CHAPTER 10 one had already formed anti-E and another formed anti-Fya Fourteen additional subjects, all of whom were K negative, were injected with K-positive blood Four showed a collapse curve and all of these were given a second injection from their first donor: in two cases the red cells were more rapidly destroyed and one of the two formed anti-E; in the other two recipients the survival of a second injection was normal or only slightly subnormal Eight out of the 14 who showed normal survival of the first lot of red cells were given a second injection; one showed rapid destruction and formed anti-K In summary, collapse curves were noted following 14 out of 45 first injections of red cells and occurred on average on the sixteenth day after injection (range –33 days) In 13 out of these 14 cases a second injection was given Normal or only slightly subnormal survival was found in eight cases and in these no alloantibodies were found In the remaining five, red cell destruction was observed following a second injection and in three of these, alloantibodies (two examples of anti-E and one of anti-Fya) were detected Several other studies in humans have shown an incidence of collapse curves similar to that observed by Adner and co-workers (1963) For example, in one series the incidence was reported as 20% (Brown and Smith 1958) When further cases were added to the series the incidence of collapse curves became 15 out of 40 survival measurements in 32 subjects who had almost always received 10 –20 ml of blood (5 –10 ml of red cells) The diminished survival at first appeared to be related to passage of red cells through a pump oxygenator, but this factor was later deemed irrelevant (GS Eadie, personal communication) In a series studied by ER Giblett (1956, unpublished), red cells from 1– ml of blood obtained from the placenta of normal infants were labelled with 51Cr and injected into normal adults ‘Collapse’ was observed in three of nine cases and none of these cells was circulating at 20 days (see Fig 10.20) In another series, ‘collapse’ occurred in 10 out of 41 crosstransfusions of small volumes of 51Cr-labelled red cells, usually at about the fourteenth day after transfusion The remaining cells were usually eliminated within the following week (Kaplan and Hsu 1961) In summary, when transfusions of 1–10 ml of Rh D-compatible red cells are given to previously untransfused subjects, collapse curves are observed in approximately 30% of cases (Table 10.3) When therapeutic amounts of blood are transfused to previously untransfused subjects, premature curtailment of survival is much less common From a review of more than 100 cases in which the survival of transfused red cells had been followed for not less than 60 days after the transfusion of at least 400 ml of blood, unexpected shortening of survival was found in about 5% of cases (Mollison 1954) A similar incidence was apparent in a series described by Szymanski and Valeri (1971) Forty-four survival studies were carried out by automated differential agglutination in 39 subjects following a transfusion of 450 ml of blood Two collapse curves were observed: accelerated destruction developed on about the tenth day after transfusion and all Table 10.3 Incidence of collapse of the red cell survival curve following the transfusion of 1–10 ml of red cells to previously untransfused adult recipients Reference Donors Amount of red cells transfused (ml) Incidence of collapse Adner and Sjölin (1957) Unpublished observations made by ER Giblett Kaplan and Hsu (1961) Adner et al (1963)* Newborn infants Newborn infants Newborn infants Adults 4–10 1–8 2–7 3–9 Adults 5–10 5/15 3/9 10/41 14/45 32/110 (29.1%) 15/40† GS Eadie (personal communication) * Following the red cell injections, alloantibodies were detected in three cases in the series of Adner et al (1963) and in three cases in the series of Brown and Smith (1958), but in no cases in the other series † Not included in total above because 40 experiments were carried out in 32 recipients 444 RED CELL INCOMPATIBILITY IN VIVO the cells had been eliminated by day 30 There is some evidence that collapse curves are less common in non-responders to Rh D than in responders, possibly indicating that non-responders to Rh D lack some recognition mechanism for allogeneic red cells (Mollison 1981) What causes subnormal red cell survival without demonstrable antibodies? It is tempting to suppose that collapse curves represent primary immunization In support of this belief, the following observations can be cited: (1) in the series of Adner and co-workers (1963), a collapse curve following a first injection of red cells was sometimes followed by the rapid clearance of a second dose of red cells from the same donor with production of an alloantibody; (2) following a first injection of D-positive red cells to D-negative subjects, collapse curves are commonly observed in responders, whereas they are found rarely or not at all in non-responders (Mollison 1981) Evidence opposed to the hypothesis that collapse curves represent primary immunization is the following: (1) in the series of Adner and co-workers (1963), in about 50% of cases, a collapse curve following a first injection of red cells was followed by normal survival of a second dose of red cells from the same donor; (2) collapse curves are not found invariably during primary immunization to strong alloantigens, such as D and K – examples of the normal survival of a first dose of D-positive red cells at 28 days despite the formation of anti-D within the following months were supplied by Samson and Mollison (1975), and an example of the normal survival of a first dose of K-positive red cells for at least 47 days in a subject who formed anti-K within 10 days of a second injection was described by Adner and co-workers (1963); and (3) in rabbits, when Hg (A+) red cells were transfused to Hg (A–) animals, the incidence of collapse curves was 41 out of 51 (Smith and Mollison 1974), but was also high (10 out of 16) when Hg (A–) red cells were transfused to Hg (A–) rabbits The possible role of cell-bound antibody in causing red cell destruction in the absence of detectable antibody in the serum Griffiths and co-workers (1994) have suggested that in circumstances in which the ratio of specific antibody to non-specific plasma IgG is expected to be high, macrophages may become ‘armed’ with antibody in the spleen where antibody-secreting plasma cells and FcR-bearing macrophages are in close proximity This idea is an extension of the previous suggestion that antibody-mediated destruction may be favoured in the spleen because plasma IgG has less of an opportunity to compete with IgG-coated red cells for binding to Fc receptors on macrophages (Engelfriet et al 1981) The concept that there may be macrophages to which antibody has been bound would provide an explanation for the specific destruction of red cells in the absence of detectable antibody in the plasma Destruction of recipient’s red cells by transfused antibodies Passively acquired antibodies may destroy the recipient’s own red cells or, when blood from more than one donor is transfused, the red cells of another donor In either case, the mechanism of red cell destruction is similar to that brought about by actively produced antibodies, although the effects tend to be much less severe due to dilution of the transfused antibody by the recipient’s plasma and, in the case of anti-A and anti-B, by the inhibiting effect of A and B substances in the plasma and in tissues other than red cells In this chapter, some experimental observations are described; haemolytic reactions arising accidentally are considered in the following chapter Destruction of recipient’s red cells following the transfusion of plasma containing potent anti-A or anti-B Ottenberg (1911) first suggested that persons of group O could be used as ‘universal donors’ He argued that the anti-A and anti-B agglutinins in the donor’s plasma, although theoretically capable of damaging the patient’s red cells if the patient belonged to group AB, A or B, would in fact be so diluted in the recipient’s plasma as to be harmless The use of group O blood for transfusion to patients of all groups spread rapidly Throughout the Second World War, enormous numbers of transfusions of group O blood were given to patients of all groups, without any preliminary matching tests In the vast majority of cases there was no evidence of undesirable effects Any risks that the 445 CHAPTER 10 procedure carried were considered small compared with the potential problems of attempting to group recipients and crossmatch blood under the extremely hazardous field conditions Nevertheless, the transfusion of group O plasma to group A recipients sometimes causes severe red cell destruction Acute haemolysis has been reported following ABO-incompatible singledonor platelet concentrates and may be more common than is appreciated (Larsson et al 2000; see also Chapter 11) Transfusion to human volunteers The first systematic attempt to assess the effect of transfusions of incompatible plasma was made by Aubert and co-workers (1942), who transfused plasma containing potent anti-A alloagglutinins to volunteers of group A By eliminating the complicating factor of the donor’s red cells the investigators could be certain that any haemolysis was due to destruction of the patient’s own cells They observed varying degrees of haemoglobinaemia, ‘intravascular agglutination’ and hyperbilirubinaemia followed by a progressive reduction in red cell count The lowest titre of anti-A agglutinin associated with signs of blood destruction was 512 In their hands, such anti-A titres were found in the plasma of 40% of group O donors and they concluded that only about 60% of group O individuals could be considered suitable as universal donors Similar findings were reported by Tisdall and coworkers (1946a) In their hands, about 23% of group O persons had anti-A or anti-B titres of 640 or higher The transfusion of 250 ml of plasma with an alloagglutinin titre of 600 – 4000 frequently caused haemoglobinaemia In one case, a volunteer who received plasma with a titre of only 600 developed a sufficient degree of haemoglobinaemia to produce haemoglobinuria Many patients developed elevation of serum bilirubin, although none became jaundiced Tisdall and coworkers also noted the phenomenon described by Aubert and co-workers (1942) as ‘intravascular agglutination’, the presence of agglutinates in saline suspensions of blood taken from patients immediately after the transfusions In a further series of experiments Tisdall and colleagues (1946b) took blood from a group B volunteer who had been immunized by the injection of group A substance, so that his anti-A titre was 2500 On one occasion, injection of as little as 25 ml of this plasma 446 into a group A volunteer produced haemoglobinuria However, after the addition of group-specific substances to the plasma in vitro, as much as 250 ml could be injected without producing signs or symptoms As the donor of the plasma was group B, the anti-A was probably mainly IgM In a series of cases, 10 ml of the group-specific substances were added to 250-ml amounts of plasma containing potent agglutinins and transfused to volunteers No signs of red cell destruction were observed in any of the recipients One of the difficulties in interpreting these observations is the uncertainty as to whether the high-titre agglutinins were IgM or IgG IgM anti-A and anti-B are far more easily inhibited by soluble blood group substances than are IgG antibodies, so that it would be unwise to conclude that plasma from group O donors, containing potent anti-A and anti-B, can necessarily be rendered safe by the addition of blood group substances In fact in at least one case a haemolytic transfusion reaction in a group A patient transfused with group O blood occurred despite the addition of AB substance to the plasma before transfusion (Ervin and Young 1950) Observations made by Ervin and co-workers (1950) first called attention to the importance of ‘immune’ characteristics of anti-A and anti-B in causing red cell destruction in vivo These authors investigated four severe haemolytic reactions in group A patients following the transfusion of group O blood and showed that in all four cases the donor’s plasma contained anti-A, which was difficult to inhibit and had a higher indirect antiglobulin titre than saline–agglutinin titre Signs of red cell damage persisted for relatively long periods after transfusion Thus osmotic fragility was increased for 8–11 days and microspherocytes were seen on blood films for approximately weeks after transfusion The same authors reported the results of two transfusions of group O plasma to a group A volunteer The first transfusion was of plasma with a moderate saline–agglutinin titre (640), which was readily inhibited by A substance in vitro and was presumably wholly or predominantly IgM No signs of red cell destruction developed By contrast, when the same volunteer received plasma that had a scarcely higher saline–agglutinin titre (1280) and probably contained a potent IgG antibody, as it was not readily inhibited by AB substance, the recipient developed severe intravascular haemolysis and his haematocrit fell from 43% to 24% in days RED CELL INCOMPATIBILITY IN VIVO Summary of effects of incompatible plasma (anti-A and anti-B) Destruction of recipient’s red cells by transfused or injected anti-D The following summary is based partly on the experimental work described above, partly on unpublished observations made with H Chaplin, H Crawford (Morton) and M Cutbush (Crookston) in 1952 and partly on various transfusion accidents described in the following chapter Haemoglobinaemia tends to be slight and haemoglobinuria is unusual Jaundice occurring within a few hours of transfusion has been noted occasionally; see, for example, the infants observed by Gasser (1945), referred to in the following chapter Progressive anaemia is the most commonly observed sign of red cell destruction; PCV may continue to fall for at least week after transfusion; see Fig 11.1 Spontaneous agglutination of whole blood samples withdrawn from the recipient is an invariable feature Agglutination occurs even when plasma containing relatively weak anti-A is transfused (In a group A subject transfused over 20 with 340 ml of blood from a donor whose serum had a saline–agglutinin titre of 64 and a haemolysin titre of 4, a sample of blood taken immediately after transfusion showed strong spontaneous agglutination; this was best demonstrated by spreading a drop of blood on an opal tile; h later, the sign was still present but was weaker; the next day it was not present In patients transfused with potent anti-A, spontaneous agglutination of blood samples in vitro may persist for more than 24 h.) The direct antiglobulin test becomes positive in group A subjects transfused with plasma containing even weak immune anti-A In the subject referred to in the paragraph above, the DAT was definitely positive immediately after transfusion, was weakly positive the next day and was negative thereafter In patients transfused with potent immune anti-A the DAT may remain positive for as long as week The osmotic fragility of the patient’s red cells increases when incompatible plasma is transfused After transfusion, maximal osmotic fragility occurs after 24 h and is almost maximal after h (Ervin et al 1950) It may remain elevated for at least 11 days and during this period peripheral blood films show microspherocytosis Only experimental observations are described in this section; for accidents in clinical practice see Chapter 11 Transfusion of plasma containing anti-D The transfusion, to D-positive subjects, of 200 ml of plasma containing anti-D with a titre of 256 (IAT) produced a positive DAT and spherocytosis within 24 h, and a fall in Hb concentration of 2.5 g /dl during the following week In a second case, in which 250 ml of plasma with a titre of 128 was transfused, the recipient’s bilirubin concentration rose to 2.5 mg /dl at the end of h and the Hb concentration fell by 2.6 g /dl in week In several recipients who were transfused with plasma containing antibodies with titres between and 16, no definite evidence of red cell destruction was detected (Jennings and Hindmarsh 1958) In a series in which normal volunteers received 250 ml of plasma containing moderately potent anti-D (or anti-c or anti-K or anti-M), there were no definite signs of red cell destruction (Mohn et al 1961) The blood volume of these recipients was considerably greater than that of the subjects studied by Jennings and Hindmarsh (1958) In one subject who was transfused with serum containing an exceptionally potent anti-D (indirect antiglobulin titre of 1000; serum albumin titre of 400 000), the PCV fell from 0.48 to 0.22 in 12 days, and spherocytosis and mild haemoglobinaemia persisted for weeks (Bowman et al 1961) Injections of anti-D immunoglobulin In one series of D-positive adults with AITP, who were injected with 750– 4500 µg of intramuscular or intravenous anti-D over a period of 1–5 days, the red cells became coated with 3.2–7.7 µg of IgG/ml cells, but only minimal signs of red cell destruction developed (Salama et al 1986) In another series, in which the AITP was related to AIDS, 13 µg of IgG anti-D/kg were given daily for days, followed by 6–13 µg/kg weekly In five out of six patients, Hb fell by 6–44 g/l (Cattaneo et al 1989) As described in Chapter 5, the intravenous injection of anti-D in doses between 10 and 15 µg /ml of red cells is sufficient to clear 200 – 600 ml of D-positive red 447 CHAPTER 10 cells from the circulation of D-negative subjects in 2–8 days In a case in which approximately 3000 µg of anti-D were infused to a man with AITP over the course of days, corresponding to a dose of less than µg of anti-D/ml red cells, Hb concentration fell from 155 to 74 g / l and it was estimated that about 1600 ml of red cells had been destroyed No cause could be determined for this surprising amount of red cell destruction (Barbolla et al 1993), although in a patient with severe thrombocytopenia, internal haemorrhage is difficult to exclude IVIG preparations may contain potent anti-D (titre 64 –256) and unexpected haemolytic reactions have been reported (Thorpe et al 2003) Other red cell alloantibodies that cause sensitization but minimal haemolysis have been detected in these preparations as well (Moscow et al 1987) References Adner PL, Sjölin S (1957) Unexpected blood group incompatibility revealed by Cr51-labelled red cells Scand J Clin Lab Invest 9: 265 Adner PL, Foconi S, Sjölin S (1963) Immunization after intravenous injection of small amounts of 51Cr-labelled red cells Br J Haematol 9: 288 Akeroyd JH, O’Brien WA (1958) Survival of group AB red cells in a group A recipient Vox Sang 3: 330 Anderson G, Gray LS, Mintz PD (1991) Red cell survival studies in a patient with anti-Tca Am J Clin Pathol 95: 87–90 Andorka DW, Arosemena A, Harris JL (1974) Neutralization in vivo of Lewis antibodies Report of two cases Am J Clin Pathol 62: 47 Ashby W (1919) The determination of the length of life of transfused blood corpuscles in man J Exp Med 29: 267 Athkambhira S, Chiewsilp P (1978) Neutralization of Lewis antibodies in vivo Communications of the 17th Congress of the International Society of Hematology, Paris Atkinson JP, Frank MM (1974a) Complement-independent clearance of IgG-sensitized erythrocytes: inhibition by cortisone Blood 44: 629 Atkinson JP, Frank MM (1974b) Studies on the in vivo effects of antibody Interaction of IgM antibody and complement in the immune clearance and destruction of erythrocytes in man J Clin Invest 54: 339 Aubert EF, Boorman KE, Dodd BE (1942) The universal donor with high titre iso-agglutinins; the effect of anti-A iso-agglutinins on recipients of group A BMJ i: 659 Badakere SS, Vasantha K, Bhatia HM (1980) High frequency of Ina antigen among Iranians and Arabs Hum Hered 30: 262–263 448 Baldwin ML, Barrasso C, Ness PM (1983) A clinically significant erythrocyte antibody detectable only by 51Cr survival studies Transfusion 23: 40–44 Baldwin ML, Ness PM, Barrasso C (1985) In vivo studies of the long-term 51Cr red cell survival of serologically incompatible red cell units Transfusion 25: 34–38 Ballas SK, Sherwood WC (1977) Rapid in vivo destruction of Yt(a+) erythrocytes in a recipient with anti-Yta Transfusion 17: 65–66 Ballas SK, Dignam C, Harris M (1985) A clinically significant anti-N in a patient whose red cells were negative for N and U antigens Transfusion 25: 377–380 Barbolla L, Nieto S, Llamas P (1993) Severe immune haemolytic anaemia caused by intravenous immunoglobulin anti-D in the treatment of autoimmune thrombocytopenia Vox Sang 64: 184–185 Basta M, Langlois PF, Marques M (1989a) High-dose intravenous immunoglobulin modifies complement-mediated in vivo clearance Blood 74: 326–333 Basta M, Kirshbom P, Frank MM (1989b) Mechanism of therapeutic effect of high-dose intravenous immunoglobulin J Clin Invest 84: 1974–1981 Basta M, Fries LF, Frank MM (1991) High doses of intravenous Ig inhibit in vitro uptake of C4 fragments onto sensitized erythrocytes Blood 77: 376–380 Bettigole R, Harris JP, Tegoli J (1968) Rapid in vivo destruction of Yt(a+) red cells in a patient with anti-Yta Vox Sang 14: 143 Blundell J (1824) Researches Physiological and Pathological London: E Cox and Son Borst-Eilers E (1972) Rhesusimmunisatie: onstaan en preventie Thesis, University of Amsterdam Bowman HS, Brason FW, Mohn JF (1961) Experimental transfusion of donor plasma containing blood-group antibodies into compatible normal human recipients II Induction of iso-immune haemolytic anaemia by a transfusion of plasma containing exceptional anti-CD antibodies Br J Haematol 7: 130 Brendemoen OJ, Aas K (1952) Hemolytic transfusion reaction probably caused by anti-Lea Acta Med Scand 141: 458 Brown DJ (1988) A rapid method for harvesting autologous red cells from patients with hemoglobin S disease Transfusion 28: 21–23 Brown DL, Lachmann PJ, Dacie JV (1970) The in vivo behaviour of complement-coated red cells: studies in C6-deficient, C3-depleted and normal rabbits Clin Exp Immunol 7: 401 Brown E (1983) Studies in splenectomy In: Immunoglobulin G Fc Receptor-mediated Clearance in Autoimmune Diseases Ann Intern Med 98: 206–218 Brown I Jr, Smith WW (1958) Hematologic problems associated with the use of extra-corporeal circulation for cardiovascular subjects Ann Intern Med 49: 1035 RED CELL INCOMPATIBILITY IN VIVO Buchholz DH, Bove JR (1975) Unusual response to ABO incompatible blood transfusion Transfusion 15: 577 Burton MS, Mollison PL (1968) Effect of IgM and IgG iso-antibody on red cell clearance Immunology 14: 861 Cattaneo M, Gringeri A, Capitanio AM et al (1989) Anti-D immunoglobulins for treatment of HIV-related immune thrombocytopenic purpura Blood 73: 357 Chaplin HJ (1959) Studies on the survival of incompatible cells in patients with hypo-gammaglobulinemia Blood 14: 24 Chaplin HJ, Cassell M (1962) The occasional fallibility of in vitro compatibility tests Transfusion 6: 375 Chaplin HJ, Coleman ME, Monroe MC (1983) In vivo instability of red-blood-cell-bound C3d and C4d Blood 62: 965–971 Chaplin HJ, Hunter VL, Rosche ME (1985) Long-term in vivo survival of Rh(D)-negative donor red cells in a patient with anti-LW Transfusion 25: 39–43 Christian RM, Stewart WB, Yuile CL (1951) Limitation of hemolysis in experimental transfusion reactions related to depletion of complement and isoantibody in the recipient Blood 6: 142 Clancey M, Bond S, Van Eys J (1972) A new example of anti-Lan and two families with Lan-negative members Transfusion 12: 106 –108 Contreras M, Mollison PL (1981) Failure to augment primary Rh immunization using a small dose of ‘passive’ IgG anti-Rh Br J Haematol 49: 371–381 Contreras M, Mollison PL (1983) Rh immunization facilitated by passively-administered anti-Rh? Br J Haematol 53: 153 –159 Contreras M, De Silva M, Teesdale P (1987) The effect of naturally occurring Rh antibodies on the survival of serologically incompatible red cells Br J Haematol 65: 475 – 478 Cooper AG, Brown DL (1971) Haemolytic anaemia in the rabbit following the injection of human anti-I cold agglutinin Clin Exp Immunol 9: 99 Crawford DH, Azim T, Daniels GL et al (1988) Monoclonal antibodies to the Rh antigen In: Progress in Transfusion Medicine Cash JD (ed.) 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individual with the Oh (Bombay) phenotype Transfusion 18: 738–742 Davey RJ, Gustafson M, Holland PV (1980) Accelerated immune red cell destruction in the absence of serologically detectable alloantibodies Transfusion 20: 348–353 Davey RJ, Rosen SR, Holland PV (1982) In vitro thermal characteristics of anti-Leb antibodies as predictors of their in vivo significance Abstracts of the 17th Congress of the International Society of Blood Transfusion, Budapest Dobbs JV, Prutting DL, Adebahr ME et al (1968) Clinical experience with three examples of anti-Yta1 Vox Sang 15: 216–221 Dzik WH, Blank J (1986) Accelerated destruction of radiolabelled red cells due to anti-Colton b Transfusion 26: 246–248 Eckrich RJ, Mallory D, Sandler SG (1995) Correlation of monocyte monolayer assays and posttransfusion survival of Yt(a+) red cells in patients with anti-Yta Immunohematology 11: 81–84 Eklund J, Nevanlinna HR (1971) Immuno-suppression therapy in Rh-incompatible transfusion BMJ iii: 623 Engelfriet CP, von dem Borne AEGKr, Beckers T (1972) Autoimmune haemolytic anaemias V Studies on the resistance against complement haemolysis of red cells of patients with chronic cold agglutinin disease Clin Exp Immunol 2: 255 Engelfriet CP, von dem Borne AEGKr, van der Meulen FW et al (1981) Immune destruction of red cells In: A Seminar on Immune-Mediated cell Destruction Chicago, IL: Am Assoc Blood Banks Ervin DM, Young LE (1950) Dangerous universal donors Observations on destruction of recipient’s A cells after transfusion of group O blood containing high titer of A antibodies of immune type not easily neutralizable by soluble A substance Blood 5: 61 Ervin DM, Christian RM, Young LE (1950) Dangerous universal donors II Further observations on the in vivo and in vitro behaviour of isoantibodies of immune type present in group O blood Blood 5: 553 Evans RS, Turner E, Bingham M (1965) Studies with radioiodinated cold agglutinins of ten patients Am J Med 38: 378 Evans RS, Turner E, Bingham M (1967) Chronic hemolytic anaemia due to cold agglutinins: the mechanism of resistance of red cells to C′ hemolysis by cold agglutinins J Clin Invest 46: 1461 449 CHAPTER 10 Evans RS, Turner E, Bingham M (1968) Chronic hemolytic anemia due to cold agglutinins II The role of C′ in red cell destruction J Clin Invest 47: 691 Fagge CH, Pye-Smith PH (1891) Textbook of the Principles and Practice of Medicine, vol London: J & A Churchill, p 648 Fehr J, Hofman V, Kappeler U (1982) Transient reversal of thrombocytopenia in idiopathic thrombocytopenic purpura by high-dose intravenous gamma globulin N Engl J Med 306: 1254 –1258 Ferguson DJ, Gaal GD (1988) Some observations on the Inb antigen and evidence that anti-Inb causes accelerated destruction of radiolabeled red cells Transfusion 28: 479– 482 Fleer A, van Schaik MLJ, von dem Borne AEGKr (1978) Destruction of sensitized erythrocytes by human monocytes in vitro: effects of cytochalasin B, hydrocortisone and colchicine Scand J Immunol 8: 515 Frank MM, Hamburger MI, Lawley TJ (1979) Defective Fc-receptor function in lupus erythematosus N Engl J Med 300: 518–523 Fudenberg HH, Allen FJ (1957) Transfusion reactions in the absence of demonstrable incompatibility N Engl J Med 256: 1180 Garratty G, Nance SJ (1990) Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry Transfusion 30: 617–621 Garratty G, Arndt PA (1999) Applications of flow cytofluorometry to red blood cell immunology Cytometry 38: 259–267 Gasser C (1945) Akute haemolytische Krisen nach PlasmaTransfusionen bei dystrophischtoxischen Saeuglingen Helv Paediatr Acta 1: 38 Gesellius F (1874) Zur Thierblut-Transfusion beim Menschen St Petersburg and Leipzig: Eduard Hoppe Gobel U, Drescher KH, Pottgen W et al (1974) A second example of anti-Yta with rapid in vivo destruction of Yt(a+) red cells Vox Sang 27: 171–175 Gorick BD, Hughes-Jones NC (1991) Relative functional binding activity of IgG1 and IgG3 anti-D in IgG preparations Vox 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(1986) Intravascular hemolytic transfusion reaction without detectable anti-bodies: a case report and review of literature Vox Sang 51: 96–101 van der Hart M, Engelfriet CP, Prins HK (1963) A haemolytic transfusion reaction without demonstrable antibodies in vitro Vox Sang 8: 363 van der Hart M, Szaloky A, van der Berg-Loonen EM (1974) Présence d’antigènes HL-A sur les hématies d’un donneur normal Nouv Rev Fr Hematol 14: 555 Hasse O (1874) Die Lammblut-Transfusion beim Menschen – Erste Reihe: 31 eigene Transfusionen umfassend St Petersburg and Leipzig: Eduard Hoppe Heistö H, Myhre K, Vogt E (1960) Haemolytic transfusion reaction due to incompatibility without demonstrable antibodies Vox Sang 5: 538 Heistö H, Myhre K, Börresen W (1962) Another case of haemolytic transfusion reaction due to incompatibility without demonstrable antibodies Vox Sang 7: 470 Hewitt WC Jr, Wheby M, Crosby WH (1961) Effect of prednisolone on incompatible blood transfusions Transfusion 1: 184 Hopkins JG (1910) Phagocytosis of red blood-cells after transfusion Arch Intern Med 6: 270 Hossaini AA (1972) Neutralization of Lewis antibodies in vivo and transfusion of Lewis incompatible blood Am J Clin Pathol 57: 489 Howard JE, Winn LC, Gottlieb CE (1982) Clinical significance of the anti-complement component of antiglobulin sera Transfusion 22: 269–272 Hsu TCS, Jagathambal K, Sabo BH (1975) Anti-Holley (Hy): characterization of another example Transfusion 15: 604 Huchet J, Cregut R, Pinon F (1970) Immuno-globulines anti-D Efficacitée‚ comparée des voies intra-musculaire et intra-veineuse Rev Fr Transfusion 13: 231 Hughes-Jones NC, Mollison PL, Veall N (1957) Removal of incompatible red cells by the spleen Br J Haematol 3: 125 Hughes-Jones NC, Mollison PL (1963) Clearance by the RES of ‘non-viable’ red cells In: Role du Système Réticuloendothelial dans l’Immunit‚ antibacterienne et antitumorale Paris: Edition du CNRS Hughes-Jones NC, Mollison PL (1968) Failure of a relatively small dose of passively administered anti-Rh to suppress primary immunization by a relatively large dose of Rhpositive red cells BMJ i: 150 RED CELL INCOMPATIBILITY IN VIVO ICSH (1980) Recommended methods for radioisotope redcell survival studies Br J Haematol 45: 659–666 Issitt PD, Valinsky JE, Marsh WL (1990) In vivo red cell destruction by anti-Lu6 Transfusion 30: 258–260 Jaffe CF, Atkinson JP, Frank MM (1976) The role of complement in the clearance of cold agglutinin-sensitized erythrocytes in man J Clin Invest 58: 942 Jandl JH (1955) Sequestration by the spleen of red cells sensitized with incomplete antibody and with metallo-protein complexes (Abstract) J Clin Invest 34: 912 Jandl JH, Greenberg MS (1957) The selective destruction of transfused ‘compatible’ normal red cells in two patients with splenomegaly J Lab Clin Med 49: 233 Jandl JH, Simmons RL (1957) The agglutination and sensitization of red cells by metallic cations: interactions between multivalent metals and the red-cell membrane Br J Haematol 3: 19 Jandl JH, Kaplan ME (1960) The destruction of red cells by antibodies in man III Quantitative factors influencing the patterns of hemolysis in vivo J Clin Invest 39: 1145 Jandl JH, Jones AR, Castle WB (1957) The destruction of red cells by antibodies in man I Observations on the sequestration and lysis of red cells altered by immune mechanisms J Clin Invest 36: 1428 Jennings ER, Hindmarsh C (1958) The significance of the minor crossmatch Am J Clin Pathol 30: 302 Jensen K, Freiesleben E, Sorensen SS (1965) The survival of red cells incompatible with Rh antibodies demonstrable only by enzyme technique Communication of the 10th Congress of the European Society of Haematology, Strasbourg Jouvenceaux A, Adenot N, Berthoux F (1969) Gammaglobuline anti-D lyophilisé intra-veineuse pour la prévention de l’immunisation anti-Rh Rev Fr Transfusion 13 (Suppl.): 341 Kaplan E, Hsu KS (1961) Determination of erythrocyte survival in newborn infants by means of Cr51-labelled erythrocytes Pediatrics 27: 354 Kaplan ME, Jandl JH (1961) Inhibition of red cell sequestration by cortisone J Exp Med 114: 921 Kelley CM, Karwal MW, Schlueter AJ et al (1998) Outcome of transfusion of K:11 erythrocytes in a patient with antiK11 antibody Vox Sang 74: 205–208 Kissmeyer-Nielsen F, Jensen KB, Ersbak J (1961) Severe haemolytic transfusion reactions caused by apparently compatible red cells Br J Haematol 7: 36 Kohan AI, Niborski RC, Rey JA et al (1994) High-dose intravenous immunoglobulin in non-ABO transfusion incompatibility Vox Sang 67: 195–198 Kumpel BM (1997) In vitro functional activity of IgG1 and IgG3 polyclonal and monoclonal anti-D Vox Sang 72: 45–51 Kumpel BM, Judson PA (1995) Quantification of IgG anti-D bound to D-positive red cells infused into D-negative subjects after intramuscular injection of monoclonal anti-D Transfusion Med 5: 105–112 Kumpel BM, Austin EB, Lee D et al (2000) Comparison of flow cytometric assays with isotopic assays of (51)chromium-labeled cells for estimation of red cell clearance or survival in vivo Transfusion 40: 228–239 Kumpel BM, De Haas M, Koene HR et al (2003) Clearance of red cells by monoclonal IgG3 anti-D in vivo is affected by the VF polymorphism of Fcgamma RIIIa (CD16) Clin Exp Immunol 132: 81–86 Kurlander RJ, Rosse WF, Logue GL (1978) Quantitative influence of antibody and complement coating of red cells on monocyte-mediated cell lysis J Clin Invest 61: 1309 Kurtz SR, Ouellet R, McMican A et al (1983) Survival of MM red cells during hypothermia in two patients with anti-M Transfusion 23: 37–39 Lachmann PJ, Pangburn MK, Oldroyd RG (1982) Breakdown of C3bi to C3c, C3d and a new fragment-C3g J Exp Med 156: 205–216 Lampe TL, Moore SB, Pineda AA (1979) Survival studies of Lan-positive red blood cells in a patient with anti-Lan (Abstract) Transfusion 19: 640 Larsson LG, Welsh VJ, Ladd DJ (2000) Acute intravascular hemolysis secondary to out-of-group platelet transfusion Transfusion 40: 902–906 Lau PYL, Jewlachow V, Leahy MF (1994) Successful transfusion of Yka-positive red cells in a patient with anti-Yka Vox Sang 64: 254–255 Lawley TJ, Hall RP, Fauci AS (1981) Defective Fc-receptor function associated with the HLA-B8/DRw3 haplotype Studies in patients with dermatitis herpetiformis and normal subjects N Engl Med J 304: 185–192 Lee D, Remnant M, Stratton F (1984) ‘Naturally occurring’ anti-Rh in Rh(D) negative volunteers for immunization Clin Lab Haematol 6: 33–38 Lewis SM, Dacie JV, Szur L (1960) Mechanism of haemolysis in the cold-haemagglutinin syndrome Br J Haematol 6: 154 Lin-Chu M, Broadberry RE (1990) Blood transfusion in the para-Bombay phenotype Br J Haematol 75: 568–572 van Loghem JJ (1965) Some comments on autoantibody induced red cell destruction Ann NY Acad Sci 124: 465 Loutit JF, Mollison PL, Young IM (1943) Citric acid-sodiumcitrate-glucose mixtures for blood storage Quart J Exp Physiol 32: 183 MacKenzie FAF, Elliot DH, Eastcott HHG (1962) Relapse in hereditary spherocytosis with proven splenunculus Lancet i: 1102 McSwain B, Robins C (1988) A clinically significant anti-Cra (Letter) Transfusion 28: 289–290 Maynard BA, Smith DS, Farrar RP (1988) Anti-Jka, -C and -E in a single patient, demonstrable only by the manual 451 CHAPTER 10 hexadimethrine bromide (polybrene) test, with incompatibilities confirmed by 51Cr-labelled red cell studies Transfusion 28: 302–306 Mohn JF, Lambert RM, Bowman HS (1961) Experimental transfusion of donor plasma containing blood-group antibodies into incompatible normal human recipients I Absence of destruction of red-cell mass with anti-Rh, antiKell and anti-M Br J Haematol 7: 112 Mollison PL (1943) The investigation of haemolytic transfusion reactions BMJ i: 529–559 Mollison PL (1951) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Mollison PL (1954) The life-span of red blood cells Lectures on the Scientific Basis of Medicine 2: 269 Mollison PL (1956) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Mollison PL (1959a) Blood group antibodies and red cell destruction BMJ ii: 1035 Mollison PL (1959b) Factors determining the relative clinical importance of different blood group antibodies Br Med Bull 15: 92 Mollison PL (1959c) Further studies on the removal of incompatible red cells from the circulation Acta Haematol (Basel) 10: 495 Mollison PL (1961) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Mollison PL (1962) Destruction of incompatible red cells in vivo in relation to antibody characteristics In: Mechanism of Cell and Tissue Damage Produced by Immune Reactions Basel: Schwabe, p 267 Mollison PL (1970) The effect of isoantibodies on red-cell survival Ann NY Acad Sci 169: 199 Mollison PL (1972) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Mollison PL (1979) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Mollison PL (1981) Determination of red cell survival using 51Cr In: A Seminar on Immune-Mediated Cell Destruction Chicago, IL: Am Assoc Blood Banks Mollison PL (1983) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Mollison PL (1985) Antibody-mediated destruction of foreign red cells In: Antibodies; Protective, Destructive and Regulatory Role F Milgrom, CJ Abeyounis, B Albini (eds) Basel: Karger, pp 65–74 Mollison PL (1986) Survival curves of incompatible red cells An analytical review Transfusion 26: 43–50 Mollison PL (1989) Further observations on the patterns of clearance of incompatible red cells Transfusion 29: 347– 354 Mollison PL, Cutbush, M (1955) The use of isotope-labelled red cells to demonstrate incompatibility in vivo Lancet i: 1290 452 Mollison PL, Hughes-Jones NC (1958) Sites of removal of incompatible red cells from the circulation Vox Sang 3: 243 Mollison PL, Hughes-Jones NC (1967) Clearance of Rh positive cells by low concentration of Rh antibody Immunology 12: 63 Mollison PL, Polley MJ, Crome P (1963) Temporary suppression of Lewis blood-group antibodies to permit incompatible transfusion Lancet i: 909 Mollison PL, Crome P, Hughes-Jones NC (1965) Rate of removal from the circulation of red cells sensitized with different amounts of antibody Br J Haematol 11: 461 Mollison PL, Frame M, Ross ME (1970) Differences between Rh(D) negative subjects in response to Rh(D) antigen Br J Haematol 19: 257 Mollison PL, Johnson CA, Prior DM (1978) Dose-dependent destruction of A1 cells by anti-A1 Vox Sang 35: 149 Mollison PL, Engelfreit CP, Contreras S (1987) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Moore HC, Issitt PD, Pavone BG (1975) Successful transfusion of Chido-positive blood to two patients with antiChido Transfusion 15: 266 Morel PA, Garratty G, Perkins HA (1978) Clinically significant and insignificant antibodies in blood transfusion Am J Med Technol 4L: 122 Moscow JA, Casper AJ, Kodis C et al (1987) Positive direct antiglobulin test results after intravenous immune globulin administration Transfusion 27: 248–249 Myllyla G, Furuhjelm U, Nordling S (1971) Persistent mixed field polyagglutinability Electrokinetic and serological aspects Vox Sang 20: Nance SJ, Arndt P, Garratty G (1987) Predicting the clinical significance of red cell alloantibodies using a monocyte monolayer assay Transfusion 27: 449–452 Nicholson-Weller A, Burge J, Fearon DT (1982) Isolation of a human erythrocyte membrane glycoprotein with decay accelerating activity for C3 convertases of the human complement system J Immunol 129: 184–189 Niejadlik DC, Lozner EL (1974) Cooling mattress induced acute hemolytic anemia Transfusion 14: 145–147 Nordhagen R, Aas M (1978) Association between HLA and red cell antigens VII Survival studies of incompatible red cells in a patient with HLA-associated haemagglutinins Vox Sang 35: 319 Noyes WD, Bothwell TH, Finch CA (1960) The role of the reticulo-endothelial cell in iron metabolism Br J Haematol 6: 43 Ottenberg R (1911) Studies in isoagglutination I Transfusion and the question of intravascular agglutination J Exp Med 13: 425 Panzer S, Mueller-Eckhardt G, Salama A (1984) The clinical significance of HLA antigens on red cells Survival studies in HLA-sensitized individuals Transfusion 24: 486–489 RED CELL INCOMPATIBILITY IN VIVO Pelosi MA, Bauer JL, Langer A (1974) Transfusion of incompatible blood after neutralization of Lewis antibodies Obstet Gynecol 4: 590 Perrault RA, Hogman CF (1972) Low concentration red cell antibodies III ‘Cold’ IgG anti-D in pregnancy: incidence and significance Acta Univ Uppsaliensis 120 Peters B, Reid ME, Ellisor SS (1978) Red cell survival studies of Lub incompatible blood in a patient with anti-Lub (Abstract) Transfusion 18: 623 Polesky HF, Swanson JL (1966) Studies on the distribution of the blood group antigen Doa (Dombrock) and the characteristics of anti-Doa Transfusion 11: 162 Ponfick P (1875) Experimentelle Beitäge zur Lehre von der Transfusion Virchows Arch Pathol Anat 62: 273 Report from Collaborating Laboratories (1991) Results of tests with different cellular bioassays in relation to severity of Rh D haemolytic disease Vox Sang 60: 225–229 Rosenfield RE, Vitale B, Kochwa S (1967) Immune mechanisms for destruction of erythrocytes in vivo II Heparinization for protection of lysin-sensitized erythrocytes Transfusion 7: 261 Sabo B, Moulds JJ, McCreary J (1978) Anti-JMH: another high titer-low avidity antibody against a high frequency antigen (Abstract) Transfusion 18: 387 Salama A, Kiefel V, Mueller-Eckhardt C (1986) Effect of IgG anti-Rho(D) in adult patients with chronic autoimmune thrombocytopenia Am J Haematol 22: 241–250 Samson D, Mollison PL (1975) Effect on primary Rh immunization of delayed administration of anti-Rh Immunology 28: 349 Sausais L, Krevans JR, Townes AS (1964) Characteristics of a third example of anti-Xga (Abstract) Transfusion 4: 312 Schanfield MS, Stevens JO, Bauman D (1981) The detection of clinically significant erythrocyte alloantibodies using a human mononuclear phagocyte assay Transfusion 21: 571–576 Schmidt PJ, Nancarrow JF, Morrison EG (1959) A hemolytic reaction due to the transfusion of Ax blood J Lab Clin Med 54: 38 Schneider J, Preisler O (1966) Untersuchungen zur serologischen Prophylaxe der Rh-Sensibilisierung Blut 12: Schreiber AD, Frank MM (1972) Role of antibody and complement in the immune clearance and destruction of erythrocytes In vivo effects of IgG and IgM complementfixing sites J Clin Invest 51: 575 Shirey RS, Oyen R, Heeb KN (1988) 51Cr radiolabelled survival studies in a patient with anti-Lu12 Transfusion 28: 375 Shirey RS, Boyd JS, King KE et al (1998) Assessment of the clinical significance of anti-Dob Transfusion 38: 1026 –1029 Shore GM, Steane EA (1977) Survival of incompatible red cells in a patient with anti-Csa and three other patients with antibodies to high-frequency red cell antigens Atlanta, GA: Commun Am Assoc Blood Banks Sieg vW, Borner P, Pixberg HJ (1970) Die Elimination Rh-positiver fetaler Erythrozyten aus der Blutbahn Rhnegativer Erwachsener durch korpereigene Isoagglutinine Blut 21: 69 Silvergleid A, Wells RF, Hafleigh EB (1978) Compatibility test using 51chromium-labelled red blood cells in crossmatch positive patients Transfusion 18: Sistonen P, Nevanlinna HR, Virtaranta-Knowles K (1981) Nea, a new blood group antigen in Finland Vox Sang 40: 352–357 Smith GN, Mollison PL (1974) Responses in rabbits to the red cell alloantigen HgA Immunology 26: 865 Stewart JW, Mollison PL (1959) Rapid destruction of apparently compatible red cells BMJ i: 1274 Strumia MM, Colwell LS, Dugan A (1958) The measure of erythropoiesis in anemias I The mixing time and the immediate post-transfusion disappearance of T-1824 dye and of Cr51-tagged erythrocytes in relation to blood volume determination Blood 8: 128 Swisher SN, Young LE (1954) Studies of the mechanisms of erythrocyte destruction initiated by antibodies Trans Assoc Am Phys 67: 124 Szymanski IO, Valeri CR (1971) Lifespan of preserved red cells Vox Sang 21: 97 Thomson A, Contreras M, Gorick B (1990) Human monoclonal IgG3 and IgG1 anti-Rh D mediate clearance of D positive red cells Lancet ii: 1147–1150 Thorpe SJ, Fox BJ, Dolman CD et al (2003) Batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti-Rh D activity Vox Sang 85: 80–84 Tilley CA, Crookston MC, Haddad SA (1977) Red blood cell survival studies in patients with anti-Cha, anti-Yka, antiGe and anti-Vel Transfusion 17: 169 Tisdall LH, Garland DM, Szanto PB (1946a) The effects of the transfusion of group O blood of high iso-agglutinin titer into recipients of other blood groups Am J Clin Pathol 16: 193 Tisdall LH, Garland DM, Wiener AS (1946b) A critical analysis of the value of the addition of A and B group-specific substances to group O blood for use as universal donor blood J Lab Clin Med 31: 437 Todd C, White RG (1911) On the fate of red blood corpuscles when injected into the circulation of an animal of the same species: with a new method for the determination of the total volume of blood Proc R Soc Lond B 84: 255 Tregellas WM, Moulds JJ, South SF (1978) Successful transfusion of a patient with anti-LW and LW positive blood (Abstract) Transfusion 18: 384 453 HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN the time of the first delivery but anti-D was produced in detectable amounts only after the further stimulus of a second pregnancy; (2) primary immunization to D occurred only during the second pregnancy It seems that the second explanation is usually the right one; the evidence is that in these cases the antibody develops only towards the end of the second pregnancy, for example in the last weeks in 50% of the cases in one series (Eklund 1978) Effect of different amounts of anti-D given immediately after delivery In a ‘blind’ trial conducted by a working party of the Medical Research Council (MRC) and begun in 1967 in the UK, different doses of anti-D were given to four groups of women (about 450 women in each group) Over the dose range 200 to 20 µg there was a significant, although small, trend towards an increase in the failure rate months after a first pregnancy (see Table 12.1) Approximately 200 women in each of the four groups were followed through a second pregnancy with a D-positive infant; the differences in the failure rates in the different groups were not statistically significant, although failures were suggestively more frequent with the 20-µg dose than with the larger doses Doses of anti-D for postpartum injection Evidence that 20 µg of anti-D/ml red cells is effective in suppressing primary immunization has been reviewed earlier To allow a margin of safety, 25 µg/ml has been recommended (WHO 1971) A dose of 300 µg of antiD has long been the standard postpartum dose in the USA and many other countries, although 100 µg has been used in the UK A dose of 300 µg covers TPHs of about 15 ml or less, that is to say the amounts found in approximately 99.8% of women at the time of delivery, whereas 100 µg covers TPHs of ml or less, as found in about 99% of women The dose of 300 µg was chosen to cover the vast majority of TPHs and to make screening tests for the occasional very large TPH inessential; nevertheless, a screening test capable of testing TPHs of 15 ml or more of red cells is mandatory in the USA (AABB 1991) With the 100-µg dose, the screening test is obviously even more important and must detect TPHs of ml or more Furthermore, when screening tests are positive, an estimate must be made of the size of the TPH so that an appropriate extra dose of anti-D can be given Flow cytometry can be used both to detect TPHs of ml or more (see above) and to provide an accurate estimate of their size, but this technique is not widely available In detecting TPHs of 15 ml or more, the rosetting test is probably the most convenient and can be used to provide semi-quantitative estimates The acid-elution technique can also be used to provide quantitative estimates (Appendix 13) In women in whom an unusually large TPH has been detected and who have been given extra anti-D Ig, tests are sometimes done to confirm that an adequate dose has been given One practice is to look for the presence of anti-D in the maternal plasma but this is unsound in principle, as even when the antigen concentration is low, not all the antibody will be bound, however little is given Furthermore, the method has been shown to be of no practical value (Ness and Salamon 1986) The method that seems most likely to be worthwhile is to test for clearance of fetal red cells from the maternal circulation, for example by using a rosetting test Although a relationship between clearance and immunosuppression has yet to be firmly established, the two seem at least to be associated; see Chapter for further discussion Doses of anti-D following premature termination of pregnancy In women undergoing termination of pregnancy, usually at about months, a common practice is to give 50 µg of anti-D In a series of 3080 women treated with this dose following therapeutic abortion, only 13 (0.42%) were found to be immunized during a second pregnancy (I Simonovits, personal communication), suggesting a very low failure rate of suppression The dose which should be given between about 12 and 20 weeks is uncertain; some give the same amount as for a termination at 12 weeks and others give the same dose as would be given at 28 weeks There is general agreement that from 20 weeks onwards the dose used should be that normally used at 28 weeks (e.g in the USA, 300 µg) Antenatal administration of anti-D As described above, a single pregnancy with a Dpositive, ABO-compatible infant initiates primary Rh D immunization in about one in six (17%) of D-negative women When anti-D immunoglobulin is 521 CHAPTER 12 given postnatally, the incidence of primary Rh D immunization falls to about 1.5%, as judged by the development of anti-D by the time of delivery of a second D-positive infant Thus, in some 90% of cases Rh D immunization that would otherwise follow pregnancy can be prevented by giving anti-D postnatally As described below, it seems likely that most of the remaining 10% of cases can be prevented by antenatal treatment Safety of antenatal treatment Although it was at first believed that injecting anti-D into a D-negative woman pregnant with a D-positive fetus was potentially dangerous, further consideration and practical experience show that the fear is groundless In D-positive infants born to mothers who are actively immunized to Rh D, the total amount of antiD in the infant is only about 10% of that in the mother (Hughes-Jones et al 1971) Accordingly, when 300 µg of anti-D is injected into the mother, even if equilibration across the placenta occurred immediately, not more than about 30 µg would be expected to be in the infant In fact, as discussed earlier, IgG is transferred relatively slowly across the placenta Accordingly, by the time that equilibrium is reached a considerable amount of the anti-D which has been injected into the mother will have been catabolized and the total amount reaching the infant will be less than 30 µg In trials in which 300 µg of anti-D were given to Dnegative women at the twenty-eighth week and again at the thirty-fourth week of pregnancy, although as many as 28% of ABO-compatible D-positive infants had a weakly positive DAT the cord serum bilirubin concentration never exceeded 3.4 mg/dl (58 µmol/l) and no infant developed hyperbilirubinaemia severe enough to require phototherapy (Bowman et al 1978) In a few cases in which D-negative women were given four doses of 300 µg of anti-D between about the twelfth and thirty-fourth weeks of pregnancy in an attempt to turn off the immune response following the discovery of low concentrations of maternal anti-D reacting only with enzyme-treated cells, not all the D-positive infants born subsequently had a positive DAT (JM Bowman, personal communication) Results observed with antenatal treatment A review of 11 published trials (seven non-randomized trials with historical or geographical controls, one 522 randomized control trial, one quasi-randomized controlled trial, one community intervention trial and one retrospective before-and-after study) concluded that routine antenatal anti-D prophylaxis is effective in reducing the number of D-negative women who are sensitized during pregnancy (Chilcott et al 2003) These authors suggest that introduction of routine antenatal anti-D prophylaxis in England and Wales would reduce the sensitization rate from 0.95% to 0.35% Furthermore, such an intervention is costeffective, at least in the UK (Chilcott et al 2004) In one series in which almost 10 000 D-negative women carrying D-positive fetuses were given an injection of anti-D at 28 weeks (either 300 µg i.m or 240 –300 µg i.v.), less than 0.1% developed anti-D by full term, whereas previous experience indicated that without antenatal anti-D the incidence would have been 1.8% (Bowman 1984) As explained on p 505, the figure of 1.8% is substantially greater than that reported by others, but the discrepancy can be explained In another series in which, unlike the one just referred to, only primiparae were included, of 1238 D-negative women carrying D-positive fetuses who were injected at both 28 and 34 weeks with 100 µg of anti-D, only 0.16% developed anti-D by the time of delivery In an earlier series from the same centre in which women were given anti-D only after delivery, 0.9% formed anti-D by the end of pregnancy (Tovey et al 1983) The foregoing figures give a slightly too favourable impression of the success of antenatal immunoprophylaxis with anti-D because in both series a few additional women developed anti-D either months after delivery or during a second pregnancy In the first series, in which most of the women were tested at months, there were two in whom anti-D was detected for the first time, an incidence of very approximately 0.02%; of an unknown number who had a second D-positive pregnancy there were another two who developed anti-D before the twenty-eighth week (Bowman 1984) In the second of the two series quoted above anti-D developed during a second pregnancy with a D-positive infant in out of 325 women, but in neither case did the antibody appear to cause clinically significant haemolytic disease Amount of anti-D for antenatal injection From the data of Bowman and co-workers (1978) it appears that treatment should be given as early as 28 weeks In the USA and some other countries, the only antenatal HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN dose of anti-D is 300 µg, injected at 28 weeks In nine women who had received this dose, plasma levels of anti-D 9–10 weeks later were between 0.7 and 2.6 ng/ml, corresponding to a total residue of 6–21 µg of anti-D At term, some women had no detectable anti-D (Bowman and Pollock 1987) Better survival of injected IgG anti-D, indicating that an average of 19 µg should remain 12 weeks after a dose of 250 µg, was found in another investigation, although there was substantial variation between different women (Eklund et al 1982) It must be concluded that a single dose of 300 µg given at 28 weeks may be too small to protect all women (Bowman and Pollock 1987) The administration of 300 µg of anti-D at 28 weeks is convenient in countries where 300 µg of or thereabouts is the standard dose to be given postpartum, as the same preparation can then be given on the two occasions On the other hand, in countries where the standard postpartum dose is 100 µg it is convenient to give two doses of 100 µg during pregnancy, at 28 weeks and 34 weeks respectively This dosage will in fact result in a rather higher concentration of anti-D in the mother’s plasma immediately before delivery than when a single dose of 300 µg is given at 28 weeks (seventh edition, p 395), will result in a certain saving of anti-D and, again, will avoid the confusion of having two different sized doses to be administered antenatally and postnatally When anti-D is given antenatally, the serum concentration at term may still be high enough to be detectable by the IAT In women who have been given anti-D Ig antenatally, the finding of a positive IAT at the time of delivery must not be regarded as a contraindication to giving the usual postpartum dose Intravenous administration of anti-D As discussed in Chapter 5, it is possible that when anti-D is given intravenously at the same time as an injection of D-positive red cells, the dose of antibody required for the suppression of primary Rh D immunization may be smaller than when the anti-D is injected intramuscularly There is therefore a potential advantage in giving antiD intravenously rather than intramuscularly immediately following delivery when it is likely that any D-positive red cells in the circulation have arrived there only recently In practice, because of the increased risk of transmitting viral infections when Ig is given intravenously (see Chapter 16) and because the methods used for viral inactivation in some preparations are suspect, the i.m route should be used whenever possible When giving anti-D antenatally there does not seem to be any potential advantage in i.v administration, as the object is to protect against Rh D immunization over a period of many weeks and, within days of giving anti-D, plasma levels are the same whether the injection has been given intravenously or intramuscularly Occasional reactions have been reported following the i.v injection of anti-D immunoglobulin prepared by fractionation on DEAE Sephadex (Hoppe et al 1973); at one centre, only seven reactions, two of which were severe enough to require treatment, were noted following injections to 80 000 women (H Hoppe, personal communication) In another series, transient marked flushing and mild chest discomfort were noted in out of 2792 women The reactions were thought to be due to aggregated material in vials that had too high a moisture content (Bowman et al 1980) In a third series, not a single untoward reaction was reported amongst 120 000 recipients (JR O’Riordan, personal communication) In a fourth series involving 261 women no adverse reactions were reported (MacKenzie et al 2004) Monoclonal/recombinant anti-D for prophylaxis Although human monoclonal anti-D derived from Epstein–Barr virus (EBV)-transformed B lymphocytes has been available since 1984 (Crawford 1983), it has not yet emerged as a licensed product capable of replacing the tried and tested polyclonal product described above Two monoclonal antibodies, one IgG1 (BRAD5) and one IgG3 (BRAD3), have been used as a blend in trials and shown to be capable of protecting D-negative male volunteers from D immunization (Kumpel et al 1995; Smith et al 2000) BRAD5 can be quantified in human serum using a monoclonal anti-BRAD5 idiotype by ELISA (Austin et al 2001) Monoclonal anti-D produced by EBV transformation of B lymphocytes can be manipulated using recombinant DNA technology to optimize affinity and Fc effector functions (Proulx et al 2002) and expressed in alternative expression systems for manufacture (Edelman et al 1997; Miescher et al 2000) A recombinant IgG1 anti-D produced in CHO cells was shown to prevent D immunization in male volunteers (Miescher et al 2004) 523 CHAPTER 12 Immunosuppression of D sensitization by induction of tolerance Successful induction of tolerance to peptides corresponding to regions of the D polypeptide in transgenic mice presages the development of a new approach to prevention of haemolytic disease resulting from D immunization (Hall et al 2005; see also Chapter 3) Paternal leucocyte therapy Lam and co-workers (2003) investigated the feasibility of isolating paternal mononuclear cells free of red cell contamination with a view to using these cells as a treatment for severe D alloimmunization Changes in the incidence and mortality of haemolytic disease As described in Chapter 3, before the introduction of immunoprophylaxis of Rh D haemolytic disease, the frequency of the disease, both in England and in North America was about per 170 births Assuming an average family size of more than two children, this figure is close to expectation: the frequency of the disease in first pregnancies is very low (less than one per 1000 births), in second pregnancies is about one per 100 births, and in subsequent pregnancies is somewhat greater Immunoprophylaxis became available in 1968–70 In one North American hospital (dealing with a predominantly white population), in which postnatal anti-D was introduced in 1968, and antenatal anti-D added in 1985, the frequency with which (immune) anti-D was found in pregnant women declined from in 238 in 1974 (Walker 1984) to in 963 in 1988 (Walker and Hartrick 1991) and to in 1663 in 1990–92; in the last period, HDN was diagnosed in only in 2419 births (Walker et al 1993) In one English region (East Anglia), the figures for 1993–95 were as follows: anti-D was found in one per 584 pregnant women but in almost 80% of cases the anti-D was not detected at the time of the first antenatal visit and was subsequently present at a concentration of less than iu/ml (0.2 µg/ml) Less than one infant per 2000 had clinical signs of HDN (A Rankin, personal communication) It should be noted that antiD is given antenatally in only approximately 12% of hospitals in England and Wales (Chilcott et al 2003) 524 The decline in the death rate from Rh haemolytic disease has been substantially greater than the decline in the incidence of the disease In England and Wales, registered deaths fell from in 2180 births in 1953 (Walker 1958) to in 5400 in 1977 and to in 62 500 in 1990 (Clarke and Mollison 1989; Hussey and Clarke 1992) It must be emphasized that these figures are not for total deaths, as up to September 1992 deaths occurring before the twenty-eighth week of pregnancy were not registered in England and Wales (Since October 1992, deaths from 24 weeks onward have been registered.) Chilcott and coworkers (2003) put the figure at one death in every 20 800 births The most important steps in preventing Rh D immunization by pregnancy First, in order to minimize the risk that, due to a technical or clerical error, a D-negative woman will be reported as D positive and thus not be given anti-D immunoglobulin after delivery, the D group of all so far unimmunized women should be determined (using two different anti-Ds) on at least two separate occasions: (1) at the time of the first antenatal visit (booking); and (2) at 28–34 weeks In addition, the following steps are recommended for all so far unimmunized D-negative women: As anti-D immunoglobulin is sometimes withheld because a report on the infant’s D group has not yet been received, anti-D immunoglobulin (100 µg or more) should be given to all D-negative women within 72 h of delivery, unless the infant has been shown to be D negative After delivery, if the infant is D positive, a screening test for fetal red cells should be made on a sample of the mother’s blood; if this is positive a quantitative estimate of the number of cells present should be made (see Appendix 13) and extra anti-D Ig given if indicated All D-negative women should be given a dose of anti-D immunoglobulin after induced abortion The case for giving anti-D after spontaneous abortions in the first trimester is not strong (see p 505) and practice varies After obstetric interference, for example amniocentesis or other potentially sensitizing event, anti-D immunoglobulin should be given, whether or not fetal red cells can be demonstrated in the mother’s blood HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN In women who are 20 or more weeks’ pregnant, a test for fetal red cells should be made and extra anti-D immunoglobulin given when indicated If the pregnancy continues, further anti-D should be given, for example 300 µg at 28 weeks or 100 µg at 28 weeks and again at 34 weeks Women with partial D A woman with partial D may develop anti-D as a result of a pregnancy with a fetus whose red cells carry the D epitopes which she lacks In two reported cases (Davey 1976b; Lacey et al 1983), the mother’s red cells belonged to category VI and, in both, the mother had apparently been immunized by pregnancy alone In the second of the two cases, the infant was severely affected and died, although mild HDN is the rule In the years 1976–85, 28 pregnant D-positive women with anti-D in their serum were tested at various centres of the National Blood Service in the UK; most of the women either belonged to category VI or had red cells which reacted too weakly with anti-D to be categorized; 26 out of the 28 infants were normal or only mildly affected; in most of these cases the titre of anti-D in the mother’s serum was low although in a few cases it was in the range 20 –256; of the remaining two infants, one required an exchange transfusion and one died in utero at 37–38 weeks (maternal anti-D titre in this case was 128) When a woman is grouped as weak D (Du) for the first time after delivery it is essential to a screening test for fetal red cells, as she may in fact be D negative with a substantial number of D-positive fetal red cells in her circulation and not only need anti-D immunoglobulin but require more than the standard dose Administration of anti-D Ig to women with partial D Most partial D red cells absorb little anti-D Accordingly, after the injection of anti-D Ig to a woman with partial D recently delivered of a Dpositive fetus, there should be enough circulating antibody to suppress Rh D immunization (Lubenko et al 1989) Incidentally, in subjects with ‘high-grade Du’ red cells injected with D-positive red cells, followed by IgG anti-D, clearance was about twice as slow as in D-negative subjects (Schneider 1976) D-negative woman with a partial-D infant A D-negative woman, whose infant was typed as D positive, was given 100 µg of anti-D immunoglobulin after it had been estimated that her circulation contained approximately ml of fetal red cells Despite a second dose of 100 µg of anti-D 72 h later, the fetal red cells were not cleared from the maternal circulation and it was then shown that the infant’s red cells not only reacted weakly with anti-D but failed altogether to react with some anti-D sera, indicating that they were partial D In vitro, the red cells reacted relatively weakly with the anti-D Ig that had been injected (Revill et al 1979, supplemented by personal communication from P Tippett) Haemolytic disease due to antibodies other than anti-D, anti-A and anti-B A list of the various red cell alloantibodies that can cause haemolytic disease is given near the beginning of this chapter; after anti-D, anti-c and anti-K are easily the most important Rh antibodies other than anti-D Haemolytic disease due to anti-c Of all deaths from HDN not due to anti-D registered in England and Wales for the years 1977–90, 32 out of 49 were associated with anti-c, with or without anti-E (Clarke and Mollison 1989, supplemented by personal communication from CA Clarke) Anti-c, with or without anti-E, is found in about 0.7 per 1000 pregnant women, for example 177 out of 280 000 (Bowell et al 1986); 65 in about 90 000 (Tovey 1986) The frequency of haemolytic disease due to anti-c is substantially lower than this for two reasons: first, as 40 –50% of pregnant women with anti-c have been immunized by transfusion (Astrup and Kornstad 1977; Bowell et al 1986), the fetus is relatively often c negative; and second, as the antibody is often present in low titre, a substantial number of c-positive infants have a negative DAT For example, of 42 c-positive infants born to mothers with anti-c, 10 had a negative DAT; in out of the 10 the antibody was detectable only with enzyme-treated red cells In 24 out of 42 infants, including the 10 with a negative DAT, the titre of anti-c was or less (Astrup and Kornstad 1977) In 525 CHAPTER 12 another series, the titre of anti-c was or less in 129 out of 177 (Bowell et al 1986) In two series, only about 20% of c-positive infants born to mothers with anti-c required exchange transfusion (Astrup and Kornstad 1977; Hardy and Napier 1981) In the several series referred to above no stillbirths and only one neonatal death were recorded In contrast, in another series, of 62 c-positive infants born to c-immunized women over a 40-year period, 20 required transfusion and eight were either stillborn (with hydrops) or died in the neonatal period (Wenk et al 1985) In a study from the USA, covering the period 1967 to 2001, fetuses from 46 out of 55 pregnancies with anti-c had a positive direct antiglobulin test and eight of the affected neonates had HDN requiring fetal transfusion An antibody titre of 1:32 or greater or the presence of hydrops identified all affected fetuses There were no perinatal deaths in this study (Hackney et al 2004) In England and Wales in 1977–87, among approximately 000 000 births, there were 26 infants recorded as dying from HDN due to anti-c (Clarke and Mollison 1989), that is approximately one per 250 000 births; even at the end of this period, that is in 1987, the death rate from HDN due to anti-D was still very much higher, i.e one per 25 600 births Several reliable methods for c typing of fetal DNA are available, based on the polymorphism at nucleotide 307 of RHCE (reviewed in Van der Schoot et al 2003; see Chapter 5) Quantitative real-time PCR can be used for c typing of cell-free fetal DNA from maternal blood in pregnancies at risk from HDN due to anti-c [Finning K, Martin P, Daniels G, cited in Daniels et al (2004)] Other Rh antibodies Anti-C was found in 38 out of 280 000 pregnancies (0.14 per 1000); it was about five times commoner in DccEE than in DccEe women; of infants tested at birth, two out of three had a negative DAT and there were no deaths from HDN (Bowell et al 1988) Anti-Cw is almost always associated with unaffected or mildly affected infants, although it has caused kernicterus (Lawler and van Loghem 1947); the incidence of HDN due to anti-Cw is probably less than in 50 000 pregnancies (Bowman and Pollock 1993) Anti-E alone is the Rh antibody found most commonly after anti-D In one series, it was present in 97 of about 90 000 pregnancies (Tovey 1986) The antibody is often naturally occurring and is then sometimes 526 detectable only with enzyme-treated red cells Anti-E very seldom causes haemolytic disease In a review of 283 pregnancies affected by anti-E alone during the period from 1959 to 2004 there was one perinatal death attributable to anti-E HDN (Joy et al 2005) Anti-e is a very rare cause of HDN; the disease is usually mild (Moncharmont et al 1990) Anti-Rh29 in Rh-null pregnant women has been associated with both mild (Bruce et al 1985) and severe HDN (Lubenko et al 1992) Anti-Rh17 in D–/D– women has been associated with both moderate (Lenkiewicz and Zupanska 2000; Brumit et al 2002) and severe HDN (Denomme et al 2004) In the case described by Denomme and coworkers, maternal ABO-mismatched blood was successfully used for intrauterine transfusion Kell antibodies Anti-K is found in about one per 1000 pregnant women, for example 127 out of 127 000 (Caine and Mueller–Heubach 1986); 27 out of 40 000 (Tovey 1986, estimates for 1982 only); 407 out of 350 000 (Mayne et al 1990) The frequency of HDN due to anti-K is much lower, as in most cases the mother has been immunized by a transfusion of K-positive blood and not by pregnancy and the fetus is K negative A history of previous transfusion was elicited from 88% of K-immunized pregnant women in one series (Mayne et al 1990) and from 80% in another (Pepperell et al 1977) In one more series, of 396 infants born to women with anti-K, only 20 were K positive, with a positive DAT In the period concerned there were somewhat more than 800 000 deliveries, giving an overall incidence of HDN due to anti-K of about one in 40 000 births (Bowman 1994, supplemented by personal communication) From this figure the incidence of HDN due to anti-K can be deduced as approximately in 20 000 pregnancies As the finding of anti-K during pregnancy is due in most cases to immunization by a previous transfusion rather than immunization by TPH from the fetus, and in most cases the fetus is K negative, the fetal genotype (i.e Kk or kk) should be determined The results of PCR-based tests on DNA obtained from amniotic fluid or chorionic villi were in complete agreement with those of subsequent tests on red cells after birth (Lee et al 1996) Several PCR assays for K typing of fetuses have been described (reviewed by Van der Schoot et al HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN 2003; see also Chapter 8) Quantitative real-time PCR can be used for K typing of cell-free fetal DNA from maternal blood in pregnancies at risk of HDN from anti-K [Finning K, Martin P, Daniels G, cited in Daniels et al (2004)] The frequency of very severe haemolytic disease (stillbirth or hydrops) in K-positive infants born to mothers with anti-K was out of 13, out of 10 and out of 20 in three series (Caine and Mueller-Heubach 1986; Mayne et al 1990; Bowman 1994), giving an approximate mortality rate of one per 100 000 births for HDN due to anti-K This estimate agrees very poorly with registered deaths Over the period 1977–90, there were approximately 000 000 births in England and Wales and 15 deaths from HDN in which the mother’s serum contained anti-K, either alone (11 cases) or with antibodies other than anti-D, giving a mortality of one in 600 000 The discrepancy between this figure and the one deduced above can only partly be explained by the fact that deaths before the twenty-eighth week of pregnancy were not recorded HDN due to anti-K has been found to be more severe when immunization is due to a previous pregnancy rather than a previous transfusion, even when the mother has had no previously affected infant (Pepperell et al 1977; Caine and Mueller-Heubach 1986) There is evidence that fetal anaemia caused by antiK is due partly to suppression of erythropoiesis More than one observer has noted that the levels of reticulocytes and of amniotic fluid bilirubin are lower than expected in relation to the degree of anaemia (see eighth edition, p 685) In 11 cases of HDN due to antiK with moderate or severe anaemia, levels of reticulocytes, erythroblasts and amniotic bilirubin levels were significantly lower than in 11 matched cases due to anti-D (Vaughan et al 1994) Kell antigens are present on early erythroblasts [ J Valinsky, cited by Marsh and Redman (1990); Southcott et al (1999)] Both monoclonal and polyclonal anti-K inhibit K-positive red cell progenitors but, with anti-D, there is no consistent inhibition of D-positive progenitors (Vaughan et al 1998) Inhibition of erythropoiesis by anti-K may result from immune destruction of the erythroid precursors (Daniels et al 1999) As D is expressed much later than K during erythropoiesis the lack of inhibition of erythropoiesis in cases of HDN caused by anti-D (and other Rh antibodies) can be explained Inhibition of myeloid and platelet progenitors by antiK in vitro has also been reported and correlated with thrombocytopenia in three cases of HDN attributable to anti-K (Wagner et al 2000a,b) The mechanism for these effects is unclear Another feature of haemolytic disease due to anti-K, which has been noted occasionally, is a poor correlation between the severity of the disease and the titre of antibody in the mother’s serum In one case, hydrops fetalis occurred despite an anti-K titre of only two at the thirty-seventh week of pregnancy (L McDonnell, personal communication) In another, hydrops at 23 weeks, associated with an Hb concentration of 22 g/l, was associated with a maternal anti-K titre of (Bowman et al 1989; see also Fleetwood et al 1996) A quantitative method for the measurement of anti-K in the serum of alloimmunized women is described by Ahaded and co-workers (2002) The method is based on elution of anti-K from K-positive red cells at acid pH and quantification of the antibody (and subclass) by enzyme-linked immunosorbent assay (ELISA) Anti-k is a very rare cause of HDN As in HDN due to anti-K, the disease may be severe despite a low maternal antibody titre For example, in a case in which the mother’s anti-k titre was 16, the infant’s Hb concentration at 31 weeks was only 60 g/l and three intravascular transfusions were given to the infant in utero (Bowman et al 1989) In another case, despite a maternal anti-k titre of only 8–16, the infant, after being born spontaneously at 33 weeks, had an Hb concentration of 76 g/l (Anderson et al 1990) Other antibodies of the Kell system Anti-Kpa, -Jsa and -Ku have been implicated in HDN of varying severity (for references, see Daniels 2002); anti-Jsb has caused hydrops fetalis (Gordon et al 1995) In two cases of severe HDN due to anti-Kpb, the mother had presumably been primarily immunized by transfusion in childhood (Dacus and Spinnato 1984; Gorlin and Kelly 1994) Other antibodies Anti-Fya Anti-Fya usually causes mild haemolytic disease, although among 11 cases reviewed by Greenwalt and colleagues (1959) there were two deaths In a more recent study of 68 pregnancies when anti-Fya was detected, three severely anaemic fetuses were identified, two of which required intrauterine 527 CHAPTER 12 transfusions (Goodrick et al 1997) These authors suggest that pregnancies in which anti-Fya is detected at titres > 1:64 should be closely monitored Mild haemolytic disease due to anti-Fy3 has been reported (Albrey et al 1971) Anti Jka Anti Jka very rarely causes severe HDN; kernicterus has once been reported (Matson et al 1959) and the authors know of a single case of hydrops fetalis: a grossly oedematous infant born by caesarean section at 36 weeks, with a positive DAT and a cord Hb of 39 g/l The mother, who had had two previous infants, had an anti-Jka titre that varied from 16 to 128 (SL Barron, E Hey and M Reid, personal communication) Haemolytic disease due to anti-M When anti-M develops during pregnancy, it may fail to affect the fetus (see Bowley and Dunsford 1949; De YoungOwens et al 1997) In rare cases, haemolytic disease develops and is occasionally responsible for hydrops fetalis (Stone and Marsh 1959; Matsumoto et al 1981) In both of these cases, the anti-M in the mother’s serum was very potent In cases in which the infant is mildly or moderately affected with haemolytic disease due to anti-M there are two features that resemble ABO haemolytic disease rather than Rh haemolytic disease: first, the DAT is only weakly positive but unwashed red cells agglutinate spontaneously in a colloid medium, and second, the osmotic fragility of the red cells may be greatly increased (Stone and Marsh 1959; Freiesleben and Jensen 1961) In a pregnant woman with relatively potent anti-M and a history of stillbirths, plasma to a total volume of 51 l was withdrawn, absorbed with M-positive red cells and reinfused Although there was only a small fall in antibody titre, the woman was delivered of a normal infant (Yoshida et al 1981) Haemolytic disease due to anti-S and anti-s This is very occasionally severe or even fatal (Levine et al 1952; Issitt 1981, p 43) Haemolytic disease due to anti-U This may be severe or even fatal (Bürki et al 1964) In a review of 15 cases, severe HDN occurred only when the maternal anti-U had a titre of 1:512 or more However, titres as high as 1:4000 were not necessarily associated with significant haemolysis (Smith et al 1998) 528 Haemolytic disease due to anti-PP1Pk HDN of varying severity has been reported In one case the IgG component of the antibody in the mother’s serum had a titre of 16 (Hayashida and Watanabe 1968) In another, in which the infant had an Hb concentration of 8.7 g/dl, the DAT was only weakly positive (Levene et al 1977) Antibodies to low-frequency antigens These have occasionally caused HDN but very few severe cases have been reported In a case due to anti-ELO, exchange transfusion was needed (Better et al 1993) Haemolytic disease of the newborn due to anti-A and anti-B (ABO haemolytic disease) Anti-A and anti-B occurring in group B and A subjects are predominantly IgM, but in O subjects are at least partly IgG In 15% of all pregnancies in white people, the mother is O and her infant is A or B, but clinically obvious haemolytic disease is comparatively rare There seem to be two main reasons for this finding: first, the A and B antigens are not fully developed at birth, and second, A and B substances are not confined to the red cells so that only a small fraction of IgG anti-A and anti-B which crosses the placenta combines with the infant’s red cells The protective effect of A and B determinants in fluids and tissues other than red cells was discussed by Tovey (1945) and Wiener and co-workers (1949) Using sensitive methods, small amounts of IgG anti-A or anti-B are commonly found on the red cells of group A and B infants born to group O mothers (Hsu et al 1974) Although severe HDN due to anti-A and anti-B is relatively rare, minor degrees of red cell destruction are common, as shown by the incidence of neonatal jaundice and by the slight lowering of the Hb concentration in ABO-incompatible infants compared with ABO-compatible infants Incidence ABO haemolytic disease must be defined before its incidence can be estimated For example, taking the criterion of the development of jaundice within 24 h of birth, the incidence was estimated to be one in 180 (Halbrecht 1951); taking the faintest trace of HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN Table 12.2 Effect of ABO incompatibility between infant’s red cells and mother’s serum on cord blood findings (from Rosenfield and Ohno 1955) Mean values No of cord blood samples* Haemoglobin concentration (g/dl) Reticulocytes (%) Bilirubin concentration (mg/dl)† 2256 16.05 4.34 2.11 558 15.82 4.53 2.22 89 14.85 5.94 2.96 A Infant’s red cells compatible with mother’s serum B Infant’s red cells incompatible with mother’s serum: infant’s direct antiglobulin test negative C As B, but direct antiglobulin test positive * Haemoglobin estimates were made on all samples, and reticulocyte counts and bilirubin estimates on almost all samples † mg/dl = 17 µmol/l jaundice in the first 24 h as the criterion, the incidence was as high as one in 70 in another series (Valentine, 1958) In two series in which the Hb concentration and bilirubin concentration of cord blood were measured in ABO-compatible and ABO-incompatible infants born to group O mothers, it was found that values for Hb concentration were slightly lower, and those for bilirubin concentration slightly higher, in ABOincompatible infants In the first of these series the DAT was positive in 14% of the ABO-incompatible infants and in these the Hb concentration was distinctly lower and the bilirubin concentration distinctly higher than in the infants with a negative antiglobulin test (Table 12.2) In the second series, a positive DAT was found in as many as 32.8% of the ABOincompatible infants and anti-A or anti-B could be eluted from the infant’s red cells in a further 38%, but clinically significant disease, judged by a peak bilirubin concentration of more than 12 mg/dl (204 µmol/l) together with anaemia and reticulocytosis, was diagnosed in only 27 out of the 680 infants, i.e in in 125 of all newborn infants or in 25 ABO-incompatible infants (Desjardins et al 1979) Cases of HDN due to anti-A or anti-B that are severe enough to need exchange transfusion are relatively rare, for example none amongst 1500 newborn infants (Rosenfield 1955); out of 14 000 consecutive births (second edition, p 505); out of 8000 births (Ames and Lloyd 1964); and out of 5704 infants born to group O mothers (Voak and Bowley 1969) The foregoing figures are from Europe or North America; a higher incidence has been found in some other populations Higher incidence in certain populations Black people There is apparent disagreement about the relative frequency of ABO haemolytic disease in black people and in white people The disease was found to be commoner in black people by Kirkman (1977), but in another series, although a positive DAT was commoner in black than in white infants, there was no difference in bilirubin levels or in the need for phototherapy (Peevy and Wiseman 1978) In assessing the relative frequency of ABO haemolytic disease in black people and white people, a complication is introduced by the fact that in ABOcompatible infants hyperbilirubinaemia is commoner in white than in black people Accordingly, if, for example, all ABO-incompatible infants whose serum bilirubin concentration exceeds 170 µmol/l are considered to have ABO haemolytic disease, the disease will appear to be much commoner in white people than it really is One way of assessing the effect of ABO incompatibility is to compare various indices, i.e the DAT, the age at the onset of jaundice and the maximum recorded serum bilirubin concentration, in ABOincompatible and ABO-compatible infants When this was done, in a very careful study in which black and white infants were being treated in the same nursery, ABO haemolytic disease was found to be clearly 529 CHAPTER 12 commoner in black than in white people, the relative frequency being between and 6:1, depending on the particular criteria chosen for diagnosis (Kirkman 1977) In a survey in Nigeria, the serum of about one-third of group O subjects was found to have strong lytic activity for A or B cells, anti-B lytic activity being commoner than anti-A lytic activity; moderate or severe jaundice, defined as a serum bilirubin concentration exceeding 10 mg/dl (170 µmol/l with a positive DAT was found to develop in about one-third of infants whose red cells were lysed in vitro by their mother’s serum (Worlledge et al 1974) These figures indicate that the frequency of ABO HDN in Nigeria is about 5% of births A factor that might be expected to produce relatively severe HDN in black people is the relatively strong expression of A and B that they have (see Chapter 4) Although the relative potency of anti-A and anti-B has in the past been suspected of being racially determined, recent work indicates that environmental factors may be more important (see Chapter 4) Arabs From a survey of almost 3000 newborn Arab infants it was concluded that ABO haemolytic disease was about as common in Arabs as in black people and that the disease tended to be more severe in Arabs than in Europeans: exchange transfusion for ABO haemolytic disease was carried out in one in every 500 newborn Arab infants (Al-Jawad et al 1985) Selected populations in Central and South America In a comparison between populations in Puerto Rico and North Carolina, the incidence of ABO HDN was found to be substantially higher in Puerto Rico (Huntley et al 1976); a similar increase has been found in Venezuela, i.e about 30% of ABO-incompatible infants have signs of haemolytic disease compared with 20% or less in European and North American populations (Cariani et al 1995) About one in 300 of all newborn infants in Venezuela need exchange transfusion (Romano et al 1994) The higher incidence and greater severity of ABO HDN in Venezuela may be due to raised anti-A titres associated with infestation with Ascaris (Romano et al 1988) Relative frequency in group A and B infants If group A and B infants were equally liable to the disease, the ratio A/B in white affected infants should be 530 approximately 2.7:1 In the series of Fischer (1961) the ratio was 3.7:1, suggesting that, in white people, group B infants are slightly less liable than group A infants to develop haemolytic disease On the other hand, in another study a positive DAT was found to be relatively commoner in group B than in group A infants, in both white and black people (Peevy and Wiseman 1978) Familial incidence In about 50% of families in which ABO haemolytic disease is diagnosed, the first ABO-incompatible infant in the family is affected (first edition, p 391) In families in which ABO haemolytic disease is mild, an affected infant may be followed by a clinically unaffected infant On the other hand, when severe disease occurs it is likely to be followed by similarly severe disease in subsequent infants of the same blood group Mrs Bak: First infant born February 1960; rapidly developed jaundice and found to have erythroblastaemia; diagnosed as having haemolytic disease due to anti-A; exchange transfusion given but infant died Second infant born April 1961; cord Hb 11.6 g/dl, DAT weakly positive; blood film showed numerous normoblasts and microspherocytes; osmotic fragility: 50% lysis in 0.590% NaCl (grossly increased) The infant was given one exchange transfusion and made excellent progress Third infant born November 1962; cord blood findings almost identical to those of the second infant, for example cord Hb 11.8 g/dl; DAT weakly positive, infant was treated by exchange transfusion and recovered uneventfully As the findings indicate, the degree of severity of the haemolytic process appeared to be almost identical in the second and third infants The titre of IgG anti-A in the mother’s serum, estimated by the method of Polley and co-workers (1965), was virtually the same at the time of birth of these two infants, namely 4096 and 8192 respectively Serological findings in mothers ABO group Mothers of infants with ABO haemolytic disease almost invariably belong to group O (Rosenfield 1955), evidently because IgG anti-A and anti-B occur far more commonly in group O than in group B and A mothers (Rawson and Abelson 1960; Kochwa et al 1961) In one series of 45 cases the mother HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN was group O in 43 instances and subgroup A2 in the remaining two; A2 mothers produced much stronger ‘incomplete’ anti-B than A1 mothers (Munk-Andersen 1958) IgG anti-A and anti-B The simplest and most satisfactory test is to treat the mother’s serum with a reducing agent (e.g DTT) to inactivate IgM antibodies and then determine the antiA or anti-B titre by IAT using an anti-IgG serum (Voak and Bowley 1969) Using this method, a titre of 512 or more was found to be very suggestive of haemolytic disease An earlier study, using a modification of the partial neutralization test of Witebsky, had shown that in ABO haemolytic disease the indirect antiglobulin titre was almost always in the range 64 –16 000, and was 1000 or more in 13 out of 18 cases in which an infant needed an exchange transfusion (Polley et al 1965) IgG subclasses of anti-A and anti-B Anti-A and anti-B from pregnant women are at least partly IgG2 (see Chapter 4) Macrophages carrying the highaffinity FcIIa receptor mediate lysis of red cells sensitized with IgG2 anti-A, although not that of cells sensitized with IgG2 anti-Rh D (Kumpel et al 1996) Presumably, therefore, IgG2 anti-A and -B play a part in red cell destruction in those infants with ABO HDN whose macrophages carry the high-affinity FcRIIa receptor Antibody-dependent cell-mediated cytotoxicity assays with monocytes In a large number of cases in which an ADCC(M) assay gave negative results, i.e < 10% lysis, the infant never showed signs of red cell destruction; in three cases in which the ADCC assay was strongly positive, i.e > 45% lysis, the infant was severely affected and needed more than one exchange transfusion In cases in which there was 10 – 45% lysis in the assay, the degree of affection could not be predicted The discrepancy between the results of the assay and the severity of red cell destruction in the infant appeared to be due to using standard adult red cells in the assay When the infant’s red cells were used, the degree of lysis was strongly affected by the number of A or B sites on the red cells (Brouwers et al 1988a) Serological findings in infants The direct antiglobulin test using anti-IgG In infants with relatively severe ABO haemolytic disease the amount of antibody on the cells was found to be less than 220 molecules per cell (0.6 µg IgG/ml red cells) in 10 of 15 cases (Romano et al 1973) As, when the spin-tube antiglobulin test is used, the minimum number of antibody molecules that can be detected is about 100 –150 (see Chapter 8), it is not surprising that in mildly affected infants the DAT may be negative When a very sensitive method is used some anti-A or anti-B can be demonstrated on the red cells of virtually all ABO-incompatible infants When the DAT was carried out in an autoanalyzer, using a lowionic-strength medium with enhancing agents, the red cells of 13 A1 and eight B infants, all apparently healthy, gave positive results The authors calculated that there were between eight and 85 molecules of IgG per red cell (Hsu et al 1974) Incidentally, the DAT was positive in only one of seven A2 infants It is evident that there is an apparent substantial discrepancy between the findings in haemolytic disease due to anti-D, on the one hand, and to anti-A and antiB, on the other In Rh D haemolytic disease, infants may have a strongly positive DAT without showing any clinical signs of disease, whereas in ABO haemolytic disease they may be clinically affected but have a negative or only very weakly positive test It has been suggested that in ABO haemolytic disease the findings in DATs not indicate correctly the amount of antibody bound to the red cells in vivo Romans and coworkers (1980) showed, for single examples of IgG anti-A and anti-B, that these antibodies must be bound by both combining sites (monogamous bivalency) to be detectable in the antiglobulin test The presence of A antigen sites on unbranched chains of glycolipids or glycoproteins on the surface of the red cells of newborn infants would make it difficult for molecules to bind bivalently because the sites on unbranched chains are not close enough together Incidentally it would also make it difficult for molecules binding univalently to co-operate in activating C1 (see also Chapter 3) Although the foregoing explanation is intellectually satisfying, it may not be correct For one thing, the claim that the binding of anti-A to adult group A red cells is predominantly bivalent has been challenged by 531 CHAPTER 12 Romano and co-workers (1983), who found that the binding constants of anti-A and its Fab derivative were similar with both adult and newborn group A red cells Other observations that appear to be incompatible with the hypothesis of Romans and co-workers (1980) have been discussed previously (seventh edition, p 695) To take one example, if cord group A1 red cells are coated with 125I-labelled human IgG anti-A, some 90% of the antibody appears to be firmly bound by the red cells (NC Hughes-Jones, personal communication, 1981) It seems that further experimental work is needed to define precisely the extent and nature of the binding of IgG anti-A and anti-B to the red cells of newborn infants The direct antiglobulin test using anti-C3d Even in severe ABO haemolytic disease the infant’s red cells not react with anti-C3d (Mollison 1983, p 694) This finding is due partly to the weak expression of A and B antigens on the red cells of newborn infants but also to the relatively low level of complement in the serum of newborn infants (Brouwers et al 1988b) Elution of antibody from infant’s red cells In ABO haemolytic disease, when the DAT is only weakly positive or even negative, eluates from the infant’s red cells may give strong indirect antiglobulin reactions with adult A1 red cells (Voak and Bowley 1969) The explanation for the finding seems to be that the elution procedure results in a considerable concentration of antibody (Voak and Williams 1971; Romano et al 1973) There is a relation between crossreactivity of eluates, for example reaction of an eluate from group A cells with B cells as well as with A cells, and severity, simply because there is a relation between crossreactivity and potency (see Chapter 4) Spontaneous agglutination of red cells Red cells from infants with ABO haemolytic disease tend to clump spontaneously when suspended in plasma (Wiener et al 1949) Similarly, in observations on a series of moderately severely affected infants it was noticed that ‘blood freshly drawn from the infant formed large clumps which were easily seen when the 532 blood was allowed to flow down the side of a tube or was examined on an opal glass tile’; the authors pointed out that if blood is taken from a patient of group A1 who has received a transfusion of plasma containing potent anti-A, it behaves in the same way (Crawford et al 1953) The tendency of red cells taken from infants with ABO haemolytic disease to clump spontaneously was re-investigated by Romano and Mollison (1975) Red cells coated with small amounts IgG anti-A and then washed were found to be almost as readily agglutinated by ABO-compatible plasma as by anti-IgG Plasma was very much more effective than serum in potentiating agglutination In a series of infants suspected of having ABO haemolytic disease, a test for autoagglutination, performed by mixing red cells with their own plasma on a slide, was positive in 23 out of 25 cases, whereas the DAT was positive in only 20 of the cases (Romano et al 1982) The observation described by Lewi and Clarke (1960) presumably also demonstrates some change in the surface charge of the red cells of affected infants Washed red cells from infants suspected of having HDN due to ABO incompatibility were suspended in polyvinyl pyrrolidone (PVP) and were found to sediment far more rapidly than similarly treated red cells from normal infants Reactivity of infant’s red cells with anti-A and anti-B in vitro As described in Chapter 4, the red cells of newborn infants react relatively weakly with anti-A and anti-B in vitro HDN due to anti-A is observed only in infants who are genetically A1 (Zuelzer and Kaplan 1954) At the time of birth the red cells of such infants may fail to react with anti-A1, although samples taken when the infant is a few months old react (Crawford et al 1953) No case of HDN due to anti-A in an infant unequivocally belonging to subgroup A2, as shown by tests at least many months after birth, has yet been described One puzzling observation is that, whereas the red cells of healthy infants who are genetically A1 are agglutinated at the time of birth by an extract of Dolichos biflorus, red cells from genetically A1 infants with haemolytic disease due to anti-A may fail to be agglutinated by an extract of Dolichos (Gerlini et al 1968) The cause of this finding has never been HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN demonstrated but blocking of antigen sites by bound IgG anti-A seems to be a possibility Premature infants seem to be protected from ABO haemolytic disease, presumably because there are fewer A and B sites on the red cells than on the cells of full-term newborn infants (Schellong 1964) osmotic fragility is almost always above normal limits, at least in moderately severe cases, whereas in Rh D haemolytic disease, even in severe cases, only minor increases in osmotic fragility are found and spherocytosis is unusual (Crawford et al 1953) In ABO haemolytic disease, the changes in osmotic fragility may persist for as long as or weeks after birth Secretor status of infant It seems that the secretor status of the infant plays little or no part in protecting it against ABO haemolytic disease In fact, the ratio of secretors to non-secretors is slightly higher than expected; see Voak (1969) who also quotes several earlier papers in support The excess of secretors may be related to the fact that secretor infants are more prone to induce immune responses in their mothers (see Chapter 4) Changes in bilirubin concentration Although the rise in serum bilirubin concentration is usually only moderate and can be controlled by phototherapy, occasional cases of kernicterus have been reported (Grumbach and Gasser 1948) so that, as mentioned above, early exchange transfusion is occasionally indicated Management of ABO haemolytic disease Haematological findings Routine antenatal tests not indicated Haemoglobin concentration In moderately severe ABO haemolytic disease, the Hb concentration of cord blood may be below normal limits (Mollison and Cutbush 1949b; see also the cases described above) After birth, due to the wider range of normal Hb values that then prevail, it is unusual to find infants who are definitely anaemic Compared with Rh D haemolytic disease, ABO haemolytic disease is a shortlived affair and it is unusual for anaemia to be found after the first weeks or so of life Reticulocytosis and erythroblastaemia A slight increase in reticulocytes is a common feature in HDN due to ABO incompatibility (Rosenfield 1955) In the series of fairly severe cases collected by Crawford and co-workers (1953), the reticulocyte count exceeded 15% in out of 11 cases In five of these cases there were 30 or more nucleated red cells per 100 leucocytes Spherocytosis and changes in osmotic fragility Microspherocytes are frequently prominent in blood films from infants with ABO haemolytic disease (Grumbach and Gasser 1948) Similarly, red cell ABO incompatibility seldom causes severe haemolytic disease and routine antenatal tests to assess the potency of anti-A and anti-B are not indicated In women who have a history suggesting that a previous infant has been affected with ABO haemolytic disease, cord blood should be taken and tested as soon as possible after birth The authors of the tenth edition of this book report finding only two convincing cases of hydrops fetalis due to ABO incompatibility In both, abnormal swelling of the mother’s abdomen at 32–34 weeks prompted an examination of the fetus by ultrasonography and revealed fetal hydrops In the first case, the infant was born by caesarean section at 32 weeks and found to be group A with a strongly positive DAT; haemoglobinuria was noted The cord PCV was reported to be 0.43, but h after birth was 0.30 Packed group O red cells were transfused but the infant died at 20 h Two previous infants had had severe neonatal jaundice The mother’s IgG anti-A titre was 4000 (Gilja and Shah 1988) In the second case, the infant was born by caesarean section at 34 weeks and found to be group B with a positive DAT The cord blood PCV was 0.20, identical to that of an umbilical sample taken while the infant was still in utero At the time of the report the infant remained gravely ill The mother’s anti-B titre was reported as 65 536 (Sherer et al 1991) 533 CHAPTER 12 Exchange transfusion in ABO haemolytic disease As severe anaemia is very uncommon, the main indication for exchange transfusion is the threat of serious hyperbilirubinaemia, leading to kernicterus Moderate hyperbilirubinaemia can be controlled by phototherapy When exchange transfusion is judged to be necessary, group O blood should be used Provided that the donor’s plasma has been screened so as to exclude donors with potent anti-A or anti-B, the antibodies in the transfused plasma are unlikely to exacerbate the haemolytic process A better solution is to use group O red cells suspended in group AB plasma, preferably from an ABH secretor Phototherapy The mode of action of phototherapy in lowering serum bilirubin concentration is described briefly on p 519 In those full-term infants with ABO haemolytic disease whose serum bilirubin concentrations threaten to rise to dangerous levels, phototherapy is often sufficient to control the situation Some recommendations about monitoring bilirubin levels and indications for phototherapy were given as follows: if the cord serum bilirubin concentration is known to have been > mg/dl, estimate bilirubin every h; otherwise, estimate bilirubin 6-hourly Phototherapy should be begun if the bilirubin level reaches 10 mg/dl within 12 h, or 12 mg/dl (68 µmol/l) within 18 h, or 14 mg/dl within 24 h or 15 mg/dl thereafter (Osborn et al 1984) Of 44 infants diagnosed by these authors as having ABO haemolytic disease, only four required phototherapy Use of intravenous immunoglobulin In a study from Saudi Arabia, the value of phototherapy plus intravenous immunoglobulin G (IVIG) was compared with phototherapy alone (control group) in reducing the requirement for exchange transfusion of babies with hyperbilirubinaemia due to ABO haemolytic disease Each group comprised 56 babies Exchange transfusion was carried out on four babies in the study group compared with 16 babies in the control group No adverse effects of IVIG were reported in the study group (Miqdad et al 2004) 534 Alternatives to exchange transfusion A case in which an ABO-incompatible term infant with hyperbilirubinaemia was successfully treated with oral D-penicillamine (300 mg / kg per day divided in three doses for days), phototherapy, intravenous fluids and recombinant human erythropoietin (200 units/kg subcutaneously on every second day for weeks) is described by Lakatos and co-workers (1999) This alternative treatment regime was undertaken because the infant’s parents were Jehovah’s Witnesses and would not agree to exchange transfusion Thirteen infants with ABO HDN, with a pretreatment serum bilirubin level of 14.6 mg/dl, were treated with A or B trisaccharides, usually in a dose of 45 mg Within 5– days bilirubin levels were normal in 10 out of 13 A comparable group not treated with trisaccharides showed no fall in bilirubin levels after days (Romano et al 1994) Haemolytic reactions after transfusing A or B red cells When red cells from adult group A or group B donors are transfused, the red cells will almost always have more A and B sites than the infant’s own red cells and are likely to undergo more rapid destruction In one case in which an infant with HDN was given an exchange transfusion with A1 blood, the infant developed haemoglobinuria and died (Carpentier and Meersseman 1956) In two other group A infants with haemolytic disease due to anti-A, the use of group A blood, for exchange transfusion in the first case, and for a simple transfusion in the second, led to a substantial increase in jaundice and the development of kernicterus The authors noted that the DAT became positive only after the transfusion of group A blood and then gave a mixed-field appearance, evidently because only the transfused group A red cells from adult donors were agglutinated (Sender et al 1971) Danger of transfusing A or B red cells from adult donors to A or B premature infants born to group O mothers As described above, A and B antigens are particularly weak in premature infants; maternal IgG anti-A or anti-B may therefore be present without causing a haemolytic syndrome However, a haemolytic transfusion reaction may be produced if group A or B red cells HAEMOLYTIC DISEASE OF THE FETUS AND THE NEWBORN from adult donors are transfused In three cases described by Falterman and Richardson (1980), haemolytic reactions were observed in infants with birthweights of between 1280 and 1560 g (30 –32 weeks’ gestation) In all three infants the DAT was negative in the first days of life but, after transfusion of red cells of the infant’s ABO group, hyperbilirubinaemia developed in all three cases and haemoglobinuria occurred in two The authors described the infants as suffering from unrecognized ABO haemolytic disease, but to the present authors that seems to be a misconception Haemolytic disease due to anti-H Following the birth of an unaffected group B infant, two successive group O infants were born to an Oh mother, whose serum contained lytic IgG anti-H Both infants had cord bilirubin levels of 45 µmol/l and needed exchange transfusion; lytic anti-H was demonstrated in cord serum (Moores et al 1994) Exchange transfusion is not always necessary when group O infants are born to an Oh mother In a case described by Battacharya and co-workers (2002) an Oh lady with potent anti-H gave birth to two group O children in consecutive pregnancies and neither child was affected by HDN These authors suggest the low expression of H antigen on cord blood and/or absorption of anti-H by H antigen on the placenta may account for the lack of disease in this case References AABB (1991) Standards for Blood Banks and Transfusion Services, 14th edn Arlington, VA: Am Assoc Blood Banks Ackerman BD, Dyer GY, Leydorf MM (1970) Hyperbilirubinemia and kernicterus in small premature infants Pediatrics 45: 968 Ahaded A, Brossard Y, Debbia M et al (2002) Quantitative determination of anti-K (KEL1) IgG and IgG subclasses in the serum of severely alloimmunized pregnant women by ELISA Transfusion 40: 1239–1245 Albrey JA, Vincent EER, Hutchinson J et al (1971) A new antibody, anti-Fy3, in the Duffy blood group system Vox Sang 20: 29–35 Alie-Daran SJ, Dugoujon J-M, Fournie A (1992) Gm typing, IgG subclasses of anti-Rh (D) and severity of hemolytic disease of the newborn Vox Sang 62: 127–128 Al-Jawad ST, Keenan P, Kholeif S (1985) Incidence of ABO haemolytic disease in a mixed Arab population Saudi Med J 7: 41– 45 Allen FH Jr (1957) Induction of labor in the management of erythroblastosis fetalis Q Rev Pediat 12: Allen FH Jr, Diamond LK, Vaughan VC III (1950) Erythroblastosis fetalis, VI Prevention of kernicterus Am J Dis Child 80: 779 Ames AC, Lloyd RS (1964) A scheme for the ante-natal prediction of ABO haemolytic disease of the newborn Vox Sang 9: 712 Anderson NA, Tandy A, Westgate J et al (1990) Haemolytic disease of the newborn caused by anti-k (Abstract) Transfus Med 1(Suppl 1): 58 Arce MA, Thompsen ES, Wagner S et al (1993) Molecular cloning of RhD cDNA derived from a gene present in RhDpositive, but not RhD-negative individuals Blood 82: 651–655 Armitage P, Mollison PL (1953) Further analysis of controlled trials of treatment of haemolytic disease of the newborn J Obstet Gynaecol Br Emp 60: 605 Astrup J, Kornstad L (1977) Presence of anti-c in the serum of 42 women giving birth to c positive babies: serological and clinical findings Acta Obstet Gynecol Scand 56: 185 Austin EB, Smith LC, Walker RY (2001) An anti-idiotopic antibody-based enzyme-linked immunosorbent assay for the quantification of the monoclonal anti-D BRAD-5 Vox Sang 80: 179–183 Barrie JV, Quinn MA (1985) Selection of donor red cells for fetal intravenous transfusion in severe haemolytic disease of the newborn (Letter) Lancet i: 1327–1328 Bartsch FK (1972) Fetale Erythrozyten im mütterlichen Blut und Immunprophylaxe der Rh-Immunisierung Klinische und experimentelle Studie Acta Obstet Gynecol Scand 20: (Suppl.): 1–128 Battacharya S, Makar Y, Laycock RA et al (2002) Outcome of consecutive pregnancies in a patient with Bombay (Oh) blood group Transfus Med 12: 379–382 Bayliss KM, Kueck BD, Johnson ST et al (1991) Detecting fetomaternal hemorrhage: a comparison of five methods Transfusion 31: 303–307 Bennett PR, Le Van Kim C, Colin Y et al (1993) Prenatal determination of fetal RhD type by DNA amplification N Engl J Med 329: 607–610 Better PJ, Ford DS, Frascarelli A et al (1993) Confirmation of anti-ELO as a cause of haemolytic disease of the newborn Vox Sang 65: 70 Bishop GJ, Krieger VI (1969) The timing of rhesus immunization and the prevention of antibody response using anti-Rh immune globulin Aust NZ J Obstet Gynaecol 9: 228 Blackburn GK (1976) Massive fetomaternal haemorrhage due to choriocarcinoma of the uterus J Pediatr 89: 680 Boorman KE, Dodd BE, Mollison PL (1945) Iso-immunisation to the blood-group factors A, B and Rh J Pathol Bact 57: 157 Booth PB, Dunsford I, Grant J et al (1953) Haemolytic disease in first-born infants BMJ ii: 41 535 ... 482 involved, Rh system: anti-D or -D, -E, 3; -c, 5; -c, -E, 6; -E, 12; -C or -Cw, 3; -C, -E, 1; -e, 1; Jk system: 27 (-Jka, 24; -Jkb, 23); Fy system: 13 (all -Fya); Kell system: 12 (all anti-K);... red-cell survival Ann NY Acad Sci 169 : 199 Mollison PL (1972) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications Mollison PL (1979) Blood Transfusion in Clinical Medicine. .. et al 1 967 ); anti-U (Meltz et al 1971; Rothman et al 19 76) ; anti-HI, -Jkb, -S and -Fyb (Giblett et al 1 965 ); anti-E, -K, -S and -Fya (Moncrieff and Thompson 1975); and anti-c, -E and -Jkb (Joseph