Immunohematology

Journal of Blood Group Serology and Education

Volume 26, Number 4, 2010

Immunohematology Journal of Blood Group Serology and Education Volume 26, Number 4, 2010 Contents

Review 133 Polylactosamines, there’s more than meets the “Ii”: a review of the I system L. Cooling

Original Report 156 Attempts to support an immune etiology in 800 patients with direct antiglobulin test–negative hemolytic anemia R.M. Leger, A. Co, P. Hunt, and G. Garratty

Review 161 The pathophysiology and prevention of transfusion-related acute lung injury (TRALI): a review D.C. Mair and T. Eastlund

Original Report 174 Comparison of gel test and conventional tube test for antibody detection and titration in D-negative pregnant women: study from a tertiary care hospital in North India M.K. Thakur, N. Marwaha, P. Kumar, S.C. Saha, B. Thakral, R.R. Sharma, K. Saluja, H.K. Dhawan, and A. Jain

Review 178 The Rh and RhAG blood group systems S.T. Chou and C.M. Westhoff

Communication 187 Letter to the editors Anti-Vel and cold-reactive autoantibody

Communication 188 Letter from the editors Thank you to the contributors of the 2010 issues 189 Index — Volume 26, Nos. 1, 2, 3, 4, 2010 193 Announcements 195 Advertisements 199 Instructions For Authors Editor-in-Chief Editorial Board Sandra Nance, MS, MT(ASCP)SBB Philadelphia, Pennsylvania Patricia Arndt, MT(ASCP)SBB Paul M. Ness, MD Pomona, California Baltimore, Maryland Managing Editor James P. AuBuchon, MD Joyce Poole, FIBMS Cynthia Flickinger, MT(ASCP)SBB Seattle, Washington Bristol, United Kingdom Philadelphia, Pennsylvania Martha R. Combs, MT(ASCP)SBB Mark Popovsky, MD Durham, North Carolina Braintree, Massachusetts Senior Medical Editor Geralyn M. Meny, MD Geoffrey Daniels, PhD Marion E. Reid, PhD, FIBMS Bristol, United Kingdom New York City, New York Philadelphia, Pennsylvania Anne F. Eder, MD S. Gerald Sandler, MD Technical Editors Washington, District of Columbia Washington, District of Columbia Christine Lomas-Francis, MSc George Garratty, PhD, FRCPath Jill R. Storry, PhD New York City, New York Pomona, California Lund, Sweden Dawn M. Rumsey, ART (CSMLT) Brenda J. Grossman, MD David F. Stroncek, MD Glen Allen, Virginia St. Louis, Missouri Bethesda, Maryland Christine Lomas-Francis, MSc Associate Medical Editors New York City, New York Emeritus Editorial Board Delores Mallory, MT(ASCP) SBB David Moolten, MD Gary Moroff, PhD Supply, North Carolina Philadelphia, Pennsylvania Rockville, Maryland Ralph R. Vassallo, MD John J. Moulds, MT(ASCP)SBB Philadelphia, Pennsylvania Shreveport, Louisiana

Editorial Assistant Immunohematology is published quarterly (March, June, September, and December) by the Sheetal Patel American Red Cross, National Headquarters, Washington, DC 20006. Immunohematology is indexed and included in Index Medicus and MEDLINE on the MEDLARS Copy Editor system. The contents are also cited in the EBASE/Excerpta Medica and Elsevier BIOBASE/Current Mary L. Tod Awareness in Biological Sciences (CABS) databases. The subscription price is $40.00 (U.S.) and $50.00 (foreign) per year. Proofreader Subscriptions, Change of Address, and Extra Copies: Lucy Oppenheim Immunohematology, P.O. Box 40325 Philadelphia, PA 19106 Production Assistant Marge Manigly Or call (215) 451-4902 Web site: www.redcross.org/immunohematology Electronic Publisher Copyright 2010 by The American National Red Cross Paul Duquette ISSN 0894-203X

On Our Cover “Les yeux clos” (1889–1905), Odilon Redon Odilon Redon created several versions of Les yeux clos (Closed Eyes) between 1889 and 1905. The striking radiance of the woman’s face hints at both the mystery and the significance of inner vision. With her eyes shut, she is unable to see the world, but because the eyes are so central to human identity, the world is also unable to truly see her. Is she asleep or dead? What are her dreams, her thoughts? The subject of the painting was Redon’s wife, although he may have also been inspired by a Michelangelo sculpture of a dying Roman slave. The Ii blood group is the subject of a review article in this issue, which provides insight into the clinical and basic science of polylactosamines.

——David Moolten, MD Review

Polylactosamines, there’s more than meets the “Ii”: a review of the I system

L. Cooling

Key Words: polylactosamine, I blood group, cold agglutinins mutations in the I gene, IGnT (GCNT2).7–10 Serologic studies of family members, who presumably are heterozygous, History can have an intermediate I+i+ phenotype with elevated i The unofficial birth of the I blood group system can and weakened I expression relative to normal controls.2,11 1 be traced to a seminal case report in 1956. Wiener and In certain kindreds, iadult is associated with congenital colleagues at New York University and Jewish Hospital cataracts.12–14 Based on older serologic studies, the 1,15 reported a fatal case of cold agglutinin syndrome caused by a incidence of iadult ranges from 1 in 4400 to 1 in 17,000. The potent autoantibody they named “anti-I” for “individuality.” prevalence of mutant IGnT alleles in the general population The patient was a 62-year-old woman with a 5-year history is unknown but is presumably rare. of anemia and episodes of acute life-threatening hemolysis. Increased i expression can be observed with several Although the patient’s serum was compatible with donor inherited and acquired hemolytic disorders as a sign of RBCs at 37°C, she had periodic acute hemolytic transfusion stressed erythropoiesis. Increased i expression is found reactions with “compatible” RBCs, even after warming the on reticulocytes, and also occurs in megaloblastic anemia, blood before transfusion. In an attempt to provide compatible erythroleukemia, thalassemia, paroxysmal nocturnal blood, the patient’s serum was eventually tested against hemoglobinuria, and other hemolytic disorders.16–18 family members, 22,000 blood donors, and an assortment Increased i is also a common finding in hereditary of animals including rabbit, sheep, horse, and cow. A total of erythroblastic multinuclearity with positive acidified five compatible donors were eventually identified, and their serum lysis test (HEMPAS) disease, a rare hematopoietic RBCs were successfully transfused to the patient during the disorder characterized by dyserythropoiesis, altered RBC next 2 years. Because these donors appeared to lack I, their glycosylation, and hemolysis.19 Unlike other hemolytic RBCs were designated i phenotype. In a desperate moment disorders, the increased i observed on HEMPAS RBCs when “compatible” human blood was unavailable, the patient reflects abnormal Golgi trafficking and glycosylation.20,21 was transfused with cow blood, which had demonstrated only weak activity at 4°C. Not surprisingly, the transfusion Biochemistry was aborted within 10 minutes, after the patient developed Ii Structure anxiety, acute dyspnea, and feelings of “impending doom”! I and i are structurally and biosynthetically related The serologic relationship between I and i phenotypes oligosaccharide chains, composed of repeating units was solidified with the identification of an anti-i by Dr. of N-acetyllactosamine (Fig. 1; [Galβ14GlcNAcβ1-] , Lawrence Marsh.2 The antibody was strongly reactive with n LacNAc). I and i are ubiquitously expressed on human cord RBCs and i RBCs but not normal adult RBCs. More adult tissues and membrane structures. On RBC glycoproteins, importantly, Marsh was able to demonstrate a progressive polylactosamines are expressed as asparagine- or N-linked age-dependent decrease in i on infant RBCs, suggesting a glycans, ranging from biantennary to large, polyvalent developmental relationship between I and i. tetraantennary structures (Fig. 1B). On gut, leukocytes, Ii System and other tissues, polylactosamine may also be expressed As noted by Marsh, I and i are developmentally regulated as O-linked glycans via covalent linkages on serine and antigens on RBCs. At birth, cord RBCs serologically type as threonine residues (Fig. 1C). Polylactosamines are also I–i+. A discernible increase in I expression is observed by 3 found on membrane glycosphingolipids (Fig. 1A), including months, accompanied by parallel decreases in i expression, massive polyglycosylceramides ranging from 20 to 50 with an adult-type I+ phenotype observed by 18 months.2 carbohydrate residues in size.22 Increases in I mirror increases in ABO expression, which Structurally, i is defined as a linear, unbranched type typically reach adult levels by 2 to 3 years of age (see section 2 chain structure bearing at least two successive LacNAc on HDFN).3 Using human anti-I sera, the number of available residues (Fig. 1A).23 I is a branched polylactosamine derived I sites is estimated to be anywhere from 30,000 to 140,000 from i by the addition of a β1,6-linked polylactosamine side on untreated adult RBCs,4–6 but increases nearly threefold chain.23 Antibodies against I, therefore, must recognize after enzyme treatment (see section on Ii antibodies).6 GlcNAcβ16Galβ-R as part of the immune epitope.23,24

The iadult phenotype is a rare, autosomal-recessive trait Although I is often depicted with binary, terminal β13 and characterized by an absence of I on RBCs as a result of β16 LacNAc epitopes, in reality, β1,6 branching can occur

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Fig. 1 Structures of Ii oligosaccharides on cell membranes. (A) Structure of i-active and I-active glycosphingolipid (N-acetyl ceramide). Endo-ß-endoceramidase can cleave i-active oligosaccharides; however, I-active oligosaccharides are resistant. (B) Structure of biantennary and tetraantennary glycans on N- or asparginine-linked glycoproteins. (C) O-glycan structures. (D) Terminal modification of Ii or type 2 chain oligosaccharides. Cer = ceramide; Gal = galactose; GalNAc = N-acetylgalactosamine; Glc = glucose; GlcNAc = N-acetylglucosamine; Man = mannose. anywhere along the polylactosamine backbone, assuming such as ABO, LeX, sLeX, LeY, and sialo-Ii activity (Fig. the presence of at least two successive LacNAc residues (see 1D).27 On human RBCs, more than 95 percent of all ABO later section). The best example of the latter is the massive antigens are type 2 chain oligosaccharides on N-glycans N-glycan on Band 3 of adult RBCs, which contains 40 to and glycolipids.28 Not surprisingly, some examples of anti- 50 oligosaccharides including five, short β1,6 LacNAc side I/i have complex reactivity, reacting more strongly with chains (see section on HDFN).25 RBCs of specific ABO types.18 Ii expression can be modified by enzyme treatment of RBCs. Endo-β-galactosidase from Bacteroides fragilis Polylactosamine Biosynthesis and Escherichia freundii cleave internal, unsubstituted The Golgi and Glycosyltransferases Galβ14R linkages.23 The enzyme will readily cleave i-active Proteins and lipids are initially synthesized in oligosaccharides in the absence of branching or modification. the endoplasmic reticulum (ER, Fig. 2A), followed by Internal galactose residues bearing substitutions, such as posttranslation glycosylation in the Golgi. The regulatory fucose (ABH, LeX; Fig. 1D) or β16GlcNAc (I) are resistant mechanisms directing the passage and modification of to enzyme cleavage.26 As a result, endo-β-galactosidase will substrates through the Golgi is still not entirely understood destroy i reactivity and reduce, but not eliminate, I because owing to its inherent dynamic and structural complexity. of the presence of substituted galactose at β1,6 branch points Grossly, the Golgi is composed of stacked membranous (Fig. 1A). In contrast, Ii expression tends to increase after cisternae arranged into three regions; cis-, medial-, and digestion with proteases and neuraminidase.26 Proteases trans-Golgi. Cargo (proteins, lipids) is shuttled between can increase accessibility to Ii expressed on glycolipids and the ER and various Golgi compartments by membrane polyglycosylceramides. Neuraminidase, on the other hand, budding, followed by targeting and fusion of vesicles to can remove terminal sialic acid on sialylated type 2 glycans, another Golgi compartment or plasma membrane. The resulting in a terminal LacNAc epitope (see Fig. 1D). process is dependent on GTP, coat complex proteins (COPI, Polylactosamines serve as scaffold structures for the COPII), SNARE proteins, membrane tethering proteins, synthesis of other type 2 chain carbohydrate antigens and lipid transfer proteins.29 Mutations in COPII proteins

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have recently been linked to altered glycosylation in GlcNAc residue by β3GnT5, a rate-limiting, gateway enzyme HEMPAS disease.21 regulating type 2 chain glycolipid synthesis (Fig. 2B).35–37 Glycosyltransferases and other Golgi processing Once formed, Lc3 is rapidly galactosylated by β4GalT1 to enzymes are typically localized within specific regions of form paragloboside (nLc4),37 which then is further extended the Golgi, with many glycosyltransferases co-localizing to by β3GnT1 (iGnT), or possibly β3GnT5,35,36 to form nLc5. The the same Golgi region and forming heterodimer complexes. latter is followed by β4GalT1 to form nLc6, the first i-active C2GnT, a β1,6 glucosaminyltransferase responsible for glycolipid bearing two successive LacNAc motifs.23 Because branched polylactosamines on O-glycans, is located in β3GnT5 is specific for short-chain glycolipid substrates,35,36 the cis-medial Golgi as a dimer.30 β3GalT1 (iGnT) and further elongation of nLc6 would be initiated by either β4GalT1 specifically colocalize to the trans-Golgi and may β3GnT1 or, possibly, β3GnT2 (Table 1). 31 synergistically participate in polylactosamine regulation. The formation of I proceeds from the addition of a β3GnT2 and β3GnT8, two β3 glucosaminyltransferases β16 GlcNAc to i by IGnT (GCNT2 by Human Genome important in polylactosamine synthesis on complex Organisation [HuGO] nomenclature).7,38 Enzyme studies 32 N-glycans, preferentially exist as a heterodimer. with purified oligosaccharides indicate the enzyme requires at least two successive LacNAc motifs for binding Synthesis of Ii on Glycolipids and Glycoproteins 27 As carbohydrate antigens, Ii are synthesized by and activity. This is also confirmed by detailed analysis a regulated, stepwise addition of sugars by a series of of I-active oligosaccharides, which typically demonstrate 22 glycosyltransferases, many of which are tissue-specific. This at least two LacNAc epitopes per β1,6 branch (Fig. 2C). is particularly true for the β1,3 glucosaminyltransferases The enzyme can form branches on both distal and centrally (β3GnT), which initiate or elongate polylactosamine placed galactose, as long as the prerequisite for two adjacent 27 chains. As shown in Table 1, up to eight different β1,3 LacNAc motifs is satisfied. The enzyme will not recognize glucosaminyltransferases have been identified capable LacNAc motifs replaced with fucose, neuraminic acid, or of either initiating or elongating polylactosamine-type α-linked galactose. As a result, sialylation and fucosylation glycans. The spectrum of polylactosamine structures can be considered regulators of polylactosamine synthesis, ultimately synthesized by any cell will reflect the complex dictating both the length and potential number of β1,6 interplay of tissue-specific transcription, enzyme kinetics, branch points (Fig. 1D). Detailed studies have shown an substrate specificity, and acceptor availability. inverse reciprocal relationship between sialylation and An example of how substrate specificity and polylactosamine chain length in immortalized cell lines.39 transcriptional regulation can direct polylactosamine There have been attempts to dissect the contributions of synthesis is the synthesis of type 2 chain glycosphingolipids. enzyme activity, substrate specificity, and Golgi localization Synthesis of i-active glycosphingolipids proceeds from in regulating polylactosamine synthesis. Kinetic studies lactosylceramide (LacCer, CDH) by the addition of a β13 with purified enzyme extracts and defined oligosaccharide

Table 1. Human ß1,3 and ß1,6 N-acetylglucosaminyltransferases (GnT)

Size (aa)* N-glycan Enzyme Alias Chromosome Total TM sites Substrate Comments

ß1,3 Glucosaminyltransferases ß3GnT1 iGnT 11q13.2 415 28 2 Galß14GlcNAc with LARGE, ß4GaT1 in trans-Golgi

† ß3GnT2 ß3GnT1 2p15 397 11 5 [Galß14GlcNAc]n=2-5 N-glycans; GSLs? ß3GnT3 13p13.1 372 21 3 Galß13GalNAc-O-Ser/Thr core 1 extension ß3GnT4 12q24 378 20 3 ß3GnT5 Lc3 synthase 3q28 378 12 4 Galß14Glc-Cer Lc3, ± Lc5 synthesis ß3GnT6 C3GnT 11q13.4 353 19 3 GalNAcO-Ser/Thr core 3 synthesis

ß3GnT7 2q37.1 401 24 1 [Galß14[(SO4)6GlcNAcß1]R keratan sulfate ß3GnT8 19q13.31 397 19 1 Galß1-4GlcNAcß1-R tetraantennary N-glycans; activates ß3GnT2 in WBC ß1,6 Glucosaminyltransferases IGnT GCNT2 6p24.2 402 19 5 Galß14GlcNAcß1-4 I antigen synthesis C2GnT1 GCNT1 9q13 428 23 2 Galß13GalNAc-O-Ser/Thr core 2, 4 O-glycans, Cis-medial Golgi *Amino acid (aa) length of the total protein and transmembrane domain (TM). †ß3GnT133 was renamed ß3GnT2 after resequencing and comparison of the two enzymes revealed they encoded the same enzyme.34

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substrates are mixed.27 It is clear that the synthesis of β13 GlcNAc by β3GnT is a critical rate-limiting step, with many β3GnT displaying specific substrate specificities (Table 1). On N-glycans, LacNAc synthesis requires the involvement of at least three different β3GnTs: β3GnT1,40 β3GnT2,34,41–43 and β3GnT8.32,44 β3GnT1 reacts well with linear and branched acceptors although the recombinant enzyme may have slightly higher activity with β1,6 branched acceptors.27,40 β3GnT2, like β3GnT1, has broad acceptor reactivity, whereas β3GnT8 is quite specific for large tetraantennary substrates on N-glycans (Fig. 1).44 β3GnT8 and β3GnT2 may cooperatively enhance polylactosamine synthesis, particularly on leukocytes.32 In HL60 cells, β3GnT8 has been shown to activate β3GnT2, increasing the

Vmax/Km ratio tenfold with a fourfold increase in β3GnT2 activity.32 β3GnT1 and β4GalT1 colocalize within the trans-Golgi, suggesting a possible coregulatory role for β4GalT1.31 Although β4GalT1 is not a rate-limiting step, studies indicate subtle enzyme preferences that may influence the length and degree of branching.27 With simple trisaccharide substrates, β4GalT1 preferentially reacts with GlcNAcβ1-3LacNAc (i type) over GlcNAcβ1- 6LacNAc (I type), suggesting a concerted effort by β3GnT1 and β4GalT1 toward synthesis of long i-type chains. Slightly different results are obtained when β4GalT1 is tested against branched substrates offering both β1,3 GlcNAc and β1,6 GlcNAc termini.27 Unlike linear substrates, β4GalT1 will preferentially recognize the β1,6 GlcNAc branch early in the reaction, although this tended to inhibit further galactosylation of the remaining β1,3 branch. In contrast, initial galactosylation of the β1,3 GlcNAc permitted a second galactosylation to form an I-type structure with both β1,3 and β1,6 LacNAc termini. Like sialylation and fucosylation, a distal β1,6 branch near the nonreducing galactose terminus may inhibit further chain elongation.

Molecular Biology The i Gene The gene generally referred to as iGnT is β3GnT1, a β1,3-N-acetylglucosaminyltransferase, which transfers a GlcNAc β13 to lactose and 40 Fig. 2 Biosynthesis of Ii oligosaccharides. (A) Transport of proteins and LacNAc termini. The gene resides on chromosome lipids from the endoplasmic reticulum (ER) through the cis-, medial-, and 11q13.2 and encodes a 415-amino acid, 47-kDa trans-Golgi. Inset shows the orientation and topical structure of a typical type II transmembrane protein. The enzyme glycosyltransferase. (B) Synthesis of i- and I-active glycosphingolipids. (C) contains two potential N-glycosylation sites and The structure of erythroglycan, an RBC polylactosaminylceramide. Note that each ß1,6 branch point is preceded by at least two lactosamine motifs an unusually long, 28-amino acid transmembrane (LacNAc). Cer = ceramide; Gal = galactose; Glc = glucose; GlcNAc = domain.40 The gene shares little sequence homology N-acetylglucosamine. with other β1,3 glucosaminyltransferases.34

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The enzyme is reportedly capable of both initiating the depending on which exon 1 is used (Fig. 3B, 3C).7,8 IGnTA synthesis of polylactosamines and elongating existing (GCNT2A, IGnT2), the most common transcript found in polylactosamine oligosaccharides. As discussed earlier, human tissues, is encoded by exons E1A, E2, and E3. The β3GnT1 colocalizes with β4GalT1 in the trans-Golgi, IGnT2B (GCNT2B) uses exon E1B and is the only mRNA where β4GalT1 may co-associate and stabilize β3GnT1 transcript identified in purified human lens epithelium.8 It retention.31 β3GnT1 also complexes with LARGE, an is also highly expressed in fetal brain and adult cerebellum. unusual glucosaminyltransferase implicated in O-linked IGnTC (GCNT2C), on the other hand, is nearly unique glycans on α-dystrophin.45 By Northern blot, the enzyme to human reticulocytes and is responsible for I antigen is ubiquitously expressed in most human tissue tested; expression on RBCs.7,8 This has been confirmed by studies however, expression is extremely weak in WBCs, thymus, in hematopoietic stem cells.53 IGnT expression is extremely neural tissue, lung, and liver.40 The relative absence of low in both adult and cord CD34+ cells. On erythroid detectable RNA in WBCs is noteworthy: granulocytes and differentiation, a parallel increase in IGnTC and I antigen monocytes express primarily type 2 chain glycans on both expression is observed.53 Mutations in exons E1C, E2, and 46,47 7–10 glycoproteins and glycolipids. E3 have all been associated with the iadult RBC phenotype. Seven additional β3GnTs have been cloned and Although encoded by the same gene, IGnTA, IGnTB, characterized (Table 1). The literature regarding these and IGnTIGnTC share only 66 to 73 percent homology β3GnTs is a bit confusing as a result of duplicate because of sequence differences in exon 1, which may publication and name changes. All seven β3GnTs share account for differences in enzyme activity among the three significant homology with each other and are related to a enzyme isoforms.8 In enzyme assays, IGnTC has nearly larger family of β1,3 galactosyltransferases.34,44 β3GnT2 twofold higher activity than either the IGnTA or IGnTB (originally named β3GnT133 to distinguish it from iGnT), was isolated from human uterus and bladder.33,34,41 Like β3GnT1, β3GnT2 was able to initiate and elongate poly-N- acetyllactosaminoglycans on a variety of substrates and displays wide expression.33 Data from β3GnT2 knockout mice indicate a major role for β3GnT2 in polylactosamine synthesis on N-linked glycans.42,43 β3GnT2 appears to physically associate with β3GnT8,32 an elongating β3GnT specific for the β1,6 branch on tetraantennary N-glycans.44 In HL60 cells, myeloid differentiation is accompanied by a parallel increase in β3GnT8 mRNA and polylactosamine chain length.32 β3GnT8 is highly expressed in bone marrow, spleen, pancreas, and small intestine with lower level expression in other tissues.44 β3GnT5 is specific for initiating type 2 chain synthesis on glycolipids,35,36 which contributes significantly to Ii and sialo agglutinin expression on human RBCs. β3GnT5 is upregulated during myeloid differentiation,36 coincident with the synthesis of type 2 chain glycolipids with LeX, sLeX, and VIM activity (Fig. 1D).46,48 Polylactosamine synthesis on O-linked glycans and keratan is directed by β3GnT3, β3GnT6, C2GnT, and β3GnT7 (Fig. 1C, Table 1).34,49,50 β3GnT3 and β3GnT6 are related glycosyltransferases responsible for initiating core 2 and core 3 O-glycan synthesis and are highly expressed in gastrointestinal tissues (Fig. 1C).34,49 Core 3 O-glycans can be further modified by C2GnT (Fig. 1C), an I-like β1,6 branching enzyme located in the cis- Fig. 3 Structure of the IGnT gene. (A) Location of IGnT on 30,51 medial Golgi. β3GnT7 recognizes and elongates sulfated chromosome 6. (B) The structure of IGnT showing the arrangement LacNAc oligosaccharides found on keratan sulfate and is and size (bp) for exons E1A, E1B, E1C, E2, and E3. The size of highly expressed in placenta, colon, stomach, and small the intervening introns is also shown. (C) The composition of the intestine.50,52 three IGnT isoforms and their tissue distribution. Triangles denote mutations associated with iadult phenotype. Solid triangles affecting exons E2 and E3 result in i with congenital cataracts. (D) The The I Gene adult IGnTC promoter region showing transcription binding sites for I (IGnT, GCNT2) is located on chromosome 6p24.2 Oct 2, Sp1, and C/EBPa. Functional binding of C/EBPa protein (Fig. 3A).38 It consists of five exons (E1A, E1B, E1C, E2, E3), to IGnTC promoter requires altered phosphorylation of serine and which give rise to three related isoforms of the enzyme threonine residues.

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isoform.8 Exon E1 encodes nearly 77 percent of the active translation initiation site.53 As shown, the IGnTC promoter enzyme (Fig. 4), including the transmembrane domain, in erythroid cells contains consensus sequences for Oct-2, stem region, and part of the catalytic domain containing Sp1, and C/EBPα transcription factors (Fig. 3D).53 the nucleotide binding site. E2 and E3, which are shared by Although both Oct-2 and Sp1 bind the IGnTC promoter, all three isoforms, encode the carboxy-terminal end of the C/EBPα binding is critical for IGnTC transcriptional enzyme. activation.53 How C/EBPα regulates IGnTC is complex, apparently involving posttranslational phosphorylation of Transcriptional Control The identification of three tissue-specific IGnT/GCNT2 C/EBPα protein. In K562 erythroleukemia cells, butyrate isoforms strongly suggests transcriptional regulation of induces changes in C/EBPα phosphorylation, a prerequisite exon 1 by cis-regulatory sequences in the 5′ untranslated for functional C/EBPα binding to the IGnTC promoter (Fig. region (5′UTR) or promoter region. This possibility is 3D). Once bound, the phosphorylated-C/EBPα drives IGnTC also supported by the structure of IGnT, in which exons transcription irrespective of Oct-2 and Sp1 binding. Similar E1A, E1B, and E1C are each separated by relatively large results can be observed during in vitro differentiation intronic sequences that may each harbor binding sites for of adult and cord stem cells.53 Loss of phosphoserine tissue-specific transcription factors.7,8 This was recently residues may be critical for C/EBPα-mediated transcription confirmed by studies in K562 cells, which identified a activation: serine phosphorylation has been shown to promoter sequence –318 to –251 base pairs upstream of the repress C/EBPα during early hematopoiesis.54

Fig. 4 Structure-function of IGnT. The IGnTC isoform, expressed in reticulocytes, is a 402–amino acid glycoprotein protein, possessing up to five N-glycosylation sites and nine conserved cysteine residues. Exon 1, which shows only 65 percent homology among the three IGnT isoforms, encodes nearly 76 percent of the enzyme. Like all glycosyltransferases, the molecule contains a short 19-amino acid hydrophobic transmembrane region (TM domain) that anchors the molecule within the Golgi membrane, a stem region, and a large catalytic domain. IGnT shares three regions of significant homology with C2GnT, a related ß-1,6-N-acetylglucosaminyltransferase ( ) whose three-dimensional structure has been characterized. Using C2GnT as a template, the location of four potential disulfide bonds (- - -), nucleotide-binding site, and acceptor-binding sites were extrapolated. IGnTC and C2GnT share significant homology in ß1, ß2, ß3, and ß4 strands that incorporate the UDP or nucleotide donor–binding site. Of six nucleotides critical for acceptor binding by C2GnT (E243, K251, R254, E320, W356, Y358; ⇓, bold), only two homologous sequences were identified in IGnT (E298, W328). Conserved sequences, including a lysine, along the ß7 strand may play a role in binding phosphate of the UDP donor.

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IGnT (GCNT2) Protein Structure-Function four β strands were localized to a 126–amino acid stretch The translated protein varies from 400 (IGnTB) to 402 in exon 1 that shares significant homology with C2GnT. amino acids (IGnTA, IGnTC).7,38 Like all glycosyltransferases, Less conservation was observed in the acceptor binding the enzyme is a type 2 transmembrane glycoprotein domain. IGnT contained glutamic acids (E298, E367) and possessing several potential N-glycosylation sites and a tryptophan (W328) corresponding to E320, E401, and 16– to 19–amino acid hydrophobic transmembrane domain W356 residues in C2GNT; however, no clear homologs near the amino terminus (Fig. 4).38 It is presumed to for E243, K251, and K254 were identified. Interestingly, possess significant tertiary structure based on exhibiting E243, K251, and K254 reside downstream of the C2GNT β5 nine conserved cysteine residues.38,55 strand, a region that shares minimal sequence homology IGnT shares sequence homology with C2GnT, a related between IGnT and C2GnT genes. β1-6 N-acetylglucosaminyltransferase that transfers UDP- GlcNAc to Galβ1-3GalNAc-O-Ser/Thr, transforming simple iadult Phenotype and Cataracts core 1 and core 3 O-glycans to branched, core 2 and core The iadult phenotype is a rare, autosomal-recessive 4 O-glycans, respectively (Fig. 1C).55,56 Mucin-type C2GnT phenotype caused by a mutation in the IGnT/GCNT2 gene. (C2GnT-M) also displays IGnT-like activity with synthesis The linkage of iadult with congenital cataracts was originally 12 of I antigen on N-glycans.56 A comparison of IGnT and reported by Ogata et al. and primarily limited to Asian kindreds. The etiology behind both i and congenital C2GnT reveals three regions sharing approximately adult 60 percent homology: a large 126–amino acid stretch cataracts was finally resolved after mapping of the IGnT incorporating the putative UDP-GlcNAc binding site and gene and identification of three distinct IGnT isoforms (Fig. 3).7–10 Mutations in exon E1C, which is the predominant two smaller regions (32 and 22 amino acids) in the carboxy- isoform expressed in erythroblasts and reticulocytes, leads terminal acceptor binding domain of the enzyme.57,58 to an i RBC phenotype without congenital cataracts. Unlike most glycosyltransferases identified to date, neither adult Although IGnT activity is missing in RBCs, IGnT activity is IGnT nor C2GnT possesses a DXD motif upstream of the intact in lens and other tissues via IGnTA and IGnTB (Fig. UDP-GlcNAc binding site.57 DXD motifs help coordinate 3C). In contrast, mutations in exons E2 and E3, which are interactions of the enzyme with the phosphate group of the common to all three IGnT isoforms, result in a tissue-wide nucleotide donor (UDP) and a divalent cation such as Mn2+ loss of IGnT activity including RBCs and lens. Mutations in or Mg2+.57 E2 and E3 are responsible for i with congenital cataracts. The x-ray crystallography structure of mouse C2GnT adult A summary of the specific mutations and phenotypes is was recently determined and identified C2GnT as a novel listed in Table 2. metal ion–independent GT-A glycosyltransferase.58 Like An I+i+ phenotype might occur in some heterozygous most GT-A type glycosyltransferases, C2GnT has an α/β/α carriers of mutant IGnT alleles. Early family studies of structure with a mixed β-sheet containing seven topological i individuals reported decreased I expression among β strands. Strands β1 through β4 bind the nucleotide adult many first-degree family members.2,11 Although these (UDP) of the UDP-GlcNAc donor: acceptor binding occurs relatives still typed as I+, titration scores with anti-I and downstream of the UDP site and involves amino acids -i sera were intermediate between those of normal and i located within the β5, β6, α5, and β7 regions (Fig. 4). adult RBCs, resulting in an “Iwi+-like” phenotype. Examples of Critical amino acids necessary for acceptor binding include haploinsufficiency and weak antigen expression have been glutamic acid (E243, E320), lysine (K251), tryptophan observed with other glycosyltransferases, including the (W256), and tyrosine (Y258). E320 corresponds to a related C2GnT.59,60 Heterozygosity for a single missense structurally conserved catalytic base required for activity mutation in C2GnT (S158C) results in a marked decrease in in GT-A glycosyltransferases. E243, E320, and Y258 have core 2 O-glycans on T cells, accompanied by resistance to  direct interactions with the Galβ1 3GalNAc acceptor, galectin-mediated cell apoptosis.59 whereas K251 helps stabilize a stacking interaction between 7–10 the acceptor and W256. Two additional basic amino acids, Table 2. IGnT mutations associated with iadult phenotype arginine 378 (R378) and lysine 401 (K401), are structurally Mutation Phenotype aligned to interact with UDP-phosphate residues, possibly Exon Nucleotide Amino acid i RBC Cataracts substituting for the DXD motif and divalent cation. C2GnT adult also contains four disulfide bonds. E1C 243T>A N81L + – Using the C2GnT sequence and crystallography data E1C 505G>A A169T + – as a template, a potential structure-function model of E1C 683G>A R228Q + – IGnTC enzyme was constructed (Fig. 4). Like C2GnT, IGnT E2 983G>A W328X + + possesses nine, positionally conserved cysteine residues E2 1005G>A G336R + + and is therefore capable of four disulfide bonds. IGnTC E3 1049G>A G350E + + also possesses homologous sequences for β1, β2, β3, and β4 strands implicated in UDP binding. Not surprisingly, all E3 1154G>A R385H + +

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Antibodies to Ii Anti-i Anti-I Naturally occurring and monoclonal antibodies Naturally occurring antibodies against I are common (MAbs) against i are relatively uncommon. As described 61 2 and have even been reported in cord blood samples. Anti-I by Marsh, anti-i reacts strongly with cord and iadult but increases progressively during childhood, with peak titers not adult RBCs. The minimal epitopes necessary for anti-i around 10 years of age.62 In adults, titers are generally binding are two repeating LacNAc motifs (Figs. 1A, 5).23 low, even at 4°C (<64). Titers can increase in the setting Like anti-I, different anti-i clones may recognize slightly of infection, particularly after infection with Mycoplasma different antigens based on the number of antigen sites per 23 pneumoniae or Streptococcus pneumonia or with rubella. RBC, particularly when cord and iadult RBCs are compared By definition, anti-I reacts strongly with adult RBCs (Table 3). 4–6 Anti-i is sensitive to endo-β-galactosidase (Fig. but not cord or iadult RBCs. Some examples of anti-I can 1) but can be enhanced by protease and neuraminidase display complex reactivity, reacting more strongly with digestion.23,26 Bombay cells do not display enhanced 18 26 RBCs of specific ABO or P1 types. Adult Bombay (Oh) reactivity with anti-i. cells, which lack ABH, have the strongest reactivity with Infectious mononucleosis is commonly associated anti-I.63,64 Anti-I reactivity can be enhanced by protease with increased anti-i titers and can occasionally result and neuraminidase but is resistant to endo-β-galactosidase in immune-mediated hemolysis.18 Lymphoid cell lines treatment.26 The amount of antibody binding sufficient derived from Burkitt’s lymphoma patients, and after to induce hemagglutination is clone- and temperature- EBV transformation in vitro, can produce MAbs with dependent. At 4°C, as few as 50 to 100 molecules per RBC apparent anti-i specificity.68–71 When tested against specific are required for hemagglutination but that number can glycolipids, some of these “anti-i” were sialo agglutinins, increase to more than 10,000 per RBC at 37°C.5 reactive with sialylated linear type 2 chain structures (see The exact epitope recognized by anti-I is clone- later section).68 specific, although nearly all clones examined recognize a GlcNAcβ16Gal as part of the epitope.23 Feizi et al.23,24 Anti-Ii were able to identify at least three distinct epitopes by Monoclonal antibodies strongly reactive with both testing a series of human monoclonal anti-I against a panel adult and cord cells have been described (Fig. 5). Some of oligosaccharides (Fig. 5). However, even among clones investigators have referred to these antibodies as “anti-j” sharing a common binding pattern, subtle differences (because j is next in the alphabet).72 Roelcke et al.72 described were evident.24 The heterogeneity of binding epitopes, two cases (ZI, BR) that reacted equally well with adult and and therefore physiologic receptors, is also inferred by the cord cells. Hemagglutination was reduced or inhibited by range in antigen binding sites when different anti-I clones linear and branched type 2 chain oligosaccharides, as well are tested (Table 3).4–6 Anti-I displaying both I and ABH as endo-β-galactosidase.72 Dube et al.73 also investigated activity (e.g., anti-HI) recognize a branched epitope bearing a monoclonal cold agglutinin with I and i reactivity. both LacNAc and ABH epitopes (Fig. 5).65–67 Loss of the ABH Inhibition studies indicated that the active epitope included epitope is associated with loss of antibody reactivity.23,66,67 both GlcNAcβ16Gal and linear, i-active sequences.

Table 3. Difference in antigen sites recognized by monoclonal anti-I/i 4–6

Antigen sites per RBC Adult RBC

† Specificity Name* Untreated Protease iadult Cord Anti-I Kau 30,000 ± 10,000 90,000 ± 20,000 ND ND Fla 130,000 ± 10,000 ND <1000 ND Loi-1‡ 115,000 ± 10,000 ND <1000 ND Loi-2‡ 50,000 ± 5,000 ND <1000 ND AJ 32,000 ND ND ND Anti-i Fou <1000 ND 30,000 ± 5000 55,000 ± 10,000 Min 0 ND 60,000 ± 10,000 25,000 ± 5000 MP 30,000 ND 110,000 75,000 Anti-Ii Abg 100,000 ± 20,000 ND 45,000 ± 10,000 40,000 ± 5000 Anti-A 0.8–1.7 × 106 250–370,000 ND = not done. *Name of human monoclonal antibody. †O adult RBCs digested with papain. ‡Two different antibody binding sites and densities were observed with the Loi antibody.4

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Fig. 5 Epitopes for examples of anti-Ii. Anti-i requires at least two LacNAc residues. Several different epitopes have been identified among anti-I (lines), although all include a GlcNAcß1→6Gal epitope. Examples of anti-Ii with HI and sialo agglutinin activity are also shown. Cer = ceramide; Fuc = fucose; Gal = galactose; GlcNAc = N-acetylglucosamine; NeuAc = neuraminic acid.

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Sialo Agglutinins Sialo agglutinins represent a subgroup of I/i antibodies that require a terminal sialic acid for activity. They are distinguished from anti-I and anti-i by their sensitivity to neuraminidase. Dieter Roelcke26 broadly subclassified the sialo agglutinins into two major classes, based on whether the antibodies recognize linear (Sia-l1) or branched (Sia-b1) sialooligosaccharides. Like anti-I, Sial-1b includes antibodies against branched structures with ABH activity (anti-FL).67 Sialo agglutinins with Sia-1b reactivity have been linked with both CMV and Mycoplasma infections.74,75 A third category of sialo agglutinins are Sia-lb1 and Sia-lb2, which recognize sialylated glycosphingolipids or gangliosides (Gd, or glycolipid-dependent).26 Sia-lb1,2 bind linear and branched type 2 chain gangliosides and are resistant to protease digestion. Inhibition studies indicate the minimal epitope for Sia-lb1,2 is the terminal mono-disaccharide incorporating sialic acid with or without galactose (Fig. 5). Not surprisingly, some Sia-1b1,2 antibodies also bind ganglioside GM3 (sialyl-lactose) and sialylparagloboside.26 As discussed earlier, anti-i produced by EBV-transformed human lymphocytes may recognize linear type 2 gangliosides.68,69 Sialylated lactosamines have been shown to be receptors for M. pneumoniae.76 Mycoplasma has been shown to bind sialyl-polylactosamines along the apical aspect of ciliated bronchial epithelium and microvilli.77 The latter could serve as a trigger for autoantibody formation and may contribute to the prevalence of sialo-I (Sia-1b) after Mycoplasma Fig. 6 Structure-function of anti-Ii antibodies. (A) A schematic of 74,78 human IgM showing five antigen binding sites per molecule. (B) infection. The structure of an IgM heavy chain and light chain pair showing constant (C , C ) and variable region (V , V ) gene segments. The Antibody Structure-Function H L H L VH and VL regions encode the antigen binding site. The hinge region Cold Agglutinin Disease along CH2 is a site for F(ab´)2 autoantibodies. (C) Autoantibodies MAbs from patients with cold agglutinin disease have against Ii tend to use VH gene segments from specific families. been invaluable in dissecting the pathologic autoimmune Polyclonal benign anti-Ii tend to use VH gene segments from the VH3 and V 4 families, whereas the V 4-34 gene segment (historically response to Ii. Multiple independent investigators have H H V 4-21) accounts for the majority of monoclonal cold agglutinins. confirmed that IgM binding to Ii is primarily mediated by H VH4-34 is also common among IgM anti-Rh monoclonal sera. (D) the IgM heavy chain via the VH or variable heavy domain. The VH gene segment consists of four framework regions (FR) and Of the 123 to 129 heavy chain VH genes available, the VH4-34 three complementarity determining regions (CDR). For VH4-34, FR1 is required for Ii binding. Sequence variation in CDR 3 may gene segment (VH4-21 or VH4.21 in older papers) is almost H exclusively used by monoclonal cold agglutinins with Ii contribute to the fine specificity of the antibody (I or i). MAb 9G4 79 antibody recognizes an epitope in FR1 of VH4-34. autoreactivity (Fig. 6). VH4-34 is also overrepresented in many, but not all, B-cell lymphoproliferative disorders including follicular lymphoma, mantle cell lymphoma, and 80 determined to be important in antigen binding. As shown Waldenström’s macroglobulinemia. In contrast, VH4-34 is unusual in chronic lymphocytic leukemia.80,81 In normal in Figure 6D, three hypervariable or complementarity determining regions (CDR) are flanked by so-called individuals, VH4-34 is identified in 3 to 6 percent of adult and fetal B cells but constitutes less than 0.5 percent of framework regions (FR). Among VH4-34 monoclonal the total circulating IgM and IgG.80 Antibodies bearing cold agglutinins, binding to either I or i is mediated by the VH4-34 gene segment can be identified with the anti- the N-terminal framework region 1 (FR1). This has been idiotypic antibody MAb 9G4.79,82 verified by recombinant IgH clones using an FR1 from 79 Detailed sequence analysis, coupled with construction a different VH4 subgroup. The 9G4 epitope is located in of recombinant immunoglobulins, has uncovered the basis FR1 (amino acids 23–25).79 Although MAb 9G4 can inhibit 70 of Ii binding by VH4-34. Although only 110 to 120 amino RBC agglutination, the 9G4-binding site lies outside the 83 acids in length, the VH region has distinct subdomains antigen-binding site. It is theorized MAb 9G4 may inhibit

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binding by steric hindrance or induced conformational in the peritoneal cavity. Infection of mice with a murine change.83 Mycoplasma strain resulted in B-cell activation and loss Recombinant IgH clones also revealed a role for CDR of tolerance with markedly increased cold agglutinin domains, mutational “hot spots” known to direct antigen synthesis, room temperature hemagglutination, and binding and specificity. Of three CDR domains, evidence increased reticulocytosis.88 suggests that CDRH3, in concert with light chains, may Cross-reactivity and molecular mimicry may also define the fine specificity (anti-I or anti-i) of the antibody.79 contribute to a rise in cold agglutinins after infection. The

The ability of CDRH3 to distinguish I from i, however, is not polysaccharide of S. pneumoniae is a linear trisaccharide mediated by a specific sequence motif: individual B-cell sequence of Gal, GlcNAc, and Glc, with short, β1,4-linked 79,82 clones show significant sequence variability in CDRH3. Gal side chains that is similar to the I and i epitopes (Fig. Crystallography studies of a human monoclonal anti-I 7).23 Lactosamine epitopes are also expressed on the show that CDRH3 and two light chain variable regions lipooligosaccharide (LOS) of some Neisseria gonococcus (VκCDRL1 and CDRL3) line the antigen-binding pocket of and meningococcus strains and can stimulate antibodies 83 the antibody. capable of cross-reacting with Ii glycolipids on RBCs The role of light chains in Ii specificity is less clear- (Fig. 7).89 As discussed earlier, M. pneumoniae binding to cut. There is a preference for Vκ, particularly Vκ3, among branched sialo agglutinins on bronchial epithelium may 82 examples of human monoclonal anti-I. Kappa light chain stimulate cross-reactive antibodies.77,78 restriction is also observed among monoclonal anti-Pr cold sialo agglutinins, with V 4 and V 3 accounting for 61 percent κ κ Monoclonal IgM Rh and Other Antibodies of clones.84 Interestingly, V 4 was linked to recognition κ Not surprisingly, other IgM MAbs using the V 4-34 of sialo agglutinins bearing an α23 neuraminic acid.84 H gene segment often display anti-i activity.90,91 This has been Monoclonal anti-i uses both V and V , with higher V κ λ λ well documented with several IgM anti-D MAbs. Thorpe et usage relative to polyclonal antibodies.82 V usage is also λ al.90 showed that 59 percent of all IgM anti-D MAbs carried relatively common in other autoimmune diseases, including the V 4-34 gene segment.90 Of these, 84 to 94 percent antibodies against dsDNA, phospholipid, rheumatoid H demonstrated anti-i activity when tested against papain- factor, and histone.85 treated RBCs at 4°C.90 Similar “cold-agglutinin” activity has been demonstrated with other V 4-34–derived MAbs Infection and Naturally Occurring Autoantibodies H against endotoxin lipid A, DNA, and IgG (rheumatoid Unlike monoclonal anti-I/i, polyclonal anti-I/i does factor). Sequence analysis suggests that the apparent I/i not display light or heavy chain restriction. Early studies reactivity of many V 4-34 anti-Rh MAbs is, in fact, charge- using the 9G4 MAb showed that only a fraction of naturally H dependent.91 Most clones display a strong net positive occurring polyclonal anti-Ii carried the V 4-34 gene H charge at pH 7.5 and will react with RBCs, vimentin, and segment.82,86 This was also supported by the inability of even cell nuclei at neutral pH and low ionic strength. MAb 9G4 to inhibit hemagglutination with polyclonal anti- 86 I/i. Naturally occurring anti-Ii uses a mix of VH segments 86 from the VH3 and VH4 families. VH3 and VH4 family genes account for 65 percent of the total B-cell repertoire and nearly 78.7 percent of all circulating immunoglobulin.85

Infection may increase the concentration of VH4-34 antibody.70 In patients with infectious mononucleosis and

M. pneumoniae, there was an increase in VH4-34 antibody in the majority of patients. Antibody binding to RBCs was directly associated with total circulating VH4-34 antibody levels. When individual B-cell clones from infected patients were examined, only clones using the VH4-34 gene segment secreted anti-i IgM capable of RBC hemagglutination.70 There was no association between hemagglutination and light chain (κ, λ) usage. The ability of infection to stimulate anti-I/i has been studied in a transgenic mouse model of cold agglutinin Fig. 7 Structure of the oligosaccharides on Streptococcus disease carrying the sequence for a human sialo agglutinin pneumoniae23 and Neisseria species89 (N. gonorrhea and N. (Sia-1b).87,88 Normally, transgenic mice did not demonstrate meningitidis). Note that S. pneumoniae, a linear repeating trisaccharide, has cross-reactive motifs with structural autoimmune hemolytic anemia attributable to a depletion of similarity to i and I.23 Gal = galactose; Glc = glucose; GlcNAc “autoreactive” B cells in the bone marrow and periphery.87 = N-acetylglucosamine; Hep = heptose; KDO = 2-keto-3- Low-level anti-Siab1 was only produced by rare B cells deoxyoctulosonic acid.

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Regulatory Autoantibodies to Cold Agglutinins the B cells of a patient with metastatic adenocarcinoma Cold autoantibodies may be autoregulated by naturally of the lung.99 The antibody strongly and preferentially occurring anti-F(ab′)2 IgG antibodies directed against a agglutinated cord RBCs at 4°C with no observable short peptide epitope in the hinge-region.92,93 The Roelke agglutination at 37°C. Typical of anti-I, epitope mapping laboratory demonstrated an inverse correlation between confirmed that the antibody was specific for a linear anti-F(ab′)2/hinge antibodies and monoclonal cold oligosaccharide containing at least two LacNAc residues. agglutinin titers. There was no correlation with hinge The antibody would tolerate terminal substitutions, binding antibodies and polyclonal cold agglutinins. It is sialo-i and long-chain, linear A-active glycolipids (nor-Ac). hypothesized that these hinge antibodies may suppress When tested against human tissues, NCC-1004 recognized B-cell and autoantibody production. The relatively poor carcinoma of the thyroid, esophagus, lung, embryonal response of cold agglutinin disease to IVIG suggests that carcinoma, basal cell epithelium, mantle cell lymphocytes, any downregulation by circulating hinge antibodies is and endothelium. moderate at best. MAb MH21-134 is a human monoclonal IgG3 antibody derived from a patient with squamous cell carcinoma Reagents Against Lactosamine and Related of the lung.100 The antibody specifically recognized Epitopes glycolipids possessing a linear, polylactosamine structure. Monoclonal Antibodies Unlike NCC-1004, MH21-134 will not bind sialylated i. Anti-Ii In hemagglutination assays, MH21-134 will only weakly MAbs IB2, FeA5, and H11 are three murine IgM MAbs agglutinate neuraminidase-treated cord cells in the that recognize unmodified, terminal lactosamine residues antiglobulin test: no agglutination was observed with on either linear or branched, I-active structures (Table 4).94– unmodified cord and adult RBCs by immediate-spin. 96 MAbs H11 and 1B2 were raised against paragloboside Among normal tissues, MH21-134 only reacted with and i-active glycolipids, whereas FeA5 was stimulated granulocytes and bronchial and pancreatic epithelial against murine testicular germ cells. In hemagglutination cells. MH21-134 reacted with several gastrointestinal tract assays, MAbs FeA5 and 1B2 behave as an anti-I with malignancies. little or no reactivity against unmodified cord cells. Both MAbs will strongly agglutinate adult and cord cells after Anti-I neuraminidase digestion.94,97 No hemagglutination data are Three anti-I murine MAbs are reported in the literature; available for MAb H11.116 MAb 1B2 is available as cells from Fe-C6,95 M18.3, and M39.6.101 Initially identified as a stage- the American Type Culture Collection (ATCC, Manassas, specific antigen on murine embryos, MAb Fe-C6 was VA). MAb FeA5 is available as a hybridoma supernatant subsequently shown to recognize a binary structure, requiring from the Developmental Studies Hybridoma Bank held at both an intact β1-3 and β1-6 terminal lactosamine epitope as the University of Iowa. shown in Table 4. Its reactivity is similar to the human MAbs In addition to these antibodies, two MAbs developed Phi, Da, Sch, and Low (Fig. 5).24,95 MAb Fe-C6 is available as a against Neisseria gonococcus and meningococcus LOS hybridoma supernatant from the University of Iowa. also have lactosamine activity (Fig. 7).89 MAbs 3F11 and MAbs M18.3 and M39.6 were raised against 06B4 recognize a paragloboside epitope expressed on human milk globule membranes as potential immune most gonococcal LOS. In addition to paragloboside, markers for breast tissue.101 Epitope mapping indicates both antibodies will recognize linear and branched that MAbs M18.3 and M39.6 primarily recognize the polylactosamine epitopes on RBC glycolipids. MAbs 3F11 Galβ14GlcNAcβ13GlcNAc epitope, with evidence of and 06B4 preferentially agglutinate adult RBCs at 4°C. β16 branching. Its reactivity is reminiscent of the human Hemagglutination with both adult and cord RBCs can be Step, Gra, Ver, and Ful antibodies (Fig. 5).24,95 MAbs 18.3 observed after neuraminidase or trypsin digestion. and M39.6 recognize breast epithelial cells: M39.6 may also We have successfully used both 1B2 and FeA5 against recognize dendritic cells. In breast cancer, M18.3 staining glycoproteins and glycolipids by Western blotting, high- is masked by increased sialylation.102 performance thin-layer chromatography (HPTLC), and flow cytometry.48,98 Although these antibodies are specific Anti-N-acetylglucosamine for Galβ14GlcNAcβ-R termini, they can be used to MAb J1 was developed against murine testicular cells identify sialylated polylactosamines when coupled with and recognizes a stage-specific differentiation antigen neuraminidase.98 expressed on high molecular weight polylactosaminyl- glycans on sperm.103 Using a panel of glycolipids, J1 was Anti-i shown to recognize terminal anti-N-acetylglucosamine Two MAbs with anti-i specificity have been isolated by (GlcNAc) residues on growing type 2 chain oligosaccharides heterohybridization of human B cells with murine myeloma (GlcNAcβ13/6Gal). J1 preferentially binds long-chain cells.99,100 MAb NCC-1004 is a human IgMλ produced from and multivalent epitopes. We have tested MAb J1 against

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Table 4. Anti-lactosamine reagents reactive with Ii-like structure

Monoclonal antibodies89,94–105 Clone Isotype/Species Epitope Reactivity Comments IB2 IgM, mouse Galß1-4GlcNAc-R I or i FC, Western, HPTLC FeA5 IgM, mouse Galß1-4GlcNAc-R I or i FC, Western, HPTLC H11 IgM, mouse Galß1-4GlcNAc-R I or i HPTLC, RIA 3F11 IgM, mouse Galß1-4GlcNAc-R I or i HPTLC, Western/LOS 06B4 IgM, mouse Galß1-4GlcNAc-R I or i HTPLC, Western/LOS

NCC-1004 IgMλ, human R-(Galß1-4GlcNAcß1-3)n≥2 -R i, si HA, HC, HPTLC, RIA

MH21-134 IgG3, human (Galß1-4GlcNAcß1-3)n≥2 –R i HPTLC, HC Fe-C6 IgM, mouse Galß1-4GlcNAcß1-3Gal I HPTLC, Western, RIA 〉 Galß1-4GlcNAcß1-R Galß1-4GlcNAcß1-6Gal M18.3 IgM, mouse Galß1-4GlcNAcß1-6Gal-R I HA, HPTLC, HC M39.6 IgM, mouse Galß1-4GlcNAcß1-6Gal-R I HA, HPTLC, HC J1 IgM, mouse GlcNAcß1-R I or i HPTLC TE4 IgM, mouse GlcNAcß1-3Gal-R I or i HPTLC, RIA TE5 IgM, mouse GlcNAcß1-3Gal-R > GlcNAcß1-2/6Man i HPTLC, RIA

TE6 IgG3, mouse GlcNAcß1-2/6Man > GlcNAcß1-3Gal-R, linear I HPTLC, RIA TE7 IgM, mouse GlcNAcß1-3Galß1-4GlcNacß1-3Gal, linear i>>I HPTLC, RIA Lectins106–119

Lycopersicon esculentum (LEA) R-(3Galß1-4GlcNAcß1-)n≥3 I or i O >> A,B Erythrina cristagalli (ECA) Galß1-4GlcNAcß1-R I or i

Erythrina corallodendron (ECL) (Fucα1-2) ± Galß1-4GlcNAcß1-R I + IH O > A/B > Ocord >> O h

Aplysia depilans (ACL) unknown I Oh > adult >> OcordI Maackia amurensis (MAA) NeuAcα2-3Galß1-4GlcNAc-R sialyl-Ii Viscum album (VAA) NeuAcα2-6Galß1-4GlcNAc-R sialyl-Ii Polyporus squamosus (PSL) NeuAcα2-6Galß1-4GlcNAc-R sialyl-Ii Trichosanthes japonica (TJA-1) NeuAcα2-6Galß1-4GlcNAc/Glc sialyl-Ii Sambucus nigra (SNA) NeuAcα2-6Galß1-4GlcNAc-R, sialyl-Ii, Tn NeuAcα2-3GalNAc-O-R FC = flow cytometry; GP = glycoprotein; HA = hemagglutination; HPTLC = high-performance thin-layer chromatography; LOS = bacterial lipooligosaccharide; RIA = radioimmunoassay. neutrophil glycolipids, which express lactotriaosylceramide structures. All four antibodies will react with glycoproteins (Lc3; Fig. 2B) as well as longer type 2 chain intermediates. and glycolipids. MAb TE5 has been very useful to identify Although Lc3 reportedly binds J1 by ELISA, we could not Lc3, Lc5, and other intermediate structures in tumor lines demonstrate J1 binding to isolated Lc3, Lc5, or any other and human leukemia.46,104,105 MAbs TE4, TE5, and TE7 are neutrophil glycolipids by HPTLC (L. Cooling, unpublished murine IgM: MAb TE6 is an IgG3. observations). MAb J1 is available as a hybridoma supernatant from the University of Iowa. Lectins Holmes et al.104 and Hu et al.105 have also produced Tomato Lectin a panel of murine MAbs with GlcNAcβ1-R activity using The tomato lectin, Lycopersicon esculentum (LEA) the pentaosylceramide Lc5 as an immunogen (Fig. can hemagglutinate group O RBCs and all neuraminidase- 2B). TE4 and TE7 have disaccharide and trisaccharide treated RBCs, regardless of ABO group. Hemagglutination epitopes, respectively, on the termini of type 2 chain is reportedly inhibited by oligomers containing β-linked oligosaccharides (Table 4). MAbs TE5 and TE6 recognize N-acetylglucosamine.39 The lectin has proved quite useful terminal βGlcNAc on the growing chains of type 2 chain in the isolation and characterization of glycoproteins, where oligosaccharides and, possibly, GlcNAcβ1Man structures it preferentially binds long-chain linear polylactosamines on N-glycan intermediates. All four antibodies recognize containing at least three or more repeating lactosamine linear type 2 chain oligosaccharides, whereas only MAb residues.39 It has also been used to detect increased TE4 shows strong reactivity to branched lactosaminyl lactosamine on cells by flow cytometry.33,43

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Erythrina cristagalli to the midwest United States and Northeast China. The Erythrina cristagalli (ECA) is a lactosamine- lectin has sialo agglutinin activity, recognizing a terminal specific lectin from the seeds of the South American coral NeuAcα23 lactosamine.111,112 It has been used successfully bean tree. It will agglutinate RBCs, regardless of ABO for the isolation of glycoproteins by immunoprecipitation group. Agglutination is enhanced when tested against and lectin affinity chromatography. It has also been trypsin- or neuraminidase-treated RBCs.106 By thin-layer used to identify sialylated polylactosamines by Western chromatography, ECA specifically recognizes glycolipids with blotting, thin-layer chromatography, and flow cytometry. terminal, unsubstituted LacNAc epitopes.107 Among I-active Of note, MAA does not bind glycophorin A and only weakly glycolipids, ECA will not bind sialo lactosamines, even if agglutinates human RBCs.111 We have used fluorescein- one terminal branch bears a bare, nonsialylated lactosamine labeled MAA with ficin-treated RBCs when testing by flow terminus. Like E. corallodendron lectin (later), ECA can cytometry.113 also bind type 2 chain H structures, although ECA does not display obvious HI activity when tested against RBCs.107 Viscum album Viscum album agglutinin (VAA) or mistletoe lectin Erythrina corallodendron recognizes sialylated polylactosamines bearing a terminal Erythrina corallodendron (EcorL) is a galactophilic α26 neuraminic acid.114 VAA has been used for Western lectin isolated from the seeds of the West Indian coral tree. blotting, thin-layer chromatography, and tissue culture. In hemagglutination assays, the lectin displays both I and HI VAA strongly binds granulocytes and lymphocytes and activity. EcorL strongly agglutinates adult O RBCs, with weaker has been shown to enhance phagocytic activity, stimulate 64 activity against group O cord, iadult, A, and B RBCs. EcorL cytokine secretion, and increase natural killer cells. VAA has little or no activity when tested against AB and Bombay is a potent cytotoxin and has been studied as an adjuvant 64 114 (Oh) cells. The absence of agglutination with Bombay cells is agent in cancer trials. a conundrum because Bombay cells should have the highest expression of lactosamine and sialo lactosamine glycans. Polyporus squamosus The I and HI-like activity have been confirmed by PSL agglutinin is a product of the polypore mushroom, structural studies using well-defined oligosaccharide Polyporus squamosus, and is similar in activity to VAA.115– receptors, x-ray crystallography, and recombinant lectin 117 Unlike SNA lectin (later), PSL is specific for sialylated mutants.107–109 Like ECL, EcorL will bind terminal LacNAc lactosamines expressed on N-linked glycans: PSL will epitopes on paragloboside, type 2 chain H, linear and not bind Tn-like epitopes on O-linked glycans.115,116 In branched polylactosamine glycolipids. Fucose, however, hemagglutination assays, PSL agglutinated formalin- is the only terminal modification accepted: EcorL will not fixed human and rabbit RBCs, irrespective of blood recognize type 2 chain structures with a terminal sialic type.115 Hemagglutination was specifically inhibited by 107 acid, Galα14 (P1 antigen), A or B epitope. In solid phase the trisaccharide NeuAcα26Galβ14GlcNAc, but not assays, EcorL binds the H-active glycolipid with higher N-acetyllactosamine or its α23 sialo-derivative.116 PSL has affinity than paragloboside; however, no comparative been used for immunohistochemistry, Western blotting, binding studies were performed with i- or I-active, and thin-layer chromatography.115–117 polylactosaminyl glycolipids.108 Crystallography indicates that LacNAc, not fucose, is the active ligand.109 Sambucus nigra Sambucus nigra agglutinin (SNA) is derived from Aplysia Gonad Lectin the bark of the elderberry tree. The lectin also recognizes Aplysia gonad lectin (AGL) is a galactophilic lectin from α26 sialylated glycoconjugates, but has a broader the gonads of Aplysia depilans, a fairly large sea slug found acceptor profile than PSL or VAA.116,118 SNA readily in the northeast Atlantic and Mediterranean Sea. In early precipitates both glycophorin A and human erythroglycan, studies, AGL was shown to agglutinate RBCs of humans indicating an ability to recognize NeuAcα26GalNAc and animals, particularly after papain digestion.110 A more and NeuAcα26Gal epitopes on O- and N-linked glycans, recent study compared AGL, EcorL and human anti-I against respectively.117–119 In hemagglutination assays, SNA 64 adult, cord, and Bombay (Oh) RBCs. AGL reactivity was agglutinates human RBCs regardless of ABO group or similar to human anti-I, preferentially binding adult RBCs. enzyme modification. SNA has been used for glycoprotein The strongest agglutination was observed with Bombay isolation, Western blotting, HPTLC-immunostaining, RBCs. Unfortunately, no information is available regarding immunohistochemistry, and flow cytometry.112,113,116,117,119 the reactivity of AGL with neuraminidase-treated RBCs. Trichosanthes japonica Maackia amurensis TJA-1 is one of two lectins isolated from the roots of Maackia amurensis (MAA) is isolated from the seed the snakegourd or Chinese cucumber plant, Trichosanthes of the Amur maackia tree, a short, hardy tree common japonica. TJA-II is specific for H-like epitopes, whereas

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TJA-1 preferentially recognizes sialolactosamines.119 Like of the murine IGnT gene. As discussed earlier, mutations PSL and VAA, TJA-1 binds N-glycans bearing a terminal affecting exons E2 or E3 are associated with i phenotype NeuAcα26Galβ14GlcNAc/Glc epitope. It has been used and cataracts in humans (Table 2). for the isolation and characterization of glycoproteins.119 Contrary to expectations, IGnT-deficient mice had normal fertility and development, despite a decrease in Biologic Role embryoglycan and laminin adhesion on embryonic stem Hemolytic Disease of the Fetus and Newborn cells.123,124 More surprisingly, there was no increase in either The delay in I synthesis until several months of age may the onset or incidence of cataracts in IGnT-deficient mice.123 play a protective role against hemolytic disease of the fetus IGnT-deficient mice did display subtle abnormalities, and newborn (HDFN) caused by ABO incompatibility. Major including a decrease in B cells, increased epidermoid maternal-fetal ABO incompatibility is relatively common, cyst formation, and mild renal dysfunction, as evidenced with up to 30 percent of infants demonstrating a positive by elevated blood urea nitrogen and creatinine and DAT at birth.18 Nonetheless, the incidence of severe HDFN increased renal tubular vacuolization. The latter appears requiring exchange transfusion is quite rare (12 of 29,200 to represent an accumulation of autophagocytic vacuoles or 0.04%).18 The infrequency of clinically significant ABO- and diminished membrane repair as a consequence of HDFN is believed to reflect low ABH levels on cord cells, decreases in N-glycosylated lysosomal proteins (LAMP- which are 25 percent of adult levels (Table 3). The low density 1, LAMP-2, synaptotagmin II, synaptotagmin IV). It is of ABH epitopes on fetal RBCs may be insufficient to support hypothesized that branched polylactosamines may play a efficient complement activation by maternal IgG antibodies.18 role in stabilizing these lysoprotein proteins: inhibition of A comparison of i, I, and A strength shows that there is a LAMP-1 glycosylation by tunicamycin shortens LAMP-1 123,125 parallel increase in I and A in the first few months of life, with half-life. I and A reaching adult levels by 2 to 3 years of age (Fig. 8A– Cataracts C).2,3 These results are consistent with detailed carbohydrate The association between the i phenotype and analysis of the lactosaminyl, biantennary N-glycan present adult congenital cataracts was initially described by Ogata on Band 3, which accounts for more than 50 percent of all et al.12 in 1979, who reported cataracts in 17 of 18 ABH on RBCs (Fig 8D). On fetal RBCs, only a linear, i-active i individuals from 10 different Japanese families. polylactosamine is synthesized, with approximately 25 adult Subsequent studies by other investigators, however, were percent bearing a terminal ABH epitope (fucose) on the long, conflicting, indicating that the i phenotype did not α1-6 mannosyl branch.120 On adult RBCs, there is elongation adult always predispose to congenital cataracts, particularly in and β1,6 branching along both α1,6 and α1,3 mannosyl 13,14 25 non-Asian populations. The apparent discrepancy was branches, with up to four distal ABH epitopes. At one finally resolved with cloning of the IGnT gene,38 followed million molecules per RBC, Band 3 is capable of displaying 2 by sequencing analysis of i individuals.7–10 As already to 4 million ABH epitopes per adult RBC. adult described, the iadult phenotype without cataracts is the Animal Models consequence of mutations affecting exon E1C, leading to an isolated loss of IGnT activity in erythroid cells (Table 2, Fig. β3GnT2 3). Conversely, mutations affecting E2 or E3 result in loss of Two strains of mice lacking β3GnT2 have been bred IGnT activity in all tissues, including human lens. (β3GnT1 in some papers using the original citation by Zhou It has been presumed that loss of branched type 33 42,43 et al. ). β3GnT2–/– were shown to have altered neural 2 glycans in human lens is responsible for congenital development. There was a loss of olfactory sensory neurons cataracts. Human lens epithelial cells do synthesize type 42 early in embryonic development, decreased migration of 2 chain glycosphingolipids, including high molecular 121 gonadotropin-releasing hormone into the ventral forebrain, weight, long-chain sLeX gangliosides with repeating LeX and decreased reproduction, possibly attributable to poor motifs (Fig. 1D).48,126 In senile cataractous lens, there is olfactory recognition of estrous females.122 an increase in LeX glycolipids, presumably caused by β3GnT2–/– also experienced altered immune desialylation of sLeX structures.127 It is likely, therefore, responses. There was a marked decrease in polylactosamine that similar polylactosamines exist on lens glycoproteins. on N-glycans of CD28 and CD19, accompanied by evidence Although there are no published studies of glycoprotein of T-cell, B-cell, and macrophage hyperresponsiveness. The oligosaccharides on human lens epithelial cells, lens tissue results suggest a role for polylactosamines in regulating does express several N-linked glycoproteins, including α- basal levels of immune reactivity.43 and β-integrins, capable of I antigen expression.128,129 Altered glycosylation of β-integrins frequently accompanies cell IGnT differentiation, activation, and oncogenesis with changes A murine model of IGnT (GCNT2) deficiency has been in cell adhesion, motility, and signaling.130 constructed by Chen and colleagues.123 To produce an The absence of congenital cataracts in IGnT-deficient IGnT-deficient strain, the investigators deleted exon III mice,123 however, is perplexing and raises old questions

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Fig. 8 Correlation between I, i, A, and Band 3 N-glycan structure on fetal and adult erythrocytes. (A) i and I strength of expression on RBCs by age.2 (B) A strength of expression on RBCs, as determined by hemolysis.3 (C) Correlation between I and A expression by age.2,3 (D) Structure of Band 3 N-glycan on cord cells.120 (E) Structure of Band 3 N-glycan on adult RBCs.25

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regarding the causal link between cataract formation and subsequent differentiation, strong expression of I can be iadult phenotype. It is quite possible that IGnT deficiency plays observed in embryonic endoderm with apical polarization no direct role in cataract formation but is closely linked along differentiating epithelial cells.138 Increased Ii is to an unrelated mutant gene. Alternatively, there may be also observed in murine teratocarcinomas and some important species-specific differences in glycan expression human choriocarcinoma cell lines.137 Disseminated on lens epithelial cells, making mice a poor animal model for choriocarcinomatosis has been associated with elevated studying the effect of IGnT deficiency on lens development. anti-i in some patients.139 Indirect evidence for the latter explanation comes from Changes in Ii or their associated glycosyltransferases knockout mice lacking α13 galactosyltransferase (α3GT- have been described in several tissues, but this topic is too KO),131 an animal-specific glycosyltransferase responsible extensive for a full discussion here. Among published reports for linear B (Galα13Galβ1-R), a xenoantigen absent in are increased sialylation with masking of I expression humans and Old World apes.132,133 αGTKO mice develop in breast cancer.101,102 An increase in polylactosamines, congenital cataracts by day 36, suggesting a key role for β3GnT8 and GnTV, which catalyze synthesis of tetravalent α1,3-galactose in murine lens differentiation.131 N-glycans, is reported in colon cancer.44,100 Conversely, transitional cell carcinoma of the bladder is associated with HEMPAS an 11-fold decrease in β3GnT2.41 Particularly interesting is HEMPAS disease, or congenital dyserythropoietic the possible role of β3GnT1 in promoting tumor metastasis.45 anemia type 2 (CAD2), is a congenital and acquired disorder β3GnT1, in conjunction with LARGE glycosyltransferase, of hematopoiesis. Patients with CAD2 have a chronic mild regulates the synthesis of laminin-binding glycans on to severe hemolytic anemia, jaundice, and splenomegaly.19 α-dystroglycan. Decreased β1GnT1 could facilitate tumor Their bone marrow is striking for erythroid hyperplasia migration and metastatic tumor formation in breast and with 20 to 45 percent binucleated and multinucleated prostate cancers.45 erythroblasts. Electron microscopy of marrow erythroblasts and erythrocytes shows “membrane duplication” as a result Gene Array Data of accumulation and retention of fragmented endoplasmic For several years, commercial “gene-chips” have been reticulum.19 In the blood bank, CDAII or HEMPAS RBCs available for high-throughput screening of tissues for show increased i expression and hemolysis with acidified thousands of genes, including many glycosyltransferases donor sera. The amount of i expressed on HEMPAS RBCs involved in blood group expression. Transcriptome studies is significantly higher than that observed on cord RBCs.134 are probably the most common analysis performed. In these Detailed analysis of HEMPAS RBCs has shown studies total tissue mRNA is analyzed for global differences altered glycosylation on RBC glycoproteins (ex, band in gene expression. At this time, the NIH requires 3, glycophorin) and glycolipids.20,21 Some aspects of investigators to upload their findings into GenBank, a public HEMPAS can be duplicated in vitro by inhibiting N-glycan access database, allowing individual gene expression to be processing, leading to speculation that HEMPAS was a queried directly by other investigators. The data are located glycosyltransferase defect.21 In late 2009, the molecular in Entrez under GEO Profiles or the Gene Expression basis for HEMPAS was finally resolved after extensive Omnibus.140 Of note, the system does support Boolean genetic studies in 33 individuals from 28 unrelated logic, limiting the search to human tissue. The latter is a families.21 Schwarz and colleagues21 in Germany were able useful trick to know: an initial search of GEO Profiles for to show that all HEMPAS patients were heterozygous for IGnT resulted in 5654 entries, covering mouse, rat, and mutations in SEC23B, a Golgi COPII component protein. human arrays. COPII proteins facilitate and direct cargo vesicles among Among human studies, IGnT and iGnT array data are different Golgi compartments.29 It is possible that the available for a wide range of studies, including these: defect in HEMPAS leads to disordered trafficking in the • Cancer Golgi, with prolonged retention in some compartments • Cell differentiation and decreased trafficking to others. It is interesting to note • Organ transplantation that β4GalT1 and β3GnT1, which drive linear lactosamine • Heart failure, atrial fibrillation synthesis, are colocalized in the trans-Golgi.31 Although • Infection the Golgi location for IGnT is unknown, C2GnT, which • Rheumatoid arthritis catalyzes β1,6 branching on O-glycans, is located within the • Emphysema and cigarette smoking cis-medial Golgi.30 • Hormone replacement • Chemotherapy Embryogenesis and Neoplasia • Immunosuppression Many carbohydrate antigens are critical to normal • Heavy metal exposure embryonic development and serve as oncofetal antigens • Growth factors during neoplastic transformation.135,136 In mice, I is • Bipolar disorder expressed on unfertilized ovum up to the morulae.137 With • Social isolation (i.e., loneliness!)

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A brief review did not identify any clear disease 8. Yu L-C, Twu Y-C, Chou M-L, et al. The molecular association between IGnT and β3GalT1 levels in the studies genetics of the human I locus and molecular available. Interestingly, many tissues displayed significant background explain the partial association of the normal variation in glycosyltransferase expression between adult i phenotype with congenital cataracts. Blood individuals. An example of IGnT and β3GalT1 expression in 2003;101:2081–8. fetal and adult human reticulocytes is shown in Figure 9.141 9. Lin M, Hou M-J, Yu L-C. A novel IGnT allele responsible for the adult i phenotype. Transfusion 2006;46: 1982–7. 10. Pras E, Raz J, Yahalom V, et al. A nonsense mutation in the glucosaminyl (N-acetyl) transferase 2 gene (GCNT2): association with autosomal recessive congenital cataracts. Invest Ophthalmol Vis Sci 2004; 45:1940–5. 11. Tippett P, Noades J, Sanger R, Race RR. Further studies of the I antigen and antibody. Vox Sang 1960; 5:107–121. 12. Ogata H, Okubo Y, Akabane T. Phenotype i associated with congenital cataract in Japanese. Transfusion 1979;19:166–8. 13. Marsh WL, DePalma H. Association between the Ii blood group and congenital cataract. Transfusion 1982;22:337–8. 14. McDonald EB, Douglas R, Harden PA. A Caucasian family with the i phenotype and congenital cataracts. Vox Sang 1983;44:322–5. Fig. 9 IGnT expression arrays. Individual data for ß3GnT1 (iGnT1) (A) and IGnT (C2GnT) (B) expression in human fetal and adult 15. Jenkins WJ, Marsh WL, Noades J, Tippett P, Sanger reticulocytes. Array data from Goh et al.141 as extracted and R, Race RR. The I antigen and antibody. Vox Sang displayed on NCBI GEO Profiles.140 1960;5:97–106. 16. Navonet JM, Muller JY, Blanchard D. Expression of blood group I antigen and fetal hemoglobin in References paroxysmal nocturnal hemoglobinuria. Transfusion 1. Wiener AS, Unger LJ, Cohen L, Feldman J. Type- 1997;37:291–7. specific cold auto-antibodies as a cause of acquired 17. Kolins J, Allgood JW, Burghardt DC, Klein HG, hemolytic anemia and hemolytic transfusion reactions: McGinniss MH. Modifications of B, I, i, and biologic test with bovine red cells. Ann Internal Med Lewis b antigens in a patient with DiGuglielmo’s 1956;44:221–40. erythroleukemia. Transfusion 1980;20:574–7. 2. Marsh WL. Anti-i: a cold antibody defining the 18. Mollison PL, Engelfriet CP, Contreras M, eds. Blood Ii relationship in human red cells. Br J Haematol transfusion in clinical medicine. 10th ed. Oxford, 1961;7:200–9. England: Blackwell Science, 1997. 3. Grundbacher FJ. Changes in the human A antigen 19. Denecke J, Marquardt T. Congenital dyserythropoietic of erythrocytes with the individual’s age. Nature anemia type II (CDAII/HEMPAS): where are we now? 1964;204:192–4. Biochim Biophys Acta 2009;1792:915–20. 4. Doinel C, Ropars C, Salmon C. Quantitative and 20. Zdebska E, Golaszewska E, Fabijańska-Mitek J, et thermodynamic measurements on I and i antigens of al. Glycoconjugate abnormalities in patients with human red cells. Immunology 1976;30:289–97. congenital dyserythropoietic anaemia type I, II and 5. Oleson H. Thermodynamics of the cold agglutinin III. Br J Haematol 2001;114:907–13. reaction. Scand J Clin Lab Invest 1966; 18:1–15. 21. Schwarz K, Iolascon A, Verissimo F, et al. Mutations 6. Vigorito E, Robles A, Balter H, Nappa A, Goñi F. affecting the secretory COPII coat component SEC23B [125I]IgM (KAU) human monoclonal cold agglutinin: cause congenital dyserythropoietic anemia type II. labelling and studies on its biological activity. Appl Nat Genet 2009;41:936–40. Radiat Isot 1995;46:975–9. 22. Zdebska E, Krauze R, Kościelak J. Structure and 7. Inaba N, Hiruma T, Togayachi A, et al. A novel blood-group I activity of poly(glycosyl)-ceramides. I-branching β-1,6-N-acetylglucosaminyltransferase Carbohydr Res 1983;120:113–20. involved in human blood group I antigen expression. Blood 2003;101:2870–6. 23. Feizi T. The blood group Ii system: a carbohydrate antigen system defined by naturally monoclonal or

150 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Review of I system

oligoclonal autoantibodies of man. Immunol Commun key regulator of lacto-series glycolipid biosynthesis. J 1981;10:127–56. Biol Chem 2001;276:30261–9. 24. Feizi T, Childs RA, Watanabe K, Hakomori S-I. Three 36. Togayachi A, Akashima T, Ookubo R, et al. types of blood group I specificity among monoclonal Molecular cloning and characterization of UDP- anti-I autoantibodies revealed by analogues of a GlcNAc:lactosylceramide β1,3-N-acetylglucosaminyl- branched erythrocyte glycolipid. J Exp Med 1979;149: transferase (β3Gn-T5), an essential enzyme for 975–80. the expression of HNK-1 and Lewis X epitopes on 25. Fukuda M, Dell A, Oates JE, Fukuda MN. Structure of a glycolipids. J Biol Chem 2001;276:22032–40. branched lactosaminoglycan, the carbohydrate moiety 37. Stults CLM, Macher BA. β1–3-N-acetylglucosaminyl- of Band 3 isolated from adult human erythrocytes. J transferase in human leukocytes: properties and role Biol Chem 1984;259:8260–73. in regulating neolacto glycosphingolipid biosynthesis. 26. Roelcke D. Sialic acid-dependent red blood cell Arch Biochem Biophys 1993;303:125–33. antigens. In: Garratty G, ed. Immunobiology of 38. Bierhuizen MFA, Mattei M-G, Fukuda M. Expression transfusion medicine. New York, NY: Marcel-Dekker, of the developmental I antigen by a cloned 1994:69–95. human cDNA encoding a member of a β-1,6-N- 27. Renkonen O. Enzymatic in vitro synthesis of I-branches acetylglucosaminyltransferase family. Genes Dev of mammalian polylactosamines: generation of 1993;7:468–78. scaffolds for multiple selectin-binding saccharide 39. Merkle RK, Cummings RD. Relationship of the determinants. Cell Mol Life Sci 2000;57:1423–39. terminal sequences to the length of poly-N- 28. Wilczyńska Z, Miller-Podraza H, Kościelak J. The acetyllactosamine chains in asparagine-linked contribution of different glycoconjugates to the total oligosaccharides from the mouse lymphoma cell ABH blood group activity of human erythrocytes. line BW5147. Immobilized tomato lectin interacts FEBS Lett 1980;112:277–9. with high affinity with glycopeptides containing 29. Jackson CL. Mechanisms of transport through the long poly-N-acetyllactosamine chains. J Biol Chem Golgi complex. J Cell Sci 2009;122:443–52. 1987;262:8179–89. 30. Skrincosky D, Kain R, El-Battari A, Exner M, 40. Sasaki K, Kurata-Miura K, Ujita M, et al. Expression Kerjaschki D, Fukuda M. Altered Golgi localization cloning of cDNA encoding a human β-1,3-N- of core 2 beta-1,6-N-acetylglucosaminyltransferase acetylglucosaminyltransferase that is essential for leads to decreased synthesis of branched O-glycans. J poly-N-acetyllactosamine synthesis. Proc Natl Acad Biol Chem 1997;272:22695–702. Sci U S A 1997;94:14294–9. 31. Lee PL, Kohler JJ, Pfeffer SR. Association of β-1,3- 41. Gromova I, Gromov P, Celis JE. A novel member N-acetylglucosaminyltransferase 1 and β-1,4- of the glycosyltransferase family, β3 Gn-T2, highly galactosyltransferase 1, trans-Golgi enzymes involved downregulated in invasive human bladder transitional in coupled poly-N-acetyllactosamine synthesis. cell carcinomas. Mol Carcinog 2001;32:61–72. Glycobiology 2009;19:655–64. 42. Henion TR, Raitcheva D, Grosholz R, et al. β1,3- 32. Seko A, Yamashita K. Activation of β1,3-N- N-acetylglucosaminyltransferase 1 glycosylation is acetylglucosaminyltransferase-2 (β3Gn-T2) by required for axon pathfinding by olfactory sensory β3Tn-T8: possible involvement of β3GnT8 in increasing neurons. J Neurosci 2005;25:1894–903. poly-N-acetyllactosamine chains in differentiated 43. Togayachi A, Kozono Y, Ishida H, et al. Polylactosamine HL-60 cells. J Biol Chem 2008;283:33094–100. on glycoproteins influences basal levels of lymphocyte 33. Zhou D, Dinter A, Gutiérrez Gallego R, et al. A and macrophage activation. Proc Natl Acad Sci U S A β-1,3-N-acetylglucosaminyltransferase with poly-N- 2007;104:15829–34. acetyllactosamine synthase activity is structurally 44. Ishida H, Togayachi A, Sakai T, et al. A novel β1,3- related to β-1,3-galactosyltransferases. Proc Natl Acad N-acetylglucosaminyltransferase (β3Gn-T8), which Sci U S A 1999;96:406–11. synthesizes poly-N-acetyllactosamine, is dramatically 34. Shiraishi N, Natsume A, Togayachi A, et al. upregulated in colon cancer. FEBS Lett 2005;579: Identification and characterization of three novel β1,3- 71–8. N-acetylglucosaminyltransferases structurally related 45. Bao X, Kobayashi M, Hatakeyama S, et al. to the β1,3-galactosyltransferase family. J Biol Chem Tumor suppressor function of laminin-binding 2001;276:3498–507. α-dystroglycan requires a distinct β3-N-acetylglu- 35. Henion TR, Zhou D, Wolfer DP, Jungalwala cosaminyltransferase. Proc Natl Acad Sci USA 2009; FB, Hennet T. Cloning of a mouse β1,3 106:12109–14. N-acetylglucosaminyltransferase GlcNAc(β1,3) 46. Symington FW, Hedges DL, Hakomori S-I. Glycolipid Gal(β1,4)Glc-ceramide synthase gene encoding the antigens of human polymorphonuclear neutrophils

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and the inducible HL-60 myeloid leukemia line. J 60. Bengtson P, Larson C, Lundblad A, Larson G, Immunol 1985;134:2498–506. Påhlsson P. Identification of a missense mutation 47. Cooling LLW, Zhang DS, Naides SJ, Koerner TAW. (G329A;Arg110Gln) in the human FUT7 gene. J Biol Glycosphingolipid expression in acute nonlymphocytic Chem 2001;276:31575–82. leukemia: common expression of shiga toxin and 61. Adinolfi M. Anti-I antibody in normal human newborn parvovirus B19 receptors on early myeloblasts. Blood infants. Immunology 1965;9:43–52. 2003;101:711–21. 62. Dube VE, Zuckerman L, Philipsborn HF Jr. Variation 48. Cooling LW, De-Sheng Z, Koerner TAW. Lewis X and of cold agglutinin levels. Vox Sang 1978;34:71–6. sialyl Lewis X glycosphingolipids. Trends Glycosci 63. Doinel C. I antigenicity of the Bombay red cells [in Glycotechnol 1997;9:191–209. French]. Rev Fr Transfus Immunohematol 1976;19: 49. Iwai T, Inaba N, Naundorf A, et al. Molecular cloning 185–91. and characterization of a novel UDP-GlcNAc: GalNAc- 64. Gilboa-Garber N, Sudakevitz D, Levene C. A peptide β1,3-N-acetylglucosaminyltransferase comparison of the Aplysia lectin anti-I specificity with (β3Gn-T6), an enzyme synthesizing the core 3 structure human anti-I and several other I-detecting lectins. of O-glycans. J Biol Chem 2002;277:12802–9. Transfusion 1999;39:1060–4. 50. Kataoka K, Huh N. A novel β1,3-N-acetylglucos- 65. Stults CLM, Sweeley CC, Macher BA. Glycosphingo- aminyltransferase involved in invasion of cancer cells lipids: structure, biological source, and properties. as assayed in vitro. Biochem Biophys Res Commun Methods Enzymol 1989;179:167–214. 2002;294:843–8. 66. Gardas A. Studies on the I-blood-group-active sites 51. Yeh J-C, Hiraoka N, Petryniak B, et al. Novel on macro-glycolipids from human erythrocytes. Eur J sulfated lymphocyte homing receptors and their Biochem 1976;68:185–91. control by a Core1 extension β1,3-N-acetylglucos- 67. Kannagi R, Roelcke D, Peterson KA, Okada Y, Levery aminyltransferase. Cell 2001;105:957–69. SB, Hakomori S-I. Characterization of an epitope 52. Seko A, Yamashita K. β1,3-N-Acetylglucosaminyl- (determinant) structure in a developmentally regulated transferase-7 (β3GnT7) acts efficiently on keratan glycolipid antigen defined by a cold agglutinin Fl, sulfate-related glycans. FEBS Lett 2004;556:216–20. recognition of an α-sialosyl and α-L-fucosyl groups in 53. Twu Y-C, Chen C-P, Hsieh C-Y, et al. I branching a branched structure. Carbohydr Res 1983;120:143–57. formation in erythroid differentiation is regulated by 68. Nagatsuka Y, Watarai S, Yasuda T, Higashi H, Yamagata transcription factor C/EBPα. Blood 2007;110:4526–34. T, Ono Y. Production of human MAbs to i blood group 54. Geest CR, Buitenhuis M, Laarhoven AG, et al. p38 by EBV-induced transformation: possible presence of a MAP kinase inhibits neutrophil development through new glycolipid in cord red cell membranes and human phosphorylation of C/EBPα on serine 21. Stem Cells hematopoietic cell lines. Immunol Lett 1995;46: 2009;27:2271–82. 93–100. 55. Bierhuizen MFA, Fukuda M. Expression cloning of 69. Nagatsuka Y, Yamaki M, Watarai S, et al. The Epstein- a cDNA encoding UDP-GlcNAc:Galβ1-3-GalNAc-R Barr virus (EBV) alters the B cell glycolipid which (GlcNAc to GalNAc) β1-6GlcNAc transferase by gene is recognized by the human monoclonal antibody to transfer into CHO cells expressing polyoma large i-blood group antigen. Virus Res 1996;43:57–68. tumor antigen. Proc Natl Acad Sci U S A 1992;89: 70. Chapman CJ, Spellerberg MB, Smith GA, Carter 9326–30. SJ, Hamblin TJ, Stevenson FK. Autoanti-red cell 56. Yeh J-C, Ong E, Fukuda M. Molecular cloning antibodies synthesized by patients with infectious

and expression of a novel β-1,6-N-acetylglucos- mononucleosis utilize the VH4–21 gene segment. J aminyltransferase that forms core 2, core 4, and I Immunol 1993;151:1051–61. branches. J Biol Chem 1999;274;3215–21. 71. Riboldi P, Gaidano G, Schettino EW, et al. Two 57. Breton C, Snajdrová L, Jeanneau C, Koca J, Imberty A. acquired immunodeficiency syndrome-associated Structures and mechanisms of glycosyltransferases. Burkitt’s lymphomas produce specific anti-i IgM

Glycobiology 2006;16:29R–37R. cold agglutinins using somatically mutated VH4–21 58. Pak JE, Arnoux P, Zhou S, et al. X-ray crystal structure segments. Blood 1994;83:2952–61. of leukocyte type core 2 β1,6-N-acetylglucosaminyl- 72. Roelcke D, Kreft H, Hack H, Stevenson FK. Anti-j: transferase: evidence for a convergence of metal ion- human cold agglutinins recognizing linear (i) and independent glycosyltransferase mechanism. J Biol branched (I) type 2 chains. Vox Sang 1994;67:216–21. Chem 2006;281:26693–701. 73. Dube VE, Kallio P, Tanaka M. Specificity of the 59. Cabrera PV, Amano M, Mitoma J, et al. monoclonal anti-I antibody (Hy). Mol Immunol 1986; Haploinsufficiency of C2GnT-1 glycosyltransferase 23:217–20. renders T lymphoma cells resistant to cell death. Blood 74. Roelcke D, Kreft H, Northoff H, Gallasch E. Sia-β1 and 2006;108:2399–406. I antigens recognized by Mycoplasma pneumoniae-

152 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Review of I system

induced human cold agglutinins. Transfusion 1991;31: 88. Havouis S, Dumas G, Chambaud I, et al. Transgenic 627–30. B lymphocytes expressing a human cold agglutinin 75. Zilow G, Haffner D, Roelcke D. CMV-induced anti- escape tolerance following experimental infection of Sia-β1 cold agglutinin in an immunocompromised mice by Mycoplasma pulmonis. Eur J Immunol 2002; patient. Beitr Infusionsther Transfusionsmed 1997;34: 32:1147–56. 180–4. 89. Mandrell RE, Griffiss JM, Macher BA. 76. Loomes LM, Uemura K-I, Feizi T. Interaction of Lipooligosaccharides (LOS) of Neisseria gonorrhoeae Mycoplasma pneumoniae with erythrocyte glycolipids and Neisseria meningitidis have components that of the I and i antigen types. Infect Immun 1985;47: are immunochemically similar to precursors of 15–20. human blood group antigens. Carbohydrate sequence 77. Loveless RW, Feizi T. Sialo-oligosaccharide specificity of the mouse monoclonal antibodies that receptors for Mycoplasma pneumoniae and related recognize crossreacting antigens on LOS and human oligosaccharides of poly-N-acetyllactosamine series erythrocytes. J Exp Med 1988;168:107–26. are polarized at the cilia and apical-microvillar 90. Thorpe SJ, Boult CE, Stevenson FK, et al. Cold domains of the ciliated cells in the human bronchial agglutinin activity is common among human epithelium. Infect Immun 1989;57:1285–9. monoclonal antibodies using the V4-34 heavy chain 78. Feizi T, Loveless RW. Carbohydrate recognition variable gene segment. Transfusion 1997;37:1111–16. by Mycoplasma pneumoniae and pathologic 91. Thorpe SJ, Turner CE, Stevenson FK, et al. Human consequences. Am J Respir Crit Care Med 1996;154 monoclonal antibodies encoded by the V4-34 gene (Suppl):S133–6. segment show cold agglutinin activity and variable 79. Li Y, Spellerberg MB, Stevenson FK, Capra JD, Potter multireactivity which correlates with the predicted

KN. The I binding specificity of human VH4-34 (VH4- charge of the heavy-chain variable region. Immunology 21) encoded antibodies is determined by both VH 1998;93:129–36. framework region 1 and complementarity determining 92. Terness P, Kirschfink M, Navolan D, et al. Striking

region 3. J Mol Biol 1996;256:577–89. inverse correlation between IgG anti-F(ab′)2 and 80. Stevenson FK, Spellerberg MB, Chapman CJ, Hamblin autoantibody production in patients with cold TJ. Differential usage of an autoantibody-associated agglutination. Blood 1995;85:548–51.

VH gene, VH4-21, by human B-cell tumors. Leuk 93. Terness P, Kirschfink M, Navolan D, et al. Inverse Lymphoma 1995;16:379–84. correlation between IgG-antihinge region and 81. Ishida F, Saito H, Kitano K, Kiyosawa K. Cold antierythrocyte autoantibody in chronic benign and agglutinin disease by autoanti-i blood type antibody malignant cold agglutination. J Clin Immunol 1997; associated with B cell chronic lymphocytic leukemia. 17:220–7. Int J Hematol 1998;67:69–73. 94. Young WW Jr, Portoukalian J, Hakomori S-I. Two 82. Silberstein LE, Jefferies LC, Goldman J, et al. monoclonal anticarbohydrate antibodies directed Variable region gene analysis of pathologic human to glycosphingolipids with a lacto-N-glycosyl type II autoantibodies to the related I and i red blood cell chain. J Biol Chem 1981;256:10967–72. antigens. Blood 1991;78:2372–86. 95. Fenderson BA, Nichols EJ, Clausen H, Hakomori 83. Cauerhff A, Braden BC, Carvalho JG, et al. Three- S-I. A monoclonal antibody defining a binary dimensional structure of the Fab from a human IgM N-acetyllactosaminyl structure in lactoisooctaosyl- 6 cold agglutinin. J Immunol 2000;165:6422–8. ceramide (IV Galβ14GlcNAcnLc6): a useful 84. Leo A, Kreft H, Hack H, Kempf T, Roelcke D. probe for determining differential glycosylation Restriction in the repertoire of the immunoglobulin patterns between normal and transformed light chain subgroup in pathological cold agglutinins human fibroblasts. Mol Immunol 1986;23:747–54. with anti-Pr specificity. Vox Sang 2004;86:141–7. 96. Myoga A, Taki T, Arai K, et al. Detection of patients 85. Foreman AL, Van de Water J, Gougeon M-L, Gershwin with cancer by monoclonal antibody directed to ME. B cells in autoimmune diseases: insights from lactoneotetraosylceramide (paragloboside). Cancer analyses of immunoglobulin variable (Ig V) gene Res 1988;48:1512–6. usage. Autoimmun Rev 2007;6:387–401. 97. Fenderson BA, Hahnel AC, Eddy EM. 86. Jefferies LC, Carchidi CM, Silberstein LE. Immunohistochemical localization of two monoclonal Immunoglobulin gene use by naturally occurring cold antibody-defined carbohydrate antigens during early agglutinins. Ann N Y Acad Sci 1995;764:433–5. murine embryogenesis. Dev Biol 1983;100:318–27. 87. Havouis S, Dumas G, Avé P, et al. Negative regulation 98. Cooling LLW, Zhang DS, Koerner TAW. Human of autoreactive B cells in transgenic mice expressing platelets express gangliosides with LKE activity a human pathogenic cold agglutinin. Eur J Immunol and ABH blood group activity. Transfusion 2001;41: 2000;30:2290–9. 504–16.

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 153 L. Cooling

99. Hirohashi S, Clausen H, Nudelman E, Inoue H, 109. Elgavish S, Shaanan B. Structures of the Erythrina Shimosato Y, Hakomori S. A human monoclonal corallodendron lectin and of its complexes with mono- antibody directed to blood group i antigen: and disaccharides. J Mol Biol 1998;277:917–32. heterohybridoma between human lymphocytes from 110. Gilboa-Garber N, Susswein AJ, Mizrahi L, Avichezer regional lymph nodes of a lung cancer patient and D. Purification and characterization of the gonad lectin mouse myeloma. J Immunol 1986;136:4163–8. of Aplysia depilans. FEBS Lett 1985;181:267–70. 100. Miyake M, Kohno N, Nudelman ED, Hakomori S-I. 111. Knibbs RN, Goldstein IJ, Ratcliffe RM, Shibuya

Human IgG3 monoclonal antibody directed to an N. Characterization of the carbohydrate binding unbranched repeating type 2 chain (Galβ14GlcNAc specificity of the leukoagglutinating lectin from β13Galβ14GlcNAcβ13Galβ1R) which is highly Maackia amurensis: comparison with other sialic expressed in colonic and hepatocellular carcinoma. acid-specific lectins. J Biol Chem 1991;266:83–8. Cancer Res 1989;49:5689–95. 112. Johansson L, Miller-Podraza H. Analysis of 3- and 101. Gooi HC, Uemura K-I, Edwards PAW, Foster CS, 6-linked sialic acids in mixtures of gangliosides using Pickering N, Feizi T. Two mouse hybridoma antibodies blotting to polyvinylidene difluoride membranes, against milk-fat globules recognise the I(Ma) antigenic binding assays, and various mass spectrometry determinant β-D-Galp-(14)-β-D-GlcpNAc-(16). techniques with application to recognition by Carbohydr Res 1983;120:293–302. Helicobacter pylori. Anal Biochem 1998;265:260–8. 102. Foster CS, Neville AM. Monoclonal antibodies to the 113. Cooling LL, Hwang D, Gu Y. The LKE-negative and human mammary gland: monoclonal antibody LiCR- LKE-weak RBC phenotypes do not reflect decreased LON-M18 identifies impaired expression and excess RBC sialylation [abstract]. Transfusion 2003;43:26. sialylation of the I(Ma) cell-surface antigen by primary 114. Müthing J, Meisen I, Bulau P, et al. Mistletoe lectin breast carcinoma cells. Hum Pathol 1984;15:502–13. I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal 103. Symington FW, Fenderson BA, Hakomori S-I. Fine Neu5Acα2-6Galβ1-4GlcNAc residues. Biochemistry specificity of a monoclonal anti-testicular cell antibody 2004;43:2996–3007. for glycolipids with terminal N-acetyl-D-glucosamine 115. Mo H, Winter HC, Goldstein IJ. Purification and structure. Mol Immunol 1984;21:877–82. characterization of a Neu5Acα2-6Galβ1-4Glc/ 104. Holmes EH, Greene TG. Isolation and fine-structure GlcNAc-specific lectin from the fruiting body of the characterization of four monoclonal antibodies polypore mushroom Polyporus squamosus. J Biol reactive with glycoconjugates containing terminal Chem 2000;275:10623–9. GlcNAc residues: application to aspects of lacto-series 116. Toma V, Zuber C, Winter HC, Goldstein IJ, Roth J. tumor antigen biosynthesis. Arch Biochem Biophys Application of a lectin from the mushroom Polysporus 1991;288:87–96. squamosus for the histochemical detection of the 105. Hu J, Stults CLM, Holmes EH, Macher BA. Structural NeuAcα2,6Galβ1,4Glc/GlcNAc sequence of N-linked characterization of intermediates in the biosynthetic oligosaccharides: a comparison with the Sambucus pathway of neolacto glycosphingolipids: differential nigra lectin. Histochem Cell Biol 2001;116:183–93. expression in human leukaemia cells. Glycobiology 117. Tateno H, Winter HC, Goldstein IJ. Cloning, 1994;4:251–7. expression in Escherichia coli and characterization 106. Iglesias JL, Lis H, Sharon N. Purification and properties of the recombinant Neu5Acα2,6Galβ1,4GlcNAc- of a D-galactose/N-acetyl-D-galactosamine-specific specific high-affinity lectin and its mutants from lectin from Erythrina cristagalli. Eur J Biochem 1982; the mushroom Polyporus squamosus. Biochem J 123:247–52. 2004;382:667–75. 107. Teneberg S, Angström J, Jovall P-A, Karlsson 118. Shibuya N, Goldstein IJ, Broekaert WF, Nsimba-Lubaki K-A. Characterization of binding of Galβ4GlcNAc- M, Peeters B, Peumans WJ. The elderberry (Sambucus specific lectins from Erythrina cristagalli and nigra L.) bark lectin recognizes the Neu5Ac(α2-6)Gal/ Erythrina corallodendron to glycosphingolipids. GalNAc sequence. J Biol Chem 1987;262:1596–601. Detection, isolation, and characterization of a novel 119. Yamashita K, Umetsu K, Suzuki T, Ohkura T. glycosphingolipid of bovine buttermilk. J Biol Chem Purification and characterization of a Neu5Acα2

1994;269:8554–63. 6Galβ14GlcNAc and HSO3–6Galβ14GlcNAc 108. Moreno E, Teneberg S, Adar R, Sharon N, Karlsson specific lectin in tuberous roots of Trichosanthes K-A, Angström J. Redefinition of the carbohydrate japonica. Biochemistry 1992;31:11647–50. specificity of Erythrina corallodendron lectin based 120. Fukuda M, Dell A, Fukuda MN. Structure of fetal on solid-phase binding assays and molecular modeling lactosaminoglycan. The carbohydrate moiety of Band of native and recombinant forms obtained by site- 3 isolated from human umbilical cord erythrocytes. J directed mutagenesis. Biochemistry 1997;36:4429–37. Biol Chem 1984;259:4782–91.

154 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Review of I system

121. Bless E, Raitcheva D, Henion TR, Tobet S, Schwarting 132. Galili U, Shohet SB, Kobrin E, Stults CLM, Macher BA. GA. Lactosamine modulates the rate of migration of Man, apes, and Old World monkeys differ from other GnRH neurons during mouse development. Eur J mammals in the expression of α-galactosyl epitopes Neurosci 2006;24:654–60. on nucleated cells. J Biol Chem 1988;263:17755–62. 122. Biellmann F, Henion TR, Bürki K, Hennet T. Impaired 133. Joziasse DH, Shaper JH, Jabs EW, Shaper NL. sexual behavior in male mice deficient for the β1-3 Characterization of an α1-3-galactosyltransferase N-acetylglucosaminyltransferase-I gene. Mol Reprod homologue on human chromosome 12 that is Dev 2008;75:699–706. organized as a processed pseudogene. J Biol Chem 123. Chen GY, Muramatsu H, Kondo M, et al. Abnormalities 1991;266:6991–8. caused by carbohydrate alterations in Iβ6-N- 134. Cooling L. Increased expression of i on HEMPAS acetylglucosaminyltransferase-deficient mice. Mol red cells: a flow cytometric study. Transfusion 1997; Cell Biol 2005;25:7828–38. 37:1102–3. 124. Muramatsu H, Kusano T, Sato M, Oda Y, Kobori K, 135. Haltiwanger RS, Lowe JB. Role of glycosylation in Muramatsu T. Embryonic stem cells deficient in I β1,6- development. Annu Rev Biochem 2004;73:491–537. N-acetylglucosaminyltransferase exhibit reduced 136. Hakomori S-I. Cancer-associated glycosphingolipid expression of embyroglycan and the loss of a Lewis X antigens: their structure, organization and function. antigen, 4C9. Glycobiology 2008;18:242–9. Acta Anat (Basel) 1998;161:79–90. 125. Barriocanal JG, Bonifacino JS, Yuan L, Sandoval 137. Knowles BB, Rappaport J, Solter D. Murine embryonic IV. Biosynthesis, glycosylation, movement through antigen (SSEA-1) is expressed on human cells the Golgi system, and transport to lysosomes by an and structurally related human group antigen I is N-linked carbohydrate-independent mechanism of expressed on mouse embryos. Dev Biol 1982;93:54–8. three lysosomal integral membrane proteins. J Biol 138. Kapadia A, Feizi T, Evans MJ. Changes in the expression Chem 1986;261:16755–63. and polarization of blood group I and i antigens in 126. Ogiso M, Ohta M, Okinaga T, et al. Glycosphingolipids post-implantation embryos and teratocarcinomas in cultured lens epithelial cells from dog and rhesus associated with cell differentiation. Exp Cell Res 1981; monkey. Glycobiology 1994;4:375–82. 131:185–95. 127. Ogiso M, Komoto M, Okinaga T, Koyota S, Hoshi M. 139. Boughton BJ. Anti-i cold agglutinins in Age-related changes in ganglioside composition in choriocarcinomatosis: trophoblastic i antigen. J Clin human lens. Exp Eye Res 1995;60:317–23. Pathol 1979;32:523–7. 128. Nishi O, Nishi K, Akaishi T, Shirasawa E. Detection of 140. GEO Profiles, National Center for Biotechnology cell adhesion molecules in lens epithelial cells of human Information. http://www.ncbi.nlm.nih.gov/sites/ cataracts. Invest Ophthalmol Vis Sci 1997;38:579–85. entrez?db=geo (Last accessed 4/2010). 129. McLean SM, Mathew MRK, Kelly JB, et al. Detection 141. Goh SH, Josleyn M, Lee YT, et al. The human of integrins in human cataract lens epithelial cells reticulocyte transcriptome. Physiol Genomics 2007; and two mammalian lens epithelial cell lines. Br J 30:172–8. Ophthalmol 2005;89:1506–9. 130. Bellis SL. Variant glycosylation: an underappreciated Laura Cooling MD, MS, Associate Professor, Pathology, regulatory mechanism for β1 integrins. Biochim Associate Medical Director, Transfusion Medicine, Biophys Acta 2004;1663:52–60. University of Michigan Hospitals, 2F225 UH, Box 0054, 131. Sørensen DB, Dahl K, Ersboll AK, Kirkeby S, d’Apice 1500 East Medical Center Drive, Ann Arbor, MI 48109- AJF, Hansen AK. Aggression in cataract-bearing 0054. α-1,3-galactosyltransferase knockout mice. Lab Anim 2008;42:34–44.

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 155 Original Report

Attempts to support an immune etiology in 800 patients with direct antiglobulin test–negative hemolytic anemia

R.M. Leger, A. Co, P. Hunt, and G. Garratty

Clinical and hematologic evidence of warm autoimmune They developed a method, the complement-fixing antibody hemolytic anemia (AIHA) is present in some patients whose consumption (CFAC) test, capable of detecting this low direct antiglobulin test (DAT) is negative. The most common level of RBC-bound IgG. The authors reported that RBCs causes for AIHA associated with a negative DAT are RBC-bound from five of six patients with acquired hemolytic anemia IgG below the sensitivity threshold of the DAT, RBC-bound IgA and a negative DAT were found to have greater quantities and IgM not detectable by routine reagents, and low-affinity IgG of IgG per RBC (70–434 molecules) compared with 15 to 35 that dissociates during the testing process. Samples submitted from 800 patients with hemolytic anemia and a negative DAT molecules of IgG per RBC found on the RBCs obtained from 4 were tested by an antiglobulin sera (AGS) panel of anti-IgG, normal persons. Gilliland extended these results with an anti-C3, anti-IgM, and anti-IgA by a routine DAT. Additional tests additional nine patients; six of these nine responded to included a direct Polybrene test to detect small amounts of RBC- corticosteroid therapy. bound IgG, a cold-wash technique to detect low-affinity IgG, and Petz and Garratty5 performed extensive tests, including a DAT by gel test with anti-IgG. A positive result was obtained the CFAC, on specimens obtained from 27 patients with a with at least one method for 431 (54%) of 800 specimens tested. diagnosis of immune hemolytic anemia with a negative DAT, The AGS panel was positive for 400 (50%) of samples, with IgG or confirming that all of these patients had RBC-bound IgG C3 or both accounting for reactivity in 48 percent. IgA alone was detectable by the CFAC; 11 (41%) patients had RBC-bound C3 found on 2 percent of samples; IgM was never found alone. Low- detectable by a potent anti-C3 prepared in their laboratory affinity IgG was found on 37 (5%) samples. The direct Polybrene test was the only positive test for 15 (2%) samples. The gel anti- but not detected by the referring hospital. Because the CFAC IgG test was never the only positive test. Clinical correlations is difficult to standardize and to perform consistently, other for these data were not available; however, previously published tests have been used to detect RBC-bound IgG, including correlations suggest a positive predictive value for tests that enzyme-linked antiglobulin test (ELAT), radiolabeled anti- extend routine DAT methods in patients with DAT-negative IgG, flow cytometry, monocyte monolayer assay (MMA), AIHA. Immunohematology 2010;26:156–60. concentrated eluates, the direct Polybrene test, the direct polyethylene glycol test, solid phase, column agglutination, Key Words: autoimmune hemolytic anemia, DAT-negative, and the DAT using cold washes.2 When hematologists or Coombs-negative referring laboratories contact our laboratory, they often ask Most patients with autoimmune hemolytic anemia for a “Super Coombs Test.” There is no such test. We believe (AIHA) have IgG or complement, or both, on their RBCs the term originated as jargon to cover tests such as the detectable by the routine DAT. However, approximately 2 to ELAT and radiolabeled anti-IgG previously used to detect 11 percent of patients with hematologic and clinical findings small amounts of RBC-bound IgG. suggestive of warm AIHA have a negative DAT by routine No one test has been shown to be optimal for investigating tube technique and have no detectable serum antibodies.1 DAT-negative AIHA; we and others have found that a battery Hematologists seeking evidence supporting immune- of tests appears to be the best approach.1,6–9 By 1995 we had mediated hemolysis may request nonroutine and more decided that there was no cost benefit in performing time- sensitive tests for these patients. consuming assays such as the ELAT, so we concentrated on It has been suggested that there are at least three causes a serologic approach. We transferred DAT-negative AIHA for AIHA associated with a negative DAT: RBC-bound IgG workups from our Immunohematology Research Laboratory below the threshold of detection of the routine antiglobulin to the Immunohematology Reference Laboratory (IRL). test, low-affinity IgG that dissociates from RBCs during All samples that are submitted for a DAT-negative AIHA saline washes for the DAT, and RBC-bound IgM and IgA investigation are tested by tube test with an antiglobulin sera that are not detectable by routine reagents.1,2 The routine (AGS) panel consisting of anti-IgG, anti-C3, anti-IgM, and tube DAT can detect about 150 to 200 molecules of IgG anti-IgA. When the AGS panel is not informative, additional per RBC. Gilliland and coworkers3,4 suggested that the tests performed include a direct Polybrene test to detect inability to detect IgG on the RBCs of some patients with low amounts of immunoglobulin, a cold (4°C) LISS wash AIHA was caused by low levels of RBC-bound IgG that technique for preparation of RBCs to detect low-affinity IgG, were below the threshold of detection by the routine DAT. and a column agglutination (gel) test for IgG.

156 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 DAT-negative hemolytic anemia

We report the serologic results for a large series of controls to ascertain the persistence of agglutination. One patients’ samples that were submitted for a DAT-negative additional drop of resuspending solution was added plus AIHA evaluation. 2 drops of group AB plasma (undiluted). After mixing, the tubes were washed three times and converted to an Material and Methods antiglobulin test with anti-IgG. The AGS panel consisted of a rabbit anti-IgG (American The gel test DAT was performed as described by the National Red Cross, Washington, DC; or Ortho Clinical manufacturer (ID-MTS Anti-IgG Card, Ortho). Control Diagnostics, Raritan, NJ), anti-C3 (reagent prepared by tests were performed in parallel with the ID-MTS Buffered injecting a rabbit with purified C3), rabbit anti-human IgM Gel Card. (CLB, distributed by Research Diagnostics, Flanders, NJ; or Sanquin, distributed by Accurate Chemical & Scientific Results Corporation, Westbury, NY), goat anti-human IgA (Tago, Eight hundred specimens submitted January 1997 Burlingame, CA), and a 6% bovine serum albumin control. through December 2008 (12 years) for suspected DAT- The anti-C3, anti-IgM, and anti-IgA were standardized for negative AIHA were evaluated. All 800 samples were RBC agglutination in tube tests as previously described.1 tested by the AGS panel; 761 (95%) were tested by the 4ºC Specimens were also tested from April through December LISS wash technique, and 667 (83%) were tested by the 2008 with Gamma-clone Anti-IgG (murine monoclonal, direct Polybrene test. All three techniques were performed Immucor, Norcross, GA). For the DAT, RBCs were washed on 635 (79%) of the 800 specimens tested; the gel test was four times with PBS (pH 7.0–7.4) and resuspended to performed on 434 (54%) samples. When positive results achieve a 3 to 5% suspension; 1 drop was added to 2 drops were obtained by the AGS panel, especially with anti-IgG, of the antiglobulin reagent or control, centrifuged, and read the 4°C LISS wash or direct Polybrene test was not always immediately. Centrifugation and reading was repeated performed. Positive results were obtained with at least one after incubation at room temperature if indicated by the of the methods for 431 (54%) of the 800 specimens tested. standardization. A positive result with the 6% albumin Some specimens were reactive by one method but not by control invalidated the test. When a positive result with another; others were reactive by more than one method. the albumin control was obtained, repeat testing may have Results for the battery of serologic tests used in the DAT- been performed after washing a fresh aliquot of RBCs with negative AIHA workup are shown in Table 1. Specimens warm (37ºC) saline to remove cold autoagglutinins, or after that yielded an invalid result, i.e., a reactive negative treatment with 0.01 M dithiothreitol (DTT) to disperse IgM control, were not included in the number of specimens autoagglutinins.10 tested for that method. The number of invalid results The cold LISS wash technique was performed by obtained for each method was as follows: AGS panel = 2, washing the RBCs four times with ice-cold (4°C) LISS11 using direct Polybrene test = 2, 4ºC LISS wash technique = 11, a refrigerated centrifuge.1 The RBCs were resuspended to and DAT by gel test = 2. An initially reactive control for a 3 to 5% suspension in cold LISS; 1 drop was added to 2 the AGS panel was resolved for 10 samples by washing the drops of cold anti-IgG or cold 6% albumin, centrifuged in a RBCs with warm (37ºC) saline (n = 7) or by treating them serologic centrifuge, and read immediately. The 6% albumin with DTT (n = 3).13 served as a control for the presence of a cold autoagglutinin; Our AGS panel provided a positive DAT result in 50 a positive result with the 6% albumin control invalidated percent of cases. The distribution of the AGS panel reactivity the cold LISS wash test with anti-IgG. is shown in Figure 1. Reactivity with anti-IgG or anti-C3 The direct Polybrene test,1 a modification of the indirect accounted for positive results in 48 percent of cases; most Polybrene test,12 was performed as previously described. of this reactivity was weak (≤1+). All eight specimens that Briefly, 1 drop of 3 to 5% washed RBCs was added to 2 drops reacted with anti-IgM also reacted with anti-C3 or with anti- of 5% group AB inert plasma. In parallel, 3 drops of a 1.5% IgG plus anti-C3. IgA was detected on 13 (2%) specimens suspension of the patient’s unwashed RBCs suspended in that were nonreactive with anti-IgG and anti-C3. autologous plasma were tested. One milliliter of the low-ionic Table 1. Results for battery of serologic tests for DAT-negative medium was added to each tube, and the test was incubated autoimmune hemolytic anemia at room temperature for 1 minute. Two drops of 0.05% Polybrene (hexadimethrine bromide, Sigma Chemical, Results No. tested* No. positive (%) Co., St. Louis, MO) in unbuffered saline were added, and Any positive test 800 431 (54%) the contents were mixed. After 15 seconds, the tubes were Antiglobulin sera panel positive 800 400 (50%) 4ºC LISS wash anti-IgG positive, room centrifuged at 1000 x g for 10 seconds. The supernatant 761 37 (4.9%) was decanted, and 2 drops of sodium citrate resuspending temp wash anti-IgG negative solution were added. The tubes were gently shaken at a Direct Polybrene test only positive 667 15 (2.2%) 45-degree angle and examined for agglutination. Results Gel anti-IgG test only positive 434 0 were compared with concurrent positive and negative *Number tested does not include invalid results.

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these RBCs also reacted with anti-C3. The direct Polybrene test was also positive in the three samples that were tested. Eighteen of the 37 (49%) low-affinity IgG autoantibodies were tested by the gel test: five were reactive and 13 were nonreactive. We discontinued using the gel test in April 2005 as part of the DAT-negative AIHA workup.

Discussion We have previously reported on a 10-year series of 259 cases of hemolytic anemia in which an immune basis was suspected but the DAT was reported as negative.6 Similar to the results in the current series, 10 percent of the cases had positive DATs with commercial anti-IgG (e.g., used by hospitals) when tested in our laboratory. A battery of tests (the direct Polybrene test, direct polyethylene glycol test, DAT with cold washes, flow cytometry with fluorescein- Fig. 1 Distribution of DAT antiglobulin sera panel positive results. labeled anti-IgG, direct ELAT, a concentrated eluate, and the MMA) was used to detect RBC-bound IgG on the The direct Polybrene test was positive for 67 of 667 remaining samples. Thirty-four percent of the 234 samples (10%) specimens tested, and was the only positive test for were positive by at least one of the additional tests. Although 15 specimens. The 4ºC LISS wash technique was used for it was found that the direct Polybrene test was as efficient as 761 samples. Of these, low-affinity IgG was detected on 37 the more-complex flow cytometry or ELAT methods, some (4.9%) samples that did not react with anti-IgG using the samples that were positive by the latter were not positive by routine room temperature wash in the DAT AGS panel; the direct Polybrene test and vice versa. It was concluded 21 of these 37 samples reacted with anti-C3 in the routine that no one test is optimal, and a battery of tests would be test. The anti-IgG reactivity after the 4ºC LISS wash varied the most efficient approach for these DAT-negative AIHA from weak to 4+. Interestingly, three of these were readily cases. Subsequently, a battery of serologic tests that could detected (1+ to 2+) by the murine monoclonal anti-IgG be performed as a routine investigation in our IRL was (Immucor), but were nonreactive (n = 2) or only very weakly adopted. In the current series of 12 years of data generated (microscopically, n = 1) reactive with the rabbit anti-IgG from this battery of tests, we were able to provide some (Ortho).14 serologic evidence of an immune etiology in slightly more The anti-IgG gel test was positive in 57 of 434 (13%) than half of the cases submitted. tests, but was never the only positive test. The reactivity of Our panel of AGS detected immunoglobulin or ten samples that were positive by gel test (weak to 1+) but complement on RBCs of 50 percent of the samples that negative with anti-IgG by room temperature tube method were DAT negative at the hospital. The 13 percent positive is shown in Table 2. RBC-bound IgG was detected by the for IgG probably reflects differences in performing the DAT gel test in four samples that were nonreactive with anti-IgG between the referring and the reference laboratory; weak by tube test using room temperature or 4ºC LISS wash; agglutination can be easily shaken out and missed by less experienced technologists. Some of the 45 percent positive Table 2. Reactivity of ten samples of RBCs using anti-IgG by gel anti-C3 results may reflect a difference in technique plus test but nonreactive with anti-IgG in the routine tube test the use of a stronger rabbit anti-C3. Most of the anti-C3 Gel Anti-IgG by 4ºC Direct reactions were weak (≤1+), but 47 (13%) were moderately to Sample anti-IgG AGS panel LISS wash Polybrene test strongly reactive. RBC-bound IgM can be difficult to detect 1 1+ C3 only Negative Positive by the antiglobulin test13; fortunately, IgM antibodies 2 1+ C3 only Negative Positive associated with AIHA characteristically bind complement. 3 1+ C3 only 1+ NT IgA autoantibodies are uncommon, and for them to occur 4 1+ C3 only 4+ NT as the only detectable immunoglobulin in a patient with 1 5 1+ C3 only Micro NT AIHA occurs in only about 2 percent of AIHA. Low-affinity autoantibodies are uncommon, but they 6 1+ C3 only Negative NT may sometimes cause a false-negative DAT result because of 7 1+ Negative 1+ NT a loss of reactivity during the test process. These antibodies 8 Weak C3 only Negative Positive are susceptible to “elution” when the RBCs are washed with 9 1+ C3 only Invalid Positive room temperature or 37ºC saline. Washing RBCs with cold 10 1+ C3 only 1+ NT (4ºC) saline or LISS has been shown to enhance detection AGS = antiglobulin sera; NT = not tested. of low-affinity antibodies.1,15 In the current series, washing

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with 4ºC LISS enabled detection of IgG in 37 (5%) samples 3. Gilliland BC, Leddy JP, Vaughan JH. The detection of when the routine DAT (i.e., washing with room temperature cell-bound antibody on complement-coated human saline) was negative. red cells. J Clin Invest 1970;49:898–906. Column agglutination tests, e.g., gel test, have 4. Gilliland BC. Coombs-negative immune hemolytic sometimes yielded a positive DAT for IgG on RBCs that anemia. Semin Hematol 1976;13:267–75. are DAT-negative by routine tube test.16–20 This, however, 5. Petz LD, Garratty G. Acquired immune hemolytic may depend on the method and reagents used by the anemias. New York: Churchill Livingstone, 1980. manufacturer as various techniques and antibody sources 6. Garratty G, Postoway N, Nance S, Arndt P. Detection used in different countries are not identical. The increasing of IgG on red cells of patients with suspected direct use of the gel test in the United States may change the antiglobulin test negative autoimmune hemolytic failure rate of hospitals for detection of RBC-bound IgG anemia (AIHA). 1990 ISBT/AABB Book of Abstracts, because of poor technique when performing tube methods. p. 87. Column agglutination tests theoretically would be helpful 7. Garratty G, Leger RM, Hunt P, Co A. Serological in detecting low-affinity antibodies as the washing phase investigation of a large series of direct antiglobulin test- has been eliminated.21 There have been a few reports of low- negative hemolytic anemias (abstract). Transfusion affinity IgG detected using a cold-wash technique that were 2004;44(Suppl):121A–2A. also detected by column agglutination.17,22 In our study, two 8. Rubino M, Kavitsky DM, Nance S. Serologic testing in thirds of the low-affinity IgG detected using the 4ºC LISS autoimmune hemolytic anemia (AIHA) with negative wash were not detected by the ID-MTS gel anti-IgG test. direct antiglobulin test (DAT) (abstract). Transfusion The direct Polybrene test is rapid, but the test is 2002;42(Suppl):104S. technique-dependent. Polybrene (hexadimethrine 9. Fueger JT, Gottschall JL, Curtis BR, Johnson ST. bromide) causes nonspecific aggregation and close DAT negative immune hemolytic anemia—the role of approximation of normal RBCs permitting cross-linking enhanced techniques (abstract). Transfusion 2003; of antibody molecules. Aggregates are dispersed by 43(Suppl):103A. the addition of a sodium citrate solution, but antibody- 10. Reid ME. Autoagglutination dispersal utilizing mediated agglutination will persist after neutralization of sulphydryl compounds. Transfusion 1978;18:353–5. the Polybrene effect. The test can then be converted to an 11. Löw B, Messeter L. Antiglobulin test in low-ionic antiglobulin test (anti-IgG) if antibody is not detected after strength salt solution for rapid antibody screening and the aggregates are dispersed. The direct Polybrene test has cross-matching. Vox Sang 1974;26:53–61. previously been shown to be almost as sensitive as flow 12. Lalezari P, Jiang AF. The manual Polybrene test: a cytometry, and as sensitive as the ELAT, for detecting low simple and rapid procedure for detection of red cell levels of RBC-bound IgG.1,6,23 Others have also found this antibodies. Transfusion 1980;20:206–11. test to be useful for DAT-negative AIHA samples.8,24 13. Arndt PA, Leger RM, Garratty G. Serologic findings We had limited hematologic data and follow-up of the in autoimmune hemolytic anemia associated with patients in this study. The data reflect the results of tests on immunoglobulin M warm autoantibodies. Transfusion samples from patients who were suspected to have AIHA 2009;49:235–42. but the hospital laboratory had reported a negative DAT. 14. Arndt PA, Hunt PP, Garratty G. Suspected autoimmune The hematologists were seeking some support for their hemolytic anemia associated with RBC-bound IgG suspected diagnosis and therapy. The current study was not only detectable by 4C LISS wash method using designed to confirm earlier publications showing that such certain anti-IgG reagents (abstract). Transfusion tests are useful in the diagnosis of DAT-negative AIHA. The 2009;49(Suppl):129A. data were reviewed to see how many times in 12 years we 15. Garratty G, Arndt P, Nance S, Postoway N. Low affinity found RBC-bound proteins not detected in routine DATs autoantibodies—a cause of false negative direct performed at hospitals. The results in this large series show antiglobulin test. 1990 ISBT/AABB Book of Abstracts, that there is value in using an IRL experienced in reading p. 87. weak reactions, where special AGS are available, and where 16. Kroll H, Salama A, Berghöfer H, Mueller-Eckhardt tests to detect low-affinity and small amounts of RBC- C. The direct antiglobulin test: clinical significance bound IgG can be performed. and prospective comparison of column agglutination technology, gel test and tube test in 163 patients References (abstract). Vox Sang 1994;67(Suppl 2):65. 1. Petz LD, Garratty G. Immune hemolytic anemias. 2nd 17. Lai M, Rumi C, D’Onofrio G, Voso MT, Leone G. ed. Philadelphia: Churchill Livingstone, 2004. Clinically significant autoimmune hemolytic anemia 2. Garratty G. Immune hemolytic anemia associated with with a negative direct antiglobulin test by routine tube negative routine serology. Semin Hematol 2005;42: test and positive by column agglutination method. 156–64. Immunohematology 2002;18:109–13.

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18. Sajur J, Fournier Y, Sheridan B, Heddle NM. 23. Garratty G, Postoway N, Nance S, Brunt D. The Comparison of gel and tube techniques for detection of IgG on the red cells of “Coombs direct antiglobulin tests (abstract). Transfusion Negative” autoimmune hemolytic anemias (abstract). 1997;37(Suppl):27S. Transfusion 1982;22:430. 19. Chaudhary R, Das SS, Gupta R, Khetan D. Application 24. Owen I, Hows J. Evaluation of the manual of flow cytometry in detection of red cell bound IgG hexadimethrine bromide (Polybrene) technique in in Coombs negative AIHA. Hematology 2006;11: the investigation of autoimmune hemolytic anemia. 295–300. Transfusion 1990;30:814–18. 20. Dittmar K, Procter JL, Cipolone K, Njoroge JM, Miller J, Stroncek DF. Comparison of DATs using traditional Regina M. Leger, MSQA,MT(ASCP)SBB,CMQ/OE(ASQ) tube agglutination to gel column and affinity column (corresponding author), Research Associate, Asuncion Co, procedures. Transfusion 2001;41:1258–62. MT(ASCP)SBB, Senior Reference Specialist, Penny Hunt, 21. Lapierre Y, Rigal D, Adam J, et al. The gel test: a new MT(ASCP)SBB, Senior Reference Specialist, and George way to detect red cell antigen-antibody reactions. Garratty, PhD, FRCPath, Scientific Director, American Transfusion 1990;30:109–13. Red Cross Blood Services, Southern California, 100 Red 22. Sokol RJ, Booker DJ, Stamps R, Jalihal S, Paul B. Cross Circle, Pomona, CA 91768. Direct Coombs test-negative autoimmune hemolytic anemia and low-affinity IgG class antibodies. Immunohematology 1997;13:115–18.

Manuscripts The editorial staff of Immunohematology welcomes manuscripts pertaining to blood group serology and education for consideration for publication. We are especially interested in case reports, papers on platelet and white cell serology, scientific articles covering original investigations, and papers on new methods for use in the blood bank. Deadlines for receipt of manuscripts for consideration for the March, June, September, and December issues are the first weeks in November, February, May, and August, respectively. For instructions for scientific articles, case reports, and review articles, see Instructions for Authors in every issue of Immunohematology or on the Web at www.redcross.org/ immunohematology. Include fax and phone numbers and e-mail address with all articles and correspondence. E-mail all manuscripts to [email protected].

160 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Review

The pathophysiology and prevention of transfusion-related acute lung injury (TRALI): a review

D.C. Mair and T. Eastlund

Transfusion-related acute lung injury (TRALI) is a clinically or two-hit model.” The former has served as the basis for important complication of transfusion that is often difficult some recent preventive interventions that appear to be to diagnose, is probably underreported, and likely has a decreasing the prevalence of TRALI in the United States multifactorial origin that is incompletely understood, making it and internationally. challenging to find effective treatments and preventative steps. In this review we will address the definitions of TRALI The spectrum of its severity and clinical symptoms seems wide, but and the two commonly proposed pathogenetic mechanisms its pathogenesis is most likely associated with pulmonary damage from activated recipient neutrophils. Despite the pathogenesis of TRALI along with the central role the neutrophil plays in of TRALI being unclear, many severe cases are related to causing it, and we will specifically look at data from recent transfusion of donor WBC antibodies, and preventive measures animal models and clinical research. Finally, we will touch based on avoiding donations by multiparous donors have been on how our current understanding of TRALI formed the implemented at some sites, with early reports showing benefits. foundation of the preventive measures that have already This review will address some of the questions surrounding the been implemented. etiology of this potentially fatal reaction and how measures, predicated on many severe cases being related to transfusion of TRALI: Background and Definition plasma from multiparous donors, led to preventive steps to avoid A hallmark of TRALI is noncardiogenic pulmonary these donations. Immunohematology 2010;26:161–73. edema. When a transfusion is accompanied by severe Transfusion-related acute lung injury (TRALI) has shortness of breath, causes other than TRALI are often become one of the more frequently reported causes of the first to be considered, including hemolytic reactions, transfusion-related morbidity and mortality in several bacterially contaminated components, circulatory countries including the United States.1,2 From 2005 to volume overload, bronchospastic “anaphylactoid” allergic 2009, TRALI was the leading cause of patient death pulmonary reactions, and coincidental underlying attributable to blood components, comprising 48 percent of conditions such as pulmonary emboli, asthma, and anxiety all the fatal transfusion reactions reported to the Food and that can occur during a transfusion. Drug Administration (FDA).3 Because TRALI is suspected Reports of posttransfusion, noncardiogenic pulmonary to be underrecognized, underdiagnosed, and hence edema first surfaced in the 1950s.6,7 Since then two seminal underreported, these documented cases likely represent papers by Popovsky et al.8,9 in the 1980s described pulmonary just the tip of what has been called the TRALI iceberg.4 In reactions in patients from the Mayo Clinic and linked them other words, the true prevalence of TRALI is unknown, with donor leukocyte antibodies in what we now identify as and consequently its mortality rate may be higher than the a clinical entity called TRALI. The 1985 article depicted 36 number of reports would suggest. patients with acute respiratory distress and new infiltrates Because treatment for TRALI is basically supportive, on chest x-ray lacking an obvious cause such as circulatory and up to 10 percent of patients with it die even with overload or pneumonia.9 All of the reactions occurred within aggressive therapy,5 implementation of effective preventive 6 hours of administering the blood, and most of them took measures is urgently needed. However, preventing an illness place within 2 hours. Two of the patients died. In 89 percent usually requires a good understanding of its distinctive of the cases a WBC antibody was identified in a donor, and clinical characteristics and its causes, and the ready in 6 percent the antibody was in the patient. availability of diagnostic testing. At present the exact cause Since these landmark papers, TRALI has generally or causes of TRALI remain unclear. Its pathogenesis appears been considered to be mediated by the transfusion of complicated, and its clinical presentation is similar to that of components containing a donor antibody with specificity other illnesses and syndromes. In addition, there are no clear- to the recipient’s WBCs. Although happening rarely, cases cut diagnostic tests. Further studies into its pathogenesis are have been reported that seem to be related to a recipient needed to develop more effective therapies than the general antibody with specificity to donor WBCs contained in the supportive measures that are available today. transfusion. Because antibodies have not been identified in There are two leading theories on the pathophysiology all cases of TRALI, there has been much interest in TRALI of TRALI, the antibody-mediated theory and a “two-event, cases that do not seem to be antibody mediated.

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Studies have shown that other substances in stored groups selected the AECC definition for ALI versus the blood components may also cause TRALI, and this has clinically more severe acute respiratory distress syndrome been called the two-event, or two-hit, model of TRALI. In (ARDS), but added O2 saturation as another way to measure this model, endogenous inflammatory mediators produced hypoxemia.10–13 This gave physicians the flexibility of using in certain patients may result in priming and sequestration pulse oximetry to aid in the diagnosis of TRALI when of neutrophils in the lung. Substances that develop and an arterial blood gas had not been performed. It is also accumulate during blood component storage can activate noteworthy that these TRALI definitions rely on clinical the sequestered neutrophils of transfused recipients, information, and although the presence of WBC antibodies causing damage and alveolar permeability. This two-event in the donor or patient may support the diagnosis, neither theory was proposed as a major alternative to the antibody- definition required that testing be performed. mediated model of TRALI’s pathogenesis. Both antibody- Less severe forms of posttransfusion dyspnea are often mediated TRALI and the two-event model will be discussed not investigated for a diagnosis of TRALI, and the NHLBI in more detail in a later section. and Canadian Conference definitions did not address Since the 1980s, with improvements in infectious this less established entity called “mild TRALI.” Palfi et disease screening of donors and reduction of transfusion- al.14 demonstrated that plasma from multiparous donors, transmitted infections, TRALI gradually became recognized which was more likely to contain antibody (although the as a leading cause of transfusion-related morbidity and investigators did not test for this), induced a small, yet mortality. Partly in response to the increasing number of clinically significant, drop in the partial pressure of arterial 3 TRALI fatalities reported to agencies like the FDA, British oxygen to fraction of inspired oxygen (PaO2/FIO2) ratio Blood Services,1 etc., the National Heart, Lung, and Blood of 100 patients when compared with control plasma. The Institute (NHLBI)10 and a Canadian consensus conference11 authors suggested this slight hypoxia may represent a each convened experts to analyze the TRALI literature with milder form of TRALI. In an abstract published 3 years the ultimate goal of improving patient safety. Because the later, Palfi and associates15 noted the majority of TRALI cause of TRALI remains somewhat unclear, both forums reactions reported in south Sweden were considered to concluded that decreasing the number of TRALI cases be mild. They also acknowledged the need for an accurate posed potentially significant challenges given the current consensus definition. As research and our understanding of state of knowledge and that additional studies were needed. TRALI’s pathophysiology and incidence improve, hopefully To advance research on TRALI, a commonly accepted milder forms of transfusion-associated respiratory distress clinical description was viewed as indispensable. NHLBI will also be addressed. and the Canadian panel produced similar definitions based Retrospective “lookback” studies of recipients who on the American-European Consensus Conference (AECC) received transfusions containing donor WBC antibodies definition of acute lung injury (ALI),12 with one difference have provided evidence that donor antibodies frequently being the Canadian group created a separate entity called do not cause symptoms in transfused recipients and “possible TRALI” for patients who might have other risk also suggested there may be more subtle versions of the factors for lung damage besides transfusion (Table 1). Both reaction that qualify as mild TRALI.16,17 In these reports, an

Table 1. The American-European Consensus Conference (AECC) definition for ARDS/ALI and the National Heart Lung and Blood Institute and Canadian Consensus Conference definitions of TRALI

American-European Consensus Conference National Heart, Lung, and Blood Institute Canadian Consensus Conference definition definition of ARDS and ALI12 definition of TRALI10 of TRALI11 Acute onset New-onset ALI (i.e., no ALI exists before New-onset ALI (i.e., no ALI exists before transfusion) by AECC definition of ALI* transfusion) by AECC definition of ALI* Hypoxemia Clear temporal relationship to transfusion Clear temporal relationship to transfusion PaO2/FIO2 ≤ 200 mm Hg = ARDS PaO /FIO ≤ 300 mm Hg = ALI During or within 6 hours of completing the During or within 6 hours of completing the 2 2 transfusion transfusion Bilateral infiltrates on chest radiograph Patients may have other risk factors for ALI† No alternative risk factor for ALI PAOP ≤ 18 mm Hg when measured or no If risk factors other than transfusion are clinical evidence of left atrial hypertension Possible TRALI also present, patient’s clinical course may New onset ALI with a temporal relationship clarify whether ALI was caused by blood to a transfusion components or the alternative cause or both Lung injury also has a clear temporal relationship to an alternative risk factor for ALI†

ALI = acute lung injury; ARDS = acute respiratory distress syndrome; FIO2 = fraction of inspired oxygen; PaO2= partial pressure of arterial oxygen; PAOP= pulmonary artery occlusion pressure.

* NHLBI and the Canadian group expanded the criteria for hypoxemia to include O2 saturation of ≤ 90% on room air = ALI. † Causes of ALI other than transfusion may include aspiration, severe sepsis, pneumonia, shock, toxic inhalation, multiple trauma, lung contusion, burn injury, near drowning, acute pancreatitis, cardiopulmonary bypass, and drug overdose.

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antibody-positive donor was identified in the investigation of a severe or fatal TRALI. Evaluating prior recipients of components from these antibody-positive donors revealed previously unreported cases of milder respiratory distress. A minority of the recipients also had severe reactions that were suspicious for TRALI and should have been reported to the hospital’s transfusion service, but were not.17 This latter finding supports the contention that TRALI is often unrecognized and probably underreported.

The Pathophysiology of TRALI The Role of the Neutrophil in TRALI The clinical presentation of ARDS/ALI has much in common with the features observed in TRALI patients. Beyond the signs and symptoms there is also similarity in the histopathology insofar as neutrophils are concerned. For example, microscopic examination of postmortem lung Fig. 1. Neutrophil priming by inflammatory mediators results in samples from TRALI victims has shown sequestration enhanced superoxide production. The dashed arrow between the of neutrophils in the pulmonary vasculature and in the unprimed and primed state of the neutrophil indicates that priming alveolar space, analogous to what has been observed in may be a reversible process. fMLP = N-formyl-methionine-leucine- ARDS and ALI.18 Reviewing how the neutrophil functions phenylalanine; GM-CSF = granulocyte/macrophage colony stimulating factor; LPS = lipopolysaccharide; O2– = superoxide in pulmonary inflammatory processes, such as infection or anion; PAF = platelet-activating factor; TNFα = tumor necrosis lung injury, would be beneficial in understanding its role in factor α´. Reproduced with permission from Condliffe et al.20 either the antibody-mediated or the two-event model. The neutrophil is the first line of defense against pre-primed state. In most cases, this response to the bacterial infection, and it can generate a wide array of infection is short and limited to a level that is appropriate to molecules that are bacteriocidal and initiate inflammation remove the infecting agents without significant neutrophil- that can damage healthy cells and tissue in the same area. related damage to the surrounding tissues.22 To more efficiently eradicate infectious agents, neutrophils This same sequence of neutrophil priming, ideally go through a stage of priming first. Primed sequestration, and activation is believed to occur in the lung neutrophils have enhanced microbial killing capabilities in in TRALI, except the activated neutrophils release their response to a subsequent stimulus versus their unprimed toxic substances onto the pulmonary capillary, disrupting counterparts.19,20 Priming agents such as platelet-activating the barrier between the endothelium and lung epithelium, factor (PAF) or tumor necrosis factor ά (TNFά) potentiate and ending with edema in the alveolar space. In healthy both the oxidative and granular microbicidal arsenal when people the lung normally stores a relatively large amount the neutrophil is later exposed to an activating substance of the body’s available neutrophils (28%), and because this such as N-formyl-Met-Leu-Phe or fMLP (Figure 1).19 quantity increases even further with sequestration, the Priming starts when neutrophils home in on infected areas respiratory system may be more inclined to experience in response to chemoattractants that could be bacterially neutrophil-induced injury than other organs.22 Neutrophil derived or released when local endothelial cells become priming and activating agents that cause TRALI may activated. Increased expression of neutrophil adhesion spare nonpulmonary tissue because of the relatively low molecules occurs on the surface of both the neutrophil neutrophil burden outside of the lung. These substances and the endothelium.19,21,22 The neutrophils become less that trigger sequestration and cell damage for each of the deformable, further slowing their transit through the two TRALI theories are discussed in a later section. narrow pulmonary vascular beds, allowing more time and increased contact between the leukocytes and the now more The Role of Antibodies in TRALI receptive endothelium, leading to further accumulation of A causal relationship between the transfusion of WBC neutrophils (sequestration) at the site of infection.22 At this antibodies and ALI has been suspected for more than 50 point the neutrophils can be said to be primed.21,22 In an years. In 1957, Brittingham7 described a healthy research infection, the neutrophils then marginate into the tissue’s subject who exhibited symptoms of ALI after receiving a interstitium, where they can phagocytize the pathogens, leukoagglutinin-containing blood component. Popovsky resulting in neutrophil activation and destruction of the and colleagues8,9 in 1983 and 1985 crafted the term TRALI microbe through the release of reactive oxidative species and emphasized the association between TRALI and the and proteases.21 In this way, a primed neutrophil can passive transfusion of donor HLA or granulocyte-specific clear an infection more effectively than it could in its antibodies into recipients with corresponding antigens on

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Fig. 2. Three scenarios of how transfused antibodies might prime and activate the recipient’s neutrophils (A–C) to cause transfusion- related acute lung injury (TRALI). (A) Antibody directly binds to antigen on the neutrophil. (B) HLA class II antibody attaches to the patient’s monocytes, stimulating their release of inflammatory mediators, which ultimately affects neutrophils. C( ) Antibody to HLA class I antigen binds to pulmonary endothelium in the recipient. The neutrophil’s Fc receptor adheres to the constant region of the antibody, leading to sequestration in the lungs and activation. their leukocytes. In a minority of their cases, Popovsky’s can prime and hence enhance an fMLP-activated respiratory team found the antibodies in the patient and the reactions burst in antigen-positive leukocytes. Similarly, in another were seemingly initiated by infusion of incompatible donor experiment using a rat model, the release of reactive oxygen WBCs. species from the animal’s isolated neutrophils was increased Whether the antibody originated in the donor or the after they were incubated with antibodies that recognize patient, the interaction between the antibody and its regions of the rat MHC locus that are analogous to class I HLA neutrophil ligand can result in endothelial cell and alveolar A, B, and C loci in humans.24 This latter rodent experiment damage with capillary leak into the alveoli.22 Animal models also required fMLP to induce granulocyte activation. have provided more detailed insights into the antibody Several studies have suggested that antibodies can cause theory and have demonstrated some immunoglobulins are neutrophil activation and lung injury. In an ex vivo model, capable of both directly priming and activating neutrophils rabbit lungs developed evidence of edema (an increase in in TRALI (Figure 2A). pulmonary vascular permeability and lung weight) after Silliman and colleagues23 demonstrated in vitro that being injected with a combination of complement, human donor antibodies to the human neutrophil antigen (HNA) -3a antibody to HNA-3a, and human neutrophils positive for

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that antigen.25 No edema was seen in animals when HNA- TRALI as a result of donor HLA antibodies and evaluated 3a–negative neutrophils were substituted or if complement 18 other recipients of blood from the same donors. Of five was not added. In animals it seemed that anti-HNA-3a, plasma recipients only one was found to have undiagnosed in the presence of complement, activated antigen-positive TRALI, whereas another had an HLA type matching human neutrophils and caused lung injury. the donor antibody specificity but had no reaction. Of 13 A study by Sachs et al.,26 using another ex vivo lung RBC recipients, none developed a reaction. Kopko et al.17 rodent model, showed the antibody-antigen reaction could initiated a lookback investigation after a fatal TRALI cause lung injury independent of complement and that reaction was attributed to a granulocyte antibody directed TRALI may be more complex than simply antibody-to- against HNA-3a. This antigen is common throughout the WBC antigen recognition alone. In these experiments, rats population as it has been found in 89 percent to 99 percent were infused with human HNA-2a–positive neutrophils of tested individuals.30 The lookback study evaluated 36 and CD177 monoclonal antibody (an antibody that patients who previously received blood from that donor recognizes the HNA-2a antigen) without the addition of and identified 15 transfusion reactions in only 13 (36.1%) complement. A key variable here was that the quantity of of the recipients, and just 2 of these individuals were HNA-2a antigen expression can vary dramatically among initially diagnosed with TRALI by the treating physicians.17 different antigen-positive persons. Using quantitative Although an additional 7 of these 13 reacting patients had indirect immunofluorescence and flow cytometry, two severe respiratory problems that possibly should have been different sources of neutrophils were identified for this called TRALI, most of the 36 recipients had been transfused study: one from individuals with a high percentage of HNA- without incident. Muniz et al.31 also identified a donor with 2a–positive cells (defined as greater than 70% expression) an anti-HNA-3a whose blood had been given previously to and the second from low expressers (less than 30%). 11 patients. One experienced TRALI, another had chills, TRALI did not occur in these rats when the neutrophils but the remaining nine had no reactions. Fadeyi et al.32 came from individuals with a lower percentage of HNA- described a donor-derived anti-HNA-2a (the corresponding 2a, unless it was followed by injection of fMLP as an antigen is present in 95–99% of the population) that induced activator. Conversely, HNA-2a antibody alone appeared to dyspnea or leukopenia in 18 of 39 transfusion events, but no activate leukocytes and cause lung injury when cells from episodes of TRALI were found. As was discussed earlier, high expressers were used.26,27 On the basis of this study, variation in HNA-2a expression among antigen-positive neutrophil-activated TRALI reactions attributable to anti- individuals might explain why many of the recipients were HNA-2a appeared to be antigen dose–dependent (i.e., the asymptomatic or did not experience more severe pulmonary amount of antigen present was also important to neutrophil compromise. activation), although this could be overcome by the later Each of these lookback studies suggests that even in addition of another inflammatory mediator (e.g., fMLP). the setting of donor antibody with specificity for recipient antigen, TRALI usually does not occur. Even taking into Infusion of Donor-Derived Antibody Without Causing account potential TRALI cases that were initially missed, TRALI most recipients were asymptomatic. The reasons for this The observation made by Sachs and colleagues,26 that wide range of clinical responses are unclear, but may be infused antibody with specificity for recipient antigens related to individual variations in antibody titers or avidity, may sometimes be insufficient to induce TRALI, has also concentration of corresponding antigens on recipient cells, been demonstrated in a number of lookback studies on underlying diseases in recipients, variability in neutrophil transfused patients. In these publications donor antibody, reactivity to activated complement and cytokines, directed against antigens that most patients would be concentration of neutrophil adhesion molecules, and expected to carry on their cells, was not harmful in the reactivity of the recipient’s pulmonary vessels and alveoli. majority of recipients. Toy et al.16 identified only one patient with TRALI Antibodies to Class II HLA Antigens from among 103 recipients of blood from a donor with In addition to antibodies directed at class I HLA and multiple HLA antibodies. Fifty-four of these patients had a HNA specificities, later work showed that donor antibodies corresponding WBC antigen and no TRALI was observed. to HLA class II antigens can also be associated with Forty-two of these 103 patients were neutropenic, an TRALI,33 and these antigens are not normally expressed attribute that might have protected them against lung on neutrophils.34 Granulocytes have exhibited HLA-DR (a injury mediated by activated neutrophils. In another study, class II antigen) on their surface after exposure to agents Win and associates28 found no cases of TRALI in a lookback like granulocyte/macrophage colony-stimulating factor, analysis on 43 transfusions from six donors with leukocyte interferon (IFN)-γ, and interleukin (IL)-3,34 but this antigen antibodies, again directed against either multiple HLA or expression has not been documented in TRALI cases.22 neutrophil antigens having a relatively high frequency in Immunohistochemical stains performed on lung tissue the general population. Nicolle et al.29 reported two cases of from a TRALI fatality found HLA class II expression

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was limited only to pulmonary macrophages, despite injury did transpire if they first were infused with normal granulocyte accumulation in the capillaries and evidence Fc receptor–positive neutrophils and then received the the endothelium was activated.35 Activated pulmonary monoclonal antibody. These authors hypothesized that the endothelium may also demonstrate HLA class II antigens, infused class I MHC monoclonal antibody preferentially but pulmonary endothelium from the deceased TRALI bound to antigen present on the pulmonary endothelium, patient did not express class II antigens despite being and the antibody then sequestered circulating neutrophils positive for CD34, an indicator of endothelial activation.35 via the Fc receptor interaction, resulting in neutrophil Accordingly, some authors have postulated these antibodies activation and lung injury. The presence of Fc receptors on act indirectly on neutrophils by first binding to monocytes, neutrophils appeared to be a requisite feature for TRALI in which in turn release cytokines that sequester and activate this experiment. neutrophils to induce lung damage (Figure 2B).22 In an in Clinical evidence that TRALI can develop from vitro study in which Kopko et al.33 combined anti-HLA class transfusion of antibody with specificity to antigens on lung II antibodies with human monocytes of a corresponding tissue came from a patient who experienced unilateral HLA type, the cells became activated and produced TRALI.39 Ten weeks after a single-lung transplant, the the inflammatory cytokines IL-1β and TNFα, as well as patient became dyspneic and exhibited pulmonary tissue factor, and each of these inflammatory cytokines is infiltrates in the single transplanted lung but not in the capable of attracting and stimulating neutrophils.36 When native lung after receiving a unit of RBCs containing Nishimura and colleagues37 incubated antibodies to HLA- antibodies with HLA-B44 specificity. The recipient was DR15 with antigen-positive monocytes and cultured human HLA-B44 negative, but the transplanted lung was B44 lung microvascular endothelium, high levels of the cytokine antigen positive. Although the lung donor’s B44-positive leukocytes may have been circulating in small numbers, leukotriene B4 (a potent neutrophil attractant) were produced. Endothelial cells were also needed to interact the transfused antibody was presumed to have bound to the B44-positive transplanted lung itself. with the WBCs and antibody to generate leukotriene B4 in this model. From this it may be inferred that endothelium is The Two-Event Model and the Role of Biologically also a necessary part of the inflammatory process in TRALI. Reactive Mediators in TRALI Although much of the transfusion medicine literature Antibody Interaction With Lung Tissue and TRALI has focused on the antibody-mediated cause of TRALI, Presumably, the antigen-binding site of the offending the two-event model of ARDS has attempted to answer antibody is targeting a matching substance on either some of the questions that remain about TRALI’s origin 4 the neutrophil or the monocyte in TRALI. However, and especially how TRALI can develop in the absence 38 recent work by Looney et al., using a murine model of of an antibody.40 The first event is a preexisting clinical lung injury, argued that the antigen binding sites may condition in the patient (e.g., sepsis, recent surgery) also adhere to HLA antigens on the endothelium instead whereby the patient’s illness produces endogenous and that the Fc receptor of the circulating neutrophil generalized inflammation that affects the pulmonary interacts with the antibody’s constant region to alter endothelium. The endothelium can then become activated, the neutrophil’s circulation, leading to its sequestration expressing adherence molecules on its surface, resulting in and activation (Figure 2C). The investigators were able binding of the neutrophils to the pulmonary vasculature, to induce ALI (demonstrated by excess lung water and sequestration, and the neutrophils becoming primed.19,21,22 increased vascular permeability) in vivo by injecting the Neutrophil priming agents may be released from patient- mice with a monoclonal antibody targeting an antigen in derived sources in the postoperative period or by invading the murine class I MHC complex. Although flow cytometry infectious agents. Potential neutrophil priming substances, showed that the monoclonal antibody recognized class which may be produced by dying cells, WBCs, or activated I MHC antigen on neutrophils, this antibody to WBC pulmonary endothelium, include PAF, TNFα, and IL-8.41–43 antigen binding was not thought to have caused the lung Lipopolysaccharides (LPS) from bacterial cell walls may injury because the antibody did not directly activate the also serve in this capacity.44 cells. Microscopic examination of lung tissue from the The proposed second event is transfusion of a cellular affected mice confirmed there was marked pulmonary blood component containing various exogenous cytokines neutrophil sequestration, and immunohistochemistry or lipid-based biologically reactive mediators (also known revealed the monoclonal antibody preferentially bound as biologic response modifiers or BRMs) that stimulate to pulmonary endothelium over tissue in other organs. and activate the adherent neutrophils to release proteases, Despite other WBCs having Fc receptors, the mice did not which, as in the antibody-mediated model, causes exhibit lung injury if they were made neutropenic before endothelial cell and alveolar damage with noncardiogenic being challenged with the class I MHC antibody. TRALI pulmonary edema.40,45 also did not occur when antigen-positive mice that lacked These lipid-based substances seem to accumulate Fc receptors on their cells were given the antibody, but lung only in cellular components during storage and are not

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found in frozen plasma components.46–49 CD40 ligand, The investigators of a previously discussed murine a platelet-derived inflammatory mediator, as well as model, which argued the importance of the Fc-receptor lysophosphatidylcholines (Lyso-PCs), IL-6, IL-8, and on neutrophils, surprisingly found in later work that the others, have all been proposed to function as BRMs in this experiment was less likely to produce lung injury after the model.46–50 Older platelet components and packed RBCs mice had been moved to barrier-protected, pathogen-free contain greater amounts of these BRMs and thus appear to housing.53 The rodents from the prior study had been kept pose a greater risk of causing TRALI.5,48,51 in a “dirtier” environment and had a higher neutrophil Wyman et al.19 exposed monolayers of human count than their counterparts in the more sterile setting, pulmonary endothelium to one potential priming agent: which the authors attributed to a more primed state of bacterial endotoxin (i.e., LPS). In response, the cells the immune system in the previous group. Exposing the generated chemokines and expressed adhesion molecules. animals from the pathogen-free environment to LPS When neutrophils were later added, they became primed (analogous to a first event) restored the model’s ability to and adherent to the monolayer. Analogously to what is cause TRALI. Looney et al.53 interpreted this as evidence thought to happen in TRALI, the adherent neutrophils their experiments also represented a two-event model of were then activated by biologically active lipids from stored TRALI. Interestingly, they found not only that neutropenia packed RBCs (i.e., Lyso-PC) and damaged the endothelial was protective but that thrombocytopenia was as well. Mice cells. Blocking antibodies against the adhesion molecules pretreated with aspirin also did not experience lung injury, on the neutrophils and the endothelium prevented injury which suggests that anti-platelet therapy may have a role in to the monolayer. Also notable was that LPS and Lyso-PC the future treatment or prevention of TRALI. used individually only primed the neutrophils, but adding One limitation of the two-event model is not all them in sequence caused neutrophil activation and harm to investigators accept the premise of TRALI being limited 54 7 the monolayer. This showed that using two priming agents to the ill. For example, Brittingham’s healthy research together could result in activation. subject, appearing to lack a first event, exhibited clinical BRMs have also been demonstrated in the findings consistent with TRALI (dyspnea, fever, cyanosis, posttransfusion serum of patients who exhibited TRALI hypotension, leukopenia, and marked bilateral infiltrates reactions.40 When their post-TRALI serum was incubated on chest x-ray) after receiving just 50 mL of blood from a with neutrophils, a significantly greater superoxide release patient with a strong leukoagglutinin. Recently, a theory was observed on activation than when neutrophils were known as the threshold model attempted to explain this incubated with the same patient’s pretransfusion serum discrepancy between the Brittingham case and the two- or when the sera of recipients with febrile and urticarial event theory, while proposing answers to other TRALI reactions were used. This suggested the presence of a puzzles including the origin of mild TRALI and why antibody and corresponding WBC antigen does not produce neutrophil priming agent in these TRALI patients that TRALI in all cases. arose from the infusion of the blood component. Although BRMs are not found in frozen plasma The Threshold Model of TRALI 47–49 components, donor-derived antibodies can be found Many investigators now accept that TRALI appears to in these components. This explains the strong correlation be multifactorial with neutrophil activation and lung injury between frozen plasma transfusion and the development representing a final common pathway that can be initiated 52 of TRALI. Because antibodies can serve as the second by either an antibody or biologically active lipids.55 event, they can play a causal role in either the antibody Along these lines, the threshold model put forth by Bux hypothesis or the two-event model, and hence these two and Sachs56 attempts to merge the two TRALI theories. TRALI theories are not mutually exclusive. A recent study Consistent with its name, this model assumes neutrophils using a rat model demonstrated that either antibodies or must exceed a certain hypothetical threshold of activation BRMs can cause TRALI.24 As the first event, rats were to cause lung injury. The first and second events (and again injected intraperitoneally with LPS versus control animals the latter can be passive donor antibody) have additive that were injected with an equal volume of saline solution. inflammatory effects toward surmounting this theoretical For the second event the rodents were injected with either activation level needed to cause either a severe or a mild an antibody directed against class I MHC antigens or the TRALI reaction56 (Figure 3). Presumably, a mild TRALI extract from a 42-day-old RBC. Both antibody and the RBC case has a lower threshold than a serious one and no extract were able to produce pulmonary edema in the LPS- reaction is observed if the degree of activation falls beneath injected group but not in the control animals. Microscopic the mild level. examination in the rats with pulmonary injury documented This threshold model incorporates both of the leading neutrophil infiltration in the lungs, again demonstrating the theories and takes into account the observation that a importance of neutrophils in ALI. Furthermore, inducing broad variety of substances may be able to prime or activate neutropenia in the LPS-primed rodents was protective neutrophils. The patient’s threshold could be reached by against lung damage. the transfusion of an extremely potent antibody from a

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blood component (Figure 3: healthy research participant). thus would make a logical target for preventive action. Alternatively, the patient approaches the threshold by Previously pregnant female donors are more likely to have releasing his or her own endogenous inflammatory WBC antibodies and hence pose a greater risk than male substances as a result of a preexisting disease state, and donors of being associated with TRALI reactions. Several then transfusion of a less potent antibody or a moderate studies have supported preventing TRALI by restricting level of BRMs is sufficient to push the patient over the limit the use of plasma from parous donors. From 1996–2003 (Figure 3: patients 1 and 2). the United Kingdom’s hemovigilance program (Serious This model also addresses some of the gaps in our Hazards of Transfusion, or SHOT) analyzed 93 reported understanding of this transfusion reaction. In the rare TRALI cases and found frozen plasma and platelets circumstance of a healthy person exhibiting TRALI, the were respectively about 7 and 8 times more likely to be recipient would be assumed to contribute no endogenously the implicated component than a relatively low plasma- generated priming or activating agents to the process. containing component such as a packed RBC.57 Densmore Therefore, the blood component alone must have enough et al.58 found that 14.6 percent of female apheresis donors potency to push the recipient over the TRALI threshold who reported one or two pregnancies had HLA antibodies, without assistance from a preexisting illness. This may and this rate increased to 26.3 percent among multiparous have caused the transfusion-associated respiratory distress donors, defined as women with three or more pregnancies. in the research subject reported by Brittingham,7 and this These authors’ work was also recently confirmed in the uncommon event has been referred to by some as a “one- much larger leukocyte antibody prevalence study (LAPS), event” TRALI.4 From results of lookback studies, one which enrolled approximately 8000 donors, including could hypothesize that most patients did not experience about 5800 females. LAPS found the prevalence of HLA TRALI as a result of some combination of their not being antibodies in women with zero, one, two, three, and four ill enough and the blood donor’s antibody not being potent or more pregnancies was 1.7 percent, 11.2 percent, 22.5 enough. The dose dependency of the HNA-2a antigen in percent, 27.5 percent, and 32.2 percent, respectively.59 causing TRALI, found in the rat model used by Sachs et In October 2003, the British implemented a al.,26 may be another such example. When low expressers predominantly male-donor plasma program. SHOT data of HNA-2a antigen were used, antibody was insufficient to published after taking this initiative showed that highly overcome the threshold and fMLP had to be added to push likely or probable cases of TRALI, attributed to plasma, the neutrophils beyond the activation level needed to cause decreased from their preprogram level of 29 to a total of 2.57 It lung injury. Additional research would be needed to explore should be noted, the editorial accompanying this publication whether such variables truly play a role in the low reaction commented that the SHOT description of a highly likely or rates observed in these lookback cases. probable case was not purely clinical and relied in part on the presence of a WBC antibody.60 This differed from the NHLBI TRALI Prevention and Canadian TRALI definitions, which did not require Because treatment of TRALI is currently supportive, positive serology in the donor or patient. Accordingly, TRALI actions taken to prevent the reaction become the paramount cases caused by the other mechanisms (e.g., the two-event means of decreasing the morbidity and mortality associated model) and lacking antibody may not have been appropriately 60 with it. A very important first step for prevention of included or categorized. But given the reported success of TRALI is to avoid unneeded transfusions. Monitoring of the British male plasma initiative, and because TRALI was use, enforcement of guidelines, and education about the the leading cause of transfusion-related mortality in the judicious use of blood components should not be ignored. United States, Americans considered adopting this strategy. This is especially important for transfusions of plasma and Additionally, data from the American Red Cross was platelets that contain a larger portion of donor plasma than found to be consistent with the SHOT study’s findings. The other components. American Red Cross, which provides more than 40 percent Some of the more specific recommended approaches of the nation’s blood, analyzed 550 suspected TRALI cases, 52 for preventing TRALI have included changes to donor 72 of which were fatal, in the system for 2003 to 2005. In 71 screening practices such as gender-based exclusions or percent of these fatalities, a female, WBC antibody–positive implementing new tests. Others have suggested different donor was believed to be the cause, and in 75 percent, plasma 52 manipulations to the final component (e.g., washing), but was the transfused component. AABB followed the United regardless, all of the proposed interventions are based on Kingdom’s lead in 2006 and instituted recommendations the two leading theories of TRALI. for TRALI reduction: blood collection facilities needed to implement processes that lessen the chance of high-plasma Preventive Measures for Frozen Plasma volume–containing components being collected from donors Based on the antibody-mediated mechanism, at higher risk of having WBC antibodies.61 These processes components with the most plasma and highest antibody needed to be in place by the fall of 2007 for frozen plasma content would be more likely to cause TRALI and components (except cryoprecipitate, which was considered

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Fig. 3. The threshold model.58 Shown are three blood component recipients and the hypothetical levels of neutrophil activation needed to exceed their thresholds for a transfusion-related acute lung injury (TRALI) reaction. The gray bar represents the relative contribution of their own inflammatory mediators before transfusion, and the black bar is the contribution of mediators in the blood component, such as biologically reactive mediators (BRMs) or antibodies. In both patients 1 and 2, the blood component alone had insufficient inflammatory potential to exceed the threshold, and therefore the occurrence of either a severe (patient 1) or mild (patient 2) TRALI reaction relied on contributions from the recipients’ preexisting inflammatory mediators. The severity of the reaction is determined by the magnitude of neutrophil response. Unlike the first two, the third recipient was healthy before transfusion and thus had no endogenous mediators (i.e., lacked a first event). In this setting, all of the TRALI- producing mediators were provided by a potent blood component alone (e.g., as a strong leukoagglutinin), and hence this bar is completely black. The double arrow between the resting and primed state of the neutrophils indicates that the cells may deprime if activation does not occur.20,22,58 a low-volume, hence low-risk, component) and apheresis theoretically any single donor with a potentially harmful platelets by fall of 2008. WBC antibody should have the immunoglobulin diluted As in Britain, preliminary data in the United States below the level of clinical significance for a recipient.63 collected by the American Red Cross since establishment Studies have shown that no antibodies to HNA or HLA of a male-predominant plasma initiative revealed the class I or II antigens are detectable in this component even project has already had a positive impact.62 Plasma when they were present in some of the units of starting 64,65 distribution from male donors was 55 percent in 2006, 79 plasma. SD-plasma is not available in the United States percent in 2007, and 95 percent in 2008, and the number but has been used in Europe and no cases of TRALI have 65,66 of probable TRALI fatalities attributed to plasma in those been reported after use of over 13 million units. years were 6, 5, and then 0, respectively, with the last year Preventive Measures for Platelets being a statistically significant drop from the preceding 2 Although some were considering it at the time, none years. The quantity of nonfatal TRALI cases attributable of the countries with nationalized blood systems had yet to plasma also decreased dramatically from 26 in 2006 to implemented HLA or HNA antibody screening of their 7 in 2008. donors by the time of their participation in the 2007 An international forum published in 2007 revealed international forum.2 Testing for granulocyte antibodies that most of its participants had implemented the is generally considered to be labor intensive and does production of plasma from males only or used a solvent not readily lend itself to the high throughput needed for detergent-treated (SD) component.2 The latter is suspected processing blood.67 On the other hand, solid-phase assays to diminish the risk of TRALI because it is manufactured with shorter turnaround times and relevance to donor from pools of more than a thousand donations, so screening may become a reality in the near future now

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that several neutrophil antigens have been characterized that are less than 2 weeks old and platelets less than 3 days molecularly, including the clinically important HNA-3a.68,69 old).70,71 This might minimize accumulation of and exposure Platelet additive solutions, which have been licensed to BRMs that act as the second event in TRALI development. in some European countries, were also mentioned in the In the 2007 international forum, only Poland indicated they international forum, but because 100 mL of plasma may advise against giving blood older than 14 days to patients in remain in the final component, the boon to TRALI reduction severe clinical condition.2 However, concerns about blood is still in question.2 Several countries admitted to a variety availability and how to appropriately define fresh have of different initiatives to decrease the risk of TRALI from likely limited this practice. None of the other participants, this component, such as resuspending the pooled buffy which included representatives from South America, Japan, coat platelets in the plasma from one of the male donors, the United States, and several other European countries, whereas other countries restricted women with a history admitted to having interventions aimed at preventing TRALI of pregnancy from donating whole blood–derived platelets as a result of BRMs. Continued research is needed to more 2 (WBPs). fully understand the generation and accumulation of BRMs Recently, a number of American blood centers opted during storage of cellular blood components and to develop to meet the AABB’s 2008 deadline for apheresis platelets effective methods for their prevention or removal to reduce by implementing HLA antibody screening, as this was the risk of TRALI and other transfusion complications. technically less cumbersome than testing for granulocyte antibodies. Some use algorithms based on parity to more Conclusions strategically select which donors to perform HLA antibody testing on, and this practice is supported by the LAPS paper Many questions remain about the mechanism or likely that showed that asking about transfusion history brought mechanisms behind TRALI. Until recently, clinical research no additional benefit as there was no statistical difference in on this reaction was believed to be limited by the lack of a the rate of antibody positivity among untransfused males, commonly accepted definition. Despite these challenges, transfused males, and nontransfused females without current data suggest that TRALI has a pathogenesis that a history of pregnancy.59 As a result, blood centers might is multifactorial. It appears that transfusion of either decrease testing costs by choosing not to screen these three BRMs or antibodies can end in a final common pathway groups for HLA antibodies.58 The LAPS authors estimated of lung injury as a result of neutrophil activation. Why that deferring previously pregnant, HLA antibody–positive some patients may be more susceptible to TRALI than donors from apheresis collections would result in a others remains a mystery, but potential explanations potentially tolerable 6 percent decrease in platelet apheresis include severity of their preexisting illness or possibly donations.59 The AABB’s 2006 recommendations did not the amount of antigen expressed and therefore available suggest interventions for WBPs because any antibody to certain infused antibodies. The apparent, early success present was believed to be diluted by pooling.61 of preventive measures aimed at avoiding donations by multiparous donors suggests our understanding of TRALI Preventing TRALI as a Result of BRMs may be improving. Now that similar clinical definitions of To date, most of the actions to avoid TRALI reactions TRALI are available, and new insights have been provided have concentrated on preventing antibody-mediated by recent animal models, we can hope that forthcoming reactions. Interventions for decreasing BRMs infused clinical and laboratory research will provide even better into recipients have also been described, although not as preventive steps in the future, along with treatments that popularly implemented because of potential downsides. are curative instead of just supportive. Washing is one way to remove BRMs; however, because of the preparation time involved, washed RBCs and References platelets would likely be used only for planned transfusions and would be impractical for emergency or massive- 1. Stainsby D, Jones H, Asher D, et al. Serious hazards transfusion settings.67 Furthermore, little data exist on the of transfusion: a decade of hemovigilance in the UK. effectiveness of lipid removal by washing.67 Theoretically, Transfus Med Rev 2006;20:273–82. in a nonemergent setting, severely ill patients believed to 2. Wendel S, Biagini S, Trigo F, et al. Measures to prevent be at the highest risk for TRALI (e.g., in the intensive care TRALI. Vox Sang 2007;92:258–77. unit), could be scheduled to receive washed components, 3. U.S. Food and Drug Administration. Fatalities but this potential benefit would have to be weighed against reported to FDA following blood collection and the reduction in component quality and shelf life that is transfusion: annual summary for fiscal year 2009. caused by washing.67 Rockville, MD: U.S. Department of Health and Since cellular blood components develop higher levels Human Services, 2009. Available from: http://www. of biologically active lipids and cytokines during storage,47–49 fda.gov/BiologicsBloodVaccines/SafetyAvailability/ some investigators believe that patients with a preexisting ReportaProblem/TransfusionDonationFatalities/ illness might benefit from receiving fresher blood (e.g., RBCs ucm204763.htm.

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4. Boshkov LK. Transfusion-related acute lung injury 20. Condliffe AM, Kitchen E, Chilvers ER. Neutrophil and the ICU. Crit Care Clin 2005;21:479–95. priming: pathophysiological consequences and under- 5. Boshkov LK. Transfusion-associated acute lung injury lying mechanisms. Clin Sci (Lond) 1998;94:461–71. (TRALI): an evolving understanding of the role of 21. Silliman CC, Kelher M. The role of endothelial anti-leukocyte antibodies. Vox Sang 2002;83(Suppl activation in the pathogenesis of transfusion-related 1):299–303. acute lung injury. Transfusion 2005;45(Suppl):109S– 6. Barnard RD. Indiscriminate transfusion: a critique of 16S. case reports illustrating hypersensitivity reactions. N 22. Fung YL, Silliman CC. The role of neutrophils in the Y State J Med 1951;51:2399–402. pathogenesis of transfusion-related acute lung injury. 7. Brittingham TE. Immunologic studies on leukocytes. Transfus Med Rev 2009;23:266–83. Vox Sang 1957;2:242–8. 23. Silliman CC, Curtis BR, Kopko PM, et al. Donor 8. Popovsky MA, Abel MD, Moore SB. Transfusion- antibodies to HNA-3a implicated in TRALI reactions related acute lung injury associated with passive prime neutrophils and cause neutrophil-mediated transfer of antileukocyte antibodies. Am Rev Respir damage to human pulmonary microvascular Dis 1983;128:185–9. endothelial cells in a two-event in vitro model. Blood 9. Popovsky MA, Moore SB. Diagnostic and pathogenetic 2007;109:1752–5. considerations in transfusion-related acute lung 24. Kelher MR, Masuno T, Moore EE, et al. Plasma injury. Transfusion 1985;25:573–7. from stored packed red blood cells and MHC class 1 10. Toy P, Popovsky MA, Abraham E, et al. Transfusion- antibodies causes acute lung injury in a 2-event in vivo related acute lung injury: definition and review. Crit rat model. Blood 2009;113:2079–87. Care Med 2005;33:721–6. 25. Seeger W, Schneider U, Kreusler B, et al. Reproduction 11. Kleinman S, Caulfield T, Chan P, et al. Toward an of transfusion-related acute lung injury in an ex vivo understanding of transfusion-related acute lung lung model. Blood 1990;76:1438–44. injury: statement of a consensus panel. Transfusion 26. Sachs UJH, Hattar K, Weissmann N, et al. Antibody- 2004;44:1774–89. induced neutrophil activation as a trigger for 12. Phua J, Stewart TE, Ferguson ND. Acute respiratory transfusion-related acute lung injury in an ex vivo rat distress syndrome 40 years later: time to revisit its lung model. Blood 2006;107:1217–19. definition. Crit Care Med 2008;36:2912–21. 27. Lögdberg LE, Vikulina T, Zimring JC, Hillyer CD. 13. Ware LB, Matthay MA. The acute respiratory distress Animal models of transfusion-related acute lung syndrome. N Engl J Med 2000;342:1334–49. injury. Transfus Med Rev 2009;23:13–24. 14. Palfi M, Berg S, Ernerudh J, Berlin G. A randomized 28. Win N, Ranasinghe E, Lucas G. Transfusion-related controlled trial of transfusion-related acute lung acute lung injury: a 5-year look-back study. Transfus injury: is plasma from multiparous blood donors Med 2002;12:387–9. dangerous? Transfusion 2001;41:317–22. 29. Nicolle AL, Chapman CE, Carter V, Wallis JP. 15. Palfi M, Holthuis N. Does mild TRALI exist? Analysis Transfusion-related acute lung injury caused by two of one year transfusion complications from south donors with anti-human leucocyte antigen class II Sweden (abstract #M10.04). Vox Sang 2004;87 antibodies: a look-back investigation. Transfus Med (Suppl 3):S4. 2004;14:225–30. 16. Toy P, Hollis-Perry KM, Jun J, Nakagawa M. Recipients 30. Davoren A, Curtis BR, Shulman IA, et al. TRALI due to of blood from a donor with multiple HLA antibodies: granulocyte-agglutinating human neutrophil antigen- a lookback study of transfusion-related acute lung 3a (5b) alloantibodies in donor plasma: a report of 2 injury. Transfusion 2004;44:1683–8. fatalities. Transfusion 2003;43:641–5. 17. Kopko PM, Marshall CS, MacKenzie MR, Holland PV, 31. Muniz M, Sheldon S, Schuller RM, et al. Patient- Popovsky MA. Transfusion-related acute lung injury: specific transfusion-related acute lung injury. Vox report of a clinical look-back investigation. JAMA Sang 2008;94:70–3. 2002;287:1968–71. 32. Fadeyi EA, De Los Angeles Muniz M, Wayne AS, Klein 18. Dry SM, Bechard KM, Milford EL, Churchill WH, HG, Leitman SF, Stroncek DF. The transfusion of Benjamin RJ. The pathology of transfusion-related neutrophil-specific antibodies causes leukopenia and acute lung injury. Am J Clin Pathol 1999;112:216–21. a broad spectrum of pulmonary reactions. Transfusion 19. Wyman TH, Bjornsen AJ, Elzi DJ, et al. A two-insult in 2007;47:545–50. vitro model of PMN-mediated pulmonary endothelial 33. Kopko PM, Paglieroni TG, Popovsky MA, Muto KN, damage: requirements for adherence and chemokine MacKenzie MR, Holland PV. TRALI: correlation of release. Am J Physiol Cell Physiol 2002;283:C1592– antigen-antibody and monocyte activation in donor- 603. recipient pairs. Transfusion 2003;43:177–84.

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34. Gosselin EJ, Wardwell K, Rigby WF, Guyre NADPH oxidase through the platelet-activating-factor PM. Induction of MHC class II on human receptor. Vox Sang 1992;63:133–6. polymorphonuclear neutrophils by granulocyte/ 48. Silliman CC, Johnson CA, Clay KL, Thurman GW, macrophage colony-stimulating factor, IFN- Ambruso DR. Compounds biologically similar to gamma, and IL-3. J Immunol 1993;151:1482–90. platelet activating factor are present in stored blood 35. Kao GS, Wood IG, Dorfman DM, Milford EL, Benjamin components. Lipids 1993;28:415–18. RJ. Investigations into the role of anti-HLA class II 49. Silliman CC, Clay KL, Thurman GW, Johnson CA, antibodies in TRALI. Transfusion 2003;43:185–91. Ambruso DR. Partial characterization of lipids that 36. Bux J, Sachs UJH. Pathophysiology of TRALI. develop during the routine storage of blood and prime In: Kleinman SH, Popovsky MA, eds. TRALI: the neutrophil NADPH oxidase. J Lab Clin Med 1994; mechanisms, management and prevention. Bethesda, 124:684–94. MD: AABB Press, 2008:43–69. 50. Khan SY, Kelher MR, Heal JM, et al. Soluble CD40 37. Nishimura M, Hashimoto S, Takanashi M, Okazaki H, ligand accumulates in stored blood components, Satake M, Nakajima K. Role of anti-human leucocyte primes neutrophils through CD40, and is a potential antigen class II alloantibody and monocytes in cofactor in the development of transfusion-related development of transfusion-related acute lung injury. acute lung injury. Blood 2006;108:2455–62. Transfus Med 2007;17:129–34. 51. Silliman CC, Dickey WO, Paterson AJ, et al. Analysis 38. Looney MR, Su X, Van Ziffle JA, Lowell CA, Matthay of the priming activity of lipids generated during MA. Neutrophils and their Fcγ receptors are essential routine storage of platelet concentrates. Transfusion in a mouse model of transfusion-related acute lung 1996;36:133–9. injury. J Clin Invest 2006;116:1615–23. 52. Eder AF, Herron R, Strupp A, et al. Transfusion-related 39. Dykes A, Smallwood D, Kotsimbos T, Street A. acute lung injury surveillance (2003–2005) and the Transfusion-related acute lung injury (TRALI) in a potential impact of the selective use of plasma from patient with a single lung transplant. Br J Haematol male donors in the American Red Cross. Transfusion 2000;109:674–6. 2007;47:599–607. 40. Silliman CC, Paterson AJ, Dickey WO, et al. The 53. Looney MR, Nguyen JX, Hu Y, Van Ziffle JA, Lowell association of biologically active lipids with the CA, Matthay MA. Platelet depletion and aspirin development of transfusion-related acute lung injury: treatment protect mice in a two-event model of a retrospective study. Transfusion 1997;37:719–26. transfusion-related acute lung injury. J Clin Invest 41. Vercellotti GM, Yin HQ, Gustafson KS, Nelson RD, 2009;119:3450–61. Jacob HS. Platelet-activating factor primes neutrophil 54. Engelfriet CP, Reesink HW, Brand A, et al. responses to agonists: role in promoting neutrophil- Transfusion-related acute lung injury (TRALI). Vox mediated endothelial damage. Blood 1988;71:1100–7. Sang 2001;81:269–83. 42. Berkow RL, Wang D, Larrick JW, Dodson RW, 55. Swanson K, Dwyre DM, Krochmal J, Raife TJ. Howard TH. Enhancement of neutrophil superoxide Transfusion-related acute lung injury (TRALI): production by preincubation with recombinant human current clinical and pathophysiologic considerations. tumor necrosis factor. J Immunol 1987;139:3783–91. Lung 2006;184:177–85. 43. Daniels RH, Finnen MJ, Hill ME, Lackie JM. 56. Bux J, Sachs UJH. The pathogenesis of transfusion- Recombinant human monocyte IL-8 primes NADPH- related acute lung injury (TRALI). Br J Haematol oxidase and phospholipase A2 activation in human 2007;136:788–99. neutrophils. Immunology 1992;75:157–63. 57. Chapman CE, Stainsby D, Jones H, et al. Ten years of 44. Guthrie LA, McPhail LC, Henson PM, Johnston RB Jr. hemovigilance reports of transfusion-related acute Priming of neutrophils for enhanced release of oxygen lung injury in the United Kingdom and the impact of metabolites by bacterial lipopolysaccharide. Evidence preferential use of male donor plasma. Transfusion for increased activity of the superoxide-producing 2009;49:440–52. enzyme. J Exp Med 1984;160:1656–71. 58. Densmore TL, Goodnough LT, Ali S, Dynis M, Chaplin 45. Silliman CC, Bjornsen AJ, Wyman TH, et al. Plasma H. Prevalence of HLA sensitization in female apheresis and lipids from stored platelets cause acute lung injury donors. Transfusion 1999;39:103–6. in an animal model. Transfusion 2003;43:633–40. 59. Triulzi DJ, Kleinman S, Kakaiya RM, et al. The effect 46. Silliman CC, Voelkel NF, Allard JD, et al. Plasma of previous pregnancy and transfusion on HLA and lipids from stored packed red blood cells cause alloimmunization in blood donors: implications for a acute lung injury in an animal model. J Clin Invest transfusion-related acute lung injury risk reduction 1998;101:1458–67. strategy. Transfusion 2009;49:1825–35. 47. Silliman CC, Thurman GW, Ambruso DR. Stored blood 60. Hume HA. TRALI: moving toward prevention. components contain agents that prime the neutrophil Transfusion 2009;49:402–5.

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61. Strong DM, Lipton KS. Transfusion-related acute transfusion-related acute lung injury (TRALI). Crit lung injury. American Association of Blood Banks Care Med 2006;34(Suppl):S137–43. Bulletin#06-07, Nov 3, 2006. 68. Greinacher A, Wesche J, Hammer E, et al. 62. Eder AF, Herron RM Jr, Strupp A, et al. Effective Characterization of the human neutrophil alloantigen- reduction of transfusion-related acute lung injury 3a. Nat Med 2010;16:45–8. risk with male-predominant plasma strategy in the 69. Curtis BR, Cox NJ, Sullivan MJ, et al. The neutrophil American Red Cross (2006–2008). Transfusion 2010 alloantigen HNA-3a (5b) is located on choline Apr 30 [Epub ahead of print]. transporter-like protein 2 and appears to be encoded 63. Chapman CE, Williamson LM. UK hemovigilance and by an R>Q154 amino acid substitution. Blood 2010 the preferential use of male plasma. In: Kleinman 115:2073–6. SH, Popovsky MA, eds. TRALI: mechanisms, 70. Silliman CC, Ambruso DR, Boshkov LK. Transfusion- management and prevention. Bethesda, MD: AABB related acute lung injury. Blood 2005;105:2266–73. Press, 2008:143–60. 71. Goldman M, Webert KE, Arnold DM, Freedman J, 64. Sinnott P, Bodger S, Gupta A, et al. Presence of HLA Hannon J, Blajchman MA; TRALI Consensus Panel. antibodies in single-donor-derived fresh frozen Proceedings of a consensus conference: towards an plasma compared with pooled, solvent detergent- understanding of TRALI. Transfus Med Rev 2005;19: treated plasma (Octaplas). Eur J Immunogenet. 2004; 2–31. 31:271-4. 65. Sachs UJ, Kauschat D, Bein G. White blood cell- David C. Mair, MD (corresponding author), American reactive antibodies are undetectable in solvent/ Red Cross-North Central Region, 100 South Robert Street, detergent plasma. Transfusion. 2005;45:1628-31. St. Paul, MN 55107, and Ted Eastlund, MD, University of 66. Triulzi DJ. Transfusion-related acute lung injury: New Mexico School of Medicine, Department of Pathology, current concepts for the clinician. Anesth Analg 2009; Transfusion Medicine, Albuquerque NM. 108:770–6. 67. Mair DC, Hirschler N, Eastlund T. Blood donor and component management strategies to prevent

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IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 173 Original Report

Comparison of gel test and conventional tube test for antibody detection and titration in D-negative pregnant women: study from a tertiary-care hospital in North India

M.K. Thakur, N. Marwaha, P. Kumar, S.C. Saha, B. Thakral, R.R. Sharma, K. Saluja, H.K. Dhawan, and A. Jain

Conventional tube testing was used for antibody screening and introduced by Lapierre et al.3 in 1990 has gained popularity titration in D– pregnant women in our hospital until the recent as a result of its standardized performance, technical ease, introduction of the gel test. In this study we assessed the sensitivity stable end point, and versatility of methodology. There have of the gel test in our setup and tried to establish a correlation been many studies to show its superiority versus the tube between these tests for determining antibody titer. We collected test for antibody detection. The blood transfusion services 652 blood samples from 223 antenatal D– women during a span of 1 year. The samples were tested separately by the conventional tube in our country are gradually introducing gel technology for technique and the gel test for antibody detection and titration. The grouping, compatibility testing, and alloantibody detection tube test detected 84 (12.8%) positive samples as compared with owing to the many advantages. However, in some situations 93 (14.2%) by gel test, indicating the latter to be more sensitive such as HDFN not only the specificity of alloantibody but (p < 0.01). The gel test picked up weakly reactive anti-D that the also its titer has a direct impact on the fetus. Therefore, tube test missed. We did not use any enhancing media such as we evaluated the gel test and the conventional tube test LISS in titration studies performed by either method in an effort for antibody screening and titration to determine if there to establish a correlation. However, much higher titers (one- to is any correlation between the results obtained by using fivefold) were obtained by the gel test with no clear correlation both technologies and to find out whether critical levels with the corresponding tube values. When comparing the titer values to the finding of hydrops on ultrasound and Liley’s chart of alloantibody could be determined. We further studied OD reading on amniocentesis, a value of less than 128 (i.e., 64) obstetric management such as ultrasonography and by gel test corresponded to normal results. Through this study, intrauterine transfusion. We tried to correlate the titers we thus conclude that the gel test is more sensitive for antibody with these clinical interventions. detection, although a linear correlation could not be established for titers. Clinical correlation may point toward a critical titer of Material and Methods 64 for the gel test, but further studies need to be done to support The duration of study was from July 2005 to June 2006, this finding. Immunohematology 2010;26:174–77. during which a total of 652 clotted samples were collected Key Words: alloantibodies, antibody screening, titration, gel from 223 antenatal D– women. The study was conducted test, D-negative women after approval from the Institute Ethics Committee. A written informed consent was taken from the patients for Hemolytic disease of the fetus and newborn (HDFN) the study. was first reported in 1609,1 although the discovery of the Rh system was made in 1939 and the implication of D antigen Sample Collection in its pathogenesis in 1940.2 However, to the present day, A 3-mL clotted sample was collected, which was then researchers and clinicians alike have worked to unravel its centrifuged and the serum separated. Serum was stored in pathogenesis, effects, prevention, and treatment. a frozen state at –80°C in two aliquots each until further Anti-D is by far the most common cause of HDFN testing. The first sample was taken in the first trimester or at although its incidence has drastically fallen with antenatal the time of the first visit. Subsequent samples were taken in D immunoglobulin use as prophylaxis. Other antibodies every trimester and at 28 weeks before the administration are also implicated, and their prevalence has been studied of anti-D immunoglobulin. Immunized women were in the Western world but such data are lacking in the followed up every 3 to 4 weeks. Indian population. The antibodies can be identified as well as semiquantitated from the mother’s serum. Titer Antibody Detection determination of anti-D by tube test helps in the clinical The serum was thawed to room temperature before decision to proceed with an invasive procedure such as being tested. Antibody detection was done in parallel by the amniocentesis. conventional tube technique and the gel test. A commercially The conventional tube test has stood the test of time available three-cell screening panel (DiaScreen; DiaMed, in both antibody detection and titration. The gel test Cressier sur Morat, Switzerland) was used. For tube testing,

174 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Comparison of gel test and tube test

a 3% suspension in normal saline was used, and for gel The findings were compared with the titer values obtained testing, a 0.8% suspension in LISS was used. by both methods. All women with titer values of 16 or above by the conventional tube method were further evaluated by Tube IAT amniocentesis. The OD values were plotted on Liley’s chart5 One drop of RBC suspension was added to two drops of and compared with the titer results by both methods. serum to be tested into labeled test tubes. The tubes were Statistical analysis was performed using SPSS software incubated in a water bath at 37°C for 60 minutes. After version 13.0 (SPSS, Inc., Chicago, IL). three washes with normal saline solution, polyspecific anti- human globulin was added to the RBC button and the tube Results was centrifuged at 3000 x g for 15 seconds in an appropriate On antibody screening, the tube technique detected 84 centrifuge. The tubes were examined for agglutination. (12.8%) positive results, whereas the gel test detected 93 The reactions were graded and recorded as per the AABB (14.2%) positive results individually. On comparison, 83 of 4 Technical Manual. the 84 positive by tube were also positive by gel, whereas 1 of the 84 was missed by gel. On the other hand 10 samples that Gel Test were positive by gel had been missed by the tube technique. We dispensed 50 µL of RBCs into labeled gel cards On identification the sample missed by gel was anti-Lea in and added 25 µL of the serum to be tested. The cards were specificity, and all samples missed by tube were anti-D in incubated for 15 minutes at 37°C in specially designed specificity. Both the tests showed a positive correlation in incubators. They were then centrifuged at 1050 rpm for 10 antibody detection that was significant with a Spearman’s minutes. The reactions were graded and recorded. Antibody correlation of +0.842 (p < 0.01). However, the gel technique identification was done using commercially available panels proved to be more sensitive than the tube technique (p < (DiaMed) according to the manufacturer’s instructions. 0.01). Table 1 shows the profile of antibodies detected. Antibody Titration Table 1. Comparison of antibodies detected by both methods Titration was performed on those samples that were Sample tested Tube Gel positive on antibody screening. Serial twofold dilutions Total 652 652 were made in normal saline solution in clean test tubes. The dilutions were tested in parallel for both tests. In-house Tests positive 84 93 Anti-D (all) 73 83 prepared R1R1 RBCs from a single donor were used for D antibodies, whereas RBCs with heterozygous expression Anti–D (passive) 11 21 were used for Lea and Leb antibodies. The RBCs were washed Anti-C* 2 2 three times in normal saline solution and resuspended Anti Lea 6 5 to a final concentration of 3% and 0.8% in normal saline Anti Leb 5 5 solution for tube testing and gel testing, respectively. *Anti-C was found in association with anti-D. Critical titer was taken as 16 by the tube technique followed in our institute. Table 2 compares the titer values obtained by both methods for D antibodies. The Lewis antibody titer values Tube Test The method used was the same as that for antibody are compared in Table 3. The titers on gel were generally detection. higher than those obtained by the tube technique, and as the tube titers increased, the gel titers also increased. The titer increase with the gel test was higher when compared Gel Test The RBCs used were suspended in normal saline solution with the tube test. The values varied from onefold to fivefold to a final concentration of 0.8%, and the cards incubated at (mean, 1.6-fold). The observed differences were onefold in 37°C for 60 minutes. The remainder of the procedure was 21 sera, twofold in 32, threefold in 18, fourfold in 3, and the same as for antibody detection. Titers were taken as fivefold in 1. the highest dilution that gave 1+ agglutination. The gel test Obstetric Studies had been modified from the manufacturer’s guidelines for Ultrasonography titration. LISS was not used in the process, and the time of The titers were compared with the fetal findings on incubation was increased from 15 minutes to 60 minutes. ultrasonography, i.e., normal vs. hydropic. All the women We tested several samples in parallel with the standard method and found that deviating from the original method with alloantibodies were investigated. The mean titer value did not miss any antibodies. for those who had normal ultrasonographic findings was The women who were positive for alloantibodies were 62.39 by tube technique with a standard deviation of 114.5 investigated with ultrasonography for features of hydrops. and titers ranging from 1 to 512. By the gel technique, the

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 175 M.K. Thakur et al.

Table 2. Comparison of antibody titers on tube with those on gel test for anti-D

Tube Gel titers titers Neg 1 2 4 8 16 32 64 128 256 512 1024 2048 Neg — — 6 4 — — — — — — — — — 1 — 2 2 5 2 1 — — — — — — — 2 — — 3 3 3 — — — — — — — — 4 — — — — 2 2 2 — — — — — — 8 — — — — 2 2 — 1 1 — — — — 16 — — — — — — 1 2 1 — — — — 32 — — — — — — — 1 4 3 1 1 — 64 — — — — — — — — 1 3 2 — — 128 — — — — — — — — 1 7 4 3 — 256 — — — — — — — — — — 2 1 — 512 — — — — — — — — — — — — 2 Total samples—83

Table 3. Titer values for Lewis antibodies by both methods Table 5. Median titer values by both methods for different zones of amniocentesis Lewis Number of antibody Corresponding samples per titer Zone as per Liley’s Tube titer range Gel titer range type Tube titer value Gel titer value chart (n) (median) (median) Lea 1 Neg 1 Low zone (4) 16–64 64–512 2 2 5 (32) (128) Leb 4 4 4 Mid zone (6) 32–512 128–2048 8 4 1 (64) (256) Total 11 High zone (8) 128–512 256–2048 (128) (718) mean value was 271.04 with a standard deviation of 460.27 n = Number of women falling in each group. and a range of 2 to 2048. The mean titer value for those who had hydropic findings on ultrasound was 182.86 by tube Discussion technique with a standard deviation of 145.138 and titers Rh antigens are by far the most common cause of ranging from 128 to 512. By the gel technique, the mean alloimmunization in pregnant women. The D antigen is value was 841.04 with a standard deviation of 604.259 and the most immunogenic; yet, alloimmunization caused by it a range of 256 to 2048. has been virtually eliminated in the Western world. Use of When compared using nonparametric tests, the antibody screening and titration studies aid the obstetrician difference between the two groups was significant by the in deciding among early preventive or treatment modalities tube method (p = 0.02) and also by gel test (p = 0.03). to manage HDFN. Table 4 shows the median values for normal and hydropic Risk of HDFN can be diagnosed early in pregnancy findings using both techniques. with noninvasive serologic methods such as the indirect antiglobulin test to detect the presence of irregular Table 4. Median titer values by both methods for antenatal ultrasound (USG) showing normal vs. hydropic changes antibodies in the maternal serum. Usually the firstborn is the initiator of sensitization to antigens present on fetal Median titer Normal on USG Features of hydrops RBCs. This occurs mostly at delivery. During subsequent Tube 32 128 pregnancy reexposure to the same antigen initiates a Gel 32 256 secondary immune response and with the potential for the pathogenesis of HDFN. Antibody that has been detected in maternal serum must be identified. The antibody can Amniocentesis then be quantitated using laboratory methods such as The titer values by both methods were compared with titration studies. The titration studies are useful in guiding the need for amniocentesis, which was performed on all the timing of clinical intervention required in utero. The women with a tube titer greater than 16. The values for the noninvasive serologic methods precede the more invasive corresponding zones on Liley’s chart were compared by methods such as amniocentesis, which is associated with both methods. The comparison is shown in Table 5. fetal morbidity and mortality.

176 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Comparison of gel test and tube test

The higher sensitivity and versatility of the gel test References compared with the tube test have been reported in various 1. Bowman J. Thirty-five years of Rh prophylaxis. studies.6,7 A recent study comparing the conventional tube Transfusion 2003;43:1661–6. test with the gel technique for crossmatch also showed the 2. Landsteiner K, Wiener AS. An agglutinable factor in 8 latter to be more sensitive. Other positive points of the gel human blood recognized by immune sera for rhesus test include smaller volume of sample required, elimination blood. Proc Soc Exp Biol Med 1940;43:223–224. of washing steps, stable results, and easy readability. The 3. Lapierre Y, Rigal D, Adam J, et al. The gel test: a new tube test in our study missed 10 examples of anti-D that the way to detect red cell antigen-antibody reactions. gel test detected. However, the anti-D in these samples were Transfusion 1990;30:109–13. all attributable to antenatal anti-D immunoprophylaxis and 4. Brecher ME. Antibody detection, identification, and they became undetectable after 4 to 6 weeks. Thus, the tube test did not miss any of the significant antibodies. Data compatibility testing. In: Roback JD, Combs MR, are also presented that indicate the propensity of the gel Grossman BJ, Hillyer CD, eds. Technical manual. 16th test to miss clinically insignificant antibodies like Lea.7 The ed. Maryland: American Association of Blood Banks, gel test in our study missed one sample of anti-Lea. Nine 2008: 899-917. samples of anti-Lea and anti-Leb were, however, detected by 5. Liley AW. Liquor amnil analysis in the management both methods. The titration of antibodies by both methods of the pregnancy complicated by rhesus sensitization. showed variable results. Similar data have been published Am J Obstet Gynecol 1961;82:1359–70. by Novaretti et al.9 in which they tested gel and tube titers 6. Judd WJ, Steiner EA, Knafl PC. The gel test: sensitivity for anti-D. They found the titers to be threefold to eightfold and specificity for unexpected antibodies to blood higher. Our study showed the titers to be one- to fivefold group antigens. Immunohematology 1997;13:132–5. higher. The gel titers tended to be higher when compared 7. Delaflor-Weiss E, Chizhevsky V. Implementation of with the tube titers. However, no correlation between the gel testing for antibody screening and identification two methods could be found. in a community hospital, 3-year experience. Lab Med 10 The recommended titration study by Judd is a saline 2005;36:489–92. antiglobulin procedure with 60-minute incubation at 37°C. 8. Lange J, Selleng K, Heddle NM, Traore A, Greinacher In our study, we did not use LISS for gel titration, which A. Coombs’ crossmatch after negative antibody is a known enhancing medium, in an attempt to establish screening—a retrospective observational study a correlation, if any exists, between the conventional tube technique and the gel technique. Judd also stated that comparing the tube test and the microcolumn until substantial data are available that show correlation technology. Vox Sang 2010;98:e269–75. between gel microcolumn assay and saline tube antiglobulin 9. Novaretti MC, Jens E, Pagliarini T, Bonifácio SL, titers IgG gel column technology should not be used for Dorlhiac-Llacer PE, Chamone DA. Comparison of prenatal antibody titration. The critical tube titers of 16 conventional tube test with DiaMed gel microcolumn corresponded to gel titers ranging from 32 to 128. Hence, assay for anti-D titration. Clin Lab Haematol 2003;25: it is difficult to arrive at a definitive conclusion of critical 311–15. gel titers when a direct correlation is attempted. When the 10. Judd WJ; Scientific Section Coordinating Committee titers were compared with ultrasonography for hydrops of the AABB. Practice guidelines for prenatal and and amniocentesis OD values as per Liley’s chart, a gel perinatal immunohematology, revisited. Transfusion value of less than 128 (i.e., 64) corresponded with normal 2001;41;1445–52. ultrasonography and low zone OD values. However, this value should be interpreted with caution until more studies Manish K. Thakur, MD (corresponding author), Senior support it. Resident, and Neelam Marwaha, MD, Professor and Head, Based on the above observations, we conclude that Department of Transfusion Medicine, Praveen Kumar, the gel test is a better method than the conventional tube MD, DM(Neonatology), Additional Professor, Department test for antibody detection because of its higher sensitivity and technical safety. Titration by gel, however, should of Pediatrics, Subhash C. Saha, MD, Associate Professor, not be considered for antenatal HDFN management as Department of Obstetrics and Gynecology, and Beenu gel titers do not show linear correlation with tube titers, Thakral, MD, Assistant Professor, Ratti Ram Sharma, MD, which predict fetal outcome in RhD sensitized women. Assistant Professor, Karan Saluja, MD, Assistant Professor, Developing countries work under resource constraints, Hari Krishan Dhawan, MD, Senior Resident, Department and such studies would optimize cost-effective use of this of Transfusion Medicine, and Ashish Jain, MD, Senior technology. Resident, Department of Transfusion Medicine, Post Graduate Institute of Medical Education and Research, Sector 12, Chandigarh, India 160012.

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 177 Review

The Rh and RhAG blood group systems

S.T. Chou and C.M. Westhoff

History the International Society of Blood Transfusion (ISBT) Rh is the most well-recognized blood group system Working Party on Red Cell Immunogenetics and Blood after ABO because of the immunogenicity of the principal Group Terminology maintains a list of alleles that encode antigen, D. Discovered more than 70 years ago, the D blood group antigens (see Web Resources). antigen is well known as the target in severe and potentially fatal hemolytic disease of the fetus and newborn (HDFN).1 Terminology and Nomenclature Today, HDFN caused by anti-D is much less frequently The term “Rh” for Rhesus resulted from work by observed in developed countries owing to prevention by Landsteiner and Wiener, who found that antiserum administration of Rh immunoglobulin (RhIgG; anti-D produced by immunizing guinea pigs and rabbits with RBCs 2,3 IgG) after delivery of a D+ infant. Children of women of the Rhesus macaque agglutinated 85 percent of human of European extraction are more likely affected as D– is RBCs. Initially, they believed that a common factor “Rh” most common in Caucasians (15–17%), less common in had been identified in animals and humans.11 The antibody African Blacks (3–5%), and uncommon or rare in Asians actually detected the LW antigen, which is present on D+ 4 (<0.1%). Thus, the D antigen status is not routinely tested RBCs in greater amounts than on D– RBCs. Although for in some parts of Asia, principally China, where D– is misnomers, the terms Rh factor and anti-Rh continued considered a rare blood type. HDFN caused by anti-D is still in common usage to describe the human D antigen and seen where women have limited access to prenatal care and the D antibody. Although the use of “Rhesus” is now in countries where RhIG administration is not standard discouraged, it has been pointed out (WA Flegel, personal practice. Anti-D during pregnancy is also more often seen communication) that “Rh” can be difficult to translate into if RhIG is not administered during the third trimester of other languages. pregnancy in addition to after delivery. The policy of giving Over the years, four Rh system nomenclatures have antepartum RhIG to all D– women has not been adopted in been introduced (Table 1). Terminology established by all countries. Fisher-Race in England was based on the premise that The Rh system is a complex RBC antigen system that three closely linked genes, C/c, E/e, and D, were responsible includes more than 50 different antigenic specificities for the antigens, whereas the Wiener nomenclature (Rh- (Table 1). “Rh-positive” and “Rh-negative” refer to the Hr) was based on the belief that a single gene encoded presence or absence of the D antigen. The five principal Rh several blood group factors. The Rh system is encoded antigens—D, C, c, E, and e—are responsible for the majority 12 of clinically significant antibodies. Some of the others by two genes, RHD and RHCE, as predicted by Tippett. represent compound specificities, i.e., ce or f is an epitope Rosenfeld gave each Rh antigen a number based on the on the Rhce protein that is not present on RBCs with order of its discovery or assignment to the Rh system. The RhcE or RhCe. Many of the antigens are found primarily ISBT Working Party on Terminology for Red Cell Surface 13 in a population group or specific ethnic group; for example Antigens adopted a six-digit number for each RBC antigen. CX has the highest prevalence in Finns, and V and VS are The first three numbers represent the system (the number found primarily in African Black ethnic backgrounds 004 was assigned to the Rh system), and the remaining (summarized in Reid and Lomas-Francis5). three digits refer to the antigenic specificity (Table 1). Our understanding of the Rh system has been greatly Current terminology distinguishes the genes and the advanced since the genes were cloned in the early 1990s. The proteins from the antigens. Capital letters, with or without sequence of RHCE was first reported in 1990,6,7 and RHD italics, are used when referring to the RH genes, RHD and was subsequently sequenced in 1992.6,8 The different RHCE RHCE. Alleles of RHCE are designated after an asterisk, i.e. alleles responsible for the C or c and E or e antigens were RHCE*ce, RHCE*Ce, RHCE*cE, RHCE*CE, according to clarified in 1994.9 In the last decade, molecular genotyping which antigens they encode. Alleles of RHD are designated has revealed that the genetic diversity of the RH locus RHD*DVI, RHD*DIIIa, RHD*weak D type 2, and so on, greatly exceeds estimates predicted by serology. More than according to the partial or weak D encoded, and the alleles 170 RHD and more than 70 different RHCE alleles have have also been given numbers by the ISBT Working Party been described, and new alleles are still being discovered. A on Red Cell Immunogenetics and Blood Group Terminology directory of RHD alleles is maintained on the RhesusBase (see Table 2 for examples and Web Resources). Lastly, the website, and RHCE alleles are found on the National Center Rh proteins are indicated without italics and with the “h” for Biotechnology Information (NCBI) human blood group in lowercase, as RhD or RhCE, or according to the specific mutation Web site.10 In an effort to standardize terminology, antigens they carry, Rhce, RhCe, RhcE, or RhCE.

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Table 1. Rh and RhAG blood group systems: antigen Genetics and Inheritance terminology/nomenclature RH Genes and Rh Proteins Fisher-Race ISBT Rosenfeld Wiener Other names RH genes are inherited as codominant alleles. RHD DCE encodes the D antigen and RHCE encodes CE antigens Rh (ce, cE, Ce, or CE). Each gene has ten coding exons, and 004001 Rh1 D Rh 0 the RhD and RhCE proteins differ by 32 to 35 amino acids 004002 Rh2 C rh′ 004003 Rh3 E rh″ (depending on the specific CE allele). The large number 004004 Rh4 C hr′ of differences explains why D, when seen by the immune 004005 Rh5 E hr″ system of a D– person who lacks the protein, often induces 004006 Rh6 ce Hr a robust immune response. The large number of amino 004007 Rh7 Ce rh i acid differences also explains the numerous epitopes of 004008 Rh8 CW rhW Willis the D antigen, estimated to range from 9 to more than 004009 Rh9 CX rhX 14 004010 Rh10 V hrV 30. Although only nine or ten of the amino acid changes 004011 Rh11 EW rhW2 are predicted to be on the extracellular surface of the RBC 004012 Rh12 G rhG membrane, changes located in the transmembrane and Rh13, 14, 15, 16 obsolete cytoplasmic regions also alter the topology and epitopes 004017 Rh17 Hr 0 of the protein. Although early on it was assumed that only 004018 Rh18 HrS Shabalala 004019 Rh19 hrS Shabalala extracellular changes would prompt an immune response to 004020 Rh20 VS Rh proteins, new antigens identified by an antibody response 004021 Rh21 CG are also generated with intracellular changes. For example, 004022 Rh22 CE Rh Jarvis the Leu245Val change in Rhce proteins is not extracellular, 004023 Rh23 DW Wiel but this change results in expression of the novel RBC Rh24 and Rh25 obsolete antigens V and VS, which are also serologic markers for an 004026 Rh26 c-like Deal altered e antigen. Patients who are homozygous for altered 004027 Rh27 cE rhi 004028 Rh28 hrH Hernandez Rhce alleles encoding partial e antigens type serologically 004029 Rh29 Total Rh as e+, but are at risk for anti-e production when exposed to 004030 Rh30 DCor Gonzalez, Goa conventional Rhce protein. 004031 Rh31 hrB Bastiaan The RHCE gene encodes C/c and E/e antigens on a single N 004032 Rh32 R protein. C and c differ by four amino acids, whereas the E 004033 Rh33 Har, DHar 004034 Rh34 HrB Bastiaan and e antigens differ by only one amino acid. Although the 004035 Rh35 1114 common alleles are RHCE*ce, RHCE*Ce, and RHCE*cE, a 004036 Rh36 Bea (Berrens) large number of alleles with additional polymorphisms are 004037 Rh37 Evans found in different ethnic groups and are discussed in a later Rh38 obsolete (see Duclos below) section. 004039 Rh39 C-like 004040 Rh40 Tar, Targett 004041 Rh41 Ce-like Molecular Basis of Rh Antigens CceS, 004042 Rh42 CeS rh s D Antigen i Thornton 004043 Rh43 Crawford D Negative (Rh-Negative) 004044 Rh44 Nou Several genetic mechanisms are responsible for the 004045 Rh45 Riv 004046 Rh46 Sec D– phenotype, but deletion of the RHD gene is the primary 15 004047 Rh47 Dav background. Some exceptions include silenced RHD as a 004048 Rh48 JAL result of point mutations, nucleotide insertions, premature 004049 Rh49 STEM stop codons, or RHD-CE hybrids. In African Blacks, 66 004050 Rh50 FPTT percent of D– individuals have a 37-bp insertion in RHD 004051 Rh51 MAR causing a premature stop codon, whereas 15 percent carry 004052 Rh52 BARC 004053 Rh53 JAHK a hybrid RHD-CE-D characterized by expression of weak 16 004054 Rh54 DAK C but no D antigen. The rare D– phenotype in Asians is 004055 Rh55 LOCR often attributable to point mutations in RHD, although 10 004056 Rh56 CENR to 30 percent of Asians who type as D– have very low levels RH57 CEST 17 of D; this phenotype is termed Del. Rh58 CELO Rh59 CEAG RhAG D Positive (Rh-Positive) 030001 RhAG1 Duclos Prev 901013 Most people with D+ RBC phenotypes have 030002 RhAG2 Ola Prev 700043 conventional RhD protein, but more than 150 different 030003 RhAG3 DSLK alleles encoding changes in the amino acid sequence of the

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Table 2A. Examples of RHD alleles† weak D phenotype can occur when Ce (r′) is Phenotype(s) or Nucleotide present in trans to RHD, and depression of an category Allele designation changes Amino acid changes already weak D type by Ce (r′) can result in an

D RHD*01 apparent D– (rr′, r′r′, or r″r′) or Del phenotype RH:1 RHD in a sample that is in reality R (weak D)/r′(Ce). Partial D examples DII RHD*02 Del RHD*DII 1061C>A Ala354Asp RBCs with a Del phenotype express very low levels of D antigen detected serologically DIIIa RHD*03.01 186G>T Leu62Phe only by adsorption and elution with anti-D. RH:54 (DAK+) RHD*DIIIa 410C>T Ala137Val Del is most commonly found in Asians, who 455A>C Asn152Thr comprise 10 to 30 percent of individuals who 602C>G Thr201Arg type as D– with standard serologic testing. Del 667T>G Phe223Val is less often found in Europeans (0.027%). In DIIIb RHD*03.02 Hybrid with CE exon 2 Ile60Leu Asians, the Del phenotype most often results RH:–12 RHD*DIIIb RHD*D-CE(2)-D Ser68Asn from a nucleotide change 1227G>A, which G– Ser103Pro does not affect the amino acid sequence but Weak D example interferes with efficient splicing of exon 9, Type 1 RHD*01W.1 809T>G Val270Gly whereas a single amino change, Met295Ile, is 17,18,19 RHD*weak D type 1 the most common mutation in Europeans. D RBCs are primarily diagnosed by RHD D– examples el genotyping or adsorption and elution studies D– RHD*01N.01 Complete deletion because the RBCs type as D–. D– RHD*04N.01 37-bp insert, 609G>A, inactive RHD*Pseudogene 645G>C, 677T>G, RHD* Ψ 674C>T, 807T>G Partial D W in the allele name indicates a weak phenotype. RBCs with partial D type serologically N in the allele name indicates a null phenotype. as D+, but individuals can produce anti-D †ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology when stimulated by transfusion or pregnancy. protein, or changes in the conserved nucleotides at the ends Because the D antigen consists of numerous conformation- of coding exons required for efficient splicing of pre-mRNA dependent epitopes (epD), a person who lacks one or to generate mature transcripts, have been reported. These more D epitopes can form alloantibodies to the missing portion(s). More than 30 D epitopes have been defined result in weak D, partial D, and Del phenotypes. with monoclonal antibodies, designated epD1 through 14 Weak D epD9 with further subdivisions of each. Point mutations Although common D phenotypes have 10,000 to in RHD causing single amino acid changes, or more often 30,000 antigen sites per RBC, weak D RBCs have less hybrid genes in which portions of RHD are replaced by the than 100 to 5000 sites (summarized in Reid and Lomas- corresponding regions of RHCE, cause partial D. The novel Francis5). Historically, weak D RBCs were defined as sequences of the hybrid protein resulting from regions of requiring the IAT for detection, but with the introduction of RhD joined to RhCE result in the loss of D epitopes and monoclonal antibodies this depends on the specific reagent may generate new low prevalence or cause the loss of high used to test the RBCs. Weak D phenotypes are most often prevalence antigens. associated with single nucleotide changes in RHD.18 These encode amino acid changes that negatively affect insertion Elevated D or D– – Haplotypes of the protein in the membrane or negatively affect efficient Deletion phenotypes, designated D– –, Dc–, and DCW–, splicing of the coding exons, resulting in reduced expression (the dashes represent missing antigens) can have enhanced of D antigen on the RBCs. To date, more than 70 different expression of D antigen and no, weak, or altered C/c and E/e mutations causing weak D expression have been reported, antigens.20 Many are caused by replacement of portions of the most common encoding a Val270Gly change called weak RHCE by RHD, such that the additional RHD sequences in D type 1.18 The different weak D phenotypes have been given RHCE, along with a normal RHD, account for the increased number designation, i.e., 1 through 73 to date. (summarized D expression. However, some are caused by mutations that by Wagner, FF. RhesusBase Web site; see Web Resources) alter or silence RHCE. For example, a silenced RHCE*cE Additionally, less D antigen is present in cells that also designated RHCE*cE(907del C) encodes a stop codon and have C antigen, such that an R2R2 individual (DcE/DcE) has is associated with the D– – haplotype and the absence of c 21 more D antigen sites than an R1R1 individual (DCe/DCe). A and E antigen expression in Hispanics.

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Table 2B. Examples of RHCE alleles.† The nucleotides and amino acids C/c and E/e Antigens diagnostic for the presence of RHCE*ce are indicated shaded as the reference allele and the changes diagnostic for each altered allele are The common RHCE alleles encode the shown. C or c and E or e antigens. Of the four amino acid changes (Cys16Trp; Ile60Leu; Ser68Asn; Nucleotide Amino acid Also reported Phenotype Allele Changes changes as Ser103Pro) associated with the C to c ce‡ RHCE*01 polymorphism, Ser103Pro correlates with C/c RH:4 or c RHCE*c 307C Pro103 antigen expression. The E to e polymorphism RH:5 or e RHCE*e 676G Ala226 is caused by a single amino acid substitution, RH:6 or ce or f RHCE*ce Pro226Ala. Numerous changes in RHCE cause RHCE*01.01 48G>C Trp16Cys quantitative and qualitative changes in C/c or RHCE*ce.01 E/e antigen expression, with altered C and e RHCE*01.02 48G>C Trp16Cys most often encountered. More than 70 different RHCE*ce.02 1025C>T Thr342Ile RHCE*ceTI Ce§ RHCE*02 RHCE alleles are associated with altered, weak, RH:2 or C RHCE*C 48G>C Trp16Cys or no expression of the principal antigens. 178C>A Leu60Ile 203A>G Asn68Ser Compound Antigens (ce, Ce, cE, and CE) 307C>T Pro103Ser Compound antigens define epitopes that RH:5 or e RHCE*e 676G Ala226 depend on conformational changes that result RH:7 or Ce RHCE*Ce from amino acids associated with both C/c cE|| RHCE*03 676G>C Ala226Pro RH:4 or c RHCE*c and E/e. These were previously referred to RH:5 or e RHCE*E as cis products, to indicate the antigens were RH:27 or Ce RHCE*cE expressed from the same haplotype, but it is CE** RHCE*04 48G>C Trp16Cys now known that these are expressed on a single RH:2 or C RHCE*C 178C>A Leu60Ile protein. These include ce or f, Ce or rhi, cE or RH:3 or E RHCE*E 203A>G Asn68Ser Rh27, and the rare CE or Rh22. Serologically, RH:22 RHCE*CE 307C>T Pro103Ser RBCs with Dce/DCE or DcE/DCe haplotypes 676G>C Ala226Pro would be identical when tested with common † ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology. Rh antisera, but only the former sample would ‡ § || ce=01 Ce=02 cE=03 **CE=04 react with anti-f.

D and D-like Epitopes on Rhce Altered or Variant C and e Antigens One further complication of serologic D status In Caucasians, altered C is not common but when determination is the expression of D epitopes by the encountered is primarily associated with amino acid protein product of the RHCE gene in the absence of RHD. changes Gln41Arg or Ala36Thr, and expression of the CW Two significant examples are HAR D , found in individuals and CX antigens, respectively. RBCs that express CW and of German ancestry,22,23 and Crawford (ceCF), found in CX, in the absence of C in trans have weak expression of individuals of African ancestry.24 These two are notable C antigen. Altered C is also associated with RhCE proteins because the RBCs show strong reactivity with some expressing novel antigens, including JAHK (Ser122Leu), monoclonal anti-D reagents (3+ to 4+), but are nonreactive and JAL (Arg114Trp). The RBCs type as C+, but patients with others. Other amino acid changes in Rhce mimic can make anti-C when exposed to conventional C antigen. a D-epitope structure, designated ceSL and ceRT, and Altered RHCE alleles are relatively common in such RBCs react weakly to some but not all monoclonal individuals with African ancestry. In this population, the anti-D.25,26 Importantly, the phenotypes discussed here lack most common cause for altered C is an RHDIIIa-CE(4-7)-D the RhD protein, and individuals with them can be readily hybrid gene, which does not encode a D antigen, but encodes sensitized if transfused with D+ RBCs.22,27 altered C antigen on a hybrid protein background (Fig. 1). Approximately 25 percent of African Americans have this Frequency of Altered RHD hybrid.16 In most cases, the RBCs type C+ with commercial Approximately 2 percent of Europeans carry altered monoclonal reagents, and the altered C goes undetected. RHD, and those most often encountered will vary by Most of these patients coinherit an altered e antigen and geographic location, which reflects population diversity in have a V–VS+ phenotype; together this haplotype is the region. In our experience, weak D is found primarily in referred to as (C)ceS.28 Because altered RHCE alleles are European and Asian backgrounds. The incidence of partial not distinguished by routine serologic tests, homozygous RHD alleles in individuals with African backgrounds has patients often make anti-C and anti-e alloantibodies after not been determined but seems to be significantly higher transfusion. Additionally, homozygous individuals typically than in European populations. lack high-prevalence antigens, designated hrB and hrS.

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undetectable. Therefore, to prevent delayed transfusion reactions, some advocate avoiding c+ blood for patients alloimmunized to the E antigen. In contrast, pursuing anti-E in serum containing anti-c is not necessary as the patient will likely have been exposed to c without being exposed to E. Moreover, the vast majority of c– donor blood Figure 1: Representation of the RH gene associated with altered will be negative for E. C antigen. The RHD and RHCE genes are shown as 10 boxes representing the coding exons. Amino acid changes are indicated Rh Antibodies in Patients with Sickle Cell Disease by their single letter designation and the position of the amino acid in the protein is given. Some sickle cell transfusion programs perform Rh typing to match patients and donors for D, C, and E (in addition to K) 34,35 Anti-hrB and anti-hrS can be clinically significant,29,30 and to prevent alloimmunization. The prevalence of RH alleles finding compatible blood can be very difficult because that encode altered or variant D, C, and e antigens in African of differing RH gene backgrounds.31,32 As an additional ethnic groups often underlies the production of complex Rh complication, altered RHCE*ce are often inherited with alloantibody specificities in chronically transfused patients partial RHD (DIII, DAU, DAR, etc.). The inheritance of with sickle cell disease (SCD). Many altered Rh protein partial D with altered RHCE*ce may lead to production of epitopes cannot be distinguished serologically, and are antibodies sometimes identified as anti-HrB (RH34) and only identified by RH genotyping once a patient develops anti-Hr (RH18). apparent autoantibodies to Rh antigens. Some common examples include the partial C antigen encoded by the r′S G Antigen allele, i.e. (C)ceS, and partial DIIIa; the RBCs react 3+–4+ The G antigen is found on RBCs expressing C or D with monoclonal reagents and are not detected as altered. with a 103Ser residue present on RhD, RhCe, or RhCE. Anti-hrS, -hrB, -HrB, and -Hr can be difficult to identify Serologically, anti-G reacts as though it were anti-D plus and can cause clinically significant hemolytic transfusion anti-C. Anti-G explains the D– person transfused with D– reactions including transfusion fatalities.30 C+ blood, or the D– woman who delivered a D–C+ child, who subsequently appears to have made anti-D. To distinguish Other Rh Antibodies anti-D, -C, and -G, adsorption and elution studies can be In addition to the SCD population, D– – and Rhnull performed.33 Their results determine the need for RhIG individuals also develop alloantibodies to high-prevalence prophylaxis for obstetric patients with anti-G to prevent Rh antigens with Rh17 and Rh29 specificity, respectively. additional immunization and formation of anti-D. Lastly, autoantibodies to high-prevalence Rh antigens often occur in the sera of patients with warm autoimmune Antibodies in the Rh System hemolytic anemia and in some cases of drug-induced Most Rh antigens are immunogenic and have the autoimmune hemolytic anemia.36 The biologic explanation potential to cause clinically significant HDFN or transfusion for this apparent specificity or cross-reactivity remains to reactions. Rh alloimmunization occurs by exposure to be determined. foreign RBCs during pregnancy or after transfusion and may persist for many years. Even if serum antibody drops Clinical Considerations below detectable levels, subsequent exposure to the antigen Determination of D status can produce a rapid secondary immune response. Outside The AABB Standards for Blood Banks and Transfusion of the ABO system, the D antigen is the most immunogenic Services37 requires donor blood to be tested for weak D antigen, followed by c and E. Anti-c can cause severe expression and to be labeled as Rh-positive if the test is HDFN, whereas anti-C, -E, and -e are associated with mild positive. Hospital transfusion services are also required HDFN. Because Rh alloimmunization is IgG-mediated, to confirm the D-negative status before the unit is transfusion reactions are primarily extravascular. The released for transfusion to avoid transfusion of D+ RBCs patient may have fever, jaundice, icteric sclera, dark urine, to D– recipients. It is recognized that some very weak D or a drop in hemoglobin level. Antigen-negative RBCs antigens are not detected, and standard typing procedures should be provided to any patient with a history of Rh (including IAT) do not detect Del RBCs. Isolated cases have antibody sensitization. been reported in which very weak D and Del donor units Rh antibodies are often found together. It is not have caused anti-D alloimmunization in D– recipients.38–41 unusual for a patient with a single Rh antibody to become When determining the D type of a patient, detecting alloimmunized to additional Rh antigens if further exposed. weak D expression is not necessary unless testing RBCs of

For example, a DCe/DCe (R1R1) patient with anti-E has an infant of a D– mother at risk for D immunization. DVI usually been exposed to the c antigen as well. Anti-c may be is the most common partial D found in Caucasians, and present at the time anti-E is identified, but may be weak and anti-D produced by women with partial DVI has resulted in

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fatal hemolytic disease.42 Therefore, current FDA-licensed have included samples from diverse backgrounds. However, monoclonal IgM reagents are selected to be nonreactive the complexities and numerous alleles in the Rh blood with partial DVI RBCs in direct tests. Performing only a group system have precluded high-throughput genotyping direct test and eliminating the indirect test for female for RH other than for the common nonvariant forms of children and women of child-bearing age avoids the risk of C/c and E/e antigens. The detection of numerous silencing sensitization by classifying DVI as D– for transfusion and mutations is required for accurate typing; several regions RhIG prophylaxis. of the genes must be sampled to detect multiple alleles, and Patients who have RBCs expressing the common type new alleles are continuously being identified.48 1, 2, or 3 weak D phenotype usually do not make anti-D and Currently, RH genotyping is primarily restricted can safely receive D+ blood. Patients with the rare weak to specialized immunohematology laboratories. One D types 11 and 15 have become alloimmunized, suggesting application of RH genotyping is to find compatible donors in they have altered D epitopes.38 The risk for anti-D the American Rare Donor Program (ARDP) for patients with production in other uncommon weak D types is unknown. antibodies to high-prevalence Rh antigens.32 Genotyping In contrast to weak D, patients with partial D RBCs form can also be used to determine the RHD status of a fetus by alloanti-D directed at the missing D epitopes and should amniocentesis or chorionic villus sampling. More recently, receive D– blood and RhIG when indicated. Unfortunately, noninvasive techniques have been used to collect cell-free, serologic reagents frequently do not distinguish partial D fetal-derived DNA from maternal plasma for RHD genotype status. Many partial D RBCs, including DIIIa, the most prediction with a high-throughput method.49,50 Once high- common partial D in African Americans, type strongly throughput platforms encompassing all the Rh variants D-positive in direct tests and are only recognized after are developed and are cost effective, RH genotyping can producing anti-D. complement serologic testing for typing transfused patients, RHD zygosity determination, fetal D typing, resolution of D Sickle Cell Disease and Altered RH Alleles status, and finding compatible blood for patients with SCD. RBC alloimmunization remains a serious complication of transfusion for patients with SCD, with more than RhAG System 25 to 30 percent of chronically transfused SCD patients RHAG is the ancestral gene from which RHCE and developing RBC antibodies. Altered D, C, and e antigens RHD arose. It resides on chromosome 6 and encodes the often underlie the complex RH alloantibodies SCD patients Rh-associated glycoprotein and the antigens of the RhAG exhibit after transfusion. Although randomized controlled blood group system. The RhAG protein consists of 409 trials have not been performed, extended RBC antigen- amino acids and associates with the Rh proteins in the matching (including D, C, E, and K) has been shown in membrane to form the Rh-core complex.51 numerous single-institutional and prospective multicenter experiences to significantly reduce the incidence of alloanti- Antigens body production in SCD.43–45 Therefore, many centers The RhAG protein does not express the common Rh determine the pretransfusion RBC serologic phenotype and antigens, but it has recently been shown to carry several provide RBCs that are antigen-matched for D, C, E, and K. antigens,52 and it has been assigned system 30 by ISBT Because the high alloimmunization rates are also believed (Table 1). Two high-frequency antigens, Duclos and to be caused by antigenic disparity between African DSLK, and one low-frequency antigen, Ola, had serologic Americans and Caucasians, some programs actively recruit characteristics suggestive of expression on RhAG. This African American donors to supply blood specifically for was confirmed by gene sequencing and expression of patients with SCD.34,35 Although these approaches reduce recombinant RhAG in HEK 293 cells. The Duclos-negative the incidence of alloantibody production, some patients patient was homozygous for a nucleotide 316C>G change, still become sensitized to Rh antigens (D, C, e, and hrB, HrB, encoding Gln106Glu. The DSLK-negative patient was hrS, HrS), indicating they were not Rh antigen matched. homozygous for nucleotide 490A>C, encoding Lys164Gln. Two Ol(a+) family members of a Norwegian family were RH Genotyping heterozygous for 680C>T encoding Ser227Leu. Duclos is RBC genotyping methods were introduced to RHAG1, Ola is RHAG2, and DSLK is provisionally RHAG3. transfusion medicine a decade ago after cloning of the genes made genetic testing for blood groups possible. Genotyping Rhnull methods include amplification of target gene sequences by RhAG protein must be present for the expression of

PCR followed by analysis using RFLP, real-time PCR, or Rh antigens. Rhnull RBCs lack expression of Rh antigens, sequence-specific primer PCR. More recently, mass-scale and the phenotype most often results from mutations in genotyping technologies have been developed by several RHAG, previously termed “regulator” Rhnull. Less often, manufacturers to perform high-throughput blood group Rhnull individuals have inactivating or silencing mutations prediction.46,47 There is nearly complete concordance in RHCE and deletion of RHD previously referred to as 33 between the genotype and serologic phenotype, and studies “amorph” Rhnull.

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The Rh-core complex also interacts with band 3, 2. Freda VJ, Gorman JG, Pollack W. Rh factor: prevention glycophorins A and B, LW, and CD47 and is believed to of isoimmunization and clinical trial on mothers. contribute to the RBC membrane structure because Rhnull Science 1966;151:828–30. RBCs demonstrate abnormal morphology. The function of 3. Mollison PL, Hughes-Jones NC, Lindsay M, Wessely the Rh blood group proteins is not known, but RhAG is an J. Suppression of primary RH immunization by ammonia transporter, and the recent crystalization of the passively-administered antibody. Experiments in human RhAG homolog from the kidney, RhCG, confirms volunteers. Vox Sang 1969;16:421–39. the relationship of human Rh proteins to ammonia 4. Race RR, Sanger R. Blood groups in man. 6th ed. transporters.53 Oxford, England: Blackwell, 1975. 5. Reid ME, Lomas-Francis C. The blood group antigen Summary Perspective factsbook. 2nd ed. San Diego, CA: Academic Press, The Rh/RhAG blood group systems are some of the most 2004. complex, and in the past two decades major insights have 6. Le van Kim C, Mouro I, Chérif-Zahar B, et al. Molecular been gained into the molecular basis of the Rh system. The cloning and primary structure of the human blood genetic information has confirmed many of the predictions group RhD polypeptide. Proc Natl Acad Sci U S A of the serologists, whose primary (and often only) tools 1992;89:10925–9. were the antibodies made by immunized individuals. 7. Chérif-Zahar B, Bloy C, Le Van Kim C, et al. Molecular Exploiting adsorption and elution approaches, along with cloning and protein structure of a human blood selected RBC testing strategies, they uncovered many of group Rh polypeptide. Proc Natl Acad Sci U S A the details concerning the specificity and complexity of the 1990;87:6243–7. Rh system. The prediction that Rh antigens are encoded 8. Arce MA, Thompson ES, Wagner S, Coyne KE, by two genes, not one or three, was adeptly forecast by Ferdman BA, Lublin DM. Molecular cloning of RhD 12 Patricia Tippett based solely on serologic observations. cDNA derived from a gene present in RhD-positive, Their work is the foundation of our understanding today, as but not RhD-negative individuals. Blood 1993;82: new genetic information builds on the serologic backbone. 651–5. Serologic reactivity is still the basis for blood groups and 9. Simsek S, de Jong CA, Cuijpers HT, et al. Sequence blood transfusion practice because serology defines an analysis of cDNA derived from reticulocyte mRNAs antigen. That will not change as we use DNA-based testing coding for Rh polypeptides and demonstration of E/e methods because it is important to remember that without and C/c polymorphisms. Vox Sang 1994;67:203–9. a serologic relationship, a variation in a blood group allele 10. Blumenfeld OO, Patnaik SK. Allelic genes of blood at the DNA level is just another SNP (single nucleotide group antigens: a source of human mutations and polymorphism), and these occur once in every 100 to 300 bp in the human genome. These polymorphisms are only cSNPs documented in the Blood Group Antigen Gene of academic interest until associated with a phenotype and Mutation Database. Hum Mutat 2004;23:8–16. found to be relevant to transfusion medicine by stimulation 11. Landsteiner K, Wiener AS. An agglutinable factor in of an antibody. human blood recognized by immune sera for rhesus Molecular testing will be a powerful adjunct to serologic blood. Proc Soc Exp Biol Med 1940;43:223–4. methods and improve transfusion safety and outcomes, 12. Tippett P. A speculative model for the Rh blood groups. particularly for the chronically transfused population. Ann Hum Genet 1986;50:241–7. Which of the numerous alleles in the Rh system are 13. Daniels G, Castilho L, Flegel WA, et al. International clinically significant is yet to be fully determined. Ultimately, Society of Blood Transfusion Committee on with the development of mass-scale RH genotyping terminology for red blood cell surface antigens: Macao technology, genetically matching donor units with the report. Vox Sang 2009;96:153–6. patients could significantly reduce alloimmunization, and 14. Scott ML, Voak D, Liu W, Jones JW, Avent ND. Epitopes RHD genotyping will provide a definitive D classification on Rh proteins. Vox Sang 2000;78(Suppl 2):117–20. for donors and patients. The availability of cost-effective, 15. Colin Y, Chérif-Zahar B, Le Van Kim C, Raynal V, reliable high-throughput genotyping platforms is needed Van Huffel V, Cartron JP. Genetic basis of the RhD- for this to become incorporated into clinical transfusion positive and RhD-negative blood group polymorphism medicine practice. as determined by Southern analysis. Blood 1991;78: 2747–52. References 16. Singleton BK, Green CA, Avent ND, et al. The presence 1. Levine P, Burnham L, Katzin WM, Vogel P. The of an RHD pseudogene containing a 37 base pair role of isoimmunization in the pathogenesis of duplication and a nonsense mutation in Africans with erythroblastosis fetalis. Am J Obstet Gynecol 1941;42: the Rh D-negative blood group phenotype. Blood 925–37. 2000;95:12–18.

184 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Review of Rh and RhAG systems

17. Shao CP, Maas JH, Su YQ, Köhler M, Legler TJ. 33. Issit P, Anstee, DJ. Applied blood group serology. 4th Molecular background of Rh D-positive, D-negative, ed. Durham, NC: Montgomery Scientific Publications, D(el) and weak D phenotypes in Chinese. Vox Sang 1998. 2002;83:156–61. 34. Afenyi-Annan A, Bandarenko N. Transfusion practices 18. Wagner FF, Gassner C, Müller TH, Schönitzer D, for patients with sickle cell disease at a major academic Schunter F, Flegel WA. Molecular basis of weak D medical center. Immunohematology 2006;22:103–7. phenotypes. Blood 1999;93:385–93. 35. Sesok-Pizzini DA, Friedman DF, Smith-Whitley K, 19. Sun CF, Chou CS, Lai NC, Wang WT. RHD gene Nance SJ. Transfusion support of patients with sickle polymorphisms among RhD-negative Chinese in cell disease at the Children’s Hospital of Philadelphia. Taiwan. Vox Sang 1998;75:52–7. Immunohematology 2006;22:121–5. 20. Daniels G. Human blood groups. 2nd ed. Cambridge, 36. Petz LD. Review: evaluation of patients with immune MA: Wiley-Blackwell, 2002. hemolysis. Immunohematology 2004;20:167–76. 21. Westhoff CM, Vege S, Nickle P, Hue-Roye K, Reid ME. 37. Price TH, ed. Standards for blood banks and A single nucleotide deletion in RHCE*cE responsible transfusion services. 26th ed. Bethesda, MD: AABB for the D- - haplotype in the Hispanic population. Vox 2009:29. Sang 2010:99 (Suppl):376. 38. Denomme GA, Wagner FF, Fernandes BJ, Li W, Flegel 22. Beckers EA, Porcelijn L, Ligthart P, et al. The RoHar WA. Partial D, weak D types, and novel RHD alleles antigenic complex is associated with a limited among 33,864 multiethnic patients: implications for number of D epitopes and alloanti-D production: a anti-D alloimmunization and prevention. Transfusion study of three unrelated persons and their families. 2005;45:1554–60. Transfusion 1996;36:104–8. 39. Yasuda H, Ohto H, Sakuma S, Ishikawa Y. Secondary 23. Wallace M, Lomas-Francis C, Tippett P. The D antigen anti-D immunization by Del red blood cells. characteristic of RoHar is a partial D antigen. Vox Transfusion 2005;45:1581–4. Sang 1996;70:169–72. 40. Flegel WA, Khull SR, Wagner FF. Primary anti-D 24. Flegel WA, Wagner FF, Chen Q, Schlanser G, Frame immunization by weak D type 2 RBCs. Transfusion T, Westhoff CM, Moulds MK. The RHCE allele ceCF: 2000;40:428–34. the molecular basis of Crawford (RH43). Transfusion 41. Mota M, Fonseca NL, Rodrigues A, Kutner JM, 2006:46:1334-42 Castilho L. Anti-D alloimmunization by weak D type 25. Chen Q, Hustinx H, Flegel WA. The RHCE allele ceSL: 1 red blood cells with a very low antigen density. Vox the second example for D antigen expression without Sang 2005;88:130–5. D-specific amino acids. Transfusion 2006;46:766–72. 42. Lacey PA, Caskey CR, Werner DJ, Moulds JJ. Fatal 26. Wagner FF, Ladewig B, Flegel WA.The RHCE allele hemolytic disease of a newborn due to anti-D in an ceRT: D epitope 6 expression does not require D-specific Rh-positive Du variant mother. Transfusion 1983;23: amino acids. Transfusion. 2003;43:1248-54. 91–4. 27. Westhoff CM. Review: the Rh blood group D antigen... 43. Vichinsky EP, Luban NL, Wright E, et al. Prospective dominant, diverse, and difficult. Immunohematology RBC phenotype matching in a stroke-prevention trial 2005;21:155–63. in sickle cell anemia: a multicenter transfusion trial. 28. Daniels GL, Faas BH, Green CA, et al. The VS and V Transfusion 2001;41:1086–92. blood group polymorphisms in Africans: a serologic 44. Ambruso DR, Githens JH, Alcorn R, et al. Experience and molecular analysis. Transfusion 1998;38:951–8. with donors matched for minor blood group antigens 29. Brumit MC, Carnahan GE, Stubbs JR, Storry JR, in patients with sickle cell anemia who are receiving Reid ME. Moderate hemolytic disease of the newborn chronic transfusion therapy. Transfusion 1987;27: (HDN) due to anti-Rh17 produced by a black female 94–8. with an e variant phenotype. Immunohematology 45. Tahhan HR, Holbrook CT, Braddy LR, Brewer LD, 2002;18:40–2. Christie JD. Antigen-matched donor blood in the 30. Noizat-Pirenne F, Lee K, Pennec PY, et al. Rare RHCE transfusion management of patients with sickle cell phenotypes in black individuals of Afro-Caribbean disease. Transfusion 1994;34:562–9. origin: identification and transfusion safety. Blood 46. Hashmi G, Shariff T, Zhang Y, et al. Determination 2002;100:4223–31. of 24 minor red blood cell antigens for more than 31. Reid ME, Storry JR, Issitt PD, et al. Rh haplotypes 2000 blood donors by high-throughput DNA analysis. that make e but not hrB usually make VS. Vox Sang Transfusion 2007;47:736–47. 1997;72:41–4. 47. Hopp K, Weber K, Bellissimo D, Johnson ST, Pietz B. 32. Vege S, Westhoff CM. Molecular characterization of High-throughput red blood cell antigen genotyping GYPB and RH in donors in the American Rare Donor using a nanofluidic real-time polymerase chain Program. Immunohematology 2006;22:143–7. reaction platform. Transfusion 2010;50:40–6.

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 185 C.M. Westhoff and S.T. Chou

48. Hillyer CD, Shaz BH, Winkler AM, Reid M. Integrating Web Resources molecular technologies for red blood cell typing International Society of Blood Transfusion (ISBT) Working and compatibility testing into blood centers and Party on Red Cell Immunogenetics and Blood Group transfusion services. Transfus Med Rev 2008;22: Terminology. http://ibgrl.blood.co.uk/ISBTPages/ 117–32. ISBTHome.htm. Accessed 1/4/2011. 49. Lo YM, Hjelm NM, Fidler C, et al. Prenatal diagnosis RhesusBase. http://www.uni-ulm.de/~fwagner/RH/RB/. of fetal RhD status by molecular analysis of maternal Accessed 1/4/2011. plasma. N Engl J Med 1998;339:1734–8. NCBI Blood Group Mutation Database. http://www.ncbi. 50. Finning K, Martin P, Summers J, Massey E, Poole nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home. G, Daniels G. Effect of high throughput RHD typing Accessed 1/4/2011. of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: Stella T. Chou, MD, Assistant Professor of Pediatrics, prospective feasibility study. BMJ 2008;336:816–18. Division of Hematology, The Children’s Hospital of 51. Matassi G, Chérif-Zahar B, Raynal V, Rouger P, Philadelphia, University of Pennsylvania School of Cartron JP. Organization of the human RH50A gene Medicine, 3615 Civic Center Boulevard, ARC 316D, (RHAG) and evolution of base composition of the RH Philadelphia, PA 19104, and Connie M. Westhoff, SBB, PhD, gene family. Genomics 1998;47:286-93. Director of Immunohematology and Genomics, New York 52. Tilley L, Green C, Poole J, et al. A new blood group Blood Center, 310 East 67th Street, New York, NY 10065. system, RHAG: three antigens resulting from amino acid substitutions in the Rh-associated glycoprotein. Vox Sang 2010;98:151–9. 53. Gruswitz F, Chaudhary S, Ho JD, et al. Function of human Rh based on structure of RhCG at 2.1 A. Proc Natl Acad Sci U S A 2010;107:9638–43.

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186 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 COMMUNICATIONS To the Editors:

Anti-Vel and cold-reactive autoantibody

In Immunohematology Volume 26, Number 1, 2010, Linz and colleagues present a case report titled “Role for serial prenatal anti-Vel quantitative serologic monitoring with 2-ME serum during pregnancy.” The report includes a statement that anti-Vel can simulate the serologic profile of a cold-reactive autoantibody. The authors did not quote a previous paper with similar conclusions that further support this premise. Mechanic et al. describe a study in which eight samples of stored anti- Vel were adsorbed with rabbit erythrocyte stroma (RESt, Immucor/Gamma, Norcross GA). (Transfusion 2002;42(9):1180– 3). The antibodies were reactive at various testing phases. The adsorbed samples were tested at phases selected based on the test results of the neat samples. The results indicated that anti-Vel was either partially or completely adsorbed by RESt. These results further support the idea that one must be thorough when investigating these types of antibodies.

Joan L Maurer BS, SBB(ASCP) American Red Cross Blood Services Penn Jersey Region National Reference Laboratory for Blood Group Serology 700 Spring Garden Street Philadelphia, PA 19123

Sandra Taddie Nance, MS, MT, (ASCP)SBB American Red Cross Biomedical Services Immunohematology Reference Laboratories and Heritage Division 700 Spring Garden Street Philadelphia, PA 19123

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IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 187 COMMUNICATIONS Letter from the Editors:

To Contributors to the 2010 Issues

The journal depends on readers, authors, editorial board, peer reviewers, and our Penn-Jersey staff. We wish we could thank all of you personally, but doing so is not practical. Instead, we thank each of you as members of an honored group. First and foremost, we thank the authors for their reviews, scientific articles, case reports, book reviews, and letters to the editors that come not only from the United States but from many countries of the world. This has given the journal an international flavor. Our editorial board is a prestigious one and we depend on its members, not only for peer reviews, but for guidance in policy and suggestions for improvements. Special thanks go to our medical editors, who review every article for medical content, and to our technical editors, who read every article for technical content. The current board is listed by name in the front of each issue of the journal. Our peer reviewers did a wonderful job in 2010. They are listed below; our thanks to each.

Theresa Boyd, MD Monique Mohammad John W. Burch, MD John J. Moulds, MT(ASCP)SBB Kerry Burright-Hittner, MT(ASCP)SBB Pamela Nickle, MT(ASCP)SBB Gail E. Coghlan, RT, BSc John Nobiletti, MD Martha Rae Combs, MT(ASCP)SBB Jim Perkins, MD Geoffrey Daniels, PhD Melissa Pessin-Minsley, MD Richard Davey, MD Joyce Poole, FIBMS Greg Denomme, PhD NurJehan Quraishy, MD Willy Flegel, MD Savita Singh Janis R. Hamilton, MS, MT(ASCP)SBB Jill R. Storry, PhD, FIBMS Jay Herman, MD David Stroncek, MD Maryann Keashen-Schnell Jeff Trimble, BSc.,ART(CSMLS) MLS(ASCP) Joanne Kosanke, MT(ASCP)SBB Ralph R. Vassallo, MD Mary Kowalski, MT(ASCP)SBB Sunitha Vege, MS Geralyn M. Meny, MD James Westra, MD Dawn Michelle, MT(ASCP)SBB

We also want to thank Marge Manigly, our production assistant, and Sheetal Patel, our editorial assistant, for their help in preparing the journal for press. We also thank Christine Lomas-Francis and Dawn Rumsey, our technical editors, Mary Tod, our copy editor, Lucy Oppenheim, our proofreader; and Wilson Tang, our electronic publisher. Finally, thanks go to our readers, whose enthusiasm and interest in the journal make it all worthwhile.

Sandra Nance Editor-in-Chief

Cindy Flickinger Managing Editor

188 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Subject Index Immunohematology—Volume 26, Nos. 1, 2, 3, 4, 2010

Auto antibodies Cromer—review ...... 26(3):109 Auto-anti-D—diagnostic evidence ...... 26(1):27 Dombrock—review ...... 26(2):71 Auto-anti-D—transfusion guidance ...... 26(1):27 Duffy—review ...... 26(2):51 Auto-anti-Ge ...... 26(2):60 Gerbich—review ...... 26(2):60 Auto-anti-I—association with infection . . . . 26(4):133 I—review ...... 26(4):Cool Auto-anti-I—naturally occurring ...... 26(4):133 Knops—review ...... 26(1):2 Auto-anti-LW—diagnostic evidence ...... 26(1):27 Ok—review ...... 26(3):124 Auto-anti-LW—transfusion guidance ...... 26(1):27 Rh and RhAG—review ...... 26(4):178 Comparison of methods for detection and identification ...... 26(3):104 Case report Correlation with DAT in patients with Absence of HDFN—maternal high-titer IgG thalassemia ...... 26(3):87 anti-Ku ...... 26(3):119 Low affinity ...... 26(4):156 Alloimmunization of D with weak D type 21 . . . 26(1):27 Anti-Vel—monitoring with 2-ME serum . . . . . 26(1):8 Autoimmune hemolytic anemia (AIHA) Comparison of gel test and conventional tube Anti-Ge specificity ...... 26(2):60 test for antibody detection and titration DAT-negative ...... 26(4)156 in D-negative pregnant women ...... 26(4):174 DAT-negative hemolytic anemia ...... 26(4):156 Biochemistry Traditional method and modified PEG Biochemical physiology of the Colton glycoprotein 26(1):22 adsorption method—a comparison . . . . 26(3):104 Duffy—glycoproteins and chemokines ...... 26(2):51 Gerbich—glycophorin ...... 26(2):60 Disease associations Ii—structure ...... 26(4):133 Acute respiratory distress syndrome ...... 26(4):161 Ii—glycolipids and glycoproteins ...... 26(4):133 Antibody-mediated pathogenesis of TRALI . . .26(4):161 Lectins and I ...... 26(4):133 Capillary angioma of small intestine . . . . . 26(3):109 Polylactosamine biosynthesis ...... 26(4):133 Cold agglutinin disease ...... 26(4):133 Structure and function of the C4 protein ...... 26(1):30 Congenital cataracts and iadult phenotype . . . . 26(4):133 DARC and malaria ...... 26(2):51 Blood components DARC and renal disease ...... 26(2):51 FFP—association with TRALI ...... 26(4):161 Disease associations with low levels of C4/complement . . 26(1):30 Blood group antibodies Enteropathy—protein losing ...... 26(3):109 Anti-D—caused by weak D type 21 ...... 26(1):27 HEMPAS ...... 26(4):133 Anti-Fya and Fyb ...... 26(2):51 Paroxysmal nocturnal hemoglobinuria . . . . 26(3):109 Anti-I—review ...... 26(4):133 Positive DAT in thalassemia patients ...... 26(3):87 Anti-Ku—absence of HDFN ...... 26(3):119 Pneumonia and I ...... 26(4):133 Anti-Oka ...... 26(3):124 Rh antibodies in patients with sickle cell Anti-Vel ...... 26(1):8 disease ...... 26(4):178 a a a a b Cromer antibodies: Cr , Dr , Es , TC , Wes , TRALI—granulocyte serology ...... 26(1):11 UMC ...... 26(3):109 TRALI—noncardiogenic pulmonary edema . . .26(4):161 Rh system—review ...... 26(4):178 Sialo agglutinins ...... 26(4):133 Donors

Specificities of DAT-associated antibodies D and Del donors ...... 26(4):178 in thalassemia patients ...... 26(3):87 WBC antibodies in TRALI ...... 26(4):161

Blood group systems/antigens Genetics Chido/Rodgers—review ...... 26(1):30 C4 and Chido/Rodgers ...... 26(1):30 Colton—review ...... 26(1):22

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 189 Colton—AQP1 locus ...... 26(1):22 Monoclonal antibody immobilization of Consortium for Blood Group Genes: granulocyte antigens ...... 26(1):11 2009 report ...... 26(2):47 Qualitative screens for fetal RBCs in maternal D1*01 and D1*02—comparison of phenotyping with blood ...... 26(3):92 hemagglutination, PCR-SSP, and real-time Rh immunoprophylaxis ...... 26(3):92 PCR ...... 26(2):66 Rosette screen for D+ RBCs ...... 26(3):92 DAF gene ...... 26(3):109 Serologic findings for maternal sample with Diego—genotype and alleles in Brazilian anti-Ku ...... 26(3):119 donors ...... 26(2):66 Traditional method and modified PEG Duffy—phenotypes, prevalence, and inherited adsorption method—a comparison . . . . 26(3):104 alleles ...... 26(2):51 Ge phenotype—nucleotide and amino acid Molecular changes ...... 26(2):60 Chido/Rodgers phenotypes—prevalence in Gene array data ...... 26(4):133 random samples ...... 26(1):30 Granulocyte genotyping ...... 26(1):11 Colton-null phenotype—ancestry of the probands ...... 26(1):22 I gene ...... 26(4):133 Colton—AQP1 locus ...... 26(1):22 Knops—CR1 gene ...... 26(1):2 Consortium for Blood Group Genes (CBGG): Rh genotyping ...... 26(4):178 2009 report ...... 26(2):47 RhAG protein ...... 26(4):178 CR1-1 protein bearing Knops antigen in SCR 25 . .26(1):2 RHD and RHCE primers ...... 26(2):57 D antigen ...... 26(4):178 Hemolytic disease of the fetus and newborn Diego genotyping—real-time PCR and melting curve analysis ...... 26(2):66 (HDFN) a b Absence of HDFN despite maternal high-titer Do (DO1) and Do (DO2)—polymorphic IgG anti-Ku ...... 26(3):119 antigens ...... 26(2):71 Antibody detection and titration in D-negative Duffy—antigen receptor for chemokines . . . . .26(2):51 pregnant women ...... 26(4):174 IGnT protein structure-function ...... 26(4):133 Anti-Ge3 ...... 26(2):60 Inab phenotype ...... 26(3):109 Biologic role of I ...... 26(4):133 Knops antigen ...... 26(1):2 Rh immunoprophylaxis ...... 26(3):92 Partial c antigen—DNA and RNA isolation, RT-PCR, sequencing, and cloning ...... 26(2):57 Laboratory management HLA laboratories—future direction ...... 26(1):11 Platelets/white cells Rh immunoprophylaxis—laboratory methods . .26(3):92 Donor leukocyte antibodies ...... 26(4):161 Obstetric management of antenatal D– Granulocyte antibody detection and antigen women ...... 26(4):174 typing ...... 26(1):11 Granulocyte genotyping ...... 26(1):11 Methods ISBT working party on granulocyte AGS panel ...... 26(4):156 immunobiology ...... 26(1):11 Anti-HbF method in FMH ...... 26(3):92 Neutropenia Anti-IgG test ...... 26(4):156 —alloimmune neonatal Column agglutinin ...... 26(4):156 —autoimmune —drug induced ...... 26(1):11 Direct Polybrene test ...... 26(4):156 Role of neutrophil in TRALI ...... 26(4):161 ELAT ...... 26(4):156 Eluate HbF, elution assay ...... 26(3):92 Quality Enzyme-linked antiglobulin test ...... 26(3):92 Consortium for blood group genes (CBGG): Flow cytometry—analysis of fetomaternal 2009 report ...... 26(2):47 hemorrahage ...... 26(3):92 ISBT Working Party on Granulocyte Gel agglutinin cards ...... 26(3):92 Immunobiology ...... 26(1):11 Granulocyte agglutination test ...... 26(1):11 Granulocyte immunofluorescence test ...... 26(1):11 Reviews Chido/Rodgers blood group system ...... 26(1):30 Kleihauer-Betke acid-elution assays ...... 26(3):92 Colton blood group system ...... 26(1):22 Microscopic weak D test ...... 26(3):92 Cromer blood group system ...... 26(3):109

190 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Dombrock blood group system ...... 26(2):71 Serology Duffy blood group system ...... 26(2):51 Antibody detection and titration in D-negative Gerbich blood group system ...... 26(2):60 pregnant women—a comparison of gel test and conventional tube test ...... 26(4):174 Granulocyte serology—current concepts and clinical significance ...... 26(1):11 Anti-Vel—quantitative serologic monitoring . . . .26(1):8 Knops blood group system ...... 26(1):2 Attempts to support an immune etiology in 800 patients with DAT-negative hemolytic Laboratory methods for Rh anemia ...... 26(4):156 immunoprophylaxis ...... 26(3):92 Granulocyte serology—current concepts and Ok blood group system ...... 26(3):124 clinical significance ...... 26(1):11 Polylactosamines, there’s more than meets the Ii ...... 26(4):133 Transfusion Practices Pathophysiology and prevention of transfusion- Anti-Vel serologic monitoring with 2-ME related acute lung injury (TRALI) . . . . . 26(4):161 serum treatment ...... 26(1):8 The Rh system (Rh and RhAG) ...... 26(4):178 Etiology in 800 patients with DAT-negative hemolytic anemia ...... 26(4):156 Rh blood group system Gel test and tube test for antibody detection Alloimmunization to the D antigen by a patient and titration ...... 26(4):174 with weak D type 21 ...... 26(1):27 Pathophysiology and prevention of TRALI . . . 26(4):161 Antibody detection and titration in D-negative Significance of a positive DAT in thalassemia pregnant women ...... 26(4):174 patients ...... 26(3):87 Laboratory methods for Rh Transfusion of patients with D+ RBCs and immunoprophylaxis ...... 26(3):92 serum anti-D ...... 26(1):27 RHCE*ceAR encodes a partial c (RH4) antigen . .26(2):57 The Rh system (Rh and RhAG) ...... 26(4):178

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IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 191 Contributor Index Immunohematology—Volume 26, Nos. 1, 2, 3, 4, 2010

A Hedl, J.J...... 26(3):104 R Allen, S...... 26(1):8 Holub, M.P...... 26(3):104 Rami, J...... 26(3):119 Arinsburg, S.A...... 26(3):87 Howard-Menk, C...... 26(3):119 Reid, M.E...... 26(2):47 Hunt, P...... 26(4):156 Reid, M.E...... 26(2):57 B Reid, M.E...... 26(2):60 Bachowski, G.J...... 26(1):11 I,J,K Reid, M.E...... 26(2):71 Jain, A...... 26(4):174 C Reid, M.E...... 26(3):109 Johnson, S.T...... 26(1):8 Campbell-Lee, S...... 26(3):119 Ruiz, A.S...... 26(2):66 Kakaiya, R.M...... 26(3):119 Castilho, L...... 26(2):47 Kleinert, D...... 26(3):87 S, T Castro, V...... 26(2):47 Kumar, P...... 26(4):174 Saha, S.C...... 26(4):174 Chamone, D.A.F...... 26(2):66 Saluja, K...... 26(4):174 Chou, S.T...... 26(4):178 L Sandler, S.G...... 26(3):92 Clay, M.E...... 26(1):11 ...... Laird-Fryer, B. 26(3):104 Sathiyamoorthy, S...... 26(3):92 Co, A...... 26(4):156 ...... Leger, R.M. 26(4):156 Schuller, R.M...... 26(1):11 Cooling, L...... 26(4):133 ...... Linz, W.J. 26(1):8 Sharma, R.R...... 26(4):174 Cushing, M.M...... 26(3):87 ...... Lomas-Francis, C. 26(2):57 Skerrett, D.L...... 26(3):87 Lomas-Francis, C...... 26(2):71 D, E Smart, E.A...... 26(3):124 St-Louis, M...... 26(2):47 Dhawan, H.K...... 26(4):174 M Storry, J.R...... 26(3):109 Denomme, G.A...... 26(2):47 Mair, D.C...... 26(4):161 Storry, J.R...... 26(3):124 Dorlhiac-Llacer, P.E...... 26(2):66 Malecki, Z...... 26(3):119 Symington, D.B...... 26(3):104 Eastlund, T...... 26(4):161 Mallory, D...... 26(1):39 Thakral, B...... 26(4):174 Etem, M.E...... 26(3):104 Mallory, D...... 26(1):40 Thakur, M.K...... 26(4):174 Marwaha, N...... 26(4):174 F McGann, H...... 26(1):27 Figueroa, D...... 26(3):104 V, W, Y Mougey, R...... 26(1):30 Fuchisawa, A...... 26(2):57 Wa lker, P.S...... 26(2):60 Moulds, J.M...... 26(1):2 Fueger, J.T...... 26(1):8 Wenk, R.E...... 26(1):27 ...... Meny, G.M. 26(2):51 Westhoff, C.M...... 26(2):47 G Westhoff, C.M...... 26(4):178 ...... N, O Garratty, G. 26(4):156 Whaley, A...... 26(3):119 Novaretti, M.C.Z...... 26(2):66 ...... Giardina, P.J. 26(3):87 Yazer, M.H...... 26(3):109 P, Q H Papari, M...... 26(3):119 Halverson, G.R...... 26(1):22 Peyrard, T...... 26(1):22 Halter Hipsky, C...... 26(2):57

192 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Announcements

Announcement

The Clinical and Laboratory Standards Institute (www.clsi.org) recently published the guideline “Validation of Automated Systems for Immunohematological Testing Before Implementation; Approved Guideline” (I/LA33-A). This document provides guidance to the end user and laboratory for validation of automated systems used in immunohematologic testing before implementation. The link to this document is: http://www.clsi.org/source/orders/Product_Display.cfm?section=Shop&task=3&CATEGORY=IL&PRODUCT_TYPE=SALES&SKU =ILA33A&DESCRIPTION=&FindSpec=&CFTOKEN=19503848&continue=1&SEARCH_TYPE

The link to the press release is: http://www.clsi.org/Content/NavigationMenu/NewsandEvents/PressReleases/Jan10_ILA33A_2.pdf

For information on an upcoming teleconference from CLSI-APHL titled: New Guidance for Laboratories: Validating Automated Systems for Immunohematological Testing (588-608-10), contact the Web site at www.aphl.org/clsi For additional information, contact the Web site at www.clsi.org or Amanda Cushman Holm at (610) 688-0100 ext. 129 or [email protected].

Monoclonal antibodies available at no charge: Specialist in Blood Bank (SBB) Program The New York Blood Center has developed a wide range of monoclonal antibodies (both murine and humanized) that are The Department of Transfusion Medicine, National Institutes useful for donor screening and for typing RBCs with a positive of Health, is accepting applications for its 1-year Specialist DAT. These include anti-A1, -M, -s, -U, -D, -Rh17, -K, -k, -Kpa, in Blood Bank Technology Program. Students are federal -Jsb, -Fya, -Fy3, -Fy6, Wrb, -Xga ,-CD99, -Dob, -H, -Ge2, -Ge3, employees who work 32 hours/week. This program -CD55 (both SCR2/3 and SCR4), -Oka, -I, and anti-CD59. Most introduces students to all areas of transfusion medicine of the antibodies are murine IgG and require the use of anti- mouse IgG for detection (Anti-K, k, and -Kpa). Some are directly including reference serology, cell processing, HLA, and agglutinating (Anti-A1, -M, -Wrb and -Rh17) and a few have been infectious disease testing. Students also design and conduct humanized into the IgM isoform (Anti-Jsb). The antibodies are a research project. NIH is an Equal Opportunity Organization. available at no charge to anyone who requests them. Please visit our Web site for a complete list of available monoclonal • Application deadline is December 31, 2010 for the July antibodies and the procedure for obtaining them. 2011 class.

For additional information, contact: Gregory Halverson, New • See www.cc.nih.gov/dtm > education for brochure and York Blood Center, 310 East 67th Street, New York, NY 10021/ application. e-mail: [email protected] (phone 212-570-

3026, Fax: 212-737-4935) or visit the Web site at http://www. • For further information contact Karen M. Byrne at nybloodcenter.org >research >immunochemistry >current list of (301)451-8645 or [email protected] monoclonal antibodies available.

Important Notice About Manuscripts for Notice to Readers Immunohematology Immunohematology, Journal of Blood Group Serology and Please e-mail all manuscripts to [email protected] Education, is printed on acid-free paper.

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 193 Announcements, con’t.

Masters (MSc) in Transfusion and Transplantation Sciences at The University of Bristol, England

Applications are invited from medical or science graduates for the Master of Science (MSc) degree in Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2011 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also be opportunities to continue studies for PhD or MD following the MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the University of Bristol, Department of Pathology and Microbiology. It includes: • Scientific principles of transfusion and transplantation • Clinical applications of these principles • Practical techniques in transfusion and transplantation • Principles of study design and biostatistics • An original research project

Application can also be made for Diploma in Transfusion and Transplantation Science or a Certificate in Transfusion and Transplantation Science.

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site: http://www.blood.co.uk/ibgrl/MscHome.htm

For further details and application forms please contact: Dr Patricia Denning-Kendall University of Bristol Paul O’Gorman Lifeline Centre Department of Pathology and Microbiology Southmead Hospital Westbury-on-Trym, Bristol BS10 5NB, England Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: [email protected].

194 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Advertisements

Reference and Consultation Services IgA/Anti-IgA Testing

Antibody identification and problem resolution IgA and anti-IgA testing is available to do the

HLA-A, B, C, and DR typing following: • Identify IgA-deficient patients HLA-disease association typing • Investigate anaphylactic reactions Paternity testing/DNA • Confirm IgA-deficient donors Our ELISA for IgA detects protein to 0.05 mg/dL. For information, contact: For additional information contact: Mehdizadeh Kashi Cindy Flickinger at (503) 280-0210 at (215) 451-4909,

or write to: or e-mail: [email protected], Pacific Northwest Regional Blood Services or write to: ATTENTION: Tissue Typing Laboratory American Red Cross Blood Services American Red Cross Musser Blood Center

3131 North Vancouver 700 Spring Garden Street Philadelphia, PA 19123-3594 Portland, OR 97227 ATTN: Cindy Flickinger CLIA licensed, ASHI accredited CLIA licensed

National Reference Laboratory Donor IgA Screening for Blood Group Serology • Effective tool for screening large volumes of donors Immunohematology Reference Laboratory • Gel diffusion test that has a 15-year proven track record: AABB, ARC, New York State, and CLIA licensed 24-hour phone number: Approximately 90 percent of all donors identified as IgA (215) 451-4901 deficient by are confirmed by the more sensitive testing Fax: (215) 451-2538 methods

American Rare Donor Program For additional information: 24-hour phone number: (215) 451-4900 Kathy Kaherl Fax: at (860)678-2764 (215) 451-2538 [email protected] e-mail: Immunohematology [email protected] Journal of Blood Group Serology and Education Phone, business hours: or write to: (215) 451-4902 Fax: (215) 451-2538 Reference Laboratory [email protected] American Red Cross Biomedical Services Quality Control of Cryoprecipitated–AHF Connecticut Region Phone, business hours: 209 Farmington Ave. (215) 451-4903 Farmington, CT 06032 Fax: (215) 451-2538 CLIA licensed

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 195 Advertisements, cont.

National Platelet Serology Reference Laboratory National Neutrophil Serology Reference Laboratory

Diagnostic testing for: Our laboratory specializes in granulocyte antibody detection • Neonatal alloimmune thrombocytopenia (NAIT) and granulocyte antigen typing. • Posttransfusion purpura (PTP) • Refractoriness to platelet transfusion Indications for granulocyte serology testing • Heparin-induced thrombocytopenia (HIT) include: • Alloimmune idiopathic thrombocytopenia purpura (AITP) • Alloimmune neonatal neutropenia (ANN) Medical consultation available • Autoimmune neutropenia (AIN) Test methods: • Transfusion related acute lung injury (TRALI) • GTI systems tests Methodologies employed: — detection of glycoprotein-specific platelet antibodies • Granulocyte agglutination (GA) — detection of heparin-induced antibodies (PF4 ELISA) • Platelet suspension immunofluorescence test (PSIFT) • Granulocyte immunofluorescence by flow cytometry (GIF) • Solid phase red cell adherence (SPRCA) assay • Monoclonal antibody immobilization of neutrophil antigens • Monoclonal immobilization of platelet antigens (MAIPA) (MAINA) • Molecular analysis for HPA-1a/1b TRALI investigations also include: For further information, contact: • HLA (PRA) Class I and Class II antibody detection Platelet Serology Laboratory (215) 451-4205 For further information, contact: Maryann Keashen-Schnell (215) 451-4041 office [email protected] Neutrophil Serology Laboratory (651) 291-6797 Sandra Nance (215) 451-4362 Randy Schuller (651) 291-6758 [email protected] [email protected]

American Red Cross Blood Services American Red Cross Blood Services Musser Blood Center Neutrophil Serology Laboratory 700 Spring Garden Street 100 South Robert Street Philadelphia, PA 19123-3594 CLIA licensed St. Paul, MN 55107 CLIA licensed

196 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Advertisements, cont.

Blood Group Antigens & Antibodies A guide to clinical relevance & technical tips

by Marion E. Reid & Christine Lomas-Francis This compact “pocketbook” from the authors of the Blood Group Antigen FactsBook is a must for anyone who is involved in the laboratory or bedside care of patients with blood group alloantibodies. The book contains clinical and technical information about the nearly 300 ISBT recognized blood group antigens and their corresponding antibodies. The information is listed in alphabetical order for ease of finding—even in the middle of the night. Included in the book is information relating to: • Clinical significance of antibodies in transfusion and HDN. • Number of compatible donors that would be expected Ordering Information to be found in testing 100 donors. Variations in different The book, which costs $25, can be ethnic groups are given. ordered in two ways: • Characteristics of the antibodies and optimal technique(s) • Order online from the publisher for their detection. at: www.sbbpocketbook.com • Technical tips to aid their identification. • Order from the authors, who • Whether the antibody has been found as an autoantibody. will sign the book. Send a check, made payable to “New York Blood Center” and indicate “Pocket­ Pocketbook Education Fund book” on the memo line, to: The authors are using royalties generated from the sale of this pocketbook for educational purposes to mentor people Marion Reid in the joys of immunohematology as a career. They will Laboratory of Immunochemistry accomplish this in the following ways: New York Blood Center • Sponsor workshops, seminars, and lectures 310 East 67th Street • Sponsor students to attend a meeting New York, NY 10065 • Provide copies of the pocketbook Please include the recipient’s (See www.sbbpocketbook.com for details to apply for funds) complete mailing address.

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 197 Advertisements, cont.

Becoming a Specialist in Blood Banking (SBB)

What is a certified Specialist in Blood Banking (SBB)? • Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology (ASCP) board of registry (BOR) examination for the Specialist in Blood Banking. • This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking. Individuals who have an SBB certification serve in many areas of transfusion medicine: • Serve as regulatory, technical, procedural and research advisors • Perform and direct administrative functions • Develop, validate, implement, and perform laboratory procedures • Analyze quality issues preparing and implementing corrective actions to prevent and document issues • Design and present educational programs • Provide technical and scientific training in blood transfusion medicine • Conduct research in transfusion medicine Who are SBBs? Supervisors of Transfusion Services Managers of Blood Centers LIS Coordinators Educators Supervisors of Reference Laboratories Research Scientists Consumer Safety Officers Quality Assurance Officers Technical Representatives Reference Lab Specialist Why be an SBB? Professional growth Job placement Job satisfaction Career advancement How does one become an SBB? • Attend a CAAHEP-accredited Specialist in Blood Bank Technology Program OR • Sit for the examination based on criteria established by ASCP for education and experience However: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam. Conclusion: The BEST route for obtaining an SBB certification is … to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program Contact the following programs for more information: Onsite Phone or Online Program Contact Name Contact Email Contact Website Program

Walter Reed Army Medical Center William Turcan 202-782-6210 [email protected] www.militaryblood.dod.mil On site American Red Cross, Southern Michael Coover 909-859-7496 [email protected] none On site California Region ARC-Central OH Region Joanne Kosanke 614-253-2740 [email protected] none On site x 2270 Blood Center of Southeastern Lynne LeMense 414-937-6403 [email protected] www.bcw.edu On site Wisconsin Community Blood Center/CTS Nancy Lang 937-461-3293 [email protected] http://www.cbccts.org/education/ On line Dayton, Ohio sbb.htm Gulf Coast School of Blood Bank Clare Wong 713-791-6201 [email protected] www.giveblood.org/education/ On line Technology distance/htm Hoxworth Blood Center, Susan Wilkinson 513-558-1275 [email protected] www.hoxworth.org On site University of Cincinnati Indiana Blood Center Jayanna Slayten 317-916-5186 [email protected] www.indianablood.org On line Johns Hopkins Hospital Jan Light 410-955-6580 [email protected] http://pathology2.jhu/department/ On site divisions/tranfusion/sbb.cfm Medical Center of Louisiana Karen Kirkley 504-903-3954 [email protected] none On site NIH Clinical Center Department of Karen Byrne 301-496-8335 [email protected] www.cc.nih.gov/dtm On site Transfusion Medicine Rush University Veronica Lewis 312-942-2402 [email protected] www.rushu.rush.edu/health/dept.html On line Transfusion Medicine Center at Marjorie Doty 727-568-5433 [email protected] www.fbsblood.org On line Florida Blood Services x 1514 University of Texas Health Linda Myers 210-731-5526 [email protected] www.uthscsa.edu On site Science Center at San Antonio University of Texas Medical Janet Vincent 409-772-3055 [email protected] www.utmb.edu/sbb On line Branch at Galveston University of Texas SW Medical Barbara Laird-Fryer 214-648-1785 [email protected] http://telecampus.utsystem.edu On line Center Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org and www.aabb.org Revised August 2007

198 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 Immunohematology Journal of Blood Group Serology and Education Instructions for Authors

I. GENERAL INSTRUCTIONS c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††). Before submitting a manuscript, consult current issues of 8. Figures Immunohematology for style. Double-space throughout the manuscript. a. Figures can be submitted either by e-mail or as photographs Number the pages consecutively in the upper right-hand corner, beginning (5ʺ×7ʺ glossy). with the title page. b. Place caption for a figure on a separate page (e.g. Fig. 1 Results II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH of...), ending with a period. If figure is submitted as a glossy, LITERATURE REVIEW place first author’s name and figure number on back of each A. Each component of the manuscript must start on a new page in the glossy submitted. following order: c. When plotting points on a figure, use the following symbols if 1. Title page possible: l l s s n n. 2. Abstract 9. Author information 3. Text a. List first name, middle initial, last name, highest degree, 4. Acknowledgments 5. References position held, institution and department, and complete address 6. Author information (including ZIP code) for all authors. List country when applicable. 7. Tables III. EDUCATIONAL FORUM 8. Figures A. All submitted manuscripts should be approximately 2000 to 2500 words B. Preparation of manuscript 1. Title page with pertinent references. Submissions may include: a. Full title of manuscript with only first letter of first word 1. An immunohematologic case that illustrates a sound investigative capitalized (bold title) approach with clinical correlation, reflecting appropriate b. Initials and last name of each author (no degrees; all CAPS), e.g., collaboration to sharpen problem solving skills M.T. JONES, J.H. BROWN, AND S.R. SMITH 2. Annotated conference proceedings c. Running title of ≤40 characters, including spaces d. Three to ten key words B. Preparation of manuscript 2. Abstract 1. Title page a. One paragraph, no longer than 300 words a. Capitalize first word of title. b. Purpose, methods, findings, and conclusion of study b. Initials and last name of each author (no degrees; all CAPs) 3. Key words 2. Text a. List under abstract a. Case should be written as progressive disclosure and may 4. Text (serial pages): Most manuscripts can usually, but not necessarily, be divided into sections (as described below). Survey include the following headings, as appropriate results and review papers may need individualized sections i. Clinical Case Presentation: Clinical information and a. Introduction — Purpose and rationale for study, including differential diagnosis pertinent background references ii. Immunohematologic Evaluation and Results: Serology and b. Case Report (if indicated by study) — Clinical and/or hematologic molecular testing data and background serology/molecular iii. Interpretation: Include interpretation of laboratory results, c. Materials and Methods — Selection and number of subjects, correlating with clinical findings samples, items, etc. studied and description of appropriate controls, procedures, methods, equipment, reagents, etc. iv. Recommended Therapy: Include both transfusion and Equipment and reagents should be identified in parentheses by nontransfusion-based therapies model or lot and manufacturer’s name, city, and state. Do not v. Discussion: Brief review of literature with unique features of use patient’s names or hospital numbers. this case d. Results — Presentation of concise and sequential results, vi. Reference: Limited to those directly pertinent referring to pertinent tables and/or figures, if applicable vii. Author information (see II.B.9.) e. Discussion — Implication and limitations of the study, links to viii. Tables (see II.B.7.) other studies; if appropriate, link conclusions to purpose of study as stated in introduction IV. LETTER TO THE EDITOR 5. Acknowledgments: Acknowledge those who have made substantial contributions to the study, including secretarial assistance; list any A. Preparation grants. 1. Heading (To the Editor) 6. References 2. Title (first word capitalized) a. In text, use superscript, Arabic numbers. 3. Text (written in letter [paragraph] format) b. Number references consecutively in the order they occur in the 4. Author(s) (type flush right; for first author: name, degree, institution, text. address [including city, state, Zip code and country]; for other 7. Tables a. Head each with a brief title; capitalize the first letter of first word authors: name, degree, institution, city and state) (e.g., Table 1. Results of . . .) use no punctuation at the end of 5. References (limited to ten) the title. 6. Table or figure (limited to one) b. Use short headings for each column needed and capitalize first letter of first word. Omit vertical lines. Send all manuscripts by e-mail to [email protected]

IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 199 200 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010 201 202 IMMUNOHEMATOLOGY, Volume 26, Number 4, 2010

FIRST CLASS U.S. POSTAGE PAID SOUTHERN MD Musser Blood Center PERMIT NO. 4144 700 Spring Garden Street Philadelphia, PA 19123-3594

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