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Part 2 FRCPath - written option by published papers

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1 Part 2 FRCPath - written option by published papers
Louise Lavender Royal Surrey County Hospital, Guildford 17 January 2011 : Part 2 Written Option Workshop

2 1993 PhD in Molecular Biology 1993-2000 post-doctoral research
My background: 1993 PhD in Molecular Biology post-doctoral research developing PCR methodology for immunoglobulin and T-cell receptor genes in lymphoproliferative disorders as part of a large European consortium registered Clinical Scientist in Molecular Pathology/Haematology at Southampton General Hospital 2006 joined St Georges Molecular Genetics laboratory (for crash course in molecular genetics) 2007 took part 1 written & practical (& passed!) How to get through part 2?

3 What did I do? Considered options: My PhD was out of date (1993)
Dissertation and casebook too much like hard work Decided to try submitting my publications – nothing to lose, could always go for dissertation/casebook option if publications failed Advice I got “worth submitting just to see if it gets accepted”

4 College examinations for Fellowship
Regulations and guidelines for 2011 Candidates undertaking written projects for the Part 2 examination November 2010 (superseding all previous editions)

5 General guidance applicable to all written projects
Six options: dissertation casebook published papers PhD/MD thesis professional doctorate research thesis portfolio

6 Not to be confused with:
Fellowship on the basis of published works “The required standard is unlikely to be reached without the submission of at least 30 peer-reviewed publications…”

7 Royal College of Pathologists Candidate Information - 2010 GENETICS - Molecular Genetics
This document must be read in conjunction with the current Regulations & Guidelines available on the College website and the material sent to candidates in response to an application to sit an examination. In the event of any difference in information or guidance the current Regulations and Guidelines will apply. Content of the examinations – Part 2 Written component There are four options for the written component: a) a casebook b) a dissertation c) a minimum of three refereed published papers d) a PhD/MD thesis, normally completed during the training period.

8 Initial Proposal (*new in 2009):
Candidates must submit an initial proposal for their dissertation, casebook and published papers* prior to commencing the project The initial proposal should outline the project (background, aims, methods and significance of the study) and include approval of a relevant ethics committee where appropriate. It must be no more than 1500 words

9 Published papers Published papers may be submitted as an option for the Part 2 examination in Clinical Biochemistry, Clinical Embryology, Genetics, Haematology (clinical scientists), Histocompatibility and Immunogenetics, Immunology, Toxicology, Veterinary Clinical Pathology (old style examination) and Veterinary Pathology (old style examination) Papers in press may be accepted

10 Published papers As a general guide, a minimum of three papers is expected Three publications by the candidate as sole author might be sufficient, but proportionately more multi-author papers will be required Papers will be judged on their quality and on the candidate’s contribution and, in cases of multi-author papers, the extent and nature of the candidate’s contribution should be clearly indicated and certified by the sponsor

11 Published papers Published papers submitted for the Part 2 examination in any specialty must be in English and must demonstrate original research by the candidate, reflecting a theme or themes They should be linked by a critical commentary, written by the candidate A collection of unrelated case reports is not acceptable The bulk of the work should have been undertaken during the five-year training period in pathology When multi-author papers are submitted, the sponsor is required to comment specifically on the contribution by the candidate to each paper

12 Published papers Two copies of all publications must be submitted, with a full list in chronological order of the publications, together with a full CV of the applicant The reprints of the papers submitted should be numbered to correspond with the list

13 “Minimum of three refereed published papers”
“Three publications by the candidate as sole author might be sufficient, but proportionately more multi-author papers will be required” What does that mean? Literally, if 5 authors per paper need 15 papers! In practice, no real guidelines, dependent on individual examiner Advice I got “worth submitting just to see if it gets accepted” Fairly quick and easy to write a critical commentary

14 What did I do? List of publications (9) Critical commentary
published between 2003 and 2007 (most of work carried out before I joined the NHS) 8 themed (1 first author) on “molecular diagnostics” 1 unrelated (on “proper” molecular genetics) no way proportional to 3 sole author papers Critical commentary summarising scientific content of papers (and clinical relevance) emphasising my contribution 7 pages/ 3,300 words + references (18) Letter of support from sponsor included letter from main author of themed papers outlining my contribution (sponsor not involved in publications) Submitted July 2008 Accepted September 2008!

15 Other peoples’ experience?
Most submitted more papers and more first authorships Range of subjects is wide (don’t assume has to be “pure” molecular genetics) If in doubt ask college for advice before submitting Can appeal if doesn’t get accepted: In practice, no real guidelines, dependent on individual examiner

16 Papers submitted for the Part 2 examination in Molecular Genetics
Louise Lavender: July 2008 Paper 1: van Dongen JJM, Langerak AW, Brüggemann M, Evans PAS, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, García Sanz R, van Krieken JHJM, Droese J, González D, Bastard C, White HE, Spaargaren M, Gonzáles M, Parreira A, Smith JL, Morgan GJ, Kneba M and Macintyre EA (2003). Report of BIOMED-2 Concerted Action BMH4-CT : Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations. Leukemia Paper 2: Brüggeman M, Droese J, Lavender FL, Groenen PJTA, Mills KI, Hodges E, Takla A, Spaargaren M, Martinez B, Jasani B, Evans PAS, Carter GI, Hummel M, Bastard C, Kneba M (2003). SECTION 5. TCRB gene rearrangements: Vβ-Jβ, Dβ-Jβ. Leukemia Paper 3: Beldjord K, Delabesse E, Droese J, Evans PAS, García Sanz R, Langerak AW, Villuendas R, Lavender FL, Foroni L, Milner BJ, Bloxham D, Hummerl M, Trüper L, Delfau-Larue M-H, Macintyre EA (2003). SECTION 6. TCRG gene rearrangements. Leukemia Paper 4: Lavender FL, Hodges E, Mills KI, White HE, Flohr T, Nakao M, Langerak AW, Groenen PJTA, Villuendas R, Jennings BA, Milner BJ, Bloxham D, Droese J, Macintyre EA, Beldjord K, Davi F, Smith JL (2003). SECTION 7: TCRD gene rearrangements: Vδ-Dδ-Jδ, Dδ-Dδ, Vδ-Dδ and DδDJδ. Leukemia Paper 5: White HE, Lavender FL, Delabesse E, Macintyre EA, Harris S, Hodges E, Jones DB and Smith JL (2003). SECTION 10: Use of BIOMED-2 protocols with DNA extracted from paraffin-embedded tissue biopsies and development of control gene primer set. Leukemia Paper 6: van Krieken JHJM, Langerak AW, Macintyre ER, Kneba M, Smith JL, Garcia Sanz R, Morgan GJ, Parreira A, Molina T, Cabeçadas J , Gaulard P, Jasani B, Garcia JF, Ott M, Hannsmann ML, Berger F, Hummel M, Davi F, Brüggemann M, Lavender FL, Schuuring EMD, Evans PA, White H, Salles G, Groenen PJTA, Gameiro P, Pott C, van Dongen JJM (2007). Improved reliability of lymphoma diagnostics via PCR-based clonality testing. Report of the BIOMED-2 Concerted Action BHM4-CT Leukaemia Paper 7: Langerak AW, Molina TJ, Lavender FL, Pearson D, Flohr T, Sambade C, Schuuring E, Al Saati T, van Dongen JJM, van Krieken JHJM. PCR-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls (2007). A study from the BIOMED-2 Concerted Action BMH4-CT Leukaemia Paper 8: Othman MA, Notley C, Lavender FL, White HE, Byrne CD Lillicrap D and O’Shaughnessy DF (2005). Identification and functional characterisation of a novel 27bp deletion in the macroglycoprotein-coding region of the GP1b gene resulting in platelet-type von Willebrand Disease. Blood

17 Papers submitted for the Part 2 examination in Molecular Genetics
Louise Lavender: July 2008  PCR-based protocols for detection of clonality in suspect lymphoproliferations Diagnosis of patients with lymphoproliferative disorders is most commonly made using a combination of techniques, which include histological examination, immunohistochemistry and flow cytometry, to discriminate between malignant and reactive lymphoproliferations. In a number of cases this discrimination requires supplementary molecular diagnostic techniques which utilise the fact that cells of a malignancy have a common clonal origin. Most lymphoid malignancies originate from the B-cell lineage with only a minority being of T-cell origin. Generally all cells within a malignancy contain identical rearranged immunoglobulin (Ig) and/or T-cell receptor (TCR) genes which can then be used as markers for clonality.1 The Ig and TCR gene loci contain many different variable(V), diversity(D) and joining(J) gene segments which are rearranged during early differentiation2,3 to produce the combinatorial repertoire of Ig and TCR molecules which is estimated to be in excess of 1012 molecules.4 Southern blot analysis has been the gold standard technique for molecular clonality studies for a number of years. It is a highly reliable method but has several major disadvantages in that it is very slow and time consuming to perform, is technically demanding, requires large amounts of high quality DNA and has limited sensitivity (generally 5-10%)5 and so has been increasingly displaced by PCR techniques. These have the advantage of being quick and relatively easy to perform, requiring only small amounts of DNA (such as from fine-needle aspiration biopsies), being able to utilise low quality DNA (as extracted from formalin-fixed paraffin-embedded archive material) and having increased sensitivity (better than 1% in some cases). The introduction of PCR-based clonality studies has been hampered by two major disadvantages of the technique; false negative results occur due to mis-annealing of PCR primers and false positive results can arise due to difficulties in discrimination between monoclonal and polyclonal Ig/TCR gene rearrangement patterns. Work Package 1 Although many different PCR protocols have been developed in individual laboratories, no reliably standardised methods were available at the start of this project. Thus, the BIOMED-2 Concerted Action BMH4-CT entitled ‘PCR-based clonality studies for early diagnosis of lymphoproliferative disorders’ was initiated in 1998 to specifically address this issue. A total of 47 institutes from seven European countries were recruited to participate in this study. The activities of the group were organised into seven National Networks, each co-ordinated by a National Network leader, who arranged the collection of tissue and corresponding DNA samples for use in the study and organised the testing of the samples within the National Network.

18 The initial aim of the study was to design new PCR primers and standardised PCR protocols for detection of all theoretically possible rearrangements of the Ig genes (IGH, IGK and IGL) and TCR genes (TCRB, TCRG and TCRD) as well as the chromosomal translocations t(11;14) and t(14;18). The work for each target locus was organised by a Target Network leader. I joined the BIOMED program at its start in 1998 as part of the Southampton laboratory in the GB (South) National Network. I became acting Network Leader for the Head of Laboratory, Dr John Smith, responsible for organising the work of the laboratories of the GB South network (Southampton, Cardiff, London-UCL and London-RFH). Initially the work focussed on the design and testing of PCR primer sets for the detection of clonally rearranged Ig and TCR genes as well as the t(11;14) and t(14;18) translocations using a standard PCR protocol and a minimal number of reaction tubes. I was target network leader for the TCRD locus and therefore responsible for the design and testing of the TCRD primers (each primer pair was tested against a polyclonal DNA sample and, wherever available, a clonal DNA sample). The primers were then tested to ensure they worked in multiplex reactions (with all primers combined in a single reaction tube) and to determine the sensitivity of detection (using clonal DNA serialy diluted in polyclonal DNA to determine the threshold for detection of a clonal product). The primers then underwent a general testing phase which involved evaluating their use in a diagnostic setting with 90 Southern blot-defined samples which were known to have B-cell or T-cell malignancy or were known to be reactive lesions. In addition to the design of the TCRD primers, I personally carried out all the laboratory work involved in the testing of the primers and protocols. I also assisted the Target Network leader for the TCRB locus in the design and testing of the TCRB primers and was involved in the testing of the TCRG primers, carrying out all the laboratory work myself. The results of the general testing with the newly designed primers were compared with the results obtained by Southern blotting of the same samples. All the testing of the primers was carried out by two methods; heteroduplex analysis on polyacrylamide gels (which discriminates on the basis of fragment length and base composition) and GeneScanning (separation of fluorescently labelled PCR products in a high resolution polymer on the basis of fragment length only). In addition to the main BIOMED study, a small subgroup was set up to evaluate the use of the newly designed primers with DNA extracted from paraffin-embedded tissues. In many countries, fresh tissue material is not easily available for diagnostic purposes and paraffin-embedded tissues are the main resource available for molecular clonality studies. DNA from paraffin-embedded tissues presents particular problems with regard to the quality and quantity of DNA available and it was essential to establish whether the methodologies developed as part of the BIOMED programme would be applicable to both fresh and archival material. In 2003, the results of this first phase of the BIOMED program were published. A total of 11 papers were published in a single issue of Leukemia. As a member of the BIOMED group I participated in all aspects of the primer testing, but am listed as an author on the five papers where I made a major contribution to the work as outlined below.

19 Paper 1 describes the background to the programme, the organisation of the collaborative study, the principles of the primer design and initial testing and the general testing of the 90 Southern-blot defined samples. The results for each gene locus are detailed in the following papers. Paper 2 describes the development of the TCRB primers. Molecular analysis of the TCRB gene locus had previously proved extremely difficult, largely because of the complex nature of the locus and the number of different gene segments involved (about 65 Vβ segments, divided into approximately 30 subfamilies, 2 Dβ and 13 Jβ segments).6 Due to this large germline-encoded repertoire, the combinatorial diversity of TCRB is extensive and since TCRB gene rearrangements occur in almost all mature T-cell malignancies, as well as in a high proportion of T-ALL and a significant proportion of B-ALL,7 they are important in the assessment of clonality in a large number of suspect lymphoproliferations. Primer design for the TCRB locus required 23 Vβ primers (broadly corresponding to the Vβ families), two Dβ primers and 13 Jβ primers. These were initially tested in pairs (a total of 299 Vβ-Jβ reactions and 26 Dβ-Jβ reactions) using polyclonal samples for each pair and clonal samples where available. The primers were then tested in multiplex reactions to ensure sensitivity and specificity were maintained. It was found that a total of 3 PCR reaction tubes was required to identify all theoretically possible TCRB rearrangements. Each clonal sample was tested by serial dilution in polyclonal DNA to determine the sensitivity of detection. I worked in conjunction with the TCRB Target Network leader in Kiel, Germany, to carry out the initial testing of all the primers by heteroduplex analysis while the Kiel laboratory carried out the parallel testing by fluorescent GeneScanning. When initial testing was satisfactorily completed, the general testing of the Southern-blot defined samples was carried out by the Southampton and Kiel laboratories in conjunction with a number of other laboratories. Paper 3 describes the development of the TCRG primers. The TCRG locus is far less complex than TCRB (14 Vγ segments, although only 10 of these are known to undergo rearrangement, 5 Jγ segments and no D segments)7 and so has been used for PCR-based clonality analysis for a much longer time. It is also rearranged in a very high proportion of T-ALL and T-NHL in addition to a significant proportion of B-ALL. However, whilst the reduced recombinatorial repertoire facilitates PCR amplification, the limited junctional diversity makes the distinction between clonal and polyclonal populations more difficult. Design of TCRG primers required just 4 Vγ and 2 Jγ primers which were multiplexed into two PCR reaction tubes. I was involved in the general testing of the TCRG primers on the 90 Southern-blot defined samples using heteroduplex analysis on polyacrylamide gels.

20 Paper 4 describes the development of the TCRD primers
Paper 4 describes the development of the TCRD primers. As Target Network leader for TCRD, I was responsible for the initial design of the primers. The TCRD locus is relatively simple with just 8 Vδ segments, 3 Dδ segments and 4 Jδ segments, but the junctional diversity is increased by the possible inclusion of multiple Dδ segments. The TCRD locus is the first to rearrange in T-cell ontogeny and is often deleted in TCRαβ lineage cells with few T-cell lymphomas expressing TCRγδ. However, identification of TCRD rearrangements can be extremely useful in discriminating between the immature rearrangements found in B-ALL (Vδ2-Dδ3 or Dδ2-Dδ3)7 and the mature rearrangements found in T-ALL (Dδ2-Jδ1 and complete Vδ-Jδ).8 With this in mind, I designed the TCRD primers so that they could be multiplexed in any combination to allow discrimination of complete and partial rearrangements in separate reactions, but so that they would also work when all together in one PCR tube for simple clonality detection. A total of 12 primers were designed (6 Vδ, 2 Dδ and 4 Jδ) and each primer pair was tested with polyclonal DNA and a clonal control sample. The primers were then tested in multiplex reactions and each clonal sample was diluted in polyclonal DNA to determine the sensitivity of detection. This testing was also carried out in parallel by the laboratory in Heidelberg, Germany. General testing of the 90 Southern-blot defined samples was carried out by 10 laboratories and it was my responsibility to collate all the results and prepare the publication. To increase the information available from the single TCRD analysis, I also devised a method for differentiating the specific gene segments used by differential labelling of the TCRD primers. A 3-colour system of labelling allowed the differentiation of the major rearrangements of the TCRD locus. This is outlined in the final General Discussion section. Paper 5 describes the evaluation of the use of the BIOMED primers and protocols for DNA extracted from paraffin–embedded tissue. Since this is the major source of DNA for many diagnostic laboratories and the DNA extracted is very often of poor quality, it was vital to evaluate the suitability of the BIOMED protocols for this type of sample. The first task of the paraffin sub-group was to design a set of control gene primers which could be used to test the “amplifiability” of the DNA extracted from the paraffin-embedded samples. Five sets of primers were designed that would amplify products of exactly 100, 200, 300, 400 and 600bp. The target genes were selected to have large exons to reduce the risk of selecting polymorphic regions and were designed to multiplex together using the standardised BIOMED protocols. The initial design was carried out by the group in Paris, but it was my task to go on and optimise the amplification reaction. Because of the large variation in product size and need to generate products of approximately equal intensity, this optimisation required careful titration of the primer concentrations and MgCl2 concentration. Once optimised, the control gene primers were tested along with the other Ig/TCR primers on paraffin-embedded samples (a total of 45 of the 90 Southern-blot defined samples had corresponding paraffin-embedded samples available for testing). Overall it was concluded that the control gene primer set was a useful indicator of the amplifiability of the DNA extracted, with samples amplifying products of 300bp or more in the control gene set generally giving good results with the Ig/TCR primers.

21 Summary Work Package 1 BIOMED study:
Overall, the BIOMED study presented in the series of papers described above demonstrates a reliable method for PCR-based clonality studies with good concordance (>95%) between the results obtained by Southern blotting and the results obtained with the newly designed PCR primers and protocols. In addition, the PCR-based methods showed greater sensitivity, good discrimination of clonal and polyclonal results and a very low rate of false negative results. Work Package 2 Following on from this first series of publications, the members of the BIOMED group went on to a second phase of work which looked at a far wider range of samples to validate the newly developed protocols in a variety of disease entities. Almost 400 B-cell malignancies, 200 T-cell malignancies and more than 100 histologically reactive lesions were evaluated using all the Ig, TCR and t(11;14) and t(14;18) targets to evaluate each sample. Overall, clonality was detected in 99% of B-cell malignancies, and in 94% of T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The application of multiple complementary targets in parallel helped to achieve the high overall detection rate for clonality, with a very high percentage showing at least two clonal results. As a result of this study, a strategy was drawn up to direct clonality testing in suspect lymphoproliferations. The results of this second phase of the BIOMED program were published in A total of 5 papers were published, again in a single issue of Leukemia. As a member of the BIOMED group I participated in aspects of the entire publication, but am listed as an author on the two papers where I made a major contribution to the work, as outlined below. As Target Network Leader for TCRD, I co-ordinated the results for that locus, but also carried out a significant amount of the practical work required for the testing for several diseases, especially for the reactive group of samples. Paper 6 describes the background to this second phase of investigation. It details the clonality detection rates for the individual diseases using the Ig and TCR protocols. The Ig targets show a high degree of complementarity for clonality detection in the five disease categories tested with an overall detection rate of 99%. For the TCR targets, most diseases showed a detection rate of % with only anaplastic large-cell lymphoma (ALCL) showing a reduced detection rate (79%). However it is known that approximately 20-25% of ALCL have no TCR gene rearrangements (null ALCL)9 and if these are excluded from the calculations, the overall clonality detection rate is comparable to that of the B-cell malignancies at 99%. A strategy for efficient testing of samples was proposed so that clonality testing is carried out using the most informative targets in the first instance followed by a more complete panel of Ig and TCR targets in only a limited number of targets.

22 Paper 7 describes the evaluation of the PCR protocols in tissue samples with reactive lymphoproliferations. The samples used were selected on the basis of an initial clinical suspicion of malignant lymphoma which was then changed to reactive on the basis of histological and immunophenotypic evidence. Initial diagnosis was made by the local pathologist followed by review at national level by a panel of pathologists. A total of 106 samples were included in the study. Each DNA sample was analysed in duplicate (once by heteroduplex, once by GeneScannng) for each Ig/TCR locus by 2 independent laboratories and the results compared. In the case of discrepant results, a third laboratory carried out the analysis. Intra-laboratory variation was found to be low (~1%) and was usually resolved by the third laboratory. Overall the results showed that 79 of the 106 samples (75%) were polyclonal for all loci. Sixteen of 106 cases (15%) showed low level clonality, usually involving a minor population on a polyclonal background. However 11/106 (10%) showed clear monoclonal products in at least one locus. Eight of these showed clear clonality in multiple loci. These cases showing unexpected clonal results were reviewed by the international pathology review panel. Two were identified as clear lymphoma which had been missed on original examination. The remainder included some cases which required further characterisation, but which would have been overlooked without the molecular clonality results. Summary of BIOMED clonality studies: Overall the work presented in both series of papers represents a major advance in the use of PCR-based clonality testing in lymphoproliferative disorders. Since the publication of the first series of papers, the implementation of the BIOMED protocols for clonality testing has been widespread across Europe and the USA. The BIOMED group have entered into a collaborative agreement with InVivoScribe Technologies for the large-scale production and commercialisation of the primers and protocols, with the BIOMED Core laboratories being responsible for quality assessment. Proceeds from this commercialisation are being used to run a series of European workshops for laboratories involved in clonality testing and a Europe-wide Quality Control scheme has just been instigated. The second series of papers has produced substantial evidence of the reliability of these protocols in the diagnosis of a range of lymphoproliferative disorders and, perhaps more significantly, in the discrimination between reactive and malignant proliferations. The technology developed in this program has now been used in many different applications, most notably in improved techniques for the detection of minimal residual disease.10-12

23 Novel deletion in platelet-type von-Willebrand disease
Following on from my work as part of the BIOMED group, I was subsequently appointed as a Clinical Scientist in the Molecular Haematology section at Southampton General Hospital. As part of my work, I was responsible for the laboratory supervision of a PhD student investigating a family with apparent platelet-type von Willebrand disease. My role was to oversee her work to identify the causative mutation in the family. Paper 8 describes this study. Platelet-type von-Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder, first described in 1982, resulting from gain-of function mutations in the glycoprotein Ibα (GPIbα) gene. This leads to an abnormally high affinity interaction between the platelet membrane glycoprotein complex and von Willebrand factor (VWF) leading to characteristic platelet hyperaggregability.13 Previous reports of PT-VWD have all been due to missense mutations in the VWF binding region of the gene encoding GPIbα We studied five members of a British family with 3 affected and 2 unaffected members. The proband had been given an initial diagnosis of type IIB VWD following a long history of bleeding since the age of four. An antepartum haemorrhage late in pregnancy failed to respond to VWF/FVIII which caused her platelet count to drop dramatically, but increased when platelet concentrates were given leading to a suggested diagnosis of PT-VWD rather than type IIB. Sequencing of the proband’s GPIBA gene revealed a previously unreported in-frame deletion of 27bp coding for 9 amino acids of the macroglycopeptide region of GPIbα. The other affected family members also showed this deletion whereas the unaffected members did not. Functional characterisation of this mutant form of GPIbα showed enhanced binding to VWF compared to the wild type form confirming the causal nature of the mutation. Summary of PT-VWD: This is the first report of a mutation outside the VWF binding region causing PT-VWD. The macroglycopeptide region of GPIbα forms a rigid stalk that extends the VWF binding domain away from the platelet surface. It is unclear how deletion of 9 amino acids from this region could affect VWF binding, but it may act by restricting the mobility of this domain. Although considered extremely rare, PT-VWD may be under-diagnosed due to its similarity to type IIB VWD. Since the optimum treatment for the two types is quite different, molecular diagnosis may be used increasingly to reliably discriminate between the two types.

24 References: 1.      van Dongen JJM, Wolvers-Tettero ILM. Analysis of immunoglobulin and T-cell receptor genes. Part II: possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders. Clin Chim Acta 1991; 198: 2.      Tonegawa S. Somatic generation of antibody diversity. Nature 1983; 302: 3.      Davis MM, Björkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature 1988; 334: 4.      van Dongen JJM, Szczepanski T, Adriaansen HJ. Immunobiology of leukaemia. In: Hendersen ES, Lister TA, Greaves MF (eds) Leukemia. Philadelphia: WB Saunders Company, 2002, pp 5.      van Dongen JJM, Wolvers-Tettero ILM. Analysis of immunoglobulin and T-cell receptor genes. Part I: basic and technical aspects. Clin Chim Acta 1991; 198: 1-91. 6.      Arden B, Clark SP, Kabelitz D, Mak TW. Human T-cell receptor variable gene segment families. Immunogenetics 1995; 42: 7.      Szczepanski T, Beishuizen A, Pongers-Willemse MJ, Hählen K, van Wering ER, Wijkuijs JM et al. Cross-lineage T-cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B-acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease. Leukemia 1999; 13: 8.      Breit TM, Wolvers-Tettero ILM, Beishuizen A, Verhoeven M-AJ, van Wering ER, van Dongen JJM. Southern blot patterns, frequencies and junctional diversity of T-cell receptor δ gene rearrangements in acute lymphoblastic leukaemia. Blood 1993; 82: 9.      Foss HD, Anagnostopoulos I, Araujo I, Assaf C, Demel G, Kummer JA et al. Anaplastic large-cell lymphomas of T-cell and null-cell phenotype express cytotoxic molecules. Blood 1996; 88: 10.  Szczepański T. Why and how to quantify minimal residual disease in acute lymphoblastic leukemia? Leukemia 2007; 21: 11.  van der Velden VH, de Bie M, van Wering ER, van Dongen JJ. Immunoglobulin light chain gene rearrangements in precursor-B-acute lymphoblastic leukemia: characteristics and applicability for the detection of minimal residual disease. Haematologica 2006; 91: 12.  van der Velden VH, Brüggemann M, Hoogeveen PG, de Bie M, Hart PG, Raff T, Pfeifer H, Lüschen S, Szczepański T, van Wering ER, Kneba M, van Dongen JJ. TCRB gene rearrangements in childhood and adult precursor-B-ALL: frequency, applicability as MRD-PCR target, and stability between diagnosis and relapse. Leukemia 2004; 18: 13.  Weiss HJ, Meyer D, Rabinowitz R et al. Pseudo-von Willebrand’s disease: an intrinsic platelet defect with aggregation by unmodified human factor VIII/von Willebrand factor and enhanced adsorption of its high-molecular-weight multimers. N Engl J Med 1982; 306: 14.  Miller JL, Cunningham D, Lyle VA, Finch CN. Mutation in the gene encoding the alpha chain of platelet glycoprotein Ib in platelet-type von Willebrand disease. Proc Natl Acad Sci U S A 1991; 88: 15.  Takahashi H, Murata M, Moriki T et al. Substitution of Val for Met at residue 239 of platelet glycoprotein Ibα in Japanese patients with platelet-type von Willebrand disease. Blood 1995; 85: 16.  Russell SD, Roth GJ. Pseudo von-Willebrand disease: a mutation in the platelet glycoprotein Ibα gene associated with a hyperactive surface receptor. Blood 1993; 81: 17.  Kunishima S, Heaton DC, Naoe T et al. De novo mutation of the platelet glycoprotein Ib alpha gene in a patient with pseudo-von Willebrand disease. Blood Coagul Fibrinolysis 1997; 8: 18.  Matsubara Y, Murata M, Sugita K, Ikeda Y. Identification of a novel point mutation in platelet glycoprotein Ibα, Gly to Ser at residue 233 in a Japanese family with platelet-type von Willebrand disease. J Thromb Hemost 2003; 1:

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