Topic Update

Molecular alterations of gastrointestinal stromal tumor - Prognostic and therapeutic implications

Molecular alterations of gastrointestinal stromal tumor - Prognostic and therapeutic implications 

 

Volume 12, Issue 2 August 2017  (download full article in pdf)

 

Editorial note:

Gastrointestinal stromal tumor is the commonest mesenchymal tumor in the digestive system. It is a genetically heterogeneous disease with various mutations apart from classical activation mutations in KIT  and PDGFRA genes. In the topical update, Dr. Anthony Chan provided an overview of molecular alterations of gastrointestinal stromal tumor with emphasis on their prognostic and therapeutic significance. We welcome any feedback or suggestions. Please direct them to Dr. Anthony Chan (e-mail: awh_chan@cuhk.edu.hk) of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr. Anthony W.H. Chan
Clinical Associate Professor
Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong 

 

The gist of GIST

Gastrointestinal stromal tumor (GIST) is a rare tumor with the annual incidence rate of 10- 15/1,000,000, but it is the commonest mesenchymal tumor in the digestive system. It affects both sexes equally and presents at any age from children to elderly with the median age of mid 60s. Stomach (55.6%) is the most frequent primary tumor site followed by small intestine (31.8%), large intestine (6.0%) and esophagus (0.7%). Other uncommon primary sites, such as omentum, mesentery and liver, accounts for 5.5% of all GISTs.(1) Important milestones of GIST in diagnostic, prognostic and therapeutic aspects are briefly summarized in this section. 

In the past, GIST was regarded as leiomyoma, leiomyoblastoma or leiomyosarcoma before the era of wide availability of immunohistochemistry. In 1983, Mazur and Clark first applied the term "stromal tumor" to describe a group of gastric mesenchymal tumor lacking ultrastructural features of smooth muscle or schwann cells.(2) In 1989, a short-lived term, gastrointestinal autonomic nerve tumor (GANT), was used to describe a small subset of GIST featured by small intestinal location, epithelioid appearance and focal immunoreactivity towards neural markers (S100, neurofilament and synaptophysin).(3) In 1995, CD34 was found to be the first useful diagnostic immunohistochemical marker to differentiate GIST from leiomyoma and schwannoma although only 60-70% of all GISTs are immunoreactive to CD34.(4) In 1998, the hallmark constitutive activation mutation of KIT gene and overexpression of KIT/CD117 protein in GIST were discovered by Hirota et al.(5) This finding also suggested that GIST may be originated from interstitial cells of Cajal, pacemaker cells of intestine, which express KIT and CD34. However, activation mutation of KIT gene and overexpression of KIT are not consistently correlated. A subset of KIT positive GISTs was found to lack KIT mutation and this observation led to the subsequent discovery of gain-of-function mutation of platelet-derived growth factor receptor alpha (PDGFRA) gene in 2003.(6, 7) KIT and PDGFRA mutations are mutually exclusive. About 5-10% of GISTs, particularly those with PDGFRA mutation do not express KIT. In 2004, West et al. identified a novel gene, DOG1 (discovered on GIST-1), through cDNA microarray, and showed DOG1 protein was highly expressed in GISTs (97.8%), including those KIT negative GISTs.(8) KIT and/or DOG1 become crucial diagnostic immunohistochemical markers for GIST. A small subgroup of GISTs with immunoreactivity of KIT/DOG1 lack neither KIT or PDGFRA mutation was first designated as wild-type GISTs in the same year.(9) Wild-type GISTs are later shown to be a heterogeneous group with various mutations.(10- 13)

Prognosis of patients with GIST is shown to be correlated with tumor size and mitosis. The first consensus risk stratification was proposed by investigators in National Institutes of Health (NIH) in 2002 (Table 1).(14) Anatomical location of GIST is also an important prognostic factor and firstly integrated to the Armed Forces Institute of Pathology system in 2006 (Table 2)(15). Gastric GIST behaves more indolent than small and large bowel GIST with similar size and mitosis. Tumor rupture is an additional prognosticator for GIST patients and incorporated into the modified NIH system in 2008 (Table 3).(16) Finally, the most widely adopted tumor staging system, American Joint Committee on Cancer (AJCC), include GIST risk stratification composed of tumor size, mitosis, anatomical location, nodal and distant metastases in the 7th edition in 2010, which remains unchanged in the recently released 8th edition (Table 4 and 5).

Surgical resection remains the mainstay of curative therapy for GIST but a substantial portion of GIST patients present in advanced stage beyond surgical intervention. Imatinib, a multi-targeted tyrosine kinase inhibitor specific for c-abl, c-kit and PDGFR, was first used in a patient with metastatic GIST in 2001.(17) The dramatic clinical response from this patient and the subsequent successful phase II clinical trial in 2002 secured the first-line role of imatinib for patients with inoperable GIST and pioneered molecular targeted therapy for sarcoma.(18) Primary and acquired resistance to imatinib among GIST patients led to development of newer targeted agents. Two hallmark phase III randomized controlled trials on sunitinib (NCT00075218) and regorafenib (NCT01271712) for GIST were completed in 2006 and 2013, respectively.(19, 20) Sunitinib and regorafenib are indicated for patients with advanced GIST resistant or intolerant to imatinib. 

 

Table 1: NIH risk stratification for GIST (14) 

GROUP

SIZE (CM)

MITOSIS (/50 HPF)

Very low risk <2 ≤5
Low risk 2-5 ≤5
Intermediate risk <5 6-10
5-10 ≤5
High risk >5 >5
>10 Any
Any >10

 

Table 2: AFIP risk stratification for GIST (15) 

GROUP SIZE (CM) MITOSIS (/50 HPF) STOMACH DUODENUM JEJUNUM /ILEUM RECTUM
1 ≤2 ≤5 None None None None
2 >2-5 ≤5 Very low Low Low Low
3a >5-10 ≤5 Low Moderate - -
3b >10 ≤5 Moderate High High High
4 ≤2 >5 None High - High
5 >2-5 >5 Moderate High High High
6a >5-10 >5 High High - -
6b >10 >5 High High High High

 

Table 3: Modified NIH risk stratification for GIST (16) 

GROUP SIZE (CM) MITOSIS (/50 HPF) PRIMARY SITE
Very low risk ≤2 ≤5 Any
Low risk >2-5 ≤5 Any
Intermediate Risk >2-5 >5 Gastric
≤5 6-10 Any
>5-10 ≤5 Gastric
High risk >5 >5 Any
>10 Any Any
Any >10 Any
Any Any Tumor rupture
>2-5
>5
Non-gastric
>5-10 ≤5 Non-gastric

 

Table 4: AJCC staging system for gastric and omental GIST 

GROUP SIZE (CM) N M MITOSIS
IA ≤5 0 0 ≤5
IB >5-10 0 0 ≤5
II ≤5 0 0 >5
>10 0 0 ≤5
IIIA >5-10 0 0 >5
IIIB >10 0 0 >5
IV Any 1 0 Any
Any Any 1 Any

 

Table 5: AJCC staging system for small/large bowel, esophageal, mesenteric and peritoneal GIST 

GROUP SIZE (CM) N M MITOSIS
I ≤5 0 0 ≤5
II >5-10 0 0 ≤5
IIIA ≤2 0 0 >5
>10 0 0 ≤5
IIIB >2 0 0 >5
IV Any 1 0 Any
Any Any 1 Any

 

 

Mutational landscape of GIST

KIT and PDGFRA mutations are major driver mutations in GIST tumorigenesis. Both genes encode type III receptor tyrosine kinases with similar structures: extracellular ligand binding domain and dimerization domain, a transmembrane sequence, a juxtamembrane domain and intracellular kinase domain (Figure 1). Binding of corresponding ligands, stem cell factor and PDGFA, to c-kit and PDGFRA receptor, respectively, dimerizes and activates receptor tyrosine kinases. In GIST, activation mutations in KIT and PDGFRA lead to uncontrolled ligand- independent receptor activation. Mutation hotspots of KIT gene are located at exons 9, 11, 13 and 17, whereas those of PDGFRA gene are situated at exons 12, 14 and 18. Mutation of extracellular domain of KIT encoded by exon 9 facilitate receptor dimerization. Mutations in the juxtamembrane domain, which is encoded by exon 11 of KIT and exon 12 of PDGFRA, allow dimerization of receptor without binding of ligands. Mutations of ATP binding region of kinase domain (encoded by exon 13 of KIT and exon 14 of PDGFRA) enhance kinase activity, while mutations of activation loop (encoded by exon 17 of KIT and exon 18 of PDGFRA) promote active conformation of kinase.(21) Table 6 and Figure 2 summarize the mutational landscape of GIST based on the data from population-based studies and clinical trials.(22-29) Frequencies of PDGFRA mutations are significantly lower among patients in clinical trials (mean 1.7%) than those in population-based studies (mean 14.9%) because GIST patients with PDGFRA mutations are associated with better prognosis and earlier stage and hence do not require systemic therapy.(9, 22, 23, 29)

KIT mutation accounts for 71.5% (64.8-89.1%) of mutations in GISTs.(24, 25, 27-29) Exon 11 mutation is the commonest mutation (61.1%, range: 56.1-77.1%). Deletion, substitution and duplication contribute to 23-28%, 2-20% and 2-7%, respectively. Deletion in exon 11 is associated with younger age, larger tumor size, higher mitotic count and poor prognosis, whereas duplication is associated with female and stomach predilection and better prognosis. Exon 9 mutation is found in 7.1-10.9% of GISTs, particularly in those arising from small and large intestine, and associated with poor prognosis. Exon 13 and exon 17 are rare mutation hotspots (<1-2%) in GISTs, which are almost exclusively spindle in morphology and more frequently developed in small intestine. GISTs with exon 13 and 17 mutants are associated good and intermediate prognosis, respectively.

PDGFRA mutation accounts for 14.9% (4.7-21.1%) of mutations in GISTs.(24, 25, 27-29) About 30- 40% of GISTs without immunoreactivity of KIT/CD117 harbour PDGFRA mutation. GISTs with PDGFRA mutation generally show predilection to gastric location (>90%) and epithelioid/mixed morphology, and favourable prognosis (except non-D842V exon 18 mutation).

Wild-type GIST, which express immunoreactivity of KIT/DOG1 but lack neither KIT or PDGFRA mutation, contributes to 13-18% of adult GISTs and 85% of pediatric GIST.(10-12) As previously mentioned, it is a genetically heterogeneous group (Figure 3). Wild-type GIST can be further stratified by using succinate dehydrogenase B (SDHB) immunohistochemistry and familial syndromes. On one hand, SDHB deficient wild-type GISTs accounts for about 5% of all GISTs, and can be sporadic or related to Carney triad and Carney- Stratakis syndrome. Carney triad is a constellation of GIST, paraganglioma and pulmonary chondroma with undetermined germline mutation, whereas Carney-Stratakis syndrome is an autosomal dominant disease with dyad of GIST and paraganglioma, and germline mutations in SDHB, SDHC or SDHD genes.(30) SDHB deficient wild-type GISTs are featured by female predominance (except for Carney-Stratakis syndrome), exclusive location in stomach, multifocality, epithelioid/mixed morphology, unpredictable clinical outcome by histology, indolent clinical course despite frequent nodal metastasis, and mutation id SDH subunits (except for Carney triad). On the other hand, SDHB proficient wild-type GISTs make up 10.5% of all GISTs, and are either sporadic (9%) or syndromic (1.5%). Syndromic SDHB proficient wild-type GISTs are associated with neurofibromatosis type 1, absence of sex/age predilection, small intestine in location, multifocality, spindle morphology, and favorable prognosis. Sporadic SDHB proficient wild-type GISTs can be further classified according to BRAF mutation. Sporadic SDHB proficient wild-type GISTs with BRAF mutation usually occur in 6th decade of age and small intestine with spindle morphology. Prognosis of this subgroup is inconclusive.(10, 29, 31, 32) Sporadic SDHB proficient wild-type GISTs without BRAF mutation are also known as quadruple wild-type GISTs without any mutation in KIT, PDGFRA, SDH and genes in RAS pathway (BRAF/NF1).(12, 13) They represent the commonest subgroup (7%) of wild-type GISTs and a genetically heterogeneous subgroup harboring ETV6-NTRK3 translocation, FGFR1-TACC1 translocation, mutation of MEN1 and MAX, and overexpression of COL22A1 and CALCRL.(12, 29, 33, 34) Due to complex genetic heterogeneity, clinicopathological features of this subgroup have not been well characterized.

 

Figure 1: Schematic diagram of the structures of KIT and PDGFRA receptor tyrosine kinases 

Fig 1

 

Figure 2: Mutational landscape of GIST 

Fig 2

 

Figure 3: Classification of wild-type GIST 

Fig 3

 

Table 6: Mutational landscape of GIST

Study Region n KIT exon PDGFRA exon Wild type
9 11 13 17 12 14h 18
Wozniak 2012 (24) Poland 427 7.3% 61.1% 0.5% 0.5% 0.2% 0.7% 11.9% 17.8%
Wozniak 2014 (28) Europe 1056 7.4% 61.4% 1.8% 0.6% 0.9% 0.3% 12.8% 14.9%
Künstlinger 2013 (25) Germany 1366 9.2% 59.3% 1.8% 0.8% 1.8% 0.6% 13.8% 12.7%
Wang 2014 (27) China 275 10.9% 77.1% 1.1% 0.0% 1.1% 0.0% 3.6% 6.2%
Rossi 2015 (29) Italy 451 7.1% 56.1% 0.9% 0.7% 2.2% 1.6% 17.3% 14.2%
 
ACOSOG Z9001 (26) 507 6.9% 67.3% 1.8% 0.2% NA NA NA 12.8%
CALGB 150105 (23) 378 8.2% 72.8% 0.8% 1.1% 0.0% 0.0% 1.6% 15.3%
EORTC 62005 (22) 377 15.4% 65.8% 1.6% 0.8% 0.8% 0.0% 1.9% 13.8%

 

 

Clinical implications of mutations in GIST

Different mutations in GIST have their own characteristic prognostic and therapeutic implications. Prognostic significance of individual mutations have been described by various investigators and briefly mentioned in the previous section. Rossi et al. recently systemically analyzed the prognostic impact of mutations among 451 patients with primary localized treatment-naive GISTs.(29) By multivariable Cox regression, mutational status was an independent prognosticator in addition to patient's age, tumor location, tumor size and mitotic count. Three molecular risk groups with prognostic significance were identified: Group 1 with the most favorable outcome is composed of mutations in KIT exon 13, PDGFRA exon 12 and BRAF; Group 2 with the intermediate outcome (hazard ratio 3.06) consists of KIT/PDGFRA/BRAF triple negative, and mutations in KIT exon 17, PDGFA exon 14 and 18 (D842V); and Group 3 with the most unfavorable outcome comprises mutations in KIT exon 9 and 11, and PDGFRA exon 18 (non-D842V).

Clinical response toward imatinib among GIST patients is closely related to tumor genotype. In a phase III clinical trial (SWOG S0033/CALGB 150105), the investigators demonstrated that patients with KIT exon 11 mutation (complete response [CR]/partial response [PR] 71.7%) had better response to imatinib than those with KIT exon 9 mutation (CR/PR 44.4%) and wild-type KIT (CR/PR 44.6%).(23) They also showed that doubling the dose of imatinib (from 400 mg to 800 mg) improved response rates for patients with exon 9-mutant tumors (CR/PR 17% vs. 67%). Double dose of imatinib did not offer any better response rate among patients with exon 11 mutant or wild- type KIT. A subsequent meta-analysis of 1,640 patients with advanced GIST receiving imatinib confirmed that double dose of imatinib improved progression-free survival and objective response rate, but not overall survival, among patients with KIT exon 9-mutant GIST.(35) PDGFA exon 18 (D842V) mutation and KIT/PDGFRA wild-type are responsible for primary resistance to imatinib.(36) Among patients with advanced GIST receiving imatinib, a substantial proportion of initial responders will develop acquired resistance. Secondary mutations in exon 11 (L576P and V559A), exon 13 (V654A), exon 14 (T670I), exon 17 and exon 18 (A829P) of KIT, and exon 18 of PDGFRA are related to acquired resistance to imatinib.(36)

Clinical response to sunitinib, the second line targeted therapy after imatinib failure, is also considerably affected by primary and acquired mutations of KIT. Patients with primary KIT exon 9 mutation or wild-type KIT had better overall and progression-free survival than those with KIT exon 11 mutation, whereas patients with acquired KIT exons 13 or 14 mutations had better outcome than those with KIT exon 17 or 18 mutations.(37) Similarly, clinical response to regorafenib, the third line therapy after imatinib and sunitinib failure, is significantly influenced by tumor genotype. Regorafenib provided better clinical outcome among patients with primary KIT exon 11 mutation and SDHB deficient GIST, (38) as well as those with secondary mutation of KIT exon 17, which are resistant to both imatinib and sunitinib.(39)

 

Summary

GIST is a genetically heterogeneous tumor. Genotypes and phenotypes are closely interrelated. Specific mutations have their characteristic clinicopathological features, prognostication and therapeutic implications. Genetic analyses KIT and PDGFRA are highly recommended especially among patients with advanced diseases undergoing targeted therapy. Wild-type GISTs are recommended to be further analysed by SDHB immunohistochemistry and BRAF mutation test. 

 

Reference

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8. West RB, Corless CL, Chen X, Rubin BP, Subramanian S, Montgomery K, et al. The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status. Am J Pathol. 2004;165(1):107-13.

9. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol. 2004;22(18):3813-25.

10. Agaram NP, Wong GC, Guo T, Maki RG, Singer S, Dematteo RP, et al. Novel V600E BRAF mutations in imatinib-naive and imatinib-resistant gastrointestinal stromal tumors. Genes Chromosomes Cancer. 2008;47(10):853-9.

11. Janeway KA, Kim SY, Lodish M, Nose V, Rustin P, Gaal J, et al. Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations. Proc Natl Acad Sci U S A. 2011;108(1):314-8.

12. Nannini M, Astolfi A, Urbini M, Indio V, Santini D, Heinrich MC, et al. Integrated genomic study of quadruple-WT GIST (KIT/PDGFRA/SDH/RAS pathway wild-type GIST). BMC Cancer. 2014;14:685.

13. Pantaleo MA, Urbini M, Indio V, Ravegnini G, Nannini M, De Luca M, et al. Genome-Wide Analysis Identifies MEN1 and MAX Mutations and a Neuroendocrine-Like Molecular Heterogeneity in Quadruple WT GIST. Mol Cancer Res. 2017;15(5):553-62.

14. Fletcher CD, Berman JJ, Corless C, Gorstein F, Lasota J, Longley BJ, et al. Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol. 2002;33(5):459-65.

15. Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Semin Diagn Pathol. 2006;23(2):70-83.

16. Joensuu H. Risk stratification of patients diagnosed with gastrointestinal stromal tumor. Hum Pathol. 2008;39(10):1411-9.

17. Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D, et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med. 2001;344(14):1052-6.

18. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347(7):472-80.

19. Demetri GD, van Oosterom AT, Garrett CR, Blackstein ME, Shah MH, Verweij J, et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet. 2006;368(9544):1329-38. 

20. Demetri GD, Reichardt P, Kang YK, Blay JY, Rutkowski P, Gelderblom H, et al. Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2013;381(9863):295-302.

21. Corless CL, Barnett CM, Heinrich MC. Gastrointestinal stromal tumours: origin and molecular oncology. Nat Rev Cancer. 2011;11(12):865-78.

22. Debiec-Rychter M, Sciot R, Le Cesne A, Schlemmer M, Hohenberger P, van Oosterom AT, et al. KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumours. Eur J Cancer. 2006;42(8):1093- 103.

23. Heinrich MC, Owzar K, Corless CL, Hollis D, Borden EC, Fletcher CD, et al. Correlation of kinase genotype and clinical outcome in the North American Intergroup Phase III Trial of imatinib mesylate for treatment of advanced gastrointestinal stromal tumor: CALGB 150105 Study by Cancer and Leukemia Group B and Southwest Oncology Group. J Clin Oncol. 2008;26(33):5360-7.

24. Wozniak A, Rutkowski P, Piskorz A, Ciwoniuk M, Osuch C, Bylina E, et al. Prognostic value of KIT/PDGFRA mutations in gastrointestinal stromal tumours (GIST): Polish Clinical GIST Registry experience. Ann Oncol. 2012;23(2):353-60.

25. Kunstlinger H, Huss S, Merkelbach-Bruse S, Binot E, Kleine MA, Loeser H, et al. Gastrointestinal stromal tumors with KIT exon 9 mutations: Update on genotype-phenotype correlation and validation of a high-resolution melting assay for mutational testing. Am J Surg Pathol. 2013;37(11):1648-59.

26. Corless CL, Ballman KV, Antonescu CR, Kolesnikova V, Maki RG, Pisters PW, et al. Pathologic and molecular features correlate with long-term outcome after adjuvant therapy of resected primary GI stromal tumor: the ACOSOG Z9001 trial. J Clin Oncol. 2014;32(15):1563-70.

27. Wang M, Xu J, Zhao W, Tu L, Qiu W, Wang C, et al. Prognostic value of mutational characteristics in gastrointestinal stromal tumors: a single-center experience in 275 cases. Med Oncol. 2014;31(1):819.

28. Wozniak A, Rutkowski P, Schoffski P, Ray-Coquard I, Hostein I, Schildhaus HU, et al. Tumor genotype is an independent prognostic factor in primary gastrointestinal stromal tumors of gastric origin: a european multicenter analysis based on ConticaGIST. Clin Cancer Res. 2014;20(23):6105-16.

29. Rossi S, Gasparotto D, Miceli R, Toffolatti L, Gallina G, Scaramel E, et al. KIT, PDGFRA, and BRAF mutational spectrum impacts on the natural history of imatinib-naive localized GIST: a population-based study. Am J Surg Pathol. 2015;39(7):922-30.

30. Stratakis CA, Carney JA. The triad of paragangliomas, gastric stromal tumours and pulmonary chondromas (Carney triad), and the dyad of paragangliomas and gastric stromal sarcomas (Carney-Stratakis syndrome): molecular genetics and clinical implications. J Intern Med. 2009;266(1):43-52.

31. Hostein I, Faur N, Primois C, Boury F, Denard J, Emile JF, et al. BRAF mutation status in gastrointestinal stromal tumors. Am J Clin Pathol. 2010;133(1):141-8.

32. Huss S, Pasternack H, Ihle MA, Merkelbach-Bruse S, Heitkotter B, Hartmann W, et al. Clinicopathological and molecular features of a large cohort of gastrointestinal stromal tumors (GISTs) and review of the literature: BRAF mutations in KIT/PDGFRA wild-type GISTs are rare events. Hum Pathol. 2017;62:206-14.

33. Brenca M, Rossi S, Polano M, Gasparotto D, Zanatta L, Racanelli D, et al. Transcriptome sequencing identifies ETV6-NTRK3 as a gene fusion involved in GIST. J Pathol. 2016;238(4):543-9.

34. Shi E, Chmielecki J, Tang CM, Wang K, Heinrich MC, Kang G, et al. FGFR1 and NTRK3 actionable alterations in "Wild-Type" gastrointestinal stromal tumors. J Transl Med. 2016;14(1):339.

35. Gastrointestinal Stromal Tumor Meta- Analysis G. Comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors: a meta-analysis of 1,640 patients. J Clin Oncol. 2010;28(7):1247-53.

36. Sankhala KK. Clinical development landscape in GIST: from novel agents that target accessory pathways to revisiting non-targeted therapies. Expert Opin Investig Drugs. 2017;26(4):427-43.

37. Heinrich MC, Maki RG, Corless CL, Antonescu CR, Harlow A, Griffith D, et al. Primary and secondary kinase genotypes correlate with the biological and clinical activity of sunitinib in imatinib-resistant gastrointestinal stromal tumor. J Clin Oncol. 2008;26(33):5352-9.

38. Ben-Ami E, Barysauskas CM, von Mehren M, Heinrich MC, Corless CL, Butrynski JE, et al. Long-term follow-up results of the multicenter phase II trial of regorafenib in patients with metastatic and/or unresectable GI stromal tumor after failure of standard tyrosine kinase inhibitor therapy. Ann Oncol. 2016;27(9):1794-9.

39. Yeh CN, Chen MH, Chen YY, Yang CY, Yen CC, Tzen CY, et al. A phase II trial of regorafenib in patients with metastatic and/or a unresectable gastrointestinal stromal tumor harboring secondary mutations of exon 17. Oncotarget. 2017;8(27):44121-30. 

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Laboratory testing for Direct Oral Anticoagulants (DOACs): Are we ready?

Laboratory testing for Direct Oral Anticoagulants (DOACs):

Are we ready?

 

Volume 12, Issue 1 January 2017  (download full article in pdf)

 

Editorial note:

In this topical update, Dr Rock Leung reviews the testing strategy and quality assurance issues on laboratory testing for direct oral anticoagulant (DOACs). We welcome any feedback or suggestions. Please direct them to Dr Rock Leung (e-mail: leungyyr.ha.org.hk) of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

 

Dr. Rock LEUNG
Associate Consultant, Division of Haematology, Department of Pathology and Clinical Biochemistry
Queen Mary Hospital, Hong Kong

 

Abbreviations

DOACs Direct oral anticoagulants
FIIa Thrombin
PK Pharmacokinetics
PD Pharmacodynamics
PT Prothromhin time
APTT Activated partial thromboplastin time
TT Thrombin time
dTT Diluted thrombin time
ECT Ecarin clotting time
DRVVT Diluted Russell’s viper venom time

 

Introduction

The newly available Food and Drug Administration (FDA) -approved oral anticoagulants, namely dabigatran extexilate, rivaroxaban, apixaban and edoxaban, have been more commonly used nowadays for treatment and prophylaxis of venous thromboembolism, as well as for prevention of stroke in non-valvular atrial fibrillation. This new class of anticoagulants has been referred as novel oral anticoagulants (NOACs), target-specific oral anticoagulants (TOACs), or direct oral anticoagulants (DOACs). For the sake of standardization, the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SCC) for the control of anticoagulation recommends the term DOACs. DOACs have been shown to be at least as effective as warfarin in various clinical trials. Moreover, there was reduced incidence of intracranial haemorrhage reported in some studies when compared with warfarin [1]. Unlike warfarin, DOACs do not need routine therapeutic monitoring given their predictable pharmacokinetics (PK), pharmacodynamics (PD) and wide therapeutic windows. There are, however, clinical conditions that measurement of anticoagulation activity of DOACs is necessary or potentially useful, e.g. before invasive procedures, during adverse events like break-through bleeding or thrombosis, and pre- and post-administration of reversal therapy for patients with DOACs overdose. Thus, there is a role for laboratory, by testing for DOACs, to help clinicians on patient management. In addition, it is the responsibility of the laboratory to acknowledge the interferences of DOACs on conventional and special coagulation tests as part of the laboratory quality assurance in the era of gaining popularity of DOACs usage.

 

Mechanisms of actions of DOACs

In contrast with heparin that can only inhibit free protease, DOACs are rapidly-acting, target-specific anticoagulants that inhibit both the free and bound activated serine protease [2]. The fact that DOACs can inactivate bound serine protease explains their more robust action than warfarin or heparin. Dabigatran is a direct thrombin (IIa) inhibitor while rivaroxaban, apixaban and edoxaban are direct inhibitors of activated factor X (Xa). Most of the DOACs are cleared by liver and kidney, with the exception of dabigatran being almost exclusively excreted by kidney. DOACs reach peak plasma levels within approximately two hours and plasma trough levels within 12 hours or 24 hours depending on their frequency of administration [3]. The DOACs can be withhold a few days before elective surgery or invasive procedures due to their short half-lives and favourable pharmacokinetics.

 

To test or not to test?

Routine monitoring of DOACs is not required. Testing on patients on DOACs is generally indicated in certain clinical circumstances, including acute bleeding, suspected DOACs overdose, drug interaction, in patients with impaired renal function, before surgery or invasive procedure in patients who have taken the drug beyond 24 hours and with creatinine clearance of <50 mL/min or with extreme body weight [4]. Recently, more pharmacokinetics and pharmacodynamics data on indications of clinical testing came up. Currently it is recommended that checking of drug-specific peak and trough levels for DOACs should be performed for patients with body mass index (BMI) of >40 kg m^2 or weighted over 120 kg [5]. There is currently no consensus on when to test for DOACs activities when these drugs are to be used in women with childbearing potential. One should however note that animals studies have shown teratogenic effect of dabigatran, edoxaban and rivaroxaban, these drugs were assigned by the FDA as pregnancy category C, reflecting their potential teratogenicity. Whereas no teratogenicity has been demonstrated in animals for apixaban as of today, it was categorized as pregnancy catergory B by FDA [6]. On the other hand, the use of DOACs is considered an off-label clinical application for paediatric thromboemobolic diseases [7]. It is not unreasonable to obtain information about anticoagulation activity by laboratory assay for this special group of patients, as in the case of low-molecular-weight heparin (LMWH) usage in select paediatric patients.


Given the predictable pharmacokinetics of DOACs, it was proposed that a pharmacokinetic strategy by stopping the drug for a time frame adequate for washout of drug effect is safe before surgery or invasive procedures. This approach can only be applied for planned surgery or invasive procedures, with available information regarding patient’s renal function as well as the dose and timing of the last DOAC administration. For emergent or unplanned procedures in patients with renal insufficiency or unplanned procedures when the timing of the last DOAC administration is uncertain, measurement of residual drug level will be valuable to assist clinical decisions, including the assessment of bleeding risk and the need for antidote for prompt reversal of DOAC effect before surgery. In life-threatening bleeding associated with the use of DOACs, the measurement of drug level can supplement clinical information to determine whether the bleeding is contributed by the anticoagulation effect of DOACs and whether the administration of DOAC-specific antidotes is required. If antidote is applied, laboratory test can monitor the extent of reversal.

 

What tests to do?

The ideal test for DOACs shall be accurate, readily available on a 24-hour basis in order to accommodate emergency clinical situations, and with a reasonably short turnaround time (TAT).
Gold standard method using ultra-performance liquid chromatography – tandem mass spectrometry (UPLI-MS/MS) provides the most accurate information about the drug levels for patients on DOACs. However, the test is not readily available in most of the laboratories.


Routine coagulation screening tests, i.e., prothrombin time (PT), activated partial thromboplastin time (APTT) or thrombin time (TT), have been suggested as screening tests for DOACs. For routine coagulation screening tests to be useful and suitable for testing for DOACs, linearity and adequacy of test response to increasing dosage and amenability to standardization are prerequisites [8]. For dabigatran, TT is readily available in most laboratories and prolongation of clotting time is linearly and dose-dependently related to dabigatran concentrations. However, responsiveness is excessive. Therefore, a normal TT should rule out a dabigatran anticoagulant effect but the degree of prolongation poorly reflects drug concentration. Dilute TT (dTT), i.e., testing of TT on diluted plasma, is adequately responsive to dabigatran and suitable for assessment of dabigatran activity. Ecarin clotting time (ECT), using ecarin for the conversion of FII to meizothrombin, to assess anticoagulant effect of dabigatran was also shown to have satisfactory linearity and responsiveness to increasing dabigatran concentrations. APTT, though being demonstrated to have satisfactory responsiveness to dabigatran, lacks linearity upon increasing drug concentration and there is significant inter-reagent variability [9]. PT is insensitive to dabigatran and not suitable for testing.


Rivaroxaban prolongs the PT in a concentration-dependent manner, but the correlation is generally weak and became weaker with increasing drug concentration. Significant reagent-dependent differences in assay sensitivity are noted in multiple studies, limiting its use for assessment of rivaroxaban activity if the in-house thromboplastin reagent for routine coagulation screening is insensitive to rivaroxaban [10]. APTT is insensitive to rivaroxaban and shall not be used for assessment of rivaroxaban activity. For apixaban, both PT and APTT are insensitive to increasing drug concentrations and for edoxaban, PT performance is similar to that observed for rivaroxaban and APTT is insensitive [11].
Therefore, routine coagulation screening tests PT, APTT and TT cannot provide a reliable measurement of DOAC anticoagulant effect in most circumstances. One exception being a normal TT excludes significant residual effect of dabigatran in patients. Moreover, PT and APTT are either insensitive or show variably sensitivity to the on-therapy range of DOACs and limit their use in determining whether the drug concentration is in subtherapeutic or supratherapeutic ranges. Furthermore, these coagulation screening tests are potentially affected by the presence of lupus anticoagulants and conditions resulting in factor deficiency as in liver disease or dilutional coagulopathy. Thus, the sensitivity & specificity in reflecting the anticoagulant effect of DOACs is limited.


Anti-Xa assay is a chromogenic assay based on the measurement of residual FXa with synthetic substrates upon mixing of plasma with FXa. Although one study showed the feasibility of using of anti-Xa assay for LMWH to assess the presence of rivaroxaban [12], it is recommended to use drug-specific calibrator rather than adopting the anti-Xa assay for measurement of heparin activity due to the following reasons: 1) assays to measure indirect Xa inhibitors, e.g., LMWH, are measured in IU/ml and direct Xa DOACs are measured in ng/mL and there is no direct relationship between these two units of measure, 2) there is significant variability in measured drug concentration, as demonstrated by rivaroxaban, between various anti-Xa kits and 3) the therapeutic range, at least for apixaban and rivaroxaban, far exceeds the typical calibration range for UFH and LMWH (in the 5-9 IU/ml range) and 4) the assay is not specific for anti-Xa DOACs and will detect all anti-Xa anticoagulants2.


Commercially available drug-specific coagulation assays for testing of DOACs use calibrators and controls specific for the DOAC being measured [13-15], enabling the reporting of a drug concentration upon testing of patient’s plasma sample. Multiple calibrators and test plasma dilutions are employed to ensure the test sample responses are within the range of the calibration curve and also to allow for assessment of linearity and parallelism [16]. It was recommended that anti-Xa assay and diluted TT shall be employed when carrying out the drug-specific coagulation assay for anti-Xa inhibitor and anti-IIa inhibitor respectively, given their linear relationship and good correlation with drug concentration as measured by mass spectrometry [11]. Although an ecarin chromogenic assay (ECA) for direct IIa inhibitor and a DRVVT-based assay for both direct IIa and direct Xa inhibitors have been calibrated for testing of DOACs, ECA was shown to have suboptimal accuracy when compared UPLC-MS/MS and DRVVT-based assay would give false positive result in the presence of lupus anticoagulant [17,18]. Studies have shown that various drug-specific coagulation assays differ significantly in quantitation of the DOAC being measured when compared with UPLC-MS/MS in terms of precision and accuracy [17].

 

What is the meaning of drug concentration?

Drug-specific assay is by no means a direct measurement of drug concentration for DOACs. Instead it is an extrapolation of drug concentration by its anticoagulation activity measured by clot-based or chromogenic assay.
Therapeutic ranges of DOACs have not been validated by the manufacturing pharmaceutical companies. Moreover, there is no established range of concentrations associated with bleeding. In clinical use, expected trough and peak concentrations as predicated on prescribed dose and frequency are often taken as a reference during result interpretation of drug levels [17] (Table 1).

 

There is no consensus on whether trough level is superior to peak level when interpreting the findings during monitoring of DOACs. The sample for DOAC level is often collected at a random time during emergency clinical situations. A meaningful interpretation of drug level requires the knowledge of the time of last dose of DOAC, the drug dosage and patient’s renal and liver functions so that the trend of drug concentration over time can be better predicted.
With increasing use of DOAC assay, it is expected that DOAC plasma concentration shall be a standard study parameter in future clinical trials. This will allow the identification of drug concentration threshold associated with bleeding, the establishment of a therapeutic range for different kinds of DOACs and better definition of DOAC-induced bleeding complications.

 

Antidote for reversal of DOACs

Non-specific reversal agents like prothrombin complex concentrates, “bypassing agent” like factor eight inhibitor bypass activity (FIBA) and activated FVIIa were used for the correction of DOAC effect. They only had a general antagonizing action on the anticoagulation effect of DOAC without targeting the specific DOACs themselves. Three antidotes for the DOACs are now under various stages of development. Idarucizumab (Praxbind®), the antidote for dabigatran, is now licensed in the United States and recommended for licensing by the European Medicines Agency. Andexanet alfa, the antidote for the oral anti-Xa inhibitors, is undergoing phase III study. Ciraparantag (PER977), an agent reported to reverse the anticoagulant effects of all of the DOACs is at an earlier stage of development [19]. In life-threatening bleeding, administration of antidote or reversal agent before emergency operations shall not be delayed until the availability of test results. Otherwise, the decision on whether antidote is indicated can be guided by suitable laboratory assay as mentioned in the previous section. Drug-specific assay is considered the most suitable candidate given its superior sensitivity and probably better specificity than conventional coagulation assay and better accessibility and faster turnaround compared with mass spectrometry. Measurement of drug activity shall guide the antidote treatment and allow more effective use of this costly medicine. The importance is highlighted by one study on idaruxizumab for dabigatran reversal in which dTT was normal on study entry in nearly one quarter of the study population, indicating little or no circulating anticoagulant in this group of patients, whom benefit from the administration of idaruxizuman was minimal [20]. Although DOAC concentrations warranting the administration of antidote were recommended (e.g., a drug concentration over 50 ng/mL in serious bleeding and 30 ng/mL in patients requiring urgent intervention) [19], these actionable limits have not been validated in clinical studies.

 

Quality assurance issues on DOACs testing

Laboratories should develop customized algorithms on DOACs testing strategy for DOACs based on their need. The relative sensitivity of routine coagulation screening test, especially APTT and TT for dabigatran and PT for rivaroxaban, apixaban and edoxaban shall be validated by calibrated materials. Most published algorithms [21, 22] assume patient’s coagulation status is solely under the effect of DOACs and may not be applicable for patients with massive transfusion, disseminated intravascular coagulopathy (DIC) or presence of lupus anticoagulant that may have contributed to the abnormal coagulation screening results. Moreover, it is not practical to change the service PT and APTT reagents solely for DOACs detection.
The set up of clot-based or chromogenic drug-specific assay needs careful literature review on the performances of different commercially available assays. For example, one study reported overestimation of rivaroxaban levels with an anti- Xa assay utilizing exogenous antithrombin [23] and ISTH [4] recommended against its use. Nevertheless, the choice of commercially available assay may be limited by its compatibility with the automated coagulometers in service.
There are currently no standards or guidelines on the validation of drug-specific coagulation assays. Same principles on validation for clot-based or chromogenic coagulation assay shall follow, including testing for accuracy, within-run & between-run precisions and lower limit of quantification (LOQ). The testing of accuracy may be limited by accessibility to mass spectrometry. This can be resolved by testing accuracy against different lot of calibrators. Precision at low drug concentration is important to determine any significant residual DOAC effect in emergency setting. Assay kit with incorporation of low-level calibrators is favored over those with calibrators only covering the usual on-therapy concentration ranges. For the same reason, LOQ validation is important and the report shall report results as “less than” numerical LOQ value (ng/mL). Testing on plasma collected from normal subjects not taking DOACs shall be carried out to determine the intrinsic anti-Xa or anti-IIa activity from natural anticoagulant, e.g., antithrombin, which may also affect the lowest reportable limit of the assay.


As part of the quality assurance, PT, APTT, TT and fibrinogen activity shall be assessed for samples sent for quantitation of DOACs. When TT is prolonged, heparin contamination shall be excluded by carrying out protamine neutralization test. Before reporting the drug concentration, linearity of the calibrator curves shall be verified. Calibrator curve shall be acquired for every patient sample instead using stored calibrator curves as a control of lot-to-lot variation of calibrators for this relatively infrequent test. Results shall be reported in ng/mL, and there should be an accompanied comment about the appropriate range of results (peak or trough levels) based on publication. Drug level shall be interpreted in light of the time since last dose of DOAC intake as well as the dosage of DOAC taken. It is critical to have continuous surveillance of test performance over time. This can be achieved through enrollment in External Quality Assurance Programme (EQAP) (e.g., College of American Pathologist).


Drug-specific coagulation assay can be performed by automated coagulometers with pre-set dilution and analysis protocols with a low to moderate level on skill requirement and hence amenable to the organization of a laboratory-wide staff training programme to cater for the development of a routine 24-hour DOAC laboratory testing service. Interval refreshment training shall be organized to upkeep staff competence. Clinical pathologists shall be involved in communication with clinicians during emergency management of patients requiring DOAC testing to ensure efficient delivery of accurate information to facilitate patient management.

 

Impact of DOACs on special coagulation assay

It is important for laboratories that carry out special coagulation assay to acknowledge the interferences of DOACs on special coagulation assay. These include clot-based and chromogenic assay. ELISA-based and molecular assays are essentially not affected by DOACs (Table 2) [2, 17].

 

Conclusion

DOACs are more commonly used nowadays. While clinical indications for laboratory testing are more available, there is a pivotal role of laboratories to formulate a testing strategy for DOACs. Routine coagulation screening tests are not informative in most cases. Development of drug-specific assay for DOACs testing is needed. The interpretation of drug level generated by drug-specific assays needs to be facilitated by more data on the association between drug concentrations and bleeding risks expected in future studies.

 

Table 1. 5th – 95th percentile of peak and trough concentrations of DOACs obtained from pharmacokinetic and pharmacodynamics studies on patients prescribed with fixed dose and frequency of DOACs.

  Trough
(ng/mL)
Peak
(ng/mL)
Apixaban
2.5 mg twice daily
10 mg twice daily

20-94
30-412

36-100
122-412
Dabigatran
150 mg twice daily

31-225

64-223
Edoxaban
30 mg once daily
60 mg once daily

130-174
268-336

376-412
388-444
Rivaroxaban
10 mg once daily
20mg once daily

1-38
4-96

91-195
160-360

 

Table 2. Impact of DOACs on select special coagulation assays

Assay Anti-FIIa DOAC Anti-FXa DOAC
Clauss fibriongen May be falsely decreased No effect
One-stage APTT-based factor assays May demonstrate false decrease in factor activity May demonstrate false decrease in factor activity
One-stage PT-based factor assays May demonstrate false decrease in factor activity May demonstrate false decrease in factor activity
Chromogenic FVIII activity No effect May demonstrate false decrease in factor activity
Bethesda assay False inhibitor present False inhibitor present
AT activity: thrombin substrate May demonstrate false increase in AT activity; may mask AT deficiency No effect
AT activity: FXa substrate No effect May demonstrate false increase in AT activity; may mask AT deficiency
PC activity: clot based May demonstrate false increase in PC activity; may mask PC deficiency May demonstrate false increase in PC activity; may mask PC deficiency
PC activity: chromogenic No effect No effect
PS activity: clot-based May demonstrate false increase in PS activity; may mask PS deficiency May demonstrate false increase in PS activity; may mask PS deficiency
PS activity: chromogenic No effect No effect
PS activity: ELSA-based or LIA-based No effect No effect
LA testing Possible to misclassify as LA present Possible to misclassify as LA present
Activated PC resistance Falsely increased ratio; possible to misclassify as FV Leiden mutation absent Falsely increased ratio; possible to misclassify as FV Leiden mutation
AT, antithrombin; PC, protein C; PS, protein S, LA, lupus anticoagulant; LIA, latex immunoassay
 

References

  1. Bauer KA. Targeted anti-anticoagulants. N Engl J of Med 2015;273:569-571.
  2. Adcock DM, Gosselin R. Direct Oral Anticoagulants (DOACs) in the Laboratory: 2015 Review. Thromb Res 2015;136:7-12.
  3. Schulman S. New oral anticoagulant agents – general features and outcomes in subsets of patients. Thromb Haemost 2014;111:575-582.
  4. Baglin T, Hillarp A, Tripodi A, et al. Measuring oral direct inhibitors of thrombin and factor Xa: a recommendation from the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2013;11:756–760.
  5. Martin K, Beyer-Westendorf J, Davidson BL, et al. Use of the direct oral anticoagulants in obese patients: guidance from the SSC of the ISTH. J Thromb Haemost 2016;14:1308-1313.
  6. Desborough MJ, Pavord S, Hunt BJ. Management of direct oral anticoagulants in women of childbearing potential: guidance from the SSC of the ISTH: comment. J Thromb Haemost 2016; 27:1-2.
  7. von Vajna E, Alam R, and So TY. Current Clinical Trials on the Use of Direct Oral Anticoagulants in the Pediatric Population. Cardiol Ther 2016;5:9–41.
  8. Tripodi A. The laboratory and the direct oral anticoagulant. Blood 2013;121:4032-4035.
  9. Stangier J, Rathgen K, Stahle H et al. The pharmacokinetics, pharmacodynamics and tolerability of dabigatran etexilate, a new oral directed thrombin inhibitor, in healthy male subject. Br J Clin Pharmacol 2007;64: 292-303.
  10. Kitchen S, Gray E, Mackie I et al. BCSH committee. Measurement of non-coumarin anticoagulants and their effects on tests of Haemostasis: Guidance from the British Committee for Standards in Haematology. Br J Haematol 2014;166:830-841.
  11. Samuelson BT, Cuker A, Siegal DM et al. Laboratory Assessment of the Anticoagulant Activity of Direct Oral Anticoagulants (DOACs): A Systematic Review. Chest. 2016 Sep 13. pii: S0012-3692(16)59148-X. doi: 10.1016/j.chest.2016.08.1462.
  12. Yates SG, Smith S, Tharpe W et al. Can an anti-Xa assay for low molecular weight heparin be used to assess the presence of rivaroxaban? Transfus Apher Sci 2016;55: 212-215.
  13. Van Ryn J, Stangier J, Haertter S et al. Dabigatran etexilate – a novel, reversible, oral direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thromb Haemost 2010;103:1116–1127.
  14. Stangier J, Feuring M. Using the HEMOCLOT direct thrombin inhibitor assay to determine plasma concentration of dibigatran. Blood Coagul Fibrinolysis. 2012;23:138-143.
  15. Samama MM, Contant G, Spiro TE, Perzborn E et al. Evaluation of the anti-factor Xa chromogenic assay for the measurement of rivaroxaban plasma concentrations using calibrators and controls. Thromb Haemost 2012;107:371-387.
  16. Mackie I, Cooper P, Lawrie A et al. British Committee for Standards in Haematology. Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis. Int J Lab Hematol 2013;35:1–13.
  17. Gosselin RC, Adcock DM. The laboratory's 2015 perspective on direct oral anticoagulant testing. J Thromb Haemost 2016;14:886-893.
  18. Schmitz EM, Boonen K, van den Heuvel DJ et al. Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants. J Thromb Haemost 2014;12 :1636-1646.
  19. Levy JH, Ageno W, Chan NC et al. When and how to use antidotes for the reversal of direct oral anticoagulants: guidance from the SSC of the ISTH. J Thromb Haemost 2016;14:623–627.
  20. Pollack CV Jr, Reilly PA, Eikeboom J et al. Idaricizumab for dabigatran reversal. N Engl J Med 2015;373:511-520.
  21. Lippi G, Favaloro EJ. Recent guidelines and recommendations for laboratory assessment of the direct oral anticoagulants (DOACs): is there consensus? Clin Chem Lab Med 2015;53:185-197.
  22. Favaloro EJ, Lippi G. Laboratory testing in the era of direct or non-vitamin K antagonist oral anticoagulants: a practical guide to measuring their activity and avoiding diagnostic errors. Semin Thromb Hemost 2015;41:208-227.
  23. Mani H, Rohde G, Stratmann, G et al. Accurate determination of rivaroxaban levels requires different calibrator sets but not addition of antithrombin. Thromb Haemost 2012;108:191–198

 

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Newborn Screening: Past, Present and the Future

Newborn Screening: Past, Present and the Future

Volume 11, Issue 2 August 2016  (download full article in pdf)

 

Editorial note:

In this topical update, Dr Chloe Mak reviews the history and development of newborn screening, in particular for Hong Kong. Both benefits and limitations of expanded newborn screening were discussed. The latest pilot screening programme, as stipulated in the Policy Address by Chief Executive, was also illustrated. We welcome any feedback or suggestions. Please direct them to Dr. Sammy Chen (e-mail: chenpls@ha.org.hk) of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

 

Dr MAK Miu Chloe 

Department of Pathology, Princess Margaret Hospital 

 

Introduction

Newborn screening (NBS) is one of the most successful public health programs in the 20th century. In fact, the idea of mass screening was totally new to the society before 1960’s. When Dr Ivar Asbjørn Følling discovered the disease phenylketonuria (PKU) leading to mental retardation in many children [1] and Dr Robert Guthrie invented a simple and reliable screening test using bacterial inhibition test for blood phenylalanine [2] together with the understanding of disease pathogenesis and effective treatment to prevent mental retardation initiated during early asymptomatic phase [3], the proposal of NBS was  

born. However, criticisms were vigorously received over the uncertainties of disease nature, assay validity and long-term treatment effectiveness. To begin with, NBS for PKU was tested as a pilot service in Massachusetts in 1962 [4]. World Health Organization (WHO) issued two landmark reports about population screening: “The Principles and Practice of Screening for Disease” [5] and “The WHO Scientific Group on Screening for Inborn Errors of Metabolism (IEM), Geneva” [6]. The latter report elaborates more on screening for IEM.

After the success of PKU screening in preventing mental retardation, the legislation for mandatory screening was made in 1975 in USA. More disorders were added to the panel, such as congenital hypothyroidism (incidence 1 in 2,200) in 1976, congenital toxoplasmosis (1 in 27,800) in 1986, hemoglobinopathies (1 in 2,900) and congenital adrenal hyperplasia (1 in 19,200) in 1990, biotinidase deficiency (1 in 42,000) in 1992, medium-chain acyl-CoA dehydrogenase deficiency (1 in 21,000) and cystic fibrosis (1 in 2,900) in 1999 in the New England Newborn Screening Program of the University of Massachusetts Medical School [7]. The Centers for Disease Control (CDC) launched the Quality Assurance Program for NBS laboratories since 1978 and now with more than 200 laboratories worldwide participated.

The first wave of NBS started in other countries soon, such as Canada in 1963, Singapore in 1965, Japan in 1967, Australia in 1967, Portugal in 1979, while in other Asian areas NBS was mostly initiated after 1980s: Mainland China, Hong Kong, India, Malaysia and Taiwan in 1980s; Bangladesh, Indonesia, South Korea, Philippines and Thailand in 1990s; Mongolia, Myanmar, Palau, Pakistan, Sri Lanka and Vietnam in 2000s [8-10]. The approach adopted was one-test-one-disease and the panel was limited to a few conditions usually including PKU, congenital hypothyroidism, maple syrup urine disease, homocystinuria, galactosemia, cystic fibrosis and/or congenital adrenal hyperplasia.

 

Expanded Newborn Screening for Inborn Errors of Metabolism

IEM is a huge group of clinically and genetically heterogeneous metabolic disorders (Table 1). There are more than 1,000 diseases mainly affecting children. The cumulative incidence was reported up to 1 in 800 [11, 12]. Some IEM are amenable to timely treatment with good prognosis. Traditionally, the diagnosis replies on one or more tests for one disease. However, the advent of tandem mass spectrometry (TMS) applications in amino acids and acylcarnitines detection enables the one-test-many-diseases breakthrough in NBS for IEM [13-15]. TMS accurately identifies analytes by their fingerprint molecular mass-to- charge ratios with commendable specificity and sensitivity. It only requires 0.3 mL whole blood to test for more than 30 diseases in a single dried blood spot. The analytical time takes about two minutes for one sample allowing a high-volume throughput with rapid turnaround time in a NBS setting. Table 2 shows the advantages and disadvantages of TMS applications in NBS. 

 

Table 1. Classifications of IEM

1. Disorders of amino acid and peptide metabolism
2. Disorders of carbohydrate metabolism
3. Disorders of fatty acid and ketone body metabolism
4. Disorders of energy metabolism
5. Disorders in the metabolism of purines, pyrimidines and nucleotides
6. Disorders of the metabolism of sterols
7. Disorders of porphyrin and haem metabolism
8. Disorders of lipid and lipoprotein metabolism
9. Congenital disorders of glycosylation and other disorders of protein modification
10. Lysosomal disorders
11. Peroxisomal disorders
12. Disorders of neurotransmitter metabolism
13. Disorders in the metabolism of vitamins and (non-protein) cofactors
14. Disorders in the metabolism of trace elements and metals
15. Disorders and variants in the metabolism of xenobiotics (article link) 

 

Table 2 Advantages and Disadvantages of TMS Applications in NBS

Advantages
1. Detection of multiple analytes in the same analytical run
2. Small blood volume required (0.3 mL whole blood)
3. Fast analytical time about two minutes per sample
4. High throughput capacity
5. Accurate identification of molecular compounds by their fingerprint mass-to-charge ratios
6. Highly sensitive and specific with low false positive rate
7. Availability of commercial kits for acylcarnitines and amino acids

Disadvantages
1. High capital cost
2. Skillful expertise
3. Lack of full automation

  

In 1998, the New South Wales Newborn Screening Program was the first center to implement expanded NBS based on electrospray ionization TMS [16]. In the next year, the New England Newborn Screening Program introduced an optional metabolic panel for 19 IEM [7]. Twenty IEM patients were identified after 2.5 years screening of 200,000 newborns [17]. The prospective study showed that screened patients had shorter hospitalization and required less extra parental care. There was no significant difference in parental stress among NBS screened true positive, false positive results and normal control groups. In the same year, Germany started its extended screening with an unrestricted approach and since 2005 streamlined into 10 conditions [18]. Japan piloted TMS-based NBS from 1997 to 2007 with screening of 606,380 babies [19] and 65 IEM patients were identified with overall incidence of 1 in 9,330. Mainland China piloted TMS based NBS in Shanghai from 2003 to 2007 with 116,000 newborns screened [20]. Twenty patients were positive for six IEM with mainly PKU, maple syrup urine disease, methylmalonic acidemia and propionic acidemia. The overall incidence of IEM was 1 in 5,800. There were significant differences in the disease spectrum between northern and southern Chinese [21]. For example, classical PKU with phenylalanine hydroxylase deficiency accounts for the majority of PKU in northern Chinese, whereas, 6-pyruvovyl-tetrahydropterin synthase deficiency was much more common among southern Chinese. There were around 1,300 new cases of PKU screened in China each year. Glucose-6-phosphate dehydrogenase (G6PD) deficiency was very prevalent in Guangzhou with incidence of 1 in 28 but not in Northern Chinese [22]. In addition to expanded NBS in some advanced provinces covering more than 30 IEM, congenital hypothyroidism and PKU are mandatorily screened throughout the whole mainland stipulated in the law of maternal and infant health (launched in 1994) and its action program (launched in 2000) [22].

The International Atomic Energy Agency had devoted a total of $6.7 million USD to assist developing countries developing the infrastructure for NBS, in particular for congenital hypothyroidism [23]. In 2008, the Working Group of the Asia Pacific Society for Human Genetics on Consolidating Newborn Screening Efforts in the Asia Pacific Region was formed with representatives from 11 countries, viz. Bangladesh, China, India, Indonesia, Laos, Mongolia, Pakistan, Palau, Philippines, Sri Lanka and Vietnam. [24].

In 2006, the American College of Medical Genetics (ACMG) announced a consensus statement to standardize the NBS panel and decision matrix with recommendations of a core panel of 29 disorders and 25 additional secondary targets disorders [25]. It also provides the act sheets and confirmatory algorithms on each condition (http://www.ncbi.nlm.nih.gov/books/NBK55827/).

Wilson-Jungner criteria have been recently revisited in the context of genomic and modern medicine. The emphasis has been shifted towards more on the benefits to the affected baby and the family from early diagnosis and the availability of a satisfactory medical system for subsequent patient management [26]. Whether curative treatment is available or not, this is not a mandatory pre-requisite for NBS implementation.

 

Newborn Screening in Hong Kong

In Hong Kong, two metabolic conditions have been screened on a population basis namely congenital hypothyroidism and G6PD deficiency since March 1984 under the Neonatal Screening Unit of Clinical Genetic Service, Department of Health. The local incidence of CH is about 1 in 2,500, while that of G6PD deficiency is 4.5% in male and 0.3% in female newborn [27]. The program significantly lowered the mortality and morbidity. Apart from antenatal education through the Maternity and Child Health Centers, the Department of Health also provides follow-up and counseling to affected families.

The third was neonatal hearing screening. Language development is significantly improved if the hearing loss is treated before the age of 6 months. A local feasibility study was performed in 1999 screening 1,064 infants with an incidence of permanent deafness 1 in 355 [28]. A two-stage program was implemented in all Hospital Authority hospitals with maternity service since 2007 [29].

In 2008, a Coroner inquest was called into the acute death of a 14-year-old boy with a postmortem genetic diagnosis of glutaric acidemia type II (multiple acyl-CoA dehydrogenase deficiency) [30]. The Coroner’s report recommended that “the Department of Health, the Hospital Authority, the Faculty of Medicine of various universities and others concerned should carry out a feasibility study to see whether universal check may be carried out on all newborn babies for congenital metabolism defect” (http://www.judiciary.gov.hk/en/publications/coro ner_report_july08.pdf).

In 2012, the University of Hong Kong conducted the first territory-wide pilot study funded by the SK Yee Medical Fund Foundation (http://hub.hku.hk/cris/project/hkugrant107939). The study tested the feasibility of expanded NBS in public hospitals with an OPathPed model [31]. In 2013, a private NBS for IEM service commenced in the Chinese University of Hong Kong, sponsored by Joshua Hellmann Foundation for Orphan Disease (http://www.obg.cuhk.edu.hk/fetal-medicine/fetal- medicine-services/jhf-newborn-metabolic- screening-program/).

In 2015, the Policy Address by the Chief Executive announced that a working group was established between the Department of Health and Hospital Authority to study the feasibility and logistics of expanded NBS for IEM in the public healthcare system (http://www.info.gov.hk/gia/general/20150 1/14/P201501140477.htm).

The feasibility study in the form of a pilot study was officially initiated on 1 October 2015 and lasts for 18 months, testing in two public hospitals with the collaboration between the Department of Health and the Hospital Authority. The aim of this pilot study is to demonstrate the feasibility of implementing NBS for IEM while developing and optimising education on IEM to public and healthcare professional, the screening tests, laboratory algorithms, clinical management and follow-up algorithms and programme evaluation. Twenty four conditions are included (Table 3). Educational materials were distributed to public and healthcare professionals (figure 1). A video was broadcasted in antenatal clinics and postnatal wards (CantoneseMandarin and English version). 

 

Table 3 Screening Panel of Government-initiated Pilot Study

Disorders of Amino Acids
Classical phenylketonuria 6-pyruvoyl-tetrahydropterin synthase deficiency Argininosuccinic acidemia

Maple syrup urine disease Citrullinemia type I Citrullinemia type II Tyrosinemia Type I Homocystinuria

Disorders of Organic Acids

Multiple carboxylase deficiency Glutaric acidemia type I Methylmalonic acidemia Propionic acidemia

Isovaleric acidemia 3-hydroxy-3-methylglutaryl-CoA lyase deficiency Beta-ketothiolase deficiency

Disorders of Fatty Acid Oxidation

Carnitine uptake deficiency
Carnitine-acylcarnitine translocase deficiency Carnitine palmitoyltransferase II deficiency Medium-chain acyl-CoA dehydrogenase deficiency Very long-chain acyl-CoA dehydrogenase deficiency Glutaric acidemia type II

Others

Congenital adrenal hyperplasia Biotinidase deficiency
Classic galactosemia 

 

Pros and Cons of Expanded Newborn Screening

NBS for IEM enables early diagnosis and treatment, prevents morbidity and mortality, avoids unnecessary investigations, alleviates family’s anxiety, predicts prognosis and provides valuable information for family planning and genetic counseling. In addition, some maternal diseases with treatment implications can also be detected during NBS, such as primary carnitine deficiency, PKU and vitamin B12 deficiency. The storage of DBS on a population scale can be a valuable asset in quality assurance, biomedical researches and forensic investigations.

NBS is shown to be cost- effective. Although randomized clinical trial on clinical utility and cost-effectiveness is difficult due to the rarity of individual IEM, cost- effectiveness in PKU [32-34], congenital hypothyroidism [35-37] and MCADD [34, 38, 39] were well documented. Table 4 shows some examples of studies on the outcome comparison between screened and unscreened patients.

There are also limitations in expanded NBS. First, because of the short history of expanded NBS developed only in the last two decades, long-term evaluation is still lacking. Recently, the Southeastern Newborn Screening Genetics Collaborative and the Public Health Informatics Institute collaborated to address the long-term issue through international effort. Second, patients with early symptom onset before release of NBS result would not benefit. False negative can happen to patients with mild or atypical presentation or use of non-standardized cutoff values and testing strategies. Third, since TMS allows one-test-multiple-diseases, some diseases which are not required by the program would also be unraveled. Conditions which are benign or with doubtful pathological significance may be identified, for examples, 3-methylcrotonyl-CoA carboxylase deficiency and short-chain acyl-CoA dehydrogenase deficiency. Detection and disclosure of carrier status such as in sickle cell disease and cystic fibrosis may create confusion to the parents [40, 41]. Fourth, although screening is available and even mandatory in some countries, treatment is not and not all screened positive children received proper treatment. Some treatments require special drugs and milk formulae. The clinical follow-up system may not be as well established as the screening program. Fifth, NBS results can be false positive or inconclusive. The overall sensitivity and

specificity of TMS-based NBS is already commendable more than 99% with false positive rate from 0.07% to 0.33%, positive predictive values from 8% to 18% [20, 42-46]. False positive may lead to unnecessary hospitalizations and parental anxiety [47]. Measures such as better education and communication, algorithmic interpretation rules and two-tier testing system, can be implemented to reduce false positive rates andpotentialadverseeffects.

 

Conclusion

NBS represents the highest volume of genetic testing. It is more than a test and it requires a comprehensive healthcare system from pre- analytical, analytical to post-analytical phase involving expertise from public health, healthcare management, clinical, pathology and information technology. The field of NBS and IEM is still expanding. More disorders are under evaluation and covered such as severe combined immunodeficiency [48, 49] and X-linked adrenoleucodystrophy [50]. Various different or new technologies are applied to enhance the diagnostic performance, increase throughput, allow more automation and decrease costs [51-54]. Although a genomic approach for NBS is technically feasible, it entails a lot of difficult technical, clinical, social and ethical issues with hazards more than good [55]. On the other hand, using SNP array approach to detect a large panel of well-known pathogenic mutations on a wide spectrum of disorders would be more pragmatic. Expanded NBS is shown to be economically valid with significant reduction in critical care and chronic medical care expenditures. Last but not the least, NBS saves lives. 

 

Table 4 Outcome comparison between screened and unscreened IEM patients

Reference Study Results
Wilcken et al. [56] Screening more than two million babies The handicap rate 1 in 74,074 in the clinical group versus 1 in 232,558 in NBS group
Boneh et al. [57] Six babies with glutaric acidemia type I detected by NBS These patients benefited from mild protein restriction and carnitine supplement. All patients except one had normal cognitive and gross motor development, versus in unscreened patients with glutaric acidemia type I leads to acute encephalopathy and debilitating dyskinetic dystonia.
Klose et al. [58] 57 patients clinically diagnosed with organic acidemias and fatty acid oxidation defects Sixty-three percent of these patients presented within the first year of life and 54% suffered from acute metabolic crises with eight deaths.
Majority of these metabolic crises (93.5%) and death (87.5%) could have been prevented by expanded NBS and early treatment.
Schulze et al. [44] 250,000 neonates for 23 metabolic diseases and 106 patients with positive screening results followed for 42 months Seventy patients received proper treatment and remained asymptomatic. Six patients developed symptoms and three died. Nine patients presented earlier than the availability of screening results. Overall, 1 in 4,100 babies benefited from the early screening and subsequent treatment.
Cipriano et al. [59] Decision-analytic model analyzing 21 diseases taking into account of the disease severity, analytical sensitivity and specificity, need of confirmatory tests, specialist management, start-up and operating costs, hospital-related costs and potential deflation of future costs and benefits. Bundling PKU together with 14 diseases was the most cost-effective strategy with $70,000 Canadian dollars per life-year gain.
Seymour et al. [60] Systemic reviews published by the Health Technology Assessment in United Kingdom Recommended screening for PKU, biotinidase deficiency, congenital adrenal hyperplasia, MCADD and glutaric acidemia type I
Pollitt et al [61] Systemic reviews published by the Health Technology Assessment in United Kingdom Considered screening as many conditions as possible with the emphasis on the benefits of early diagnosis to the patients and the family. The availability of effective treatment was not a compulsory pre- requisite.
Filiano et al. [62] Cost-benefit study The lifetime costs for one cerebral palsy patient from infancy to 65 years old were $167,000 to $1 million USD as at 1998. The costs included medical charges, developmental services, special education and lost wages. Projected yearly savings of $36,600,000 (USD as at 1998) could be achieved through expanded NBS. The saving was twice of the incremental cost for NBS.
Couce et al. [63] 10-year clinical follow-up of 137 IEM patients picked up by expanded NBS The incidence was 1 in 2,060 newborns. With the long-term management, death rate was only 2.92% and majority of the survivors (95.5%) were asymptomatic after a mean observation of 54 months.
Linder et al. [64] 373 IEM patients detected from a cohort of 1,084,195 newborns studying the efficacy and outcome of 10-year experience in expanded NBS Presymptomatic diagnosis and treatment of other IEM achieved the same clinical benefits as in PKU.

 

 

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The sick returned traveller

The sick returned traveller

Volume 11, Issue 1 January 2016  (download full article in pdf)

 

Editorial note:

With increasing international travel, awareness and knowledge on the microbiology aspects of the returning traveller is essential, in order for timely diagnosis of infectious diseases acquired abroad and for administration of effective clinical management and public health control measures. In this issue of the Topical Update, Dr. Samson Wong presents a synopsis of the conditions associated with the returned traveller, which will be of practical application to any medical professional. We welcome any feedback or suggestion. Please direct them to Dr. Janice Lo (e-mail: janicelo@dh.gov.hk), Education Committee, The Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

 

Dr. Samson SY WONG
Assistant Professor, Department of Microbiology, The University of Hong Kong, Queen Mary Hospital, Hong Kong
 

Introduction

The number of international travellers has been increasing over the past 20 years. In 1995, there were 530 million international arrivals; this figure increased to 1,138 million in 2014 [1]. This rising trend has only been punctuated in 2003 and 2009, coinciding with two infectious disease epidemics, SARS and pandemic influenza, respectively. With the unprecedented volume, speed, and reach of international travel comes an increasing number of patients who developed travel-related health issues. About 15–64% international travellers may develop health problems during their travel [2–5]. The risk depends on the duration of travel, destination, behaviour of the travellers, and the use of prophylactic measures. In most studies, gastrointestinal (usually in the form of travellers’ diarrhoea) and respiratory illnesses are the commonest complaints, followed by skin problems, fever, and other conditions such as altitude sickness, envenoming, accidents and injuries. In this article, we shall focus on the concerns and precautions in the laboratory diagnosis of some important infections in the returned travellers.

 

Spectrum of infections and approach to the sick returned traveller

The spectrum of travel-related infections is diverse. A large body of information is available from individual centres and from GeoSentinel which consists of 63 travel clinics in 29 countries on 6 continents (http://wwwnc.cdc.gov/travel/ page/geosentinel. Accessed on 2 December 2015). However, similar data are lacking in Hong Kong, and one should note that the prevalence of different infections in the literature may not be applicable locally because of differences in the adoption of prophylactic measures and habits of travel. Data from the more recent GeoSentinel surveillance are consistent with earlier studies in that the commonest illnesses in returned travellers affected the gastrointestinal tract, respiratory system, skin, or presented as fever or systemic illnesses (Table 1) [6–9]. Fever is a common manifestation in such patients, which may occur as an undifferentiated febrile illness or be associated with specific symptoms such as rash, arthritis/arthralgia, or other localizing symptoms. The presence of localizing signs and symptoms helps to narrow the differential diagnoses. Most studies in the literature described malaria as one of the commonest causes of fever, followed by dengue in the more recent series (Table 2). Although malaria is certainly a diagnosis not to be missed, it is not the commonest aetiology of fever in returned travellers in Hong Kong. For example, in 2014, 23 cases of malaria and 112 cases of dengue were notified to the Department of Health [10]. Given that both diseases are primarily imported from endemic countries, dengue would be commoner as a cause of fever in the travellers in Hong Kong.

The clinical approach should always begin with a thorough history including a detailed itinerary (with stopovers), potential exposure history, and prophylactic measures. Despite the long list of differential diagnoses to each clinical syndrome, the most likely causes can often be suggested by the geographical areas visited, the likely incubation period of the disease, and the relevant exposure history. Some important infections associated with specific exposures are listed in Table 3. Subsequent choice of organ imaging and laboratory investigations is guided by the most likely diagnosis. It is important that after the initial assessment, one must not miss conditions that are clinically severe and potentially treatable, as well as those that have a high risk of hospital or community transmission. Severe infections must be investigated and treated urgently, such as sepsis, severe malaria, haemorrhagic fevers, and central nervous system infections. Examples of diseases that require prompt infection control precautions include viral haemorrhagic fevers, Middle East respiratory syndrome (MERS), avian influenza and infections caused by other novel influenza viruses.

 

Important clinical syndromes and laboratory investigations

Malaria

Malaria must always be considered as a potential cause of fever occurring in anyone who develops fever seven days after travelling to an endemic area [11]. Missing a case of malaria, especially falciparum malaria, can lead to serious and often fatal outcomes which in turn, may lead to medicolegal litigations. There are no pathognomonic clinical signs and symptoms of malaria. Patients are sometimes erroneously diagnosed to have influenza or gastroenteritis initially because of the non-specific clinical symptoms [12–15]. The textbook description of periodic fever is only present in 8–23% of malaria patients [12, 13]. Appropriate laboratory testing must be performed in any patient with a compatible travel history.

The diagnosis of malaria is conventionally made by examination of the thin and thick blood films. The four species of human Plasmodium, P. vivax, P. ovale, P. malariae, and P. falciparum are distinguished morphologically. In the past decade, the simian malaria P. knowlesi has emerged as an important cause of human malaria in some Southeast Asian foci, especially in Malaysian Borneo. P. knowlesi infection of travellers has been well reported. The difficulty with P. knowlesi is that its morphology closely resembles other human plasmodia, especially P. malariae. Definitive speciation can generally be made using molecular techniques [16]. Quantification of the level of parasitaemia is essential for falciparum malaria both upon initial diagnosis and serial examination of the blood smear because the level of parasitaemia carries prognostic significance and failure to reduce the level of parasitaemia after antimalarial treatment could signify drug resistance.

Any positive blood smear results must be conveyed to the attending clinician immediately. This is especially critical for P. falciparum which is a medical emergency in the non-immune travellers. It is important to remember that one single negative blood smear cannot exclude malaria. It is generally recommended that in patients with a negative blood smear but with a high clinical suspicion for malaria, at least three blood smears must be repeated over 48 hours to exclude the diagnosis [17–19]. Alternatives to microscopic diagnosis of malaria include antigen detection and nucleic acid amplification tests (NAAT) from peripheral blood. Immunochromatographic antigen detection kits are widely accepted as a form of rapid diagnostic test [20]. These are particularly useful as a form of point-of-care testing and in situations where experienced microscopists are not available. The major drawbacks include the limited sensitivity in patients with low level parasitaemia and their inability to differentiate all four species of human plasmodia. Speciation is clinically essential because P. vivax and P. ovale infections require radical cure with primaquine. NAAT is currently the most sensitive method for detection of bloodborne parasites and mixed infections, and also allows definitive speciation in problematic cases, including P. knowlesi infection [21]. Availability is, however, currently limited to a few centres and the turnaround time is often too long for routine diagnostic purposes.

Arboviruses

The arthropod-borne viruses are fast becoming some of the most important causes of emerging and re-emerging infectious disease outbreaks in tropical and subtropical countries. This is contributed by the global climate and environmental changes, as well as the geographic spread of the vectors, especially mosquitoes. There is a long list of arboviruses, many of which belong to the families of Togaviridae (mosquito-borne; e.g. Chikungunya, Ross River, Eastern, Western, and Venezuelan equine encephalitis viruses), Flaviviridae (e.g. mosquito-borne dengue, yellow fever, Japanese encephalitis, Zika, Murray Valley encephalitis viruses; tick-borne encephalitis virus), and Bunyaviridae (e.g. mosquito-borne Rift Valley fever virus; tick-borne Crimean-Congo haemorrhagic fever virus; sandfly-borne Toscana and sandfly fever viruses) [22].

The global incidence and geographical extent of dengue have been growing over the past five decades with regular outbreaks in different parts of the world [23]. The most recent outbreak is the ongoing epidemic (at the time of writing) in Taiwan, with 39,350 indigenous cases in 2015 (as of 1 December 2015) [24]. This is also the commonest notifiable arbovirus infection in Hong Kong with occasional local transmissions. Dengue is a relatively common cause of fever in returned travellers, causing 2–16.5% of the cases [25]. The disease is traditionally classified into uncomplicated dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. The last two entities are usually associated with secondary infections due to serotypes of the virus that are different from the one causing the first episode of infection. Since 2009, the World Health Organization re-classified the disease into dengue fever and severe dengue, the latter being characterized by severe plasma leakage, bleeding, and organ impairment [23].

Chikungunya is another arbovirus infection that has gained much attention in the past decade since an outbreak started in Kenya in 2004, with subsequent spread to the Indian Ocean islands till 2006, and infected over one third of the population in La Réunion [26]. Outbreaks of this togavirus have been repeatedly reported in recent years, affecting countries in Asia, Africa, the Pacific islands, Central and South Americas. Likewise, infections due to Zika virus received little attention until it caused large outbreaks in the Yap State of Federated States of Micronesia in 2007 and the French Polynesia in 2013 [27]. Zika virus is endemic in various countries in Africa, Asia, Oceania, the Pacific islands, and since 2014, in Latin and South America (especially Brazil, but also Chile, Colombia, Suriname, Jamaica, and Dominican Republic) [27, 28].

Given the large number of arboviruses, the choice of diagnostic tests should be guided by the geographical area(s) of travel and clinical syndrome. Arbovirus infections commonly manifest as systemic febrile illnesses with or without rash, arthralgia or arthritis, encephalitis or meningoencephalitis, or viral haemorrhagic fever (Table 4) [22, 29]. Many of the viruses are geographically restricted. Requests for investigations against specific viruses should be guided by the travel history and clinical manifestations. Viral culture can be performed for some viruses, but this is generally not the test of choice in most circumstances. Viral serology, preferably with paired sera for antibody testing, is one of the options of investigation, though the availability of serological tests for rarer infections is limited. Antibody testing is readily available for arboviruses such as dengue, Japanese encephalitis, and chikungunya. It should be remembered that antibody testing may be negative in the very early stage of disease, and a second serum should always be obtained. The paired antibody profile in dengue patients can also help to differentiate primary from secondary infections. Another drawback in viral antibody testing is the potential cross reactivity between different viruses, which is common among flaviviruses for example. In terms of dengue diagnostics, detection of the viral NS1 antigen in serum is superior to IgM antibody detection in the first two to three days after disease onset, a window period where IgM is often negative [30]. A combined NS1 antigenaemia and IgM antibody testing is currently a common approach to initial diagnosis of dengue. The use of NS1 lateral flow assay kits may even allow point-of-care testing for dengue, and if the roles of urine and saliva NS1 are substantiated by further studies, this will facilitate the diagnosis of dengue in resource-limited settings [31, 32]. NAAT is another option for early diagnosis of dengue which also permits detection of co-infection by different serotypes (and with other arboviruses) and genotyping of the infecting viral strains [33–35]. Antibody detection (IgM and IgG) and NAAT can also be used for the diagnosis of chikungunya and Japanese encephalitis. Consultation with clinical virologists should be made in order to choose the most appropriate tests, especially when unusual viral agents are suspected.

Enteric syndromes

Important enteric infections encountered in returned travellers include travellers’ diarrhoea, enteric fever, and amoebiasis. Travellers’ diarrhoea is the commonest travel-related infection, affecting 10–40% of individuals travelling from developed to developing countries [36]. It is most often a bacterial infection caused by various diarrhoeagenic Escherichia coli (especially enterotoxigenic strains) and other enteorpathogens such as Campylobacter, Salmonella, and Shigella. Other pathogens include viruses (especially Norovirus, classically associated with passenger ships) and parasites (such as Cryptosporidium, Giardia, Cyclospora) as well as mixed infections. Most cases of travellers’ diarrhoea are self-limiting. Specific microbiological investigations may not be necessary in milder cases, but should be considered in those with more severe manifestations (such as fever, dysentery, bloody diarrhoea, cholera-like symptoms), persistent symptoms, or in immunocompromised individuals [36]. Specific requests for viral agents (antigen detection or NAAT for Rotavirus, NAAT for Norovirus) or special concentration and staining for protozoa (e.g. modified acid-fast staining for Cryptosporidium, Cyclospora, and Cystoisospora) are necessary if routine bacterial cultures are unremarkable.

Enteric fever in Hong Kong can be indigenous or imported. This is most commonly due to typhoid and paratyphoid fevers, caused by Salmonella enterica Typhi and Paratyphi (A, B, C) respectively. Laboratory diagnosis of typhoid and paratyphoid fevers remains problematic. A positive culture from blood or other specimens (stool, urine, bone marrow) provides the definitive diagnosis, though this is not always possible due to the kinetics of bacterial shedding and circulation at different sites or prior antibiotic usage, and that bone marrow culture (the most sensitive type of specimen) is not routinely performed in this setting. Serological testing has been an important adjunct to diagnosis. Unfortunately, the widely available Widal’s test is neither sensitive nor specific for this purpose, especially when only a single serum is tested. Newer serological assays such TUBEX TFTM (IDL Biotech, Sweden) and TyphidotTM (Reszon Diagnostics, Malaysia) have improvements over the Widal’s test, but their performance in the field has not been encouraging [37, 38]. Perhaps more promising in the future is the detection of Salmonella Typhi and Paratyphi in peripheral blood by NAAT [39]. This approach has the additional benefit of detecting other circulating pathogens as a panel (e.g. using multiplex PCR) for systemic febrile illnesses in travellers, such as Plasmodium, Babesia, Rickettsia, Orientia, and other pathogenic bacteria and viruses [40].

Entamoeba histolytica infection most often manifests as amoebic colitis; extraintestinal amoebiasis is less frequently seen and the usual presentation is amoebic liver abscess. Intestinal infection is mainly diagnosed by faecal microscopy. As in the case of other enteric parasites, multiple stool samples (usually at least three) should be examined. E. histolytica is morphologically identical to at least three other species of Entamoeba, viz. E. dispar, E. moshkovskii, and E. bangladeshi. Definitive identification is best achieved by molecular methods. Antigen detection assays are viable alternatives for the detection E. histolytica in stool [41]. Some of these kits can concurrently detect other enteric protozoa such as Giardia intestinalis and Cryptosporidium. Not all of the antigen detection assays, however, are able to differentiate E. histolytica and E. dispar. Microscopy is less useful in amoebic liver abscesses; amoebae are seen in only 20% or less of liver aspirates [42]. Stool samples of patients with amoebic liver abscesses have positive microscopy for E. histolytica in only 8–44% of the cases [42]. Antibody testing is helpful in the diagnosis of amoebic liver abscess; a positive serology is present in over 95% of the patients [41–43]. The major drawback of serology is that it cannot differentiate active from past infections with E. histolytica, and hence it is less useful for populations in endemic areas.

Other issues

Space does not permit further discussion on other travel-related infections. Two more recent issues may require attention from clinical and laboratory colleagues. Firstly, the transmission of epidemic-prone infectious diseases through travel, and in particular, air travel, has caused much concern in the past few years. Examples include avian influenza and other novel influenza viruses, MERS-CoV (and the SARS-CoV in 2003), Ebola virus and other agents of viral haemorrhagic fevers. Although these are relatively uncommon causes of infection in returned travellers (with the exception of pandemic influenza in 2009), transnational spread of these infections remains a constant threat to non-endemic countries and contingency plans for surveillance, screening, clinical management, infection control, and laboratory diagnostics must be formulated in anticipation [44].

Secondly, the importation of antibiotic-resistant bacteria from travellers has emerged as another menace [45–47]. The main microbes of concern include extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae, multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci. These may be causing active infections or merely colonizing the travellers. Areas with the highest risks are the Indian subcontinent, Southeast Asia, and Africa [48–51]. Encounters with hospitals or medical facilities abroad could be due to medical problems that appeared during travel or being part of an increasingly popular medical tourism. Admission screening for multidrug-resistant organisms should be considered for patients with recent hospitalization in overseas facilities.

And not just for the microbiologists

Although the majority of tests for infective complications among the returned travellers are performed by the clinical microbiology laboratory, other specialties of clinical pathology may sometimes be involved in the investigation. The commonest scenario is the examination of peripheral blood films by haematology colleagues for malaria parasites. In addition to Plasmodium species, other pathogens that may be seen in the blood films include Babesia spp., Trypanosoma spp. (the African T. brucei gambiense and T. brucei rhodesiense, as well as the American T. cruzi), microfilariae, and the spirochaete bacterium Borrelia spp. The identification of Babesia spp. is sometimes mistaken for Plasmodium spp. because of the presence of intra-erythrocytic ring forms [52]. The travel history, especially when the destination is not a malaria-endemic region, should alert the microscopist to the possibility of babesiosis. Other morphological features that are suggestive of Babesia include the absence of stipplings and malarial pigments, presence of multiple pleomorphic rings within a single erythrocyte, and arrangement of the parasites in a Maltese cross appearance. Borrelia burgdorferi, the cause of Lyme disease, is possibly the best-known Borrelia species. However, B. burgdorferi is not normally seen in the peripheral blood smear. When spirochaetes are observed, the possibility of tick-borne or louse-borne relapsing fever should be considered. Tick-borne borrelioses are zoonoses caused by over 30 species of Borrelia and they have a global occurrence. Louse-borne relapsing fever, on the other hand, is restricted to countries in the Horn of Africa (Ethiopia, Eritrea, Somalia, and Sudan) nowadays. It is caused by Borrelia recurrentis which causes infection in humans only. The significance of louse-borne relapsing fever as a potential re-emerging infection is highlighted by the recent cases reported amongst asylum seekers who travelled to Europe from East Africa [53–56]. Definitive identification of Borrelia species requires molecular testing of the blood sample by sequencing of the 16S rRNA gene and other targets.

In addition to blood smear examination, the anatomical pathologist may encounter unexpected or unusual infections. Some of these infections may present as subacute or chronic lesions and may be seen in immigrants from foreign countries rather than recent travellers. Examples include colonic or liver biopsies with E. histolytica or Schistosoma; soft tissue or visceral lesions due to larval stages of nematodes (such as dirofilariasis or onchocerciasis) or cestodes (such as sparganosis, cysticercosis); skin biopsy in patients with cutaneous or mucocutaneous leishmaniasis; bone marrow, liver, spleen, or lymph node biopsies in patients with visceral leishmaniasis. Most of these are relatively rare in Hong Kong, but they may spice up our otherwise mundane everyday routines.

References

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Table 1. Illnesses in returned international travellers from GeoSentinel surveillance.

Country

USA [6]

USA [7]

Canada [8]

Global centres [9]

Years

1997–2011

2000–2012

2009–2011

2007–2011

Number of travellers studied

10,032

9,624

4,365

42,173

Systems involved

 

 

 

 

Gastrointestinal tract

45%

58.4%

43.7%

34.0%

Respiratory system

8%

10.8%

5.4%

10.9%

Skin

12%

16.6%

14.7%

19.5%

Fever or systemic illness

14%

18.2%

10.8%

23.3%

Neurological system

 

 

 

1.7%

Genito-urinary tract and gynaecological system, sexually-transmitted infections

 

 

 

2.9%

 

Table 2. Causes of fever in returned international travellers from GeoSentinel surveillance.

Country

USA [6]

USA [7]

Canada [8]

Global centres [9]

Years

1997–2011

2000–2012

2009–2011

2007–2011

Number of patients presenting with fever or systemic illness

1802

1748

675

9817

Diagnoses

 

 

 

 

Malaria

19.4%

27.4%

11.9%

28.7%

Dengue

11.1%

12%

7.1%

15.0%

Chikungunya

 

 

0.9%

1.7%

Enteric fever

 

6.1%

4.1%

4.8%

Respiratory tract infections

 

 

6.7%

 

Active tuberculosis

 

 

7%

 

Urinary tract infection

 

 

1.5%

 

Rickettsioses

 

4.7%

0.7%

3.0%

Leptospirosis

 

 

 

0.8%

Brucellosis

 

 

0.9%

0.3%

Hepatitis A and E

 

 

 

1.7%

Acute HIV infection

 

 

 

0.9%

Viral syndrome

17.1%

18.5%

 

 

Unspecified febrile illness

8.2%

 

 

 

Epstein-Barr virus infection and infectious mononucleosis-like syndrome

4.4%

8.7%

 

 

 

Table 3. Exposure history that should raise suspicion to specific infections.

Exposure

Potential infective complications

Sex, blood, body fluids, surgical operations, intravenous drug use

Hepatitis B and C, HIV infection, syphilis

Tattoos, body piercing, other body modification procedures

Hepatitis B and C, HIV infection, syphilis, non-tuberculous mycobacterial infections

Hospitalization

Antibiotic-resistant bacteria (colonization or infection)

Ingestion of raw or undercooked food

Various foodborne infections including bacterial and viral gastroenteritis, protozoal and helminth infections, brucellosis, listeriosis, toxoplasmosis, hepatitis A and E

Soil

Histoplasmosis, coccidioidomycoses, other endemic mycoses,
cutaneous larva migrans, strongyloidiasis

Freshwater

Schistosomiasis (Katayama fever), leptospirosis

Arthropod bites

Various arthropod-borne infections, such as dengue, chikungunya, Zika virus infection, rickettsioses, relapsing fevers, malaria, babesiosis, leishmaniasis, trypanosomiasis, dirofilariasis

Dog, bat and other animal bites

Rabies, bat rabies, herpes B virus infection, bite wound infections

Animals and animal products

Hantaviruses, Lassa fever, Crimean-Congo haemorrhagic fevers, avian influenza, MERS, plague, rat-bite fevers, leptospirosis, Q fever, brucellosis, tularaemia, anthrax, psittacosis.

Note that the exact risk of specific infections depends not only on the exposure history, but the geographical location of exposures.

 

Table 4. Some common arboviruses and their typical clinical syndromes.

Common clinical syndromes

Common causative agents

Systemic febrile illness ± rash

Dengue virus, West Nile virus, Yellow fever virus, Zika virus, chikungunya virus

Arthralgia, arthritis ± rash

Chikungunya virus, Ross River virus, Barmah Forest virus, dengue virus, West Nile virus, O’nyong’nyong virus

Encephalitis or meningoencephalitis

Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, West Nile virus, tick-borne encephalitis virus, California encephalitis virus, La Crosse virus, Rift Valley fever virus, Toscana virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus

Viral haemorrhagic fevers

Crimean-Congo haemorrhagic fever, Rift Valley fever virus, yellow fever virus, dengue virus, Kyasanur Forest disease virus, Omsk haemorrhagic fever, severe fever with thrombocytopenia syndrome virus

Note that the clinical syndromes and severity of disease caused by any single arbovirus can vary substantially. For example, many infections can either be subclinical or manifest as undifferentiated fever or produce a fulminant disease, such as viral haemorrhagic fever or meningoencephalitis. The likelihood of various potential pathogens depends on the exact geographical areas involved.

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Pathology of Non-Alcoholic Fatty Liver Disease

"Non-alcoholic fatty liver disease1" by Nephron - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Non-alcoholic_fatty_liver_disease1.jpg#/media/File:Non-alcoholic_fatty_liver_disease1.jpg

Volume 10, Issue 1, January 2015

Dr. Anthony W.H. Chan

Associate Professor Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a serious global health problem and associated with over-nutrition and its related metabolic risk factors including central obesity, glucose intolerance, dyslipidaemia and hypertension. It is the most common metabolic liver disease worldwide and its prevalence in most Asian countries is similar to that in the States, Europe and Australia. About 10-45% of Asian population have NAFLD. With “westernized” sedentary lifestyle, the prevalence of NAFLD in general urban population in the mainland China is about 15%. NAFLD is even more prevalent in Hong Kong. Our recent study demonstrated that NAFLD is found in 27.3% of Hong Kong Chinese adults by using proton-magnetic resonance spectroscopy. We further realized that 13.5% of Hong Kong Chinese adults newly develop NAFLD in 3-5 years. Both prevalence and incidence of NAFLD in Hong Kong are alarmingly high. Accurate diagnosis of NAFLD is crucial to allow prompt management of patients to reduce morbidity and mortality. NAFLD is composed of a full spectrum of conditions from steatosis to steatohepatitis (NASH) and cirrhosis. Various non-invasive tests, based on clinical, laboratory and radiological tests, have been developed to assess the degree of steatosis and fibrosis in NAFLD. However, liver biopsy remains the gold standard for characterizing liver histology in patients with NAFLD, and is recommended in patients with NAFLD at high-risk of steatohepati tis and advanced fibrosis (bridging fibrosis and cirrhosis), and concurrent chronic liver disease of other aetiology. This article reviews pathological features of NAFLD and highlights some practical points for our daily diagnostic work.

Download Topic Update for Pathology of Non-Alcoholic Fatty Liver Disease

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Molecular Classification and Genetic Alterations of Diffuse Large B-cell Lymphoma

"Glass ochem" by Purpy Pupple - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Glass_ochem.png#/media/File:Glass_ochem.png

Volume 9, Issue 2, July 2014

Dr CHOI Wai Lap

Department of Clinical Pathology Tuen Mun Hospital

Introduction

Diffuse large B-cell lymphoma (DLBCL) is the commonest subtype of non-Hodgkin lymphoma, accounting for about 30% to 40% of newly diagnosed non-Hodgkin lymphoma worldwide and in Hong Kong. DLBCL is heterogeneous in clinical presentation, morphology, immunophenotype, cytogenetics and prognos is. In the WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues published in 2008, several specific clinicopathological entities of DLBCL have been recognized, while leaving the rest to DLBCL, not otherwise specified, which is by far the most prevalent entity among the large B-cell lymphomas. In the following discussion, the term DLBCL will be used interchangeably with DLBCL, not otherwise specified.

Gene expression profiling and molecular classification of DLBCL

Gene expression profiling (GEP) is the simultaneous measurement of the transcription levels of thousands of genes to their corresponding messenger RNAs (mRNAs). GEP can be achieved by various technologies including DNA microarray, serial analysis of gene expression (SAGE) and most recently next generation sequencing (RNA-Seq). Using DNA microarray technology on DLBCL, two distinct molecular subgroups were discovered based on the similarity of their gene expression pattern with a possible cell of origin (COO): the germinal centre B-cell-like (GCB-cell-like, or abbreviated as GCB) and the activated B-cell-like(ABC-like, or abbreviated as ABC). These molecular subgroups showed significantly different survival rates when treated with conventional cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) chemotherapeutic regimen.

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Thyroid Dyshormonogenesis

Volume 9, Issue 1, January 2014

Dr YUEN Yuet Ping

Department of Chemical Pathology Prince of Wales Hospital

Introduction

Congenital hypothyroidism (CH) is an important preventable cause of mental retardation. To prevent irreversible brain damages caused by hypothyroidism, sufficient doses of thyroxine should be started within a few weeks after birth.(1) Since neonates with CH have no obvious or minimal clinical manifestations, biochemical screening in the newborn period has become the best public health strategy for early detection of affected neonates. In Hong Kong, a territory-wide screening programme for CH was started in 1984.(2) Cord blood samples are collected immediately after birth for measurement of thyroid stimulating hormone (TSH) by a single laboratory dedicated for newborn screening. The incidence of CH in Hong Kong was reported to be 1 in 2,404, which is comparable to that in other populations.

Causes of congenital hypothyroidism

The aetiologies of CH are summarized in Table 1.(7) Approximately 80-85% of CH are caused by thyroid dysgenesis, which is a group of congenital disorders of thyroid gland development or migration. Affected patients may have complete thyroid gland aplasia, hypoplasia or ectopic glands. The large majority of thyroid dysgenesis cases are sporadic and only about 5% has a genetic basis.(8,9) Thyroid dyshormonogenesis describes a group of inherited disorders which affect the biochemical pathway of thyroid hormone synthesis. These disorders collectively account for 10-15% of CH cases. Approximately 1/4 of patients with CH in Hong Kong have some forms of thyroid dyshormonogenesis.(10) Some neonates detected by newborn screening program have transient instead of permanent CH. Although this subgroup of patients does not require life- long thyroid hormone replacement, early identification and treatment in early years of life is equally important.(11) The time course of recovery of the hypothalamic-pituitary-thyroid axis in patients with transient CH depends on the underlying cause. Although most of the transient CH are due to acquired conditions such as iodine deficiency or maternal transfer of autoantibodies, a few genetic causes have been described.

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Epidemiology of Cervical Human Papillomavirus Infection in Hong Kong: Implications on Preventative Strategy

Volume 8, Issue 2, July 2013

Prof. Paul KS Chan

Professor, Department of Microbiology, Prince of Wales Hospital The Chinese University of Hong Kong

Introduction

The family Papillomaviridae is comprised of a large group of viruses found in many mammalian species. Infection with papillomaviruses can be asymptomatic or results in the development of benign or malignant neoplasia. Cervical cancer is the most important consequence, in terms of disease burden, of human papillomavirus (HPV) infection. To date, the genomic sequences of more than 150 HPV types have been characterized. Of these, more than 40 types can infect the female genital tract, and at least 15 types are epidemiologically linked to cervical cancer. Over the last few years, there has been a vast increase in using HPV DNA detection as an adjunctive or primary tool in cervica l cancer screening programmes. Primary prevention of cervical cancers associated with the two most common types (HPV16 and HPV18) can now be achieved by vaccination. A thorough understanding on the epidemiology of cervical HPV infection is essential to maximize the clinical benefits and cost-effectiveness of HPV-based diagnostic tests and vaccines. In this review, some key epidemiological features of HPV infection in Hong Kong are presented to assist the formulation of strategies applicable to Hong Kong.

Prevalence of infection

“How common is cervical HPV infection?” This is always the first question to ask before any advice on vaccination can be made. Local studies on “well-women” self-referred for cervical screening showed that the prevalence of cervical HPV infection (defined as having an HPV DNA-positive cervical scrape sample) was around 8% among adult women aged 26-45 years. 1,2 The figure “1 in 12” is recommended for public education. While the studies reported a significant association between number of life-time sexual partners and smoking exposure, the prevalence among those without any recognizable risk factors is high enough to recommend vaccination in general for everyone.

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Laboratory Testing For Anti-NMDAR In Autoimmune Encephalitis: The HSSA- Pathology Queensland Experience

Volume 8, Issue 1, January 2013

Bob Wilson MSc, FFS(RCPA), Kerri Prain, BSc, David Gillis, FRCPA FRACP FFS(RCPA) and Richard Wong GDM FRCPA FRACP FRCP.

Division of Immunology, Central Laboratory, HSSA-Pathology Queensland, Royal Brisbane and Women’s Hospitals, Herston, Brisbane, 4061, Australia.

Introduction

The spectrum of antibodies against intracellular, cell surface and synaptic neuronal antigens has expanded rapidly in recent years. The antigenic targets include ion channels, receptors involved in neurotransmission across synapses and proteins associated with them. There are now more than twenty anti-neuronal antibodies detected in association with neurological diseases. These antibodies may be associated with underlying malignancies and are commonly referred to as paraneoplastic antibodies (PNAs). Many PNAs have been correlated with neurological manifestations and fall into two groups: those that are cytotoxic for example anti-purkinje cell antibody-1 (PCA-1/Yo) and anti-neuronal nuclear antibody-1 (ANNA-1/Hu); and others that have functional activity, such as anti-N-Methyl-D-Aspartate receptor (NMDAR) and anti-Voltage-gated potassium channel (VGKC). Recently there has been a marked interest in both anti-NMDAR and anti-VGKC antibodies as the presence of these antibodies identify patients with treatable neurological disease.

Anti-NMDAR was initially described as a paraneoplastic antibody associated with ovarian teratoma, with a characteristic clinical picture of encephalitis with psychiatric features, cognitive dysfunction and seizures. 1 2 Although subsequent case series have confirmed that ovarian teratoma is a frequent association, it has become apparent that many patients who are positive for anti-NMDAR do not have evidence of an associated malignancy.

There is also some evidence supporting the need for rapid identification of anti-NMDAR. Patients who are diagnosed and treated with immuno-suppressive/immunomodulatory therapy within 40 days of disease onset, have been reported to have a better clinical outcome than those treated after 40 days.

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Virtual Electron Microscopy – update after one year of routine use

Volume 7, Issue 2, July 2012

Dr. King Chung Lee

Consultant Pathologist, St. Paul’s Hospital

Honorary Consultant, Queen Elizabeth Hospital

Background

Virtual microscopy using whole slide scanning has become increasingly popular in quality assurance program, teaching of pathologists and undergraduates and reproducibility studies 1-2. This concept was first extended to electron microscope (EM) about a year ago 3. This is made possible by two discoveries. Firstly, a free software component capable of stitching sequential pictures into a virtual slide that can be read by another free software. Secondly, an EM function capable of capturing up to 500 images covering a specified area automatically. Because of the simplicity acceptable degree of user intervention during the process and unsurpassed advantages over the conventional method, it was quickly adopted in routine renal biopsy diagnostic EM service and become the only routine service virtual microscopy system in Hong Kong. For those who are interested, you can download a sample from http://kvisit.com/SsKKqAQ and view it by Aperio ImageScope software, which was available for free download from the Aperio website, http://www.aperio.com/download-imagescope-viewer.asp

Summary of implementation

In the last year, over 400 renal biopsy cases were handled in our EM laboratory and over 1000 virtual ultrathin sections were generated (average 2.7 sections per case). The average EM time used in capturing is about 50 minutes per section. The average computing time is 40 minutes per section. The virtual ultrathin sections were either directly interpreted by Pathologists or screened and annotated by our EM tec hnologist before passing to Pathologists.

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