Topics in transfusion medicine - vol 5 no.1, october 1998
AUSTRALASIAN SOCIETY OF BLOOD TRANSFUSION INC.
u Editorial Board
Margaret Buring, Wendy Erber, Jim Faed, John Editor's Note This issue of topics is the first for some time and it is hoped it will be the first in a more regular series appearing at quarterly intervals throughout the year. The issue consists of a series of varied topics comprising abstracts from the recent successful annual scientific meeting held in Sydney, a historical prospective on the origins of a national blood service in Australia by Mark Cortiula and a paper on platelet immunology by Robyn Minchinton. Also included are the four abstracts that were chosen for presentation at the Presidential Symposium at this year’s meeting. The award for the best paper was presented to NA Mifsud, Australian Red Cross Blood Service, Victoria. Ken Davis Extra copies of Topics in Transfusion Medicine are available from the ASBT office for $5 each.
All Correspondence The Australasian Society of Blood Transfusion Inc 145 Macquarie Street, Sydney NSW 2000 Presidential Symposium, 32nd Annual Scientific Meeting, Sydney 1998 Abstracts from the 32nd Annual Scientific Meeting, Sydney 1998 Mechanisms of immunomodulation by blood transfusion. W Dzik Transfusion Medicine, Bi-Deaconess Medical Centre, Harvard Medical School, Boston.
The treatment of the thrombocytopenic patient: Current issues and future perspectives. MA Blajchman Departments of Pathology and Medicine, McMaster University, Hamilton, Ontario, Canada Probe testing of plasma pools MP Busch Blood Centres of the Pacific, and University of California, San Francisco CA. Thrombopoietin: implications for the diagnosis and treatment of disorders of platelet production. K Kaushansky Division of Haematology, University of Washington School of Medicine, Seattle, Washington. The molecular basis of the Kell and Duffy blood group antigens ME Reid, Immunohematology Laboratory, New York Blood Centre, New York, New York. Platelet Immunology. A not - so - trivial - pursuit.
Robyn Minchinton, Chief Scientist, ARCBS, Brisbane, Queensland.
Going back to the future: the origins of a national blood service in Australia
Mark Cortiula, University of Sydney, Sydney, NSW
Copyright of the Australasian Society of Blood Transfusion 1. PRESIDENTIAL SYMPOSIUM ASBT SYDNEY 1998 IDENTIFICATION OF A NOVEL B ALLELE IN THE ABO BLOOD GROUP SYSTEM BY MOLECULAR ANALYSIS
NA Mifsud, JA Condon, AP Haddad, RL Sparrow, Australian Red Cross Blood Service Victoria, Southbank, VIC 3006
DNA nucleotide analysis of the ABO transferase genes by various methods has enabled the discovery of novel alleles and hybrid genes in the ABO blood group system. The application of ABO genotyping can in most circumstances overcome difficulties encountered by serological typing. A suspected cis-AB sample was referred for ABO genotyping by ARCBS-NSW. Routine genotyping by PCR-Sequence Specific Oligonucleotide determined the individual be an AO1*. Further genotyping by PCR-Restriction Fragment Length Polymorphism resulted in a BO1* genotype. The discrepancy was located at nucleotide 796. A mutation in the B gene at nucleotide site 796 in exon 7 of the B transferase gene was confirmed by DNA sequencing. The mutation resulted in a nucleotide substitution of an A to a C nucleotide at site 796 (A796C) which is representative of the A allele. This novel B allele was termed B796C. The nucleotide substitution would result in an amino acid change from methionine (Met-B gene) to leucine (Leu-A gene). The critical sites for the B-specific sugar donor nucleotides are 796 and 803 and thus this nucleotide substitution and subsequent change in amino acid would alter the transferase specificity of the B gene protein transcript. Thus the ability of ABO genotyping to clarify serological difficulties was demonstrated by this example. Nucleotide assignment at the different nucleotide sites on exon 7 and the A and B transferase genes: Nucleotide B796C gene PROJECTED BENEFITS OF VIRAL NUCLEIC ACID TESTING FOR THE AUSTRALIAN BLOOD SUPPLY
A Farrugia Blood Products Unit, Therapeutic Goods Administration Laboratories, Canberra, Australia
Introduction: Despite third-generation serological screening of all blood donations, the delay in the production of detectable immunological markers following viral infection (the “window” period) results in the theoretical possibility of infectious blood entering the supply chain. The use of the Polymerase Chain Reaction (PCR) and related techniques in detecting viral genome prior to the development of serological markers has been surveyed extensively overseas. Aim of study: To estimate viral marker incidence and residual risk in overseas and Australian blood donations and to then project the expected yield of additional infective units detected using current PCR tests. Methods: Published data from international and Australian surveys was analysed to estimate: (1) Incidence of infection in repeat blood donors (2) Residual risk of infection after screening with current serological markers (3) Projected yield from PCR testing. Data for HIV, Hepatitis B Virus (HBV) and Hepatitis C virus (HCV) were analysed. Results: Australia(1) Europe(2)
1Whyte and Savoia 1997 2European Plasma Fractionation Association 1996 3Schrieber et al 1996 Conclusions: The introduction of PCR may be expected to result in a small but measurable increase in the safety of non-virally inactivated blood components, particularly for HCV. For pooled plasma products subjected to viral inactivation procedures, PCR is unlikely to further enhance viral safety. Issue of cost effectiveness and optimal test configurations must be addressed before PCR is introduced into routine transfusion practice. TRANSMISSION OF HUMAN T-LYMPHOTROPIC VIRUS, TYPE II (HTLV-II) TO AN AUSTRALIAN BLOOD DONOR BY HUMAN BITE WV Bolton1, AR Davis2, YC Ge3, DE Dwyer4, KG Kenrick1, AL Cunningham3 and NK Saksena3 1 Department of Virology and Biochemistry, and 2 Aphaeresis Unit, Australian Red Cross Blood Service, NSW and 3 Retroviral Genetics Laboratory, Centre for Virus Research, Westmead Institutes for Health Research, and 4. Centre for Infectious Diseases and Microbiology, Westmead Hospital, NSW
Human T Lymphotropic virus, type II (HTLV-II) is a human retrovirus transmissible by injecting drug use, transfusion of cellular blood products, sexual contact and breastfeeding. Soon after the introduction of routine anti-HTLV-1 screening in 1993, we identified a regular blood donor as anti-HTLV-II seropositive. Initially, we could not discern recognised risk factors for her HTLV-II infection. After laboratory and clinical investigations we could demonstrate her HTLV-II seroconversion occurred over a period contemporaneous with her suffering a severe bit from her son, who we also found to be infected with HTLV-II, probably acquired through injecting drug use in North America in 1984. Nucleotide sequencing of the proviral tax/rex gene from both mother and son demonstrated an identical sequence, phylogenetic analysis further confirming the virus to be HTLV-II subtype B. We believe this to be the first documented molecular evidence of transmission of an HTLV by human bite. This case reinforces the difficulties in determining the likelihood of HTLV infection in blood donors by risk factor analysis, and demonstrates that incident HTLV infection can occur in Australian blood donors. A MURINE MODEL OF IMMUNE THROMBOCYTOPENIA
B Dale, D Jiang, M Sturm, R Baker Haematology Department, Royal Perth Hospital, Perth, Western Australia
FcγRII is significant in humans in that it is the only Fc receptor expressed on platelets inferring an important role in the pathophysiology of immune thrombocytopenia. It has been shown to be a crucial mediatory molecule in the heparin induced thrombocytopenia (HIT) disease process and to have involvement in idiopathic thrombocytopenic purpura (ITP). Soluble FcγRII has been shown to down-regulate IgG production by B cells and recombinant FcγRII has demonstrated ability to dissociate immune complexes. Our earlier findings demonstrated that levels of soluble FcγRII were raised in conditions of immune thrombocytopenia and that recombinant FcγRII could inhibit platelet aggregation due to HIT antibody. We postulate that soluble FcγRII has a protective role in immune thrombocytopenia. A model of immune thrombocytopenia has been successfully established in C57BL/6 mice. Semilops rabbits were immunised to raise an antibody against C57BL/6 platelets. Administration of immunised rabbit serum to C57BL/6 mice resulted in rapid development of thrombocytopenia. The fall in platelet count was dose dependent and normal control rabbit serum did not cause thrombocytopenia. Protection against development of thrombocytopenia could be achieved by prior administration of anti-LY 17.2 (anti- FcγRII) or anti-LY 17.1/17.2 (anti- FcγRII/ FcγRIII) ascites. Given that murine platelets do not express Fc receptors, this finding suggests that protection of platelets is achieved by reticulo-endothelial blockade and that clearance of opsonised platelets is FcγR mediated. Refinement of this model will enable study of the efficacy of recombinant FcγRII and anti- FcγRII monoclonal antibody therapy for the treatment of immune thrombocytopenia. 2. MAJOR SYMPOSIA ABSTRACTS
2.1 MECHANISMS OF IMMUNOMODULATION BY BLOOD TRANSFUSION W Dzik Transfusion Medicine, BI-Deaconess Medical Centre, Harvard Medical School, Boston, MA
Decades of research have slowly but gradually revealed increasing evidence for an immunomodulatory effect of blood transfusion. The effect has been seen in pretransplant conditioning and tolerance induction, and has been suspected in the development of post-operative wound infection and tumour recurrence. This lecture will provide an overview of the mechanisms considered most likely to account for this effect. Three mechanisms will be discussed: clonal deletion, peripheral anergy, and T cell suppression. Clonal deletion occurs primarily as a result of a process by which developing T cells undergo apoptosis in the thymus as a result of high affinity binding of the T cell receptor to self MHC + peptide. Experimental evidence in animals documents the ability of peptide antigens to induce tolerance when delivered into the thymus. Peripheral anergy occurs when antigen is presented to T cell receptors in the absence of required co- stimulatory molecules. Recent research has focused on the potential for soluble HLA molecules to induce peripheral transplantation anergy through this mechanism. T cell suppression occurs as a result of activation of particular CD4 subsets capable of downregulating alloimmune responses. Animal models of the immunomodulatory effect of transfusion point to cytokine- mediated Th2 type subsets localised in the spleen. Studies have provided evidence of adoptive transfer of transplant tolerance by infusing these splenic T cells into recipients. These mechanisms will be put into the possible context of allogeneic blood transfusion’s immunomodulatory effect. 2.2THE TREATMENT OF THE THROMBOCYTOPENIC PATIENT: CURRENT ISSUES AND FUTURE PERSPECTIVES MA Blajchman Departments of Pathology and Medicine, McMaster University, Hamilton, Ontario, Canada
The ultimate reason to transfuse allogeneic platelet concentrates (PCs) to thrombocytopenic patients is to improve their ability to withstand a haemostatic challenge. Alterations to the standard preparative and storage parameters for PCs may result in PC preparations with retained in vitro biological function but which do not manifest in vivo haemostatic function when transfused to a thrombocytopenic recipient. Over the past few years, considerable experimental effort has been expended to try to improve platelet storage by using a variety of approaches. These include: freezing platelets; storage at 4°C; novel platelet products (ie. UVA- psoralen treated PCs); and a variety of putative platelet substitutes. The major problem with some of these developments has been the lack of appropriate methodology for assessing the haemostatic function of such alternative platelet products. While some of these novel platelet products and substitutes are already in clinical trial, most are still being evaluated in pre-clinical studies. This presentation will describe the various novel approaches being taken for the development of alternative therapeutic interventions for the treatment of bleeding thrombocytopenic patients. An outline of the various classes of products that might be available in the foreseeable future, as alternatives to liquid-stored platelets, will also be provided. Finally, an in vivo experimental animal model, which has been developed in the author’s laboratory, to evaluate the haemostatic function of these products, will be described. 2.3 PROBE TESTING OF PLASMA POOLS
MP Busch Blood Centres of the Pacific, and University of California, San Francisco CA, USA
Despite enormous progress in reducing the risk of transmission of viral infections to recipients through improved donor selection and serologic screening assays, there remains a small but significant transfusion risk for each of the major viral agents. This risk is primarily due to the failure of current serologic screening tests, which are based on antibody and antigen detection, to identify recently infected donors in the pre- seroconversion window phase of infection. In addition, for some agents there may be a subset of chronically infected persons who either fail to develop a detectable immune response or harbour antigenically variant viruses missed by serologic screening tests. Finally, a failure in the testing process itself may contribute to the risk. The absolute and relative contributions of each of these sources of risk will be the first area covered in this lecture. The risk caused by each of these types of “screen failures” could be reduced and potentially eliminated by implementation of sensitive assays targeting viral nucleic acids. A number of technologies currently exist for nucleic acid amplification (NAA) and subsequent detection of amplified target sequences. This presentation will begin with an overview of these technologies, and will then consider novel strategies/systems being development specifically for mass screening applications. Recent pressure to implement NAA testing prior to the availability of automated methods compatible with mass screening of individual donations has led to the development and evaluation of pooled sample testing strategies. The rationale for and preliminary experience with pooled NAA testing, and the complex ramifications of pooled testing for blood centres, transfusion services, donors and recipients will be discussed in detail. 2.4 THROMBOPOIETIN: IMPLICATIONS FOR THE DIAGNOSIS AND TREATMENT OF DISORDERS OF PLATELET PRODUCTION K Kaushansky Division of Haematology, University of Washington School of Medicine, Seattle, Washington, USA
Until recently, platelet production was the least understood aspect of blood cell development. This gap in our understanding was due to the scarcity of megakaryocytes and to confusion surrounding the cytokines and hormones, which support their development. The recent cloning and characterisation of thrombopoietin (TPO) has profoundly changed this. Using in vitro assay systems, several groups have shown that TPO supports the proliferation of megakaryocytic progenitor cells and their differentiation into mature platelet-producing cells. Moreover, TPO also acts in synergy with other pluripotent cytokines on the haematopoietic stem cell, and to augment the development of erythroid and myeloid progenitors. These in vitro effects correlate well with the in vivo biology of the hormone. When administered to normal animals, TPO expands the numbers of haematopoietic progenitors of all lineages, and greatly accelerates platelet production. Furthermore, when TPO or its receptor are genetically eliminated, progenitor cell levels of all lineages are reduced, and platelet production is profoundly impaired. In animals administered cytoreductive therapy, the use of TPO is associated with accelerated recovery of all haematopoietic lineage. Based on these promising in vitro and in vivo results, clinical trials of the agent in patients undergoing chemotherapy for cancer have now begun. TPO is safe, and acts to increase platelet production both before and after the administration of myelosuppressive therapy. Clinical trials are also underway to study the effect of the hormone in patients undergoing stem cell transplantation, and whether the agent can augment cell yields in platelet donors. Finally, disorders of TPO production have also been recognised. As the liver is one of the primary sources of TPO production, patients with liver failure also appear to manifest thrombocytopenia due to TPO deficiency. And several cases of TPO excess have been identified, states that lead to thrombocytosis. Thus, a better understanding of the biology of TPO has been instrumental in recent successes in the diagnosis and treatment of disorders of platelet production. 2.5 THE MOLECULAR BASIS OF THE KELL AND DUFFY BLOOD GROUP ANTIGENS ME Reid Immunohematology Laboratory, New York Blood Centre, New York, New York, U.S.A.
The 24 Kell blood group system antigens are carried on variant forms of the Kell protein. This protein of 732 amino acids is encoded by a gene with 19 exons located on chromosome 7q33. Kell is a type II membrane protein and, in the red blood cell (RBC) membrane, is linked to a multipass protein, Xk. Kell polymorphisms are associated with point mutations that are predicted to encode single amino acid substitutions. Because each point mutation ablates or creates a restriction enzyme site, it is possible to genotype for Kell variants using allele-specific primers with PCR and PCR-RFLP assays. Thus, Kell genotyping of amniocytes can be performed to identify a foetus at risk. This has particular clinical relevance because Kell incompatibility causes anaemia in the foetus by suppression of erythropoiesis rather than by haemolysis and, unlike anti-D, the strength of the antibody does not correlate with the severity of the infant’s anaemia. Fya and Fyb are encoded by the Duffy gene and differ by a single amino acid (Gly or Asp, respectively). This gene has two exons that generate two transcripts encoding Duffy proteins of 336-amino acids (major) and 338 amino acids (minor). The proteins, which differ only by the first few NH2-terminal amino acids, are predicted to span the RBC membrane seven times, are a receptor for the malarial parasite Plasmodium vivax and P. knowlesi, and function as a promiscuous chemokine receptor. The Fy(a-b-) phenotype, found in 68% of African Americans and 100% of West Africans, is caused by a point mutation with in the promoter region of the FY*B gene that abolishes binding of the erythroid-specific GATA-1 transcription factor. This silencing of Duffy in RBCs but not in other tissues is significant in transfusion medicine because Fy(b-) people with a nonfunctional GATA box should not make anti-Fyb and, therefore can be transfused with Fy(b+) RBCs without fear of eliciting an immune response to the Fyb antigen. 3. PLATELET IMMUNOLOGY:
A NOT - SO - TRIVIAL - PURSUIT. Robyn Minchinton Chief Scientist, ARCBS, Brisbane, Queensland. PLATELET ANTIGENS Newman, P.J. and Goldberger, A. (1991) Molecular genetic aspects of human platelet antigen systems. Balliere's Clinical Haematology 4, 869-888.
Advances in molecular biology have revealed the secrets of the platelet specific antigens. The platelet alloantigenic epitopes are amino acids on the platelet membrane glycoproteins. The HPA-1 (PLA) antigens are on gp IIIa, HPA-2 (Ko) on gp Ib, HPA-3 (Bak) on gp IIIb, HPA-4 (Pen) on gp IIIa and HPA-5 (Br) are on gp Ia. This knowledge has allowed platelet immunologists to undertake pre-natal screening and genetic counselling, antenatal platelet typing, better identify antibody specificities with well typed platelet panels and develop improved diagnostic tests.
Novotny, V.M.J., Huizinga, T.W.J., van Doorn, R., Briet, E. and Brand, A. (1996) HLA class I-eluted platelets as an alternative to HLA-matched platelets. Transfusion 36, 438-444.
For some years platelet immunologists have been using chloroquine or citric acid solutions to strip HLA but not platelet specific antigens from platelets to assist with the differentiation of platelet specific antibodies from HLA antibodies. Now, with careful standardisation of an aseptic, citric acid elution and washing technique, HLA antigens can be eluted from platelet concentrates and infused with good result into alloimmunized platelet transfusion refractory patients. PLATELET ANTIGEN & ANTIBODY TESTING Kiefel, V. (1992) The MAIPA assay and its applications in imunohaematology. Transfusion Medicine 2, 181-188. Brighton, T.A., Evans, S., Castaldi, P.A., Chesterman, C.N. and Chong, B.H. (1996) Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias. Blood 88, 194-201. Berchtold, P., Muller, D., Beardsley, D., Fujisawa, K., Kaplan, C., Kekomaki, R., Lipp, E., Morell-Kopp, M.C., Kiefel, V., McMillan, R., von dem Borne, A.E.K. and Imbach, P. (1997) International study to compare antigen-specific methods used for the measurement of antiplatelet antibodies. British Journal of Haematology 96, 477-483.
The monoclonal antibody immobilisation of platelet antigens (MAIPA) assay is a reliable and useful technique, which not only demonstrates the presence of platelet autoantibodies and alloantibodies, but also indicates the platelet glycoprotein targeted by the antibody. Interpretation is not influenced by the presence of even high titre HLA antibodies. These are not captured in the MAIPA except by monoclonal antibody to B2 microglobulin. MAIPA enables accurate serological platelet typing to be carried out, even with typing sera contaminated by HLA antibodies; it is useful for characterising the glycoprotein specificity of autoantibodies bound in vivo to circulating platelets in autoimmune thrombocytopenia and it is essential for the correct determination of the specificity of platelet alloantibodies. In autoimmune thrombocytopenias however, it is not any more sensitive for the detection of platelet bound immunoglobulins than other tests. In Australia, we expect the flow cytometric PIFT (platelet immunofluorescence test) to have 70-80% sensitivity in autoimmune thrombocytopenia with a lower figure for the MAIPA. Discovery of an autoantibody to a platelet glycoprotein in such patients by using the MAIPA, however, is good evidence for an autoimmune aetiology.
6. Skogen, B., Bellissimo, Hessner, M.J., Santoso, S., Aster, R.H., Newman, P.J. and McFarland, J.G. (1994) Rapid determination of platelet alloantigen genotypes by polymerase chain reaction using allele-specific primers. Transfusion 34, 955-960.
Restriction Fragment Length Polymorphism (RFLP) or Allele Specific Oligonucleotide hybridisation (ASO) are the most frequently used techniques for definitive determination of the platelet alloantigen genotypes for any individual. Specific applications are for the accurate typing of platelet panel donors for diagnostic or clinical use, for antenatal diagnosis of feto - maternal alloimmune thrombocytopenia and for confirmation of serological types in diagnostic situations. PLATELET TRANSFUSION 7. Doughty, H.A., Murphy, M.F., Metcalfe, P., Rohatiner, A.Z.S., Lister, T.A. and Waters, A.H. (1994) Relative importance of immune and non-immune causes of platelet refractoriness. Vox Sanguinis 66, 200-205. 8. Gelb, A.B. and Leavitt, A.D. (1997) Cross-match compatible platelets improve corrected count increments in patients who are refractory to randomly selected platelets. Transfusion 37, 624-629. 9. Slichter, S.J. (1994) Platelet alloimmunization. In: Anderson, K.C. and Ness, P.M. (Eds.) Scientific Basis of Transfusion Medicine. Implications for Clinical Practice. pp. 527-543. Philadelphia: W.B.Saunders]
In spite of the attention given in the literature in recent times to immune causes of platelet transfusion refractoriness, non-immune factors are actually more important. The importance of HLA antibodies has probably been overestimated. In the 1970's and early 1980's, 50-70% of multitransfused patients were found to have HLA antibodies. The use of increasingly intensive cytotoxic therapy has probably contributed to the lower currently accepted figure of 25-30% for HLA immunisation. Furthermore, the presence of HLA antibodies does not necessarily indicate a patient will be immune refractory - only 30% of such patients are actually refractory to platelet transfusions from random donors.
Similarly, the incidence of platelet antibodies, which might cause immune
destruction of transfused platelets, is quite low. Before contemplating the possibility of alloantibodies, it is prudent to first consider the influence of fever, infection and antibiotic therapy on platelet recoveries or corrected increments.
In those patients who have been shown to have circulating alloantibodies which are reactive with random donor platelets, an efficient means of selecting compatible donors is to use the patient's serum to perform prospective cross match of apheresis donor panel platelets to discover which donor is likely to provide a successful transfusion. The advantage of using a platelet cross match is that both HLA and platelet specific antigens are represented on the cross match platelets. Solid phase capture tests are most suitable for this type of screening. An alternative is to use a microtitre plate based platelet cross match method to screen all of the random single donor units held in a bank. The donors of compatible units may then be asked to return for apheresis to support a particular patient. FETO - MATERNAL ALLOIMMUNE THROMBOCYTOPENIA (FMAIT)
Kaplan, C., Morell-Kopp, M.C., Clemenceau, S., Daffos, F., Forestier, F. and Tchernia, G. (1992) Fetal and neonatal alloimmune trhombocytopenia: current trends in diagnosis and therapy. Transfusion Medicine 2, 265-271. Decary, F., L'Abby, D., Tremblay, L. and Chartrand, P. (1991) The immune response to the HPA-1a antigen: association with HLA-DRw52a. Transfusion Medicine 1, 55-62.
Alloantibodies to platelet antigen HPA-1a are most commonly found in fetomaternal alloimune thrombocytopenia and in post - transfusion purpura. A number of comprehensive studies have shown that women, who become immunised to the HPA-1a antigen, have a higher frequency of both HLA-DR3 and HLA-DRw52a. However, the prospective HLA-DR typing of HPA-1a negative women has not become routine practice worldwide.
Chow, M.P., Sun, K.J., Yung, C.H., Hu, H.Y., Tzeng, J.L. and Lee, T.D. (1992) Neonatal alloimmune thrombocytopenia due to HLA-A2 antibody. Acta Haematologica 87, 153-155. Kaplan, C., Morell-Kopp, M.C., Clemenceau, S., Daffos, F., Forestier, F. and Tchernia, G. (1992) Fetal and neonatal alloimmune trhombocytopenia: current trends in diagnosis and therapy. Transfusion Medicine 2, 265-271. Bussel, J.B., Zabusky, M.R., Berkowitz, R.L. and McFarland, J.G. (1997) Fetal alloimmune thrombocytopenia. The New England Journal of Medicine. 337, 22-26.
Fetomaternal alloimmune thrombocytopenia (FMAIT) occurs in about 1:1000 births. Initial diagnosis is made when the infant is found to be significantly thrombocytopenic with associated clinical signs ranging from bruising to intracranial haemorrhage (ICH). ICH occurs in up to 20% of affected neonates, many of who had their initial bleed in utero. In a sensitised HPA-1a negative mother, the chance of her having a subsequently affected fetus is between 85-90%. Fetal thrombocytopenia is more severe in cases of HPA-1a incompatibility between parents. In the absence of intervention the platelet count tends to fall or remain very low. With a history of antenatal cranial haemorrhage in an older sibling, the platelet count in subsequent fetuses will be at similar dangerous levels. Otherwise, sibling platelet counts are non-predictive for subsequent fetuses. The thrombocytopenia in these cases occurs as early as 20 weeks of gestation and is unremitting. For alloimmunisation to other platelet antigens, thrombocytopenia has been reported as less severe, but too few cases have been studied to be entirely predictive.
Occasionally, only HLA platelet reactive antibody
can be demonstrated in infants with sometimes severe FMAIT. There is worldwide controversy about the ability of HLA antibodies to cause this syndrome. Antenatal diagnosis is possible (via Percutaneous Umbilical Blood Sampling) and post- natally using serological techniques.
Murphy, M.F., Metcalfe, P., Waters, A.H., Ord, J., Hambly, H. and Nicolaides, K. (1993) Antenatal management of severe feto-maternal alloimmune thrombocytopenia: HLA incompatibility may affect responses to fetal platelet transfusion. Blood 81, 2174-2179. Waters, A.H., Murphy, M.F., Hambly, H. and Nicolaides, K. (1991) Management of Alloimmune Thrombocytopenia in the Fetus and Neonate. In: Nance, S.J. (Ed.) Clinical and Basic Aspects of Immunohaematology. Pp. 155-177. Arlington, VA: American Association of Blood Banks.
Two schools of thought and practice exist regarding the optimal antenatal treatment of FMAIT. They are: - •
Invasive therapy in which the fetal platelet count is determined early, around 20-24 weeks and regular umbilical vein compatible platelet transfusions are given during the course of the pregnancy.
Non-invasive therapy in which a single fetal platelet count is determined by cordocentesis at 20-24 weeks (or no count is performed at all) and the mother is treated antenatally with intravenous immunoglobulin (1g/kg/day) with or without corticosteroids.
Either approach, if uncomplicated, results in a rise in fetal platelet count, which can be maintained at levels, which at least prevent the occurrence of intracranial haemorrhage. An interesting and so far unique case has been described in which maternal HLA antibodies caused immune destruction of HPA-1a compatible platelets transfused into the fetus. The administration of HLA compatible HPA-1a negative platelets and platelets prepared from the mother resulted in improved responses. AUTOIMMUNE THROMBOCYTOPENIA (AITP) 17. Samuels, P., Bussel, J.B., Braitman, L.E., Tomaski, A., Druzin, M.L., Mennuti, M.T. and Cines, D.B. (1990) Estimation of the risk of thrombocytopenia in the offspring of pregnant women with presumed immune thrombocytopenic purpura. The New England Journal of Medicine 323, 229-235. 18. McCrae, K.R., Samuels, P. and Schreiber, A.D. (1992) Pregnancy associated thrombocytopenia: pathogenesis and management. Blood 80, 2697-2714.
Recent guidelines published by the American Society of Hematology indicate that an expert panel "strongly agreed" that platelet antibody determination in pregnant women is inappropriate or unnecessary to establish a diagnosis of autoimmune thrombocytopenia. However, the finding of a negative result for circulating maternal platelet autoantibody has been regarded by some experts as a minimal risk indicator for severe neonatal thrombocytopenia in the offspring. Similarly, women without a history of immune thrombocytopenia before pregnancy are thought to be at low risk of carrying a thrombocytopenic fetus. The usefulness of platelet immunological determinations in AITP in pregnant women remains a vexed question. Certainly, the titre of circulating platelet autoantibody is not linked to the likelihood of thrombocytopenia in the fetus. The spectre of intracranial haemorrhage remains.
George, J.N., Woolf, S.H., Raskob, G.E., Wasser, J.S., Aledort, L.M., Ballern, P.J., Blanchette, V.S., Bussel, J.B., Cines, D.B., Kelton, J.G., Lichtin, A.E., McMillan, R., Okerbloom, J.A., Regan, D.H. and Warrier, I. (1996) Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Haematology. Blood 88, 3-40. Kaplan, C. and Tchernia, G. (1995) Autoimmune Thrombocytopenias. In: Anonymous Immunopharmacology of Platelets. Pp. 167-194. New York: Academic Press] Warner, M. and Kelton, J.G. (1997) Laboratory investigation of immune thrombocytopenia. Journal of Clinical Pathology 50, 5-12.
Immune thrombocytopenia can be defined as increased platelet destruction by immune mechanisms in the setting of normal megakaryopoiesis. Its diagnosis remains largely one of exclusion. No single platelet immunological test is both wholly sensitive and specific for the diagnosis of AITP. Platelet associated IgG is usually increased in AITP, but it can also be raised in non-immune thrombocytopenias. Assays which demonstrate the glycoprotein target of the platelet autoantibody are highly specific but may not be all encompassing in their ability to detect all that is present. The usual approach in advanced platelet immunology reference laboratories is to recognise the following: •
In AITP the platelet count is the result of the balance between destruction of circulating platelets and their bone marrow production.
Cells of the reticuloendothelial system both remove antibody-coated platelets and carry many of the platelet autoantigens (and alloantigens).
Every method for detection and/or characterisation of platelet bound or circulating autoantibody has its own advantages and disadvantages - a combination approach is usually most informative.
Different haematologists will have personal "confidence limits” for the results of different platelet immunology assays.
Assays may be most usefully applied over a time period to plot changes in antibody status.
POST - TRANSFUSION PURPURA (PTP) 22. Taaning, E. and Svejgaard, A. (1994) Post-transfusion purpura: a survey of 12 Danish cases with special reference to immunoglobulin G subclasses of the platelet antibodies. Transfusion Medicine 4, 1-8. 23. Minchinton, R.M. (1990) Laboratory perspectives on the pathogenesis of post-transfusion purpura. Haematology Reviews 4, 63-78.
PTP is clinically dramatic with development of purpura, haematoma and bleeding in patients, usually women, five to twelve days after blood transfusion. The underlying severe thrombocytopenia is refractory to treatment with random donor platelet units and such transfusions may produce moderately severe transfusion reactions.
Most commonly, a platelet specific antibody to HPA-1a can be demonstrated at high titre in the
patient’s serum and the patient is subsequently discovered to be HPA-1a negative. The link between the presence of anti-HPA-1a and impressive destruction of the patient's own HPA-1a negative platelets has been a matter of debate which has not been satisfactorily resolved. Many groups suspect that an underlying platelet specific autoantibody emerging in parallel with the anamestic alloantibody is causative. As in FMAIT, there is a good association with the presence of HLA-DR3 and HLA-DRw52a. Plasmapheresis with or without the use of intravenous gammaglobulin results in prompt recovery of the platelet count and resolution of the bleeding manifestations. PTP rarely may recur in a patient re-challenged with HPA-1a positive cellular products.
DRUG - RELATED IMMUNE THROMBOCYTOPENIA Silvestri, F., Virgolini, L., Savignano, C., Zaja, F., Velisig, M. and Baccarani, M. (1995) Incidence and diagnosis of EDTA-dependent pseudothrombocytopenia in a consecutive outpatient population referred for isolated thrombocytopenia. Vox Sanguinis 68, 35-39.
Pseudothrombocytopenia results from the reaction of a naturally occurring, platelet specific autoantibody with a platelet glycoprotein conformational antigen exposed by the presence of EDTA in vitro. It is an extremely common laboratory artefact, which can be confirmed as such by re-collection of the blood in heparin or citrate. Rarely, platelet serological methods may be requested to confirm the diagnosis.
Mueller-Eckhardt, C. and Salama, A. (1990) Drug-induced immune cytopenias: a unifying pathogenetic concept with special emphasis on the role of drug metabolites. Transfusion Medicine Reviews. IV, 69-77.
Drug induced immune cytopenias can be severe and difficult to confirm by laboratory testing. Poor understanding of the mechanisms of action of the drug from commonly accepted but flawed historical hypotheses has slowed advances in this field of study. Strong new concepts are: - •
Antigen production in drug related thrombocytopenias is the result of a direct interaction between the drug and/or its metabolites and the platelet membrane.
Involvement of drug metabolites in drug induced immune cytopenias is the rule rather than the exception.
Specificity of drug dependent antibodies is determined by elements of both drug and cell membrane - antibodies cannot bind avidly to either one alone. If one is removed, the immune reaction subsides.
Drug independent antibodies are elicited by a subtle alteration of the membrane or membrane glycoproteins by the drug, but the epitope is sufficiently similar to the unaltered structure to support drug-independent binding to the patient's cells and those of normal donors. Such antibodies behave like autoantibodies.
Drug dependent antibodies precipitate an acute severe cytopenic episode, frequently mediated by complement.
Drug independent antibodies result in a prolonged, less severe cytopenic syndrome with signs of extravascular cell destruction.
Heterologous population of antibodies may co-exist in the same patient.
1998 marks the 10th anniversary of the Australasian Platelet Antibody Workshop (APAW)
Further information can be obtained from: -
Dr Robyn Minchinton Australian Red Cross Blood Service Queensland PO Box 10325, Adelaide St, Brisbane, Q, 4000 E-Mail: rminchinton@arcbs.redcross.org.au 4. GOING BACK TO THE FUTURE: THE ORIGINS OF A NATIONAL BLOOD SERVICE IN AUSTRALIA
The traditional state blood services in Australia are experiencing considerable change as the further consolidation of the Australian Red Cross Blood Service (ARCBS) fundamentally alters the way in which the blood system is managed in this country. Although the creation of the ARCBS represents a new beginning for the Australian blood service, the true origins of a loosely constructed national blood service stretch far back into the distant past. This paper traces the origins of the National Blood Transfusion Service (NBTS)- the first nationally organised wartime blood service in Australia. An examination of this organisation will show that with the establishment of ARCBS, blood collection and distribution in Australia has, to some extent, come full circle as the organisation has, once again, assumed a form that is very much national in scope. PRE-WAR BLOOD SUPPLY
There was no organised blood service operating in Australia prior to the outbreak of the Second World War. Major hospitals throughout the country maintained their own donor panels and carried out their own collection and administration of blood. Although it was a workable system in theory, hospitals quite often experienced great difficulty in securing blood particularly in critical emergencies. Dr. Beatrix Durie, a Sydney pathologist, remarked in 1939 “although we have 80 names on our hospital list we find it necessary to add fresh blood so to speak as people lose interest, change their address and so on. The main difficulty is to secure a voluntary donor in an emergency as at times 8 or 9 persons are telephoned before we can find one who can come at once”.i The panels operated by some of the larger hospitals suffered from the obvious limitation that they could not always be relied upon to supply blood. It should also be noted that many of the smaller hospitals had no transfusion services to speak of at all and were dependent upon the generosity of other hospitals or any of the other private or public blood panels then in operation. In addition to the panels organised by the hospitals there were, depending upon the locale, viable alternatives. In New South Wales, for example, private blood panels were operated by both the NSW Department of Public Health, that paid donors three £ a pint for a post- partum maternity care project it operated, as well as the Toc H Club.ii Despite the existence of these various institutional and voluntary blood donor panels, medical institutions often had difficulty in securing whole blood on short notice. Having ready access to a reliable supply of whole blood was not always a reality in the years before the Second World War. The first tentative steps toward the establishment of blood service that was national in scope can be traced to the state of Victoria, where a rudimentary blood transfusion service was established in 1929 under the auspices of the Victorian Division of the Australian Red Cross. Created largely through the relentless efforts of Lucy Bryce, a Melbourne pathologist, along with the support given by Eric Cooper, Medical Superintendent of the Melbourne Hospital who was desirous of emulating the London blood service that had been organised by the British Red Cross in 1926. The service, in its initial incarnation, provided donors without charge to the public hospitals in Melbourne.iii In addition to the established blood donor operations in Melbourne, the idea gradually spread to Western Australia, where beginning in 1935, the Red Cross Society, following the lead taken by the Victorian service, undertook to maintain and enlarge the roster of donors that existed at the Perth Hospital. The concept gradually spread to Queensland and Tasmania and during the war to New South Wales and South Australia.
WARTIME ORIGINS OF A NATIONAL BLOOD SERVICE The actual outbreak of the Second World War led to the creation of the National Blood Service-the original national blood organisation that was cooperatively organised by the Central Committee of the Australian Red Cross Society, the Commonwealth Armed Forces through the Directorate General Medical Services (DGMS) and the various state civil defence authorities. In addition to establishing a nation-wide network of donor panels, the organisation assumed responsibility for providing whole blood and serum to both military and civilian institutions. The National Blood Service was designed primarily to provide blood supplies for any possible wartime emergency, as there were fears that the ranks of donors, which during the 1930s were overwhelmingly male, would be depleted through enlistment at a time of heightened wartime demand. The establishment of a National Blood Transfusion Service was largely driven by the desire of the Australian military to have adequate supplies of whole blood and serum available for both troops and the civilian population in the event of hostilities breaking out on the home front. The formal birth of Australia’s national blood system coincided with the announcement, that the Director General of Army Medical Service, Major General F.A. Maguire had authorised, in April 1941, the establishment of twenty-five blood collecting centres in Australia. The Australian Army medical service, in conjunction with the Australian Red Cross, organised formal blood collection centres in each of the capital cities.iv These collection centres essentially functioned as Army medical units and were staffed by army personnel, and by voluntary aid workers of the Australian Red Cross. The national scheme was based upon a plan jointly drawn up by Melbourne pathologist Lucy Bryce, and the army’s Director of Pathology, Col. C.H. Kellaway, on leave from his regular position as Director of the Walter and Eliza Hall Institute. Under the terms of the agreement, the Red Cross would oversee the regular operations of the new national service through its National Emergency Blood Transfusion Committee, the forerunner to the National Blood Transfusion Committee created in December 1941 as an advisory body responsible for formulating and implementing all policies that pertained to the collection of whole blood and the production of both wet and dried serum. Unlike American and to a lesser extent British forces that showed a preference for plasma, Australia chose serum as its preferred blood substitute of choice during the war. This decision was influenced by the ability of F.G. Morgan; Director of CSL, to perfect an effective serum preparation technique that in contrast to plasma was easily filtered. v Serum was initially produced in Victoria and later in Sydney and Adelaide and was used as a blood substitute for the Australian forces in the Middle East and Pacific theatres of war. Serum was transported overseas along with the incomparable Australian-designed transfusion set that had been conceived by C.W. Ross and Ian Wood of the Walter and Eliza Hall Institute. The primary feature of this unique system was the Soluvac, a modification of the original gravity-sealed infusion sets that were popular throughout the United States and England. The primary difference was the larger capacity of the Soluvac system. It featured a 1200-millilitre as opposed to a 500-millilitre bottle that was common to the other sets. Despite concerns about larger bottle size and storage space, the Soluvac was advocated by the army medical service as a system that would better serve the wounded by encouraging the delivery of liberal quantities of transfusion fluids.vi In contrast to the multi-faceted operations performed by contemporary blood banks, the objectives of the original blood service were considerably more modest. In fact they were essentially limited to three primary goals: the collection of blood for the forces and civilian hospitals; the collection of blood for the preparation of therapeutic serum; and the establishment and maintenance of emergency bleeding centres in vulnerable areas that could be used for the collection of blood during a state of emergency.vii Although Australia’s civilian population was fortunate in not having to experience the carnage of war, on a grand scale, great efforts were made by the national blood transfusion service, to ensure that any possible wartime contingency could be met. THE POST-WAR RISE OF THE STATE SERVICES At the close of the war, the DGMS arranged with the Chair of the ARCS that the Society should take over the wartime service with the aim of establishing a peacetime service on a civilian basis. This task was facilitated by the ability of the Red Cross to retain the services of army medical officers like RJ Walsh, of NSW, Noel Gutteridge of Queensland and Cyril Fortune of Western Australia, individuals, who through the war had developed a high measure of competency in blood transfusion work.
When the Red Cross assumed responsibility for these army units, it was faced with the task of developing them into efficient civilian centres that could collect and distribute blood to meet the requirements of each state. It was from these early beginnings that the post-war emergence of RCBTS of Queensland, New South Wales, Victoria, South Australia and Tasmania is to be found. Each service operating autonomously within the state, each under the control of the state division of the Red Cross, which was in turn advised by the National and state blood transfusion committees. viii Although the post-war evolution of each of these state organisations merits further historical study, it is clear that their development as autonomous blood services has, from the start, not always been trouble free. Securing adequate space and accommodation to house operations was and still is a problem faced by some, particularly in New South Wales. Finances have also been pressing concern as the state services took on additional testing initiatives and innovative projects beyond which the ARC could realistically support. This forced the ARC to seek financial support from governmental sources, which was forthcoming beginning in 1954ix Although the post-war would usher in the development of increasingly autonomous state blood services, the origins of a national blood service extend far beyond the recent past. Revisiting the history of the blood service in Australia is one way of going back to the future. Mark Cortiula Unit for the History and Philosophy of Science University of Sydney
i Letter, B. Durie to F.S. Hansman, June 22, 1939. New South Wales Blood Bank Archives (NSWBBA)
ii Assistance by Blood Donors in a Scheme for the Reduction of Maternal Mortality, New South Wales
Department of Health Pamphlet, 1938;Letter RJ Walsh to Hon. Secretary of the British Medical Association,
iii The story of the Victoria Blood Service is recounted in, Lucy Bryce, An Abiding Gladness: The Background
to Contemporary Blood Transfusion during the Years 1929- 1959 in the Victorian Division of the Red Cross
Society, (Melbourne: Georgian House, 1965).
iv Letter, F.A. Maguire to J. Newman Morris, N.D. Australian War Memorial Archives. vF.G. Morgan, “The Preparation of Serum From Human Donors Received From Poliomyelitis” Med J. of Aust.,
viC. W. Ross, “The Collection and Storage of Blood, Blood serum and Blood Plasma, Med J. of Aust,
viiJ. Newman-Morris, “Medical Aspects of the Red Cross in the Second World War” December 1945,
viiiEdgar Thomson, “The Red Cross Blood Transfusion Service in Australia” Centenary issue of the Red Cross
ix Commonwealth Cabinet Document 655 Decision 973, March 10, 1954.National Archives, Series A 4940/1
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SUBMITTED ABSTRACTS, 2012 EMDS MEETING Description Presentation Transcriptomic analysis of blood-derived macrophages identifies 5-lipoxygenase activation short talk+ poster protein as a key tumor-induced immune molecule in glioblastoma patients. Macrophage-specific upregulation of apoE and apoCII genes by STAT1 acting on the short talk+ poster Gene expression induced by Toll-li