The residual risk of transfusion-transmitted viral infection in developed countries is considered minimal or negligible. However, zero risk remains a strong political objective. Genomic screening for HCV, HIV and HBV represents a major advance, eliminating infectious blood donations collected during the pre-seroconversion window period, rare cases of immunosilent infections and, possibly, a large spectrum of viral variants. In Western countries, HCV RNA genomic screening started on pools of 16-400 plasma samples from individual donations. Pooling may produce false-positive and false-negative results. Individual donation testing is more suitable to blood screening but requires multiplexing, automation, and affordable cost. Because donations from individuals who are HBV DNA-negative/serologically positive, or those apparently recovered from HCV infection, may remain infectious, it is unlikely that HBsAg, anti-HCV, and anti-HIV will be discontinued when genomic screening is extended to all three viruses. HIV-1 p24 antigen may prove redundant with HIV RNA screening. Anti-HTLV-I and HTLV-II will remain more effective than genomic testing.
Epstein-Barr virus (EBV) is one of eight human herpesviruses and is ubiquitous. Primary infection with EBV in childhood is generally silent, but often causes overt diseases such as infectious mononucleosis (IM) and lymphoproliferative disorders (LPD). The latter occurs in immunologically compromised individuals. Historically, EBV has been thought to be aetiologically linked to human malignancies such as EBV genome-positive Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC). Furthermore, studies using recent developments in molecular and immunological diagnostic approaches have suggested that this virus has a causative role in a spectrum of human diseases of previously unknown pathogenesis, including chronic active EBV infection syndrome (CAEBV), EBV-related haemophagocytic lymphohistiocytosis (HLH), and certain disorders such as EBV genome-positive T-cell lymphoma, natural killer (NK) cell leukaemia/lymphoma, Hodgkin's disease (HD) and gastric carcinoma. This chapter reviews recent progress regarding EBV-associated diseases.
Proteins derived from human plasma have become critically important therapeutic products since their introduction in the 1940s. In the last 20 years, the tools of molecular biology have provided alternatives to the administration of the natural products. Recombinant analogues of Factor VIII and Factor IX are commercially available, and recombinant forms of other plasma proteins are under development. Genetic engineering also provides the opportunity to modify a natural protein to improve the efficiency with which it can be produced in vitro, or to change its therapeutic profile. More efficient production systems, such as transgenic plants or animals, may yield less costly therapies and a wider availability of products that are now in limited supply. Finally, gene therapy offers the prospect of permanently correcting conditions arising from deficiencies in any one of several plasma proteins, freeing individuals from the need to undergo periodic treatments with exogenous proteins.
Hexokinase (HK) deficiency is a rare red cell enzyme deficiency associated with hereditary non-spherocytic haemolytic anaemia; to date, only 17 affected families have been reported. Human HK has four major isozymes, each of which is encoded by a separate gene. Recent studies have shown that both ubiquitously expressed type I HK (HK-I) and erythroid-specific HK-R are expressed in erythrocytes, and that these isozymes are encoded by the single HK-I gene. The human HK-I gene has 19 exons, the HK-I and HK-R transcripts being produced by using two distinct promoters. Thus, the first and second exons are specifically utilized for the erythroid-specific HK-R and ubiquitously expressed HK-I isozymes respectively. So far, only two HK variants have been analysed at the molecular level. Since the human HK-I crystal structure has recently been elucidated, the molecular analysis of the HK variants will be useful for discussing the structure-function relationship of the enzyme.
Hereditary spherocytosis (HS) is relatively common in Caucasian populations; most individuals have mild or only moderate disease. There is commonly a family history and a typical clinical and laboratory picture so that the diagnosis is usually easily made without additional laboratory tests. Atypical cases may require measurement of membrane proteins and molecular genetics to clarify the nature of the membrane disorder. It is particularly important to rule out stomatocytosis because splenectomy is contraindicated because of the thrombotic risk. Mild HS can be managed without folate supplements and does not require splenectomy. Moderately and severely affected individuals are likely to benefit from splenectomy, which should be performed after the age of 6 and with appropriate counselling about the risk of infection. In all cases careful dialogue between physician, child and the family is essential. Laparoscopic surgery can result in shorter hospital stay and less pain.
Enzyme deficiencies have been identified in all erythrocyte pathways. Their frequencies differ with respect to the affected enzyme, the severity of the clinical manifestations and the geographical distribution. Most mutations are found within the coding sequences of genes, missense mutations occurring more often than deletions, insertions, splice site defects or premature stop codons. Promoter mutations are rare. The clinical manifestations are chronic or non-chronic haemolytic anaemias. The first of these are characterized by an impairment of cell function at normal values of the external load parameters k(ATPase) and k(GSHox). Haemolysis with a non-chronic course is induced only at enhanced values of the load parameters, caused by free radical generation by oxidative drugs, fava beans, infections, fever and physical exercise. The development of secondary haemochromatosis is the most common cause of mortality in patients suffering from severe chronic non-spherocytic haemolytic anaemia. Intracellular iron deposits must be prevented by timely treatment with effective chelating agents.
Multiple interacting factors contribute to the haematological manifestations of HIV disease. The effects of HIV-1 infection influence all haemopoietic cell lineages resulting in a spectrum of haematological abnormalities. Even in the absence of other pathological processes, bone marrow morphology is invariably abnormal, and anaemia, neutropenia and thrombocytopenia are all common during the course of disease. Intercurrent opportunistic infections may cause bone marrow suppression or induce specific cytopenias. Therapies used to treat HIV and its complications are frequently implicated as the cause of haematological dysfunction, and many have significant myelotoxic side-effects. Insights into the molecular basis for many of these abnormalities have permitted a clearer understanding of the pathophysiology of HIV-1 infection. Recombinant human growth factors that may be used to treat isolated cytopenias or to ameliorate the myelotoxic effects of other essential therapies. Lymph opoietic growth factors and the use of gene modified cells provide future therapeutic strategies that may alter the course of HIV disease.
Dengue fever (DF) and dengue haemorrhagic fever (DHF) are caused by the dengue virus. The major pathophysiological hallmark that distinguishes DHF from DF is plasma leakage as a result of increased vascular permeability. Following this leakage, hypovolaemic shock occurs as a consequence of a critical plasma volume loss. Constant haematological abnormalities occurring in DHF and frequently include bone marrow suppression, leucopenia and thrombocytopenia. An enhanced immune response of the host to a secondary DV infection is a feature of DHF and leads to many consequences. These are immune complex formation, complement activation, increased histamine release and a massive release of many cytokines into the circulation, leading to shock, vasculopathy, thrombopathy and disseminated intravascular coagulation (DIC). The mechanisms underlying the bleeding in DHF are multiple. These are vasculopathy, thrombopathy and DIC. Thrombopathy consists of thrombocytopenia and platelet dysfunction. DIC is prominent in patients with shock. The most severe DIC and massive bleeding are the result of prolonged shock and cause a fatal outcome. The mechanisms of thrombopathy and DIC and the proper management of DHF are reviewed and discussed.
The development of new and often more successful regimens of treatment for childhood blood and malignant diseases has usually been associated with a parallel increase in infectious problems. This is because these successes have often, in the absence of new effective drugs, been achieved by increasing drug dose intensity to new limits. This chapter is not intended to deal with every possible infection, but rather to be a practical guide to the current management of infection. The last few years have seen major improvements in the development of haematopoietic growth factors, new antifungal agents, new antibiotics and new ways to use aminoglycosides. Attempts are being made to identify good and poor risk factors for the outcome of infection in order to facilitate shorter courses and possibly home or day care use of antimicrobial agents. In addition the different needs of children are being recognized in view of the restricted use of quinolones in this group and the different organisms and types of infection that they experience.
Childhood idiopathic thrombocytopenic purpura (ITP) is a largely trivial disorder from which over 95% of children sooner or later recover spontaneously, and for most of whom the risks of unnecessary or ineffective therapy are arguably greater than those of the untreated disease. There are, however, a few patients who continue to have very low platelet counts and remain symptomatic for many months or years. They are rare, and they present difficult management problems. Splenectomy is probably the most effective treatment but is also the most dangerous and is not always successful. It is also irreversible. Most other regimens are either ineffective, unacceptably toxic, or both. Planning management for an individual patient requires a realistic risk:benefit appraisal, a process that is impeded by inadequate epidemiological data and a scarcity of large-scale randomized clinical trials. International collaborative studies may help in the future.
Most of the metabolic needs of erythrocytes are covered by glycolysis, the oxidative pentose phosphate pathway and the glutathione cycle. Hereditary enzyme deficiencies of all these pathways have been identified, among which glucose-6-phosphate isomerase (GPI) deficiency is the second most frequent erythroenzymopathy in glycolysis, being associated with nonspherocytic haemolytic anaemia of variable severity. This autosomal recessive genetic disorder may be associated in some cases with neurological impairment. GPI is a dimeric enzyme that catalyses the reversible interconversion of fructose-6-phosphate and glucose-6-phosphate. Virtually all the mutant gene products reported are characterized by marked instability and normal substrate affinities, but altered catalytic activity and electrophoretic migration rates. At the nucleotide level, 29 mutations have been reported. This chapter reviews (a) the clinical pattern of the condition; (b) biochemical and molecular studies; (c) structure-function relationships; (d) the molecular basis of neurological dysfunctions sometimes associated with GPI deficiency; and (e) the correlation between the severity of the anaemia and the molecular defect.
In childhood acute lymphoblastic leukaemia (ALL) a number of genetic changes have been identified which provide diagnostic and prognostic information with a direct impact on patient management. The most significant abnormalities include the translocation, t(12;21)(p13;q22), giving rise to the ETV6 / AML1 gene fusion;BCR / ABL arising from t(9;22)(q34;q11); re-arrangements of the MLL gene; the E2A / PBX1 from the t(1;19)(q23;p13); re-arrangements of MYC with the immunoglobulin genes and re-arrangements of the T cell receptor genes. Chromosomal deletions, particularly those of the short arms of chromosomes 9 and 12 and the long arm of chromosome 6, have been postulated to be the sites of tumour suppressor genes (TSG). Numerical chromosomal abnormalities are of particular importance in relation to prognosis. High hyperdiploidy (50–65 chromosomes) is associated with a good risk, whereas the outlook for patients with near haploidy (23–29 chromosomes) is extremely poor. In view of the introduction of risk-adjusted therapy into the UK childhood ALL treatment trials, an interphase FISH screening programme has been developed to reveal chromosomal abnormalities with prognostic significance in childhood ALL.
Infection is one of the commonest causes of disseminated intravascular coagulation (DIC). DIC is a complex disorder that results from an imbalance of the pro- and anticoagulant regulatory pathways. This chapter will explain the cellular and molecular basis of the disorder and consider the rationale behind current and experimental treatment strategies.
Phosphoglycerate kinase (PGK) deficiency is associated with hereditary haemolytic anaemia and often with central nervous system dysfunction and/or myopathy. Twenty-three families have been discovered with this condition. Nine have manifested both symptoms, six only haemolysis, and seven central nervous system dysfunction and/or myopathy without haemolysis; one case is asymptomatic. Among them, the structural abnormalities of 14 mutants, including 11 missense mutations, 1 gene deletion, 1 gene insertion, and 1 splicing mutation, have been identified. The correlation between the phenotypic and structural differences in PGK deficiency remains to be defined. Splenectomy obviates transfusion in most patients but does not correct the haemolytic disorder. Phosphofructokinase (PFK) deficiency is associated with myopathy and/or haemolysis. More than half reported had the typical features of glycogen storage disease type VII (Tarui disease). The other cases exhibited myopathy alone, haemolytic anaemia alone, or no clinical symptom at all. Eight missense, 1 nonsense, 1 frameshift and 5 splicing mutations have been determined in the PFK-M gene. In classic PFK-M deficiency, the avoidance of undue exertion is the key to prevent muscle symptoms.
Despite the increased safety of blood components, achieved through improved donor selection and testing, transfusion recipients remain at risk of transfusion-associated diseases. Transfusion of cellular blood components has been implicated in transmission of viral, bacterial and protozoan diseases. While it is commonly recognized that hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), and retroviruses, such as human immunodeficiency virus (HIV) and the human lymphotrophic viruses (HTLV), can be transmitted through cellular components, other pathogens are emerging as potentially significant transfusion-associated infectious agents. For example, transmission of protozoan infections due to trypanosomes and Babesia have been reported. In addition to viral and protozoan infectious agents, cases of bacterial contamination of platelet and red cell concentrates continue to be reported and may be an under-reported transfusion complication. More importantly, new infectious agents continue to enter the donor population, and there is an inherent time delay before the new pathogens are definitively identified and new tests implemented in order to maintain consistent safety of the blood supply. The paradigm for this problem is the HIV pandemic. During the past decade a number of methods for inactivating infectious pathogens in platelet concentrates have been investigated as a strategy to improve the safety of platelet transfusion therapy. One method of treating platelet concentrates to inactive pathogens has now reached the advanced clinical trial phase in the United States and Europe. Similar efforts with a new class of compounds are underway for red cell concentrates, and two of these are in early phase trials. In addition to studies with allogeneic platelets and red cells, a number of laboratories have described methods for developing platelet substitutes or modified platelets to avoid the use of traditional platelet concentrates as a means to improve safety.
Direct measurement of the risk of transfusion-transmitted infection (TTI) is practical and accurate only if the level of risk is high. Historically, studies that established frozen repositories of transfusion recipient and/or blood donor samples were important in establishing the risk of many TTI agents, including the human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). However, given the current very low risk of TTI, mathematical modelling is necessary to estimate the magnitude of such a risk. For agents for which routine blood donor screening is performed, most of this risk comes from transfusion of units collected in the window period between donor infection and a positive blood screening assay. The incidence/window period model has been used to estimate the magnitude of such risks (of the order of 1:100 000 to 1:1 000 000) and for predicting the extent of risk reduction that can be expected with implementation of new tests. Direct estimation and mathematical modelling approaches are both important tools for future assessment of potential, new or emerging TTI agents.
Pyruvate kinase deficiency is the most frequent enzyme abnormality of the Embden-Meyerhof pathway causing hereditary non-spherocytic haemolytic anaemia. The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life-threatening neonatal anaemia and jaundice necessitating exchange transfusions. Splenectomy should be reserved for young patients who require regular blood transfusions. The gene encoding for pyruvate kinase (PK-LR) has been localized to the long arm of chromosome I; the cDNA of R-type is 2060 bp long and codes for 574 amino acids. More than 130 different mutations, mostly missense, have so far been described in association with PK deficiency, 1529A and 1456T being considered to be the most common mutations in Caucasians. Analysis of the three-dimensional structure of the enzyme may help in predicting the severity of the molecular defect. Further data on clinical features of homozygous patients are needed, at least for some mutations, to allow a more precise genotype/phenotype correlation.
The safety of the blood supply is critical to many parts of modern medicine. In a time when prescriber's and the public's expectations are increasing, it is essential that transfusion services globally ensure the safety of the blood supply. There are, however, many threats to this safety, one being the appearance of new infectious agents. Such agents may be truly ‘novel’, or may be existing agents, known but not routinely screened for, posing a new or increased threat. However, before an agent is considered to be a true threat to blood safety it must be well characterized, and evidence must be presented that (i) transfusion transmission is a significant route of spread, and (ii) the agent causes significant clinical disease. If either of these criteria are not met, the question has to be asked as to whether the agent is truly a threat to blood safety.
The development of an inhibitor antibody to factor VIII (or factor IX) in a child with haemophilia presents a major challenge to the paediatric haematologist. This article provides an overview of the incidence of inhibitor development in early childhood (30-52% in boys with severe haemophilia A), genetic risk factors, detection, high titre, low titre and transient inhibitors, and management. Treatment of patients with inhibitors is time-consuming and expensive. One should make every attempt to ensure that the boy's family has an understanding of inhibitors, treatment options, and just what is being recommended for their child and what this involves. Immune tolerance induction is successful in approximately 85% of boys with factor VIII inhibitors, but in only 40-50% of those with factor IX inhibitors. For treatment of bleeding episodes in children with high-titre (> or = 5 Bethesda Units) inhibitors, therapeutic options include activated prothrombin complex concentrates (APCC), rF VIIa, and (for factor VIII inhibitors) porcine factor VIII. The advantages and disadvantages of each are discussed. Although factor IX inhibitors are far less common (occurring in 2-3% of boys with haemophilia B), approximately 50% are accompanied by the occurrence of anaphylaxis or severe allergic reactions to any factor IX-containing product.
