Sukati H, Watson HG, Urbaniak SJ, Barker RN
Mapping helper T-cell epitopes on platelet membrane glycoprotein IIIa in chronic autoimmune thrombocytopenic purpura
Blood 2007; 109: 4528–38.
Chronic autoimmune thrombocytopenic purpura (AITP) is a bleeding disorder, characterised by the production of autoantibodies that mediate platelet destruction. The clinical signs include petechial haemorrhages, haemorrhagic bullae on mucous membranes, gingival or gastrointestinal bleeding, menorrhagia, retinal haemorrhages, and, most seriously, intracranial haemorrhages. Current therapeutic strategies for AITP rely on non-specific immunosuppressive agents, or intravenous immunoglobulin or anti-D, with refractory cases undergoing splenectomy to remove a major site of autoantibody production and platelet destruction.
A major focus on research into the pathogenesis of AITP has been the characterisation of the autoantibody response. Platelet membrane glycoprotein IIIa (GPIIIa) has emerged as the major autoantigen that is bound by autoantibodies in most patients. T helper (Th) cells reactive with GPIIIa, which are capable of driving the autoantibody response, are activated in AITP. The aim of the study carried out by Sukati et al. in the University, in the Regional Transfusion Centre, and in the Department of Haematology (Aberdeen Royal Infirmary, UK) was to map the fine specificity of platelet-specific Th cells from patients with chronic AITP. Samples of whole blood were obtained from 31 patients (21 women and 10 men) with AITP, who attended the outpatient haematology clinic. Samples of whole blood for isolation of peripheral blood mononuclear cells (PBMC) were also taken from 25 healthy control donors (18 women and 7 men, and from a further group of 5 patients with aplastic anaemia (4 men and 1 woman) as disease controls (because this condition responds to immunosuppression and is considered to have an autoimmune basis). The diagnosis of AITP was made by exclusion of other causes of thrombocytopenia; the majority of patients (29 of 31) were being treated with immunosuppressive drugs at the time of sampling, and 8 had undergone splenectomy.
PBMC from patients and controls were stimulated with a panel of 86 overlapping synthetic 15-mer (15-oligomers) peptides, spanning the complete sequence of GPIIIa. One or more peptides elicited recall proliferation by PBMC from 28 of patients, and, typically, multiple sequences were stimulatory. In contrast, responses in healthy control donors were rare (χ2 = 115.967; p ≤ 0.001). It was confirmed that the proliferating PBMC from patients were cells of the CD3-positive and CD4-positive helper phenotype, that were MHC class I-restricted. The presence of anti-platelet autoantibodies was serologically confirmed in 27 out of the 31 patients with AITP (serum antibodies and eluate antibodies). PBMC from disease control donors rarely proliferated when stimulated with peptides from the GPIIIa. Despite variation between different cases of AITP, particular sequences were commonly recognized, with PBMC from 24 patients (77%) responding to one or more of the four most dominant peptides.
The need for specific, effective, and safe treatment for patients with chronic AITP may be met by the development of peptide immunotherapy to re-induce Th tolerance to platelet glycoproteins. Mapping dominant autoreactive helper epitopes is the first step in new approaches to the treatment of AITP.
Kjaersgaard M, Aslam R, Kim M, et al
Epitope specificity and isotype of monoclonal anti-D antibodies dictate their ability to inhibit phagocytosis of opsonized platelets [Brief Report]
Blood 2007; 110: 1359–61.
Autoimmune thrombocytopenic purpura (AITP) is a bleeding disorder in which platelets are opsonised by autoantibodies and destroyed by Fc receptor-mediated phagocytosis. Treatment options include corticosteroids or splenectomy. Rh immune globulin (anti-D) can also be used in order to increase platelet counts in AITP; however, the competition for supplies of anti-D immune globulin for its primary indication (haemolytic disease of the newborn, HDN) has limited the use of this immune globulin for the treatment of AITP. A potential alternative to this treatment is the use of monoclonal anti-D antibodies (MoAnti-D). The D antigen is a 32-kD erythrocyte protein encoded by the RHD gene, which has several allelic variants that can generate different epitopes, thus making it necessary to produce many MoAnti-D. In fact, hundreds of MoAnti-D have been produced for phenotypic studies and many have been characterised for their ability to prevent alloimmunisation of pregnant women. With respect to AITP, however, only one study attempted to treat patients with AITP with MoAnti-D, but platelet counts were not increased (Godeau et al., Transfusion 1996; 36:328–30). The study by Kjaersgaard et al. was carried out in Denmark (Århus) and in Canada (Ottawa and Toronto). The Authors compared Rh immune globulin (polyclonal human anti-D) and six human MoAnti-D, with different isotypes and specificities, for their ability to opsonise red blood cells (RBC) and to inhibit opsonised platelet phagocytosis in vitro. Platelet phagocytosis was evaluated using a flow cytometer and the phagocytic index was calculated using the formula: median intracellular fluorescence at 37 °C/median intracellular fluorescence at 0 °C. The results demonstrated that opsonisation of RBC with polyclonal anti-D immune globulin significantly (p<0.001) reduced phagocytosis of fluorescently labelled opsonised platelets in an Fc-dependent manner. Of the MoAnti-D that shared specificity but differed in isotype, only IgG3 antibodies could significantly (p<0.001) inhibit platelet phagocytosis. In contrast, when two MoAnti-D sharing isotypes but differing in specificity were used in the assay, only one (MoAnti-D IgG1Rh113) could significantly (p<0.01) reduce platelet phagocytosis. The mechanism of how the MoAnti-D specificity affects opsonised platelet phagocytosis is unknown, but may be related to the molecule’s orientation on the RBC surface. These results suggest that MoAnti-D epitope specificity and isotypes are critical requirements for optimal inhibition of opsonised platelet phagocytosis.
In conclusion, MoAnti-D inhibition of opsonised PLT phagocytosis is dependent on isotype and epitope specificity, suggesting that preparations can be produced to mimic polyclonal anti-D and, perhaps, be therapeutically effective in AITP.
Tigue CC, McKoy JM, Evens AM, et al
Granulocyte-colony stimulating factor administration to healthy individuals and persons with chronic neutropenia or cancer: an overview of safety considerations from the Research on Adverse Drug Events and Reports project [Review]
Bone Marrow Transplant 2007; 40: 185–92
Recently, further questions have been raised about the safety of granulocyte colony-stimulating factor (G-CSF) administration to healthy (related and unrelated) donors of peripheral blood stem cells (PBSC). In this paper, investigators from the Research on Adverse Drug Events and Report (RADAR) project reviewed scientific literature on adverse events that occur when G-CSF is administered, besides to donors, to patients with severe chronic neutropenia (SCN) or with cancer. The RADAR project is a National Cancer Institute research programme that identifies clinical information on adverse drug reactions, with a particular emphasis on drugs used in haematology and oncology. The structured search was conducted using the PubMed MEDLINE interface. With the intersection of the medical subject and several keyword headings (PBSC donors, G-CSF toxicity, acute leukaemia, and so on), more than 5,000 papers were initially reviewed: only 54 studies met the inclusion criteria. Most rejected articles were excluded because they lacked detailed clinical information or were not written in English.
In several studies, in which G-CSF was administered to healthy donors for PBSC harvesting, bone pain was reported as by far the most common adverse event. The incidence of this event varies between 72% and 84% and the commonest associated symptoms are headache, fatigue and fever. Each of the consulted studies noted that these symptoms were readily treated with common analgesics (acetaminophen, ibuprofen) and resolved within 3–4 days after suspension of G-CSF administration.
There have been reports of autoimmune events associated with G-CSF administration. A German healthy donor developed severe autoimmune hyperthyroidism, and further cases of thyroid dysfunction and presence of thyroglobulin autoantibodies were reported in cancer patients who received G-CSF.
Several studies evaluated the effects of short-term administration of G-CSF on the spleen. Eleven cases of splenic rupture have been reported in individuals who received stimulating factor: four cases in healthy adult donors and seven cases in patients with neutropenia or cancer. One case in a neutropenic patient with myelodysplastic syndrome was fatal.
A few studies have reviewed the effects of G-CSF on lung function: the results indicate possible gas exchange disturbances during G-CSF administration, although these resolved once the drug was discontinued.
Among 27,000 bone marrow donors and 23,000 PBSC donors included in the European Blood and Marrow Transplant (EBMT) Registry who were evaluated for vascular disorders, there were five procedure-related deaths, with no statistical difference between marrow and PBSC donors.
Three related healthy donors, who received G-CSF before PBSC harvesting, developed acute myeloid leukaemia 1 to 5 years after drug administration. The studies reviewed did not identify an increased risk of malignancy (leukaemia, lymphoma, cancer, etc) among healthy donors who received G-CSF before PBSC harvesting. Of the over 4,000 unrelated individuals who had donated PBSC more than 1 year prior to the review, 20 subjects had developed cancer and none had developed leukaemia or lymphoma. This rate is consistent with age-adjusted cancer rates.
In conclusion, this review identifies bone pain as the most common toxicity of G-CSF. As regards a causal relationship between G-CSF administration and acute leukaemia, more long-term safety data (over 2,000 donors followed for 10 years) are needed to adequately evaluate such a possibility.
Jaing TH, Yang CP, Hung IJ, et al
Transplantation of unrelated donor umbilical cord blood utilizing double-unit grafts for five teenagers with transfusion-dependent thalassemia
Bone Marrow Transplant 2007; 40: 307–11.
Stem cell transplantation is the only cure currently available for patients with thalassaemia. Umbilical cord stem cells are increasingly used as an alternative option to bone marrow or peripheral blood stem cells for transplantation. The advantages of umbilical cord stem cells include ready availability, no risk to the donor, low rate of viral contamination and low risk of graft-versus-host disease (GvHD). The disadvantages include low stem cell dose for larger patients and lack of further stem cells for boost infusions, following the initial procedure. In multiply transfused patients (as in thalassaemic patients) rejection of donor stem cells is a major clinical problem, due to sensitisation of transplant candidates to HLA antigens.
Jaing et al. (from the University and Children’s Hospital in Taoyuan, Taiwan) hypothesised that two partially matched umbilical cord blood (UCB) units could facilitate engraftment, without crossed immunological rejection. They report their experience with this strategy.
Five patients with transfusion-dependent thalassaemia, median age 11.1 years (range: 11.0–13.1 years) each received two UCB units, after myeloablative conditioning. No patient was splenectomised before transplantation. All, but one, had received more than 100 transfusions. Searches for unrelated UCB donors were processed through the StemCyte Cord Blood Bank, in which 12,000 UCB units are available in Taiwan. The infused UCB units were 4/6-HLA-matched for four recipients and 6/6-HLA-matched unit for the fifth recipient The units contained a minimum combined pre-freezing CD34+ cell dose of 3.0x105/kg.
Engraftment occurred in all patients at a median of 15 days (range: 12–19 days). Four patients with durable trilineage engraftment showed acute, grade I–III GvHD; none developed extensive chronic GvHD, up to the date of the last contact. In patients with successful transplantation, the median time to achieve red blood cell transfusion independence was 32 days (range: 10–120 days) and the median days to achieve a platelet count >20x109/L was 49 days (range: 25–117 days). With a median follow-up of 18.5 months (range: 11–32 months), four patients, transplanted with UCB from two different, partially HLA-matched donors, were transfusion independent.
Two of the five patients succumbed to complications. One patient, a girl, developed severe haemolytic anaemia and thrombocytopenia (Evans’ syndrome) 8 months following her transplant, and died of diffuse alveolar haemorrhage 11 months post-transplantation. The second patient, a boy, had a complicated post-transplant course that included an episode of massive pericardial effusion. He eventually developed a methicillin-resistant S. aureus pericardial abscess, which necessitated surgery and multiple courses of varying combinations of antibiotics. At the moment of his death (16 months post-transplantation), he was transfusion-independent with full donor chimerism.
The study provides evidence that infusion of two, partially HLA-matched, UCB units is safe, may overcome the cell-dose barrier, and is an acceptable therapeutic approach in multiply transfused thalassaemic patients aged over 10 years old.
Sweeney J, Kouttab N, Holme S, et al
In vitro evaluation of prestorage pools consisting of mixed A and O platelet concentrates
Transfusion 2007; 47: 1154–61.
Ideally, all recipients of platelet concentrates (PC) should receive ABO-identical and Rh-matched products, but this may not always be possible. When ABO-identical and Rh-matched PC are not available, a decision needs to be made regarding the use of plasma-unmatched (e.g. O to A, B or AB) or Rh-unmatched (e.g. D+ to D-) products. Plasma-unmatched PC are the least desirable, but are sometimes transfused, in the context of HLA matching or low inventory, and, fortunately, are rarely associated with severe haemolytic reactions.
It is common practice in the USA to pool four to six whole blood PC to provide a valid dose for an adult patient. Current FDA regulations allow the prestorage pooling of PC of identical ABO type in a recent cleared platelet bag (Acrodose, Pall Medical). This is not only a logistic benefit, but allows the pool to be immediately available upon receiving an urgent order
In Europe, the standard practice is to prepare pooled PC from multiple ABO-matched donors before storage with the buffy-coat method. These products have demonstrated satisfactory storage quality, similar to apheresis or individually stored PC.
The purpose of this study, carried out by the Transfusion Medicine Research Unit in Providence (Rhode Island, USA), was to examine whether there were any effects of prestorage pooling of ABO-mixed PC on in vitro measures of platelet storage injury, surface expression of A antigen, assessment of in vitro lymphocyte activation, and any activation of the complement or coagulation systems.
PC were prepared, on day of collection, from platelet-rich plasma, filtered in line; approximately 60 mL of plasma were left in each PC. Using a sterile connecting device, prestorage pools were prepared as follows: group 0 = five group O PC per pool; group 1 = one group A and four group O PC per pool; group 2 = two group A and three group O PC per pool; group 3 = three group A and two group O PC per pool; group 4 = four group A and one group O PC per pool; group 5 = five group A PC per pool. Pools were stored in Acrodose bags and a sample was taken from the mixed pool 15 minutes after mixing, to perform the above-mentioned assays (day 1). All pools were then stored at 20–24 °C, with agitation. Subsequent samples were collected aseptically from the pools on days 5 and 7.
No differences was found on day 7 in pH, hypotonic shock response, extent of shape changes, morphology score, or white cell count, but surface P-selectin expression (an index of platelet activation) was more evident in the ABO-mixed pools (p=0.02). Small microscopic clumps were observed in all pools, but were more prominent in the ABO-mixed pools (p<0.01). Platelet counts, however, did not differ between pools, indicating that the proportion of platelets clumped was small. Surface expression of A antigen was proportionally to the number of group A PC in each pool and did not vary between study days. Complement and coagulation activation markers did not differ among pools.
In conclusion, pooling group A and O PC is associated with evidence of increased microscopic platelet clumping and activation, but these differences are not exacerbated by storage for 7 days. Other major measures of platelet quality did not differ, and there was no evidence of a mixed lymphocyte reaction. Although intentional prestorage pooling ofABO mixed pools is unlikely to become a common practice, these date are reassuring that ABO mixed pools differ minimally from ABO identical pools.
Satake M, Taira R, Yugi H, et al
Infectivity of blood components with low hepatitis B virus DNA levels identified in a lookback program
Transfusion 2007; 47: 1197–205
Nucleic acid testing (NAT) for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been implemented in developing countries for screening blood donated during the window period, and plays a critical role in preventing transfusions of infectious blood components. For hepatitis B virus (HBV), Japanese Red Cross (JRC) Blood Centres have been screening blood with haemagglutination for hepatitis B surface antigen (HBsAg) detection, combined with anti-hepatitis B core antigen (anti-HBc) and hepatitis B surface antibody (anti-HBs) testing, since 1969. In February 2000, they implemented 50-sample-pool NAT (50-NAT) for HBV, HCV, and HIV. There is still a residual risk of transfusion-transmitted infections, because of the limited sensitivity and use of pooled samples in the current NAT system. The JRC established complete repository archives of blood samples from every blood donor (5.4~6.0 million blood samples per year) since 1996, with a plan to preserve the aliquots for 11 years. This decision enabled the JRC Centres to investigate the cause of transfusion-related infections, retrospectively.
Satake and colleagues describe the incidence of HBV-containing blood among repeat donors that escaped the current screening algorithm, including HBV-NAT and anti-HBc testing. On the basis of the complete repository archives, all donations from repeat donors received from 1997 to 2004 were subjected to a lookback study, when a subsequent donation turned positive for a 50-NAT, for HBsAg or for anti-HBc. Repository samples from the immediately preceding donations of donors who became positive for the presence of the markers described above were analysed by individual donation NAT (ID-NAT). The frequency of donations that were only ID-NAT-positive and the risk of transmitting HBV by the transfusion of those blood components were investigated. The risk analysis for HBV contamination was limited to donations obtained from February 1, 2000 to January 31, 2004, all of which had been qualified by 50-NAT testing.
HBV ID-NAT was performed on 15,721 repository tubes, and 158 tubes (1.01%) were found to be positive for the presence of HBV DNA. Of these 158 donations that were only ID-NAT-positive, 95 (60%) were from carriers with low anti-HBc titres. It was possible to study 63 patients transfused with components that were only ID-NAT-positive, and 12 (19%) proved to be infected with HBV. None of the 16 identified anti-HBs-positive components showed serological evidence of infection. By analysing the outcome of patients transfused with components with a low HBV-DNA level, the study found a marked difference in the HBV infectivity of occult carrier-derived units compared to the window-period-derived units.
This lookback study showed that some of the previous donations (both ID-NAT-positive and ID-NAT-negative units) had caused HBV infection in recipients and that some have caused clinical hepatitis. The relevant donors have been detected with more sensitive anti-HBc screening or with ultrasensitive ID-NAT. The JRC is currently exploring which options remain to further reduce the risk of post-transfusion hepatitis B.
Okamura Y, Fujie T, Maruyama H, et al.
Prolonged hemostatic ability of polyethylene glycol-modified polymerized albumin particles carrying fibrinogen gamma-chain dodecapeptide
Transfusion 2007; 47: 1254–62.
Platelet transfusions play an important role in supportive therapy of thrombocytopenia, caused by cancer or haematological malignancies or in the perioperative period. The shortage of platelet concentrates (PC), their short storage life, the insufficient donations, and the greater rate of demand than supply have always been serious problems. Furthermore, the risk of viral and bacterial infections associated with PC transfusion is another critical issue. For all these reasons, various attempts have been made to develop platelet substitutes (“artificial” platelets), such as infusible platelet membrane, solubilised platelet membrane protein-conjugated liposomes (or plateletsome), fibrinogen-bonded red blood cells, fibrinogen-coated albumin microcapsules (synthocytes), liposomes bearing fibrinogen, and arginine-glycine-asparaginic acid (RGD) peptide bound red blood cells (thromboerythrocytes).
In this paper, Okamura et al. – from the Waseda and Keio Universities (both in Tokyo, Japan) – describe a novel method to prepare a second generation platelet substitute, with a longer half-life in the blood circulation than that of other substitutes and, thus, an enhanced haemostatic ability for the treatment of bleeding. They focused their attention on a dodecapeptide (H12), which is a gamma-chain carboxy-terminal sequence (γ400–411) and exists only in a fibrinogen domain.
H12 was conjugated to the surface of polymerised albumin particles (polyAlb) and modified with polyethylene glycol (PEG) chains to produce biocompatible particles (H12-PEG-polyAlb) that had a prolonged blood circulation half-time (t½) and were more stable, both in vitro and in vivo, than H12-polyAlb not modified with PEG. As is well known, PEG modification on the surface of carriers as phospholipid vesicles or biocompatible particles has been widely used to prolong the t½ or to stabilise their dispersion states.
The H12-PEG-polyAlb particles were intravenously administered into thrombocytopenic rats, and the tail bleeding time and the blood circulation t½ were measured for evaluation of the increased haemostatic effect. The tail bleeding time of the thrombocytopenic rats before administration of H12-PEG-polyAlb was 624±175 seconds. The bleeding times at 5, 60, 180, and 360 minutes after administration of H12-PEG-polyAlb were significantly reduced to 354±67, 395±75, 330±73, and 371±47 seconds, respectively. The effect lasted for at least 6 hours.
In conclusion, H12-PEG-polyAlb particles prolonged the t½ and maintained special binding ability to activated platelets. H12-PEG-polyAlb may be suitable for treatment in thrombocytopenic patients as an alternative to PC transfusions.
Kumpel BM
Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: more questions than answers [Review]
Vox Sang 2007: 93: 99–111.
The administration of anti-D immunoglobulins to D-negative women, during and after pregnancy, is very effective at preventing D immunisation by D-positive foetal red blood cells (RBC) and subsequent haemolytic disease of the foetus and newborn (HDFN). The primary mode of action of anti-D IgG is rapid clearance of foetal D-positive RBC from the maternal circulation, mediated by interactions with IgG Fc-receptors on macrophages in the spleen, but the inmost mechanism of action of anti-D immunoprophylaxis has not yet been completely elucidated.
Current anti-D immunoglobulins are produced by fractionation of IgG from pooled plasma of donors with high titres of anti-D. Originally, most donors of anti-D plasma were women immunised by pregnancy. Given the large success of immunoprophylaxis of HDFN, this source of anti-D immunoglobulins has been almost exhausted. Deliberately immunised men have also been used; titres are maintained by boosting with D positive RBC. If the supply of anti-D is compromised, unprotected women would become immunised and HDFN with its high morbidity and mortality would once again be common. An alternative safe, cheap and unlimited supply of anti-D would, therefore, be desirable.
The technology for making monoclonal anti-D first started over 25 years ago and many hundreds of monoclonal antibodies have been produced by a variety of methods. Mice do not recognise the D antigen as foreign, so conventional murine hybridoma technology cannot be used. The first monoclonal antibodies were made using an Epstein-Barr virus-transformed B-lymphoblastoid cell line. By the mid-1990s, many recombinant antibodies had been engineered and expressed in rodent cell lines, mostly in the Chinese hamster ovary (CHO) line.
Kumpel reviewed the rich literature on this matter (76 references). Twelve anti-D monoclonal antibodies were tested in eight studies for their ability to mediate clearance of autologous RBC and 13 were studied in seven trials on the clearance of D-positive allogeneic RBC injected into D-negative subjects. The activity of antibodies produced by human B-cell lines, by mouse-human heterohybridomas and by CHO differed significantly, with none being quite as effective as polyclonal human anti-D.
However, clearance mediated by recombinant antibodies produced by rat YB2/0 cells was extremely rapid, faster than that produced by polyclonal anti-D, although haemolysis and hepatic accumulation of RBC was observed in one study. Most of the clinical trials on allogeneic RBC clearance were followed by detection of serological anti-D responses for at least 6 months in the recipients, to assess whether the monoclonal or recombinant antibodies had protected the D-negative subjects from anti-D immunisation. Two human anti-D monoclonal antibodies prevented D-immunisation. In contrast, anti-D monoclonal antibodies from heterohybridomas increased the incidence and rapidity of anti-D responses. To explain these unexpected results, it has been hypothesised that unnatural glycosylation of monoclonal and recombinant antibodies produced by some cell lines affected interactions with FcãR and other receptors and molecules of the innate immune system.
Emerging information on the nature of the immune system and the structure of antibodies will be crucial in the future development of anti-D monoclonal or recombinant antibodies to replace polyclonal anti-D.
Jung HH, Danek A, Frey BM
McLeod syndrome: a neurohaematological disorder [Review]
Vox Sang 2007; 93: 112–21.
McLeod’s syndrome is an X-linked multisystem disorder, assigned to the “core” neuroacantocythosis syndromes, which also include chorea, acanthocytosis, Huntington’s disease, and pantothenate kinase associated neurodegeneration (PKAN). McLeod’s syndrome is characterised, haematologically, by the absence of Kx red blood cell (RBC) antigen, weak expression of all Kell RBC antigens, acanthocytosis, and compensated haemolysis. The McLeod phenotype may be incidentally detected in routine screening of apparently healthy blood donors. Subjects with all McLeod blood group phenotypes reported up to date have elevated serum creatine kinase and, in varying proportions, are prone to develop a severe neurological disorder resembling Huntington’s disease. The onset of neurological symptoms ranges between 25 and 60 years (mean, 30–40 years) and the penetrance of the disorder appears be high. Additional symptoms of the syndrome include generalised seizures, neuromuscular weakness and atrophy, and cardiopathy (mainly manifesting as atrial fibrillation, arrhythmias and dilated myocardiopathy). Central nervous system manifestations comprise the prototypic triad of a progressive nurodegenerative basal ganglia disease: a chorionic movement disorder, “subcortical” cognitive impairment, and psychiatric symptoms. Subjects with McLeod’s syndrome usually show a slow progression of the disease.
McLeod’s syndrome is caused by a mutation of the XK gene, encoding for the XK protein, which carries the Kx RBC antigen. The XK gene contains three exons and is located on chromosome X (in position p21.1), close to the loci for chronic granulomatous disease (CGD), Duchenne’s muscle dystrophy and retinitis pigmentosa. The Kx antigen was formerly considered part of the Kell system. At present, the Committee on Terminology for Red Cell Surface Antigens of the International Society of Blood Transfusion (ISBT) recognises 31 distinct antigens of the Kell system, numbered from K1 to K31, according to the more recent report (Cape Town Report, September 2006): two Kell antigen (K8 or Kw and K9 or KL) are considered “obsolete”. Kx is still reported among Kell system antigens in the ISBT list, in which its number is K15, but it, too, is considered “obsolete” and has been regrouped into the new antigen system (system 019).
The absence of the Kx antigen (the pathognomonic characteristic of McLeod’s syndrome) implies loss of XK protein on the RBC membrane, which might be due to mutational, transcriptional or translational deficiency of the XK gene. Kx negativity of RBC reflects the serological finding of the McLeod phenotype.
A minority of boys with X-linked CGD carry the McLeod blood group phenotype. As is well known, CGD is an inherited disorder of phagocyte function, characterised by recurrent bacterial and fungal infections and by granulomata from early childhood. Large deletions may cause a “contiguous” gene syndrome, including the loci for Duchenne’s muscular dystrophy, X-linked retinitis pigmentosa and the McLeod phenotype. Boys with CGD and McLeod phenotype are at risk of developing anti-Kx antibodies, with serious hazards when receiving multiple transfusions. Carriers of the McLeod phenotype without CGD rarely develop specific antibodies. Nevertheless, transfusion hazards must be taken into account in all carriers of McLeod phenotype and autologous blood banking should be considered before carrying out elective surgery that may necessitate transfusions.
Asymptomatic carriers of a McLeod blood group phenotype should, therefore, be given careful genetic counselling and undergo thorough neurological examinations and cardiological evaluation for the presence of a treatable myocardiopathy.
Robinson EA
The European Union Blood Safety Directive and its implications for blood services [Review]
Vox Sang 2007; 93: 122–30.
Prior to 1994, the European Union (EU) had little involvement in blood safety regulations other than the adoption, in 1987, of two original Council of Europe agreements: agreement no. 26 on the exchanges of therapeutic substances of human origin (1958) and agreement no. 39 on exchanges of blood grouping reagents (1962). The only other piece of EU legislation on blood transfusion was related to proprietary medicines, with special provision for products derived from human blood and plasma. At the high-level EU meeting held, in 1994, in Adare (Ireland) a Community blood strategy was proposed, aimed at improving blood transfusion safety and promoting self-sufficiency in Europe. As a first step of this strategy, the European Council, on behalf of the European Parliament, issued the Council Recommendation 98/463/EEC (29 June 1998) on the suitability of blood and plasma donors and the screening of donated blood in the European Community. After several years of negotiations, compromise and resubmissions, Directive 2002/98/EC was adopted on the 27th January, 2003 and published in the official Journal of the EU on the 8th February, 2003.
The objective of Directive 2002/98/EC was to set high standards of quality and safety for the collection, testing, processing, storage, and distribution of human blood and blood components. This EU Parliamentary Directive is complex piece of Community legislation, referred to as a “mother” or “core” Directive, and is now supported by three Commission (daughter) Directives. The Directive 2002/98/EC has 33 recitals (introducing legal provisions), 34 operating articles (legally binding provisions, grouped into 10 chapters), and 4 annexes (providing some technical specifications and definitions).
The main topics for blood establishments regard: inspection and control measures, personnel issues, quality management, and haemovigilance. Topics for hospital blood banks concern: personnel, quality systems based on the principle of good practice, documentation, traceability, and notification of serious adverse events and reactions.
The three Commission (daughter) Directives (2004//33/EC, 2005/61/EC and 2005/62/EC) implement the “mother” Directive and were produced through the Regulatory Committee procedures. Directives 2002/98/EC and 2004/33/EC came into force on the 8th February, 2005, while the remaining two “daughter” Directives came into force at the end of 2006.
At the September 2005 Commission meeting with the competent authorities, member States were asked to comment on any difficulties encountered in the transposition of the Directives. The following key points emerged.
The first problem was the difficulty in completing the transposition into national legislation: some countries already had a blood law and amending existing legislation or creating new legislation can be a lengthy, complex and resource hungry process.
Another problem regarded the language. Some inconsistencies in interpretation of the different translations have caused difficulties. A comment was made that good co-operation is needed between the lawyers and the medical experts to ensure that the “spirit” of the law was complied with, when debating the legal interpretations of the text.
Some of the new member States and some of the smaller countries have found it difficult to implement the Directives fully, due to constraints in both human and financial resources. In particular, problems were created by the requirement for trained and experienced Good Manufacturing Practice (GMP) inspectors, in the absence of standards and guidance on inspection and control measures.
Some member States have more than one competent authority for different aspects of the “mother” Directive. In most cases, this appears to be split between the Ministry of Health and the pharmaceutical agency responsible for inspection, licensing and ensuring compliance. This has created difficulties in determining where the final responsibility lies, particularly with regard to the legal interpretation of the Directive.
Separation of activities between blood establishments and hospital blood banks has also created difficulties because of the variety of different arrangements throughout Europe for blood transfusion services. These ranged from centrally managed and directed blood services and hospital blood banks.
While it is important to remember that the purpose of these directives is to provide the same high standards of blood quality and safety for all patients throughout the EC, a balance needs to be achieved between what technical details are absolutely necessary to implement and what may be better left out. An area of particular controversy was the inclusion of a predetermined haemoglobin level, as France had no systematic predonation haemoglobin control and the UK had a lower normal haemoglobin range than that set by the Directives.
The following specific issues were raised by member States at the Meetings of the competent Authorities: a) the requirement for the protein content of fresh frozen plasma to be not less than 50 g/L; b) the requirement for ABO and Rh labelling of autologous blood units; c) inspections of mobile collection sites managed by a blood establishment; d) nucleic acid technology (NAT) testing for human immunodeficiency virus; e) use of an anti-hepatitis B core (anti-HBc) test; f) voluntary unpaid donation.
These European transfusion safety Directives clearly pose a challenge for all blood establishments and hospital blood banks. It is, however, important to underline, once again, that its main objective is to provide the same high standards of blood quality and safety for all patients throughout the EC. This is surely something that should be welcomed.