Transfusion of HPA- 1a negative, and if possible, HPA- 5b negative, donor platelets will be compatible with the maternal HPA alloantibody in over 95% of cases. In a typical case, the platelet count should recover to normal within a week, although a more protracted recovery can occur.

Counseling

Counseling of couples with an index case about the risks of severe fetal/neonatal thrombocytopenia in a subsequent pregnancy needs to be based on the severity of disease in the index case and the outcome of immunological investigations. The following should be considered:

Thrombocytopenia in subsequent cases is as severe or

generally more severe.

Antibody specificity and titer have some correlation with severity; for example, HPA- 5b antibodies generally cause mild thrombocytopenia, which rarely results in a cerebral bleed. The latter is generally associated with HPA- 1a antibodies.

HNA antigen systems

Neutrophils, like all other cells present in blood, express polymorphic molecules that can induce strong antibodies when transfused or transplanted or as result of pregnancy. These

antigens are called human neutrophil antigens (HNAs). Five HNA antigenic systems have been described (HNA- 1 to HNA-

5), which are the result of polymorphisms except for HNA- 2. HNA- 1 is located on a glycosylphosphatidylinositol (GPI)-

anchored glycoprotein that forms the low- affinity Fcγ receptor. HNA- 1 is encoded by the FcγRIIIB (FCGR3B) gene located on chromosome 1 and mediates IgG- induced phagocytosis. Four antigens have been described for HNA- 1 (Table19.4). Five alleles of the HNA- 1 system, FCGR3B*01, FCGR3B*02, FCGR3B*03, FCGR3B*04 and FCGR3B*05 have been described. Five amino acid substitutions at positions 36, 65, 78, 82, and 106 determine the HNA- 1 antigens. Individuals genotyped positive for the FCGR3B*01 and FCGR3B*04 alleles will express the HNA- 1a epitope, while individuals positive for the FCGR3B*05 will express the epitope for HNA- 1b antibodies. Some individuals do not express the FCGR3B gene and carry a null phenotype, which is rare and is based on a double deletion of the FCGR3B gene. It is, in some cases, associated with a deletion of the FcγRIIC (FCGR2C) gene. Deficiency for the most abundant Fc receptor on neutrophils does not seem to be associated with an obvious clinical phenotype. This is in contrast with a mutation in the FcγRIIIA (FCGR3A) gene, which encodes a Leu48His substitution in the first extracellular domain of the NA cell FcγRIIIA which, although only described in one infant, was associated with recurrent and serious respiratory tract viral infection from birth.

HNA- 2 is expressed on a 58–64 kDa glycoprotein (CD177)

found on the plasma membrane and secondary granules of neutrophils. This glycoprotein is linked to the plasma membrane by a GPI anchor and is coded for by a gene located on chromosome 19. HNA- 2 is expressed in approximately 50% of the total neutrophils by 95% of individuals. Some individuals

Table19.4 Human neutrophil antigens (HNA)

Antigen system Alleles Antigens Former name Gene CD

HNA- 1 FCGR3B*01

HNA- 2 HNA- 3 SLC44A2*01

HNA- 4 ITGAM*01

HNA- 5 ITGAL*01

HNA- 2 is defined by isoantibodies.

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HNA- 1a

FCGR3B*02 FCGR3B*03 FCGR3B*04 FCGR3B*05 n/a HNA- 2 NB1 CD177 CD177

SLC44A2*02 SLC44A2*03

ITGAM*02

ITGAL*02

HNA- 1b, HNA- 1d HNA- 1c, HNA- 1b HNA- 1a HNA- 1b

var

HNA- 3a HNA- 3b HNA- 3a

var

HNA- 4a HNA- 4b HNA- 5a HNA- 5b

NA1 NA2 (HNA- 1b) SH (HNA- 1c)

5b SLC44A2 Not known

Mart ITGAM CD11b

Ond ITGAL CD11a

FCGR3B CD16

The molecular basis ofblood cell alloantigens 279

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do not express HNA- 2 due to a transcription defect in the CD177 gene. HNA- 2a alloantigen typing is based on the use of human immune antisera and immunofluorescence.

HNA- 3 is carried on the choline- transporter- like protein 2, which is a transmembrane protein encoded by the SLC44A2 gene on chromosome 19p. There have been three antigens identified, which are encoded by three alleles (SLC44A02*01, SLC44A02*02, SLC44A02*03). These are the result of two SNPs at positions 451 and 455, giving rise to amino acid changes at positions 151 and 152, respectively. A SNP (455G > A) changing an arginine to glutamine at position 152 of the protein, is responsible for the difference between HNA- 3a and HNA- 3b. SLC44A02*03 encodes the HNA- 3a epitope which is recognized by HNA- 3a antibodies but with weaker affinity.

HNA- 4 is located on the αM chain (CD11b) of the β2integrin MAC- 1. HNA- 4 is encoded by the ITGAM gene located at chromosome 16. This system has two antigens, HNA- 4a (ITGAM*01) and HNA- 4b (ITGAM*01), and is the result of a single amino acid substitution at position 61. aM/b2- integrin is a transmembrane protein expressed on the surface of many leukocytes and involved in adhesion, transmigration, phagocytosis, and cell- mediated cytotoxicity. The difference between the antigens is caused by a unique SNP at position 230 of the gene, changing an arginine into a histidine at the protein level.

HNA- 5 is located on the αL integrin unit of the leucocyte function- associated antigen (LFA)- 1, also known as CD11b. HNA- 5 is encoded by the ITGAL gene and is located at chromosome 16. aL/b2- integrin is a leukocyte- specific adhesion molecule involved in leukocyte interactions and trafficking. A single amino acid substitution at position 766 encodes the HNA- 5 antigens.

HNA allele frequency

The allele frequencies vary between populations (Table19.5). For example, the FCGR3B*03 allele (HNA 1b, 1c) is extremely rare or absent in the Chinese population, while the frequency of this allele in the African and African Caribbean heritage population is approximately 19%.

HNA detection methods

HNA- 1 and HNA- 2 antigens are uniquely expressed on neutrophils, whereas HNA- 3, HNA- 4, and HNA- 5 are also present on other cells or tissues. It is now possible to detect most of the HNA polymorphisms and FcγRIIIB null genotypes using DNA- based techniques including PCR- SSP, PCRSSOP, or DNA sequencing. However, the detection of HNAspecific antibodies is still reliant on the use of HNA- typed granulocyte panels and many of the techniques cannot determine whether antibody reactivity is due to HNA or HLA. There is a need to develop a new generation of techniques for the characterization of HNA- specific antibodies, using recombinant HNA proteins or HNA- expressing stable cell lines.

Clinical significance of HNA

Although the HNA system is not as polymorphic as the HLA and HPA systems, in circumstances where patients produce HNA antibodies, these can cause complications of transfusion and transplantation.

Neutrophils play a key role in immunity to bacteria and fungi, and antibodies that destroy or impair the function of neutrophils will increase the risk of infection.

Table19.5 HNA allele frequencies indifferent populations

Antigen system Allele

HNA- 1 FCGR3B*01

HNA- 2 n/a 86.6

HNA- 3 SLC44A2*01

HNA- 4 ITGAM*01

HNA- 5 ITGAL*01

Data generated in an unpublished NHSBT study by A. Niewiarowska, A. Poles, C. Brown, C. Navarrete, and W. Chong.

本书版权归John Wiley & Sons Inc.所有

FCGR3B*02 FCGR3B*03

SLC44A2*02

ITGAM*02

ITGAL*02

European n=386

33.9

64.5

1.7

(n=2193)

78.4

21.6

89.9

10.1

66.6

33.4

Chinese n=67

66.4

33.6 0

99.2 (n=128)

63.4

36.6

99.3

0.7 93 7

South Asian n=160

41.5

49.5

8.9

73.8

26.3

94.1

5.9

37.5

62.5

Black n=199

41.5

39.2

19.2

91 9 91 9

55.8

44.2

280 Molecular Hematology

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Neonatal alloimmune neutropenia

Neonatal alloimmune neutropenia (NAIN) is a condition similar to NAITP, where the maternal HNA antibodies cause the destruction of fetal neutrophils leaving the neonate susceptible to infection, which can be fatal and affects less than 1/1000live births. Some individuals who do not express the FCGR3B gene and carry a null phenotype can develop isoantibody of known clinical significance in cases of NAIN. The FcγRIIIB null phenotype can cause immune neutropenia in the newborn due to maternal anti- FcγRIIIB isoantibodies.

Severe but reversible neutropenia in the newborn may require treatment with antibiotics to control bacterial infection. There is no conclusive evidence that the mutations in the FcγRIIIB protein, found on peripheral blood mononuclear cells, have any functional consequences, for example the FcγRIIIB null phenotype, has not been linked with an obvious pathological phenotype. Whereas rare mutations in the FCGR3A gene product found on NK cells have been linked to susceptibility to NAIN and viral infections.

Transfusion- related acute lung injury

Transfusion Related Acute Lung Injury (TRALI) was first described as a distinct clinical entity in 1985 and is a serious, often fatal complication of transfusion where recipients of a blood transfusion experience acute dyspnea with hypoxia, bilateral pulmonary infiltrates, in the absence of circulatory overload or other causes, within 6 h of transfusion. The presence of HLA or HNA antibodies cognate with the recipient in the blood product transfused is also important as they will bind the recipients’ neutrophils and cause an immune response involving neutrophil activation, complement factors, and pro- inflammatory cytokines in the lung leading to pulmonary edema. As well as HLA class I and class II antibodies, HNAimplicated in cases of TRALI as well as HLA class I and class II antibodies.

HNA- 3a antibodies have not only been implicated in TRALI cases but also in febrile non- hemolytic transfusion reactions, where antibodies in the patient react with neutrophils in the transfused product and are less common following the introduction of universal leucodepletion of blood products in many countries.

2 and - 3a antibodies have been

discovered when renal patients were observed to be experiencing antibody- mediated rejection in the absence of donorspecific HLA antibodies. In the hemopoietic stem cell setting, both HNA- 2 and - 3a IgG alloantibodies have been associated with transplant rejection and delayed engraftment.

Red blood cell antigens andmolecular typing

More than 350 red cell antigens are now recognized and categorized into over 40 different blood group systems, genetically determined by nearly 50 genes. Exposure to “non- self” red cell antigens, through either transfusion or pregnancy, can cause hemolytic transfusion reactions (HTR) or hemolytic disease of the fetus and newborn (HDFN).

Knowledge of the molecular backgrounds of red cell antigens has allowed the development of molecular typing methods for the prediction of blood group phenotypes. Such genotyping is routinely used for the most clinically relevant blood group systems (Rh, Kell, Kidd, Duffy, and MNS), with the exception of ABO. Genotyping for ABO is not considered sufficiently accurate for routine clinical use, due to the difficulties in detecting all variants that inactivate the A and B transferase genes, and the potentially fatal consequences of an ABO- mismatched transfusion.

Molecular typing is of particular use when standard serological typing is not possible, such as for recently transfused patients (e.g. those with hemoglobinopathies) and in prediction of fetal blood group phenotype. Accurate interpretation of blood group genotype into predicted phenotype requires in- depth knowledge of the genes involved and their many allelic variants, particularly relevant in certain ethnic groups. Homology between RHD and RHCE genes of the Rh system, together with a high degree of polymorphism, leads to challenges in designing and correctly interpreting genotyping tests (also relevant to the homologous genes of the MNS system). However, genotyping offers a highly accurate method for prediction of blood group phenotype and is invaluable in situations where serology may not be possible or practical. NGS-

based methods have the potential to allow the determination of all known blood group antigens from a single test and seem likely to become the standard of blood group typing for the future.

Hemolytic disease ofthe fetus andnewborn

HNA and allograft rejection

HNA- 3a alloantibodies have also been reported to contribute to antibody- mediated rejection in renal transplants as the kidney expresses HNA- 3 on the kidney endothelium. This was

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HDFN, a disease associated with significant morbidity and mortality in the fetus and newborn, is caused by maternal alloantibodies directed against fetal blood group antigens which are not present on the maternal red cells. During

The molecular basis ofblood cell alloantigens 281

RhCE (C) mSSKYPRSVR RCLPLCALTL EAALILLFYF FTHYDASLED QKGLVASYQV GQDLTVMAAI RhCE (c) ----------

RhD

RhCE (C) GLGFLTSSFR RHSWSSVAFN LFMLALGVQW AILLDGFLSQ FPSGKVVITL FSIRLATMSA RhCE (c) -------N--

----------

RhD -------S--

----------

RhCE (C) MSVLISAGAV LGKVNLAQLV VMVLVEVTAL GTLRMVISNI FNTDYHMNLR HFYVFAAYFG RhD L-----

--------MM –I--------

RhCE

PSVNSPLLRS PIQRKNAMFN

RhCE (e)

----------

RhD -S--------

RhCE RhD

----------

RhCE AGLISIGGAK CLPVCCNRVL GIHHISVMHS IFSLLGLLGE ITYIVLLVLH TVWNGNGMIG RhD -----V----

-GA------

RhCE FQVLLSIGEL SLAIVIALTS GLLTG RhD ---------- ---------- ---------- -------E-- ---------- -------

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pregnancy, alloantibodies of the IgG class (mostly IgG1 and IgG3) can cross the placenta and bind to antigen- positive fetal red cells, causing their hemolysis. Early fetal anemia may also result from impairment of fetal erythropoiesis, due to antibody binding to erythroid progenitor cells. Untreated fetal anemia can lead to cardiomegaly, hydrops, and potential fetal death. After birth, ongoing hemolysis can cause severe hyperbilirubinemia, which may result in damage to the brain (kernicterus).

The main antigens implicated in HDFN severe enough to require antenatal intervention are RhD, K, and Rhc. In England and Wales, approximately 500 fetuses develop HDFN annually, with 25–30 perinatal deaths, and an estimated further 20 spontaneous abortions prior to 28weeks, attributed to HDFN.

Rh system

The Rh system is the most complex red cell blood group system and the most clinically important after ABO. While ABO antigens are carbohydrate in nature, with a wide tissue distribution, the Rh antigens are protein- based and specific to red cells. Rh antigens are carried on two homologous nonglycosylated proteins; RhD, carrying the D antigen, and RhCE, carrying the C, c, E, and e antigens, encoded by RHD and RHCE genes, respectively.

RH genes

The RHD and RHCE genes are located on chromosome 1 and are tightly linked but have opposite orientations. Each gene comprises 10 exons and each encodes a 417 amino acid non- glycosylated protein (although the N- terminal methionine is cleaved from the mature protein). The RHD and RHCE genes are highly homologous (93.8% over all introns and coding regions) and the encoded proteins differ only by between 32 and 35 amino acids, dependent upon RhCE polymorphisms (Table 19.6). The RhD and RhCE proteins are both highly hydrophobic and are predicted to span the red cell membrane 12 times, with 6 extracellular loops (Figure19.6). The function of the Rh proteins remains unknown.

In D- negative individuals (around 15% of white Europeans), the entire RhD protein is missing from the red cell membrane, usually arising from homozygosity for a complete deletion of the RHD gene. The pairs of antithetical antigens C/c and E/e are encoded by polymorphisms within the RHCE gene. RhC and Rhc proteins differ by four amino acids, of which Ser103Pro, located on the second extracellular loop, appears to be the most critical. RhE/e antigens are defined by a single amino acid substitution, Pro226Ala, in the fourth extracellular loop of RhCE (Table 19.6 and Figure19.6).

Table19.6 Amino acid sequence ofthe RhD andRhCE proteins

---------- -----W---- ---------- ---------- ---------- ---------

(E) LTVAWCLPKP LPKGTEDNDQ RATIPSLSAM LGALFLWMFW

---------- ---------- ---------- ---------- ---—-A----

Amino acid sequences (single letter code) showing differences between RhD and RhCE proteins, in addition to C/c (boxed) and E/e (shaded) polymorphisms. Sequence identity is indicated by dashes.

TYYALAVSVV TAISGSSLAH PQRKISMTYV HSAVLAGGVA VGTSCHLIPS PWLAMVLGLV

----V----- ---------- --G---K--- ---------- ----------

-----W---- ---------- ---------- ---------- ---------

---------- ---------- ---------- –-P-------

---------- ---------- ---------- --S-------

VD-- ---------- ---------- -N--------

--E----K--T--------- ---------- --F--A---- --E----V-

Y--G------ --P-S-I-GY N--------- -I-------D-

LLLNL KIWKAPHVAK YFDDQVFWKF PHLAVGF

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282 Molecular Hematology

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S103P (C/c)

Exon 2

11 72 75

Figure19.6 Rh proteins. Model of the Rh proteins in the membrane, showing the regions encoded by exons 1 to 10. Amino acids 16, 60, 68, and 103; highlighted in orange, differ between RhC and Rhc. The critical S103P difference between RhC and Rhc is labeled in external loop2. The P226A substitution defining RhE and Rhe, highlighted in yellow and labeled, is positioned in a proposed “vestibule” in the membrane. Positions highlighted in red show residues where RhD and RhCE proteins differ.

53 94 107 158 167 280 290 347 358282

Exon 3

131 135 186 201 263 266 321 324

Exon 1

Exon 4

P226A (E/e)

Exon 5

Exon 6

Exon 7

COOH

Exon 8

Exon 9

391

417

Exon 10

Immunogenicity of RhD and prevention ofimmunization

The complete absence of the RhD protein in D- negative individuals explains its high immunogenicity, and anti- D is highly clinically significant in both transfusion and pregnancy. Production of alloanti- D, stimulated by a D- positive fetus carried by a D- negative mother, may result in severe HDFN, associated with hydrops, kernicterus, and possible fetal death. It has been shown that this maternal anti- D production can be prevented by administration of IgG anti- D. The introduction of anti- D prophylaxis for D- negative women after delivery, or following sensitizing events during pregnancy, resulted in a steep decline in HDFN- related mortality. Before 1969, 46 deaths per 100,000 births were attributed to RhD alloimmunization, falling to 1.6/100 000 births in 1990. More recently, introduction of routine antenatal anti- D prophylaxis (RAADP) for all D- negative pregnant women in the United Kingdom has further reduced alloimmunization rates.

The discovery of cell- free fetal DNA in the maternal circulation during pregnancy has allowed the development of non- invasive fetal blood group genotyping from maternal blood. Determination of fetal RHD genotype in pregnancies at risk of HDFN allows appropriate monitoring and clinical intervention to be directed at pregnancies where the fetus is

predicted to be D-

positive. In the United Kingdom, approximately 38% of D- negative pregnant women will carry a D- negative fetus and are therefore not at risk of RhD alloimmunization. Recently, large- scale non- invasive fetal RHD screening has been introduced in the United Kingdom, and several other European countries, for all non- sensitized D- negative pregnant women to determine the individual requirement for anti- D prophylaxis. Targeting of RAADP to those women confirmed to be carrying a D- positive fetus prevents unnecessary exposure to anti- D in those carrying a

negative fetus, reducing unnecessary healthcare interven-

Dtions and costs.

In addition to anti- D, antibodies to other Rh antigens are also clinically significant, and associated with both HTR and HDFN. Anti- c is the most clinically important after anti- D and has been associated with severe HDFN, whereas anti- C, anti- E and anti- e have rarely been implicated in severe HDFN. Non- invasive fetal genotyping to predict RhC/c and E/e antigen status is useful in the management of at- risk pregnancies, although no prophylaxis is available.

Kell

The K antigen of the Kell blood group system is the most clinically important red cell antigen outside of the ABO and Rh systems. The antithetical antigens K and k are carried on

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