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    PRESS STATEMENT : Use of Over-the-counter COVID-19 Test Kits

    Thu, 2020-04-30 13:29

    PRESS STATEMENT


    Use of Over-the-counter COVID-19 Test Kits

    Download original pdf


    The College has noticed that a variety of over-the-counter Coronavirus Disease 2019 (COVID-19) self-test kits are being promoted in the market. We are concerned that the public could be misled by the results of these kits.


    These over-the-counter test kits usually utilize pinprick blood samples to detect IgG or IgM antibodies against the SARS-CoV-2 virus. Antibodies are produced over days to weeks after infection; the level and timing of response vary among individuals. The performance of these kits, including sensitivity and specificity (i.e. respectively whether the test can accurately exclude COVID-19 infection and whether a positive result is reliable to indicate actual infection), also varies among assays. Thus, the public must be aware of the risk of false negative and false positive results.


    A false negative result may cause a false sense of security, which could potentially increase transmission of the virus to others and result in delay in seeking medical consultation and management. On the other hand, a false positive result will lead to undue anxiety and unnecessary investigations, and even public health measures such as isolation of the person and his or her close contacts.


    The College reiterates that the current test of choice for the diagnosis of active COVID-19 infection remains polymerase chain reaction (PCR)-based tests detecting viral nucleic acids. The detection of antibodies is neither a suitable nor a reliable alternative. The College urges the public to seek advice from medical professionals if COVID-19 infection is suspected.


    End / Wednesday / 29 April 2020

    Issued at HKT 14:00

    hkcpath@hkcpath.org

    Tel: +852 2871 8756



    新聞稿


    就市面上出售的新型冠狀病毒快速測試之聲明

    下載原稿


    近日坊間出現各式各樣的新型冠狀病毒快速測試,並透過不同媒體銷售。惟快速測試結果存疑,有誤導公眾之嫌,本學院非常關注。


    市面上售賣之試劑盒,一般採用針刺血液樣本來檢測 SARS-CoV-2 病毒的 IgM 或 IgG 抗體。抗體於感染後數天至數週方能達至可偵測的水平,抗體之濃度及產生之時間亦因人而異。市場上不同牌子之測試劑,其敏感性 (sensitivity) 和特異性 (specificity) 不盡相同,因此市民應注意有假陰性和假陽性 結果之風險。


    假陰性結果會令市民錯誤以為自己未有受感染,從而潛在增加將病毒傳播給他人的風險,及導致延誤最佳診治的時間。另一方面,假陽性結果則會引致不必要的焦慮,多重覆檢,甚至需要採取公共衞生措施,如本人及密切接觸者的隔離檢疫。


    本學院重申,目前診斷新型冠狀病毒以聚合酶鏈反應(PCR)測試病毒核酸為首選。現時市場上出售之快速抗體測試並不適合及不可靠。本學院呼籲公眾如懷疑自己受感染,須及早向醫生求醫。


    2020 年 4 月 29 日 (星期三) 2 時正

    hkcpath@hkcpath.org

    Tel: +852 2871 8756



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    Liver injury associated with immune checkpoint inhibitors - update on clinicopathological features

    Tue, 2020-02-18 23:56

    Liver injury associated with immune checkpoint inhibitors - update on clinicopathological features


    Volume 15, Issue 1, January 2020  (download full article in pdf)


    Editorial note:


    Immune checkpoint inhibitors revolutionize the field of immuno-oncology. They have demonstrated great potential in a wide range of adult cancers by reaching long-lasting objective responses and prolonging survival. Through completed and on-ongoing clinical trials, their indications continue to expand among different cancer types. However, one of their limitations is immune-related adverse events, which are most frequently reported in skin, gastrointestinal tract, and endocrine organs. Immune-related adverse events in liver are less common hepatotoxicity but still reported up to 4 to 10% of patients receiving immune checkpoint inhibitors. This Topical Update provides a concise review on the clinicopathological features of liver injury associated with immune checkpoint inhibitors. We welcome any feedback or suggestions. Please direct them to Dr. Anthony Chan (e-mail: awh_chan@alumni.cuhk.net) 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. Regina Lo

    Department of Pathology & State Key Laboratory of Liver Research

    The University of Hong Kong



    Current applications of immune checkpoint inhibitors


    Immune checkpoint inhibitors [ICPI] have been introduced as a form of targeted therapy for human cancers. They exert anti-tumor effects by potentiating T cell functions via removing the inhibitory signals. Programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are receptors located on T cells. Ligand-receptor interactions lead to inhibition of T cell activation, therefore suppressing T cell activity against tumor cells (1). Currently, anti-PD1/PD-1 ligand (PD-L1) and anti-CTLA-4 are the two major forms of ICPI by exploiting an antagonistic approach using specific antibodies that target PD-1 and CTLA-4, respectively. Thus far, several ICPIs were approved by the US Food and Drug Administration for treating cancer (2). Nivolumab and pembrolizumab are FDA approved frontline anti-PD1 agents, while ipilimumab is an anti-CTLA-4 agent. These drugs are given either alone or in combination. Currently there are a number of on-going phase III/IV clinical trials with ICPI for various types of cancers (3).



    Clinical features of hepatotoxicity associated with ICPI


    Despite the encouraging clinical efficacy, adverse reactions related to ICPI administration have been observed, among which dermatological, gastrointestinal, endocrine manifestations were most frequently reported. These reactions are believed to result from the immune response elicited toward various organs. A meta-analysis of 17 studies revealed an increased risk of all-grade hepatotoxicity with ICPI compared with controls (pooled OR 4.10; PD-1 subgroup 1.94; CTLA-4 5.01) (4). Among all immune-related adverse reactions, hepatotoxicity was observed in a relatively small proportion of cases (up to 4-10%) in most reports (2, 5-9). Susceptibility of adverse reactions in the liver appears to be dependent on the primary cancer, regimen/dose of ICPI, and host factors. It was reported that patients receiving ICPI for HCC were at a higher risk of hepatotoxicity in terms of transaminases levels compared with lung cancer and melanoma (10). Moreover, combination therapy or a higher dose of ICPI was associated with increased risk of hepatic injury (6, 9, 11, 12). Patients may present with fever and jaundice but can also be asymptomatic (13,14). The median time from the first dose to immune-related hepatoxicity was 14.1 weeks (9.4–19.7) for anti-PD-1, 9.9 weeks (6.1–14.7) for anti-CTLA4, and 2.9 weeks for combined therapy (15). The biochemical derangement is usually of a hepatitic or mixed hepatitic/cholestatic pattern. Radiological findings most of the time do not offer additional diagnostic information. In general, hepatotoxicity associated with ICPI is classified according to Common Terminology Criteria for Adverse Events by the National Cancer Institute (CTCAE). This system comprises grades 1-5 (with grade 5 being fatal) based on the serum levels of AST, ALT, ALP, GGT and total bilirubin. Having said that, elevated bilirubin is a less frequent phenomenon than most forms of drug-induced liver injury.



    Histological features of liver injury associated with ICPI


    The commonest histological features of ICPI-associated hepatotoxicity are lobular hepatitis, portal lymphoid infiltrates and variable degrees of hepatocytic necrosis (16-19). A predominant biliary pattern has been reported but is much less frequently encountered (20, 21). Cholestasis is not commonly seen, with bland cholestasis reported in 1 of 10 cases treated with pembrolizumab (22). Two cases of ICPI-induced hepatitis histologically presenting with fibrin-ring granulomas have also been reported (23). Steatosis is rare. Some histological features may be more readily observed with the use of a specific type of inhibitor. For instance, microgranulomas and central vein endotheliitis were seen in patients who received anti-CTLA4 therapy. With anti-PD1 therapy, more prominent portal tract inflammation was encountered. In contrast to autoimmune hepatitis, plasma cells are usually low in number (24), which is line with the observation that serum IgG level is mostly normal and autoimmune serological markers are negative. Likewise, in a report comparing 7 cases of ICPI-associated hepatitis versus 10 cases of AIH and 10 cases of drug-induced liver injury (DILI) (24), hepatocytic rosettes and emperipolesis were less commonly observed than AIH. When compared with DILI, bile plugs and eosinophils were less readily seen in ICPI-associated hepatitis. On immunohistochemical delineation of the lymphoid cell population in ICPI-associated hepatitis, several reports have consistently demonstrated a predominance of CD8+ lymphocytes (17, 18, 22). This could be distinguishing feature with AIH, in which CD20+ or CD4+ lymphoid cells are frequently encountered.



    Diagnostic considerations and implications


    The diagnosis of ICPI-liver injury can seldom be made by histology alone as there are no pathognomonic features. Before attributing the cause to ICPI, potential etiologies for liver function derangement should be considered. In particular, exclusion of hepatic involvement by tumor and viral hepatitis is needed. According to a recent report, among 491 patients treated with pembrolizumab for melanoma, lung cancer or urothelial cancer, 70 developed liver injury. Among which, a probably drug-related cause was only made in 20 cases after adjudication (25). Liver histology can help to exclude some differential diagnoses and assess the severity of liver tissue injury, which could be useful to guide management plan. The treatment options for adverse reactions would depend on the severity, and include withdrawal/discontinuation of ICPI, corticosteroids (oral or IV) +/- additional immunosuppressant e.g. mycophenolate mofetil (26). The drugs are usually permanently discontinued in cases presenting with Grade 3 or Grade 4 adverse reactions. There are no standard guidelines with reference to reintroducing ICPI after recovery from Grade 1-2 adverse reactions. As far as histology is concerned, it remains an open question whether histological parameters could offer added values in the grading of ICPI-associated hepatotoxicity. Besides, further studies are awaited to better understand the histological features associated different types of ICPI, and to depict the development and progression of fibrosis in this subset of drug-induced liver injury.




    References

    1. Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen L. Immunostimulatory monoclonal antibodies for cancer therapy. Nat Rev Cancer. 2007;7(2):95-106.
    2. Nadeau A, Fecher LA, Owens SR, Razumilava N. Liver Toxicity with Cancer Checkpoint Inhibitor Therapy. Semin Liver Dis. 2018;38(4):366-78.
    3. Darvin P, Toor SM, Sasidharan Nair V, Elkord E. Immune checkpoint inhibitors: recent progress and potential biomarkers. Exp Mol Med. 2018;50(12):165.
    4. Wang W, Lie P, Guo M, He J. Risk of hepatotoxicity in cancer patients treated with immune checkpoint inhibitors: A systematic review and meta-analysis of published data. Int J Cancer. 2017;141(5):1018-28.
    5. Suzman DL, Pelosof L, Rosenberg A, Avigan MI. Hepatotoxicity of immune checkpoint inhibitors: An evolving picture of risk associated with a vital class of immunotherapy agents. Liver Int. 2018;38(6):976-87.
    6. Larkin J, Hodi FS, Wolchok JD. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015;373(13):1270-1.
    7. Motzer RJ, Rini BI, McDermott DF, Redman BG, Kuzel TM, Harrison MR, et al. Nivolumab for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial. J Clin Oncol. 2015;33(13):1430-7.
    8. Weber JS, Kahler KC, Hauschild A. Management of immune-related adverse events and kinetics of response with ipilimumab. J Clin Oncol. 2012;30(21):2691-7.
    9. Cheung V, Gupta T, Payne M, Middleton MR, Collier JD, Simmons A, et al. Immunotherapy-related hepatitis: real-world experience from a tertiary centre. Frontline Gastroenterol. 2019;10(4):364-71.
    10. Brown ZJ, Heinrich B, Steinberg SM, Yu SJ, Greten TF. Safety in treatment of hepatocellular carcinoma with immune checkpoint inhibitors as compared to melanoma and non-small cell lung cancer. J Immunother Cancer. 2017;5(1):93.
    11. Weber JS, Postow M, Lao CD, Schadendorf D. Management of Adverse Events Following Treatment With Anti-Programmed Death-1 Agents. Oncologist. 2016;21(10):1230-40.
    12. Sanjeevaiah A, Kerr T, Beg MS. Approach and management of checkpoint inhibitor-related immune hepatitis. J Gastrointest Oncol. 2018;9(1):220-4
    13. Johnson DB, Chandra S, Sosman JA. Immune Checkpoint Inhibitor Toxicity in 2018. JAMA. 2018;320(16):1702-3.
    14. De Martin E, Michot JM, Papouin B, Champiat S, Mateus C, Lambotte O, et al. Characterization of liver injury induced by cancer immunotherapy using immune checkpoint inhibitors. J Hepatol. 2018;68(6):1181-90.
    15. Gauci ML, Baroudjian B, Zeboulon C, Pages C, Pote N, Roux O, et al. Immune-related hepatitis with immunotherapy: Are corticosteroids always needed? J Hepatol. 2018;69(2):548-50.
    16. Karamchandani DM, Chetty R. Immune checkpoint inhibitor-induced gastrointestinal and hepatic injury: pathologists' perspective. J Clin Pathol 2018;71(8):665-71.
    17. Kleiner DE, Berman D. Pathologic changes in ipilimumab-related hepatitis in patients with metastatic melanoma. Dig Dis Sci. 2012;57(8):2233-40.
    18. Johncilla M, Misdraji J, Pratt DS, Agoston AT, Lauwers GY, Srivastava A, et al. Ipilimumab-associated Hepatitis: Clinicopathologic Characterization in a Series of 11 Cases. Am J Surg Pathol. 2015;39(8):1075-84.
    19. Zen Y, Yeh MM. Checkpoint inhibitor-induced liver injury: A novel form of liver disease emerging in the era of cancer immunotherapy. Semin Diagn Pathol. 2019.
    20. Kim KW, Ramaiya NH, Krajewski KM, Jagannathan JP, Tirumani SH, Srivastava A, et al. Ipilimumab associated hepatitis: imaging and clinicopathologic findings. Invest New Drugs. 2013;31(4):1071-7.
    21. Aivazian K, Long GV, Sinclair EC, Kench JG, McKenzie CA. Histopathology of pembrolizumab-induced hepatitis: a case report. Pathology. 2017;49(7):789-92.
    22. Zen Y, Chen YY, Jeng YM, Tsai HW, Yeh MM. Immune-related adverse reactions in the hepatobiliary system: Second generation checkpoint inhibitors highlight diverse histological changes. Histopathology. 2019.
    23. Everett J, Srivastava A, Misdraji J. Fibrin Ring Granulomas in Checkpoint Inhibitor-induced Hepatitis. Am J Surg Pathol. 2017;41(1):134-7.
    24. Zen Y, Yeh MM. Hepatotoxicity of immune checkpoint inhibitors: a histology study of seven cases in comparison with autoimmune hepatitis and idiosyncratic drug-induced liver injury. Mod Pathol. 2018;31(6):965-73.
    25. Tsung I, Dolan R, Lao CD, Fecher L, Riggenbach K, Yeboah-Korang A, et al. Liver injury is most commonly due to hepatic metastases rather than drug hepatotoxicity during pembrolizumab immunotherapy. Aliment Pharmacol Ther. 2019.
    26. Brahmer JR, Lacchetti C, Schneider BJ, Atkins MB, Brassil KJ, Caterino JM, et al. Management of Immune-Related Adverse Events in Patients Treated With Immune Checkpoint Inhibitor Therapy: American Society of Clinical Oncology Clinical Practice Guideline. J Clin Oncol. 2018;36(17):1714-68.
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    College Fee (updated 20200109)

    Thu, 2020-02-13 17:19

    College Fee PDF

    All Fees are in HKD

    Membership Entrance and Annual Subscription Fees

    Membership Catgory

    Subscription#

    Entrance

    Annual

    Honorary Fellows

    Nil

    Nil

    Founder Fellows*1,*3

    N/A

    $3,000

    Fellows*2,*3

    $3,000

    $3,000

    Overseas Fellows*3, *4

    $3,000

    $1,500

    Members

    $1,500

    $1,500

    Associates

    Nil

    $800

    Remarks:

    # The date of payment of annual subscription would be every 1st of January.

    # If the date of admission to membership is within 6 months from the coming 1st of January, only half of the annual subscription would be required for this period.

    # On change in the category of membership, payment of the balance of the annual subscription would be required.

    *1 For Founder Fellows residing overseas, the annual subscription would be HK$1,000. On residing in Hong Kong for more than 3 months, the annual subscription would be reverted back to the annual subscription of Founder Fellows.

    *2 For Fellows residing overseas, there would be no reduction of annual subscription. Fellows residing overseas can apply for changing their category of membership to Overseas Fellows (this category has no voting right).

    *3 For Founder Fellows, Fellows and Overseas Fellows with Retired Fellow Status as recorded in the College Register, the annual subscription would be HK$1,000 before 1st of November 2006. Afterwards, only a nominal annual subscription of HK$100 would be applied. Fellows concerned must inform the College immediately should there be any change of their retired status and the reduced rate will cease to apply thereafter.  Please refer to the form for “Application for Change of Fellowship Status” for the rights and privileges of Fellows with retired status.

    *4 For Overseas Fellows residing in Hong Kong for more than 3 months, the annual subscription would be reverted back to the annual subscription of Fellows.


    Training & Examinations Committee

    Type of Examination

    Examination / Exemption Fee

    Fellowship Assessment

    $22,000

    Membership Examination

    $18,000

    Membership Examination Exemption

    $18,000

    Supplementary Examination

    $18,000



    Description

    Administrative Fee

    Annual Trainee Registration#

    $800

    Retrospective recognition of the whole or part of laboratory training period after 6 months from the commencement of training

    $2,000

    Late Submission of Annual Report

    $500

    Remark:

    # The annual trainee registration fee is waived if the registered trainee is an Associate or Member of the College.


    Education Committee

    Description

    Administrative Fee

    Late submission of CME/CPD Record Annual Update

    $500

    Retrospective report of CME/CPD activities after submission of CME/CPD Annual Return

    $500

    Administration of CME/CPD activities for Retired Fellows

    Balance of the Annual Subscriptions between Full and Retired Fellows



    Credentials & Appeals Committee

    Description

    Administrative Fee

    Appeal against examination result

    $2,000

    Remark:

    # A written request must be submitted to Registrar within 3 months from the date of the letter notifying the examination result.



    Replacement of Certificate

    Type of Certificate

    Replacement Fee

    Membership Certificate

    $1,000/certificate

    Fellowship Certificate

    $2,000/certificate

    Remark:

    # A written request must be submitted to Registrar.



    Regalia Loan Service

    Regalia Loan Period

    Service Fee

    1 to 3 days

    $150/set

    4 to 6 days

    $250/set

    7 to 10 days

    $350/set

    Each additional day after 10 days

    $30/set/day

    Remarks:

    # The College Regalia is available for loan to all Members and Fellows.

    # Users are required to submit the Regalia Loan Form to the College Secretary either by fax, post or email at least 2 weeks before the loan period, and inform the College Secretary of any additional day of Regalia loan service beforehand.

    # Payment for Regalia loan service should be made by cheque payable to “The Hong Kong College of Pathologists”. Cheque should be sent with the Regalia Loan Form by post or in person to the College Secretary.

    # Collection of Regalia will be arranged at the Chamber of the Hong Kong College of Pathologists when both the loan form and the cheque are received. Collection time: from 9:00 am to 5:00 pm, Monday to Friday.

    # Users are responsible for keeping the Regalia in good condition and are bound by the rules and regulations stipulated in the Regalia Loan Form.

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    2019 College Annual Report and Year Book

    Thu, 2019-12-05 21:24

    2019 College Annual Report and Year Book 2019-20

    The 2019 College Annual Report and Year Book are available for download

    Annual Report 2019

    Year Book 2019/20

     

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    Results of College Exam 2019 (Council Meeting 24 September 2019)

    Tue, 2019-09-24 18:12

    Results of College Exam 2019 (Council Meeting 24 September 2019)

     

    Fellowship Assessment:

     

    EXAM NO.

    RESULT

    E19201

    PASS

    E19202

    FAIL

    E19203

    PASS

    E19204

    FAIL

    E19205

    FAIL

    E19206

    FAIL

    E19207

    FAIL

    E19208

    FAIL

    E19209

    FAIL

    E19210

    PASS

    E19211

    PASS

    E19212

    PASS

    E19213

    PASS

    E19214

    PASS

    E19215

    FAIL

    E19216

    PASS

    E19217

    FAIL

    E19218

    PASS

    E19219

    PASS

    E19220

    PASS

     

    Membership Examination:

     

    EXAM NO.

    RESULT

    E19101

    PASS

    E19102

    FAIL

    E19103

    PASS

    E19104

    FAIL

    E19105

    FAIL

    E19106

    PASS

    E19107

    PASS

    E19108

    FAIL

    E19109

    PASS

    E19110

    FAIL

    E19111

    FAIL

    E19112

    PASS

     

     

     

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    Recent Advance in Acute Lymphoblastic Leukaemia

    Wed, 2019-09-04 12:34

    Recent Advance in Acute Lymphoblastic Leukaemia

     

    Volume 14, Issue 2, August 2019  (download full article in pdf)

     

    Editorial note

    In this topical update, Dr Albert Sin reviews recent advances in diagnosis and management of acute lymphoblastic leukaemia (ALL).  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. Albert Sin
    Clinical Assistant Professor

     

    Introduction

    Acute lymphoblastic leukaemia (ALL) is an aggressive and highly fatal malignancy resulting from clonal mutations of lymphoid progenitor cells. The incidence of ALL is the most common in childhood and age after 50.18The prognosis of childhood ALL is good with long-term survival rate approaching 90% treated by intensive chemotherapy.12Although the incidence of ALL is less in adolescent, young adult as well as adult, the prognosis of ALL in those people is very poor, with only 30-40% of adult patients able to remit.18 According to the data from US database which registered all patients with diagnosed ALL from 2000 to 2007, the survival rate was 75% at 17 years old, 45% at 20 years old and 15% at 70 years old.14An increasing knowledge of disease biology of ALL transformed into insights for development of novel therapies to improve the treatment outcome of ALL.

    One of the reasons of adverse prognosis in adolescent and young adult (AYA) as well as adult patients is that they commonly harbored poor-risk genetic aberrations while less patients carried favorable genetic lesion.14This could explain the sudden drop in survival from 17 years old to 20 years old. 

     

    Ph-like ALL

    Ph-like ALL is a newly identified genetic subgroup. The genetic profile of this subgroup of ALL is similar to that of Philadelphia chromosome positive (Ph-positive) ALL but without BCR-ABL1 fusion.17They have a higher frequency of IKZF1 deletion and mutation in genes of lymphoid transcription factors with poor survival.19The incidence of Ph-like ALL increases with ages and approaching 27% of cases of adult B-ALL.14

    The nature of genetic aberration is heterogeneous. Despite its complexity, it can be simply classified into five subgroups: 1. CRLF2 rearrangement 2. Rearrangement of ABL-class gene 3. Rearrangement of JAK2 and EPOR 4. Aberrations leading to activation of JAK-STAT or MAPK pathway 5. Other rare kinase alterations.19The distribution of different types of genetic alterations are different among childhood high risk ALL, young adult and adult (Figure 1). CRLF2 rearrangement is the most common type of genetic alteration in Ph-like ALL. CRLF2 gene is responsible for producing lymphopoietin receptor and regulate the process of lymphopoiesis. Common mechanisms of CRLF2 rearrangement include 1. Translocation of CRLF2 gene into IGH gene 2. Fusion between CRLF2 gene and P2RY8 gene. 3. Point mutation F232C at CRLF2 gene. Nearly 50% of CRLF2 rearranged Ph-like ALL have concomitant JAK mutations.19


    Figure 1

    Figure 1

     

    Diagnosis of Ph-like ALL


    Genetic profiling is the gold standard for the diagnosis of Ph-like ALL. However, it is difficult to implement in routine diagnostic laboratory. 

    Cytogenetics analysis is a standard test for all cases of ALL which allows a global assessment of chromosomal abnormalities. Some of the recurrent genetic abnormalities, for example t(9;22), hyperdiploidy/hyperdiploidy,   rearrangement involving 11q23, etc, can be detected. However, most of the Ph-like ALL genetic alterations are cryptic, e.g. interstitial deletion of CRLF2, ETV6-RUNX1 fusion, etc and thus they cannot be detected by conventional cytogenetics.2

    Fluorescent in-situ hybridization (FISH) can be utilized to detect Ph-like ALL genetic abnormalities. Breakapart probes targeting genes most frequently genes including ABL1, ABL2, PDGFRB, JAK2, CRLF2, and P2RY8 are currently available. Although the positive result upon FISH study needs additional fusion probe for confirmation, it provides a readily available and useful diagnostic tool for establishing the diagnosis of Ph-like ALL. However, some of the important Ph-like ALL genetic rearrangement including intrachromosomal inversions (e.g., inv(9) resulting in PAX5-JAK2 fusion), intra-chromosomal deletions (e.g., del(X)(p22p22)/del(Y)(p11p11) resulting in P2RY8-CRLF2 fusion) are undetectable by FISH technique.2Targeted sequencing by NGS platform is an evolving technique for diagnosis.16

    Recently, antibody against CRLF2 is available for flow cytometry study. The expression of CRLF2 as detected by multiparametric flow cytometry is correlated with genetic testing for CRLF2 rearrangement.15This provides a rapid tool for identifying potential cases of Ph-like ALL before the result of genetic tests is available. 

    Most of the genetic alterations of Ph-like ALL are targetable kinase lesions, which could be treated by tailored kinase inhibitor therapy (Table 1).19This approach of therapy is current undergoing extensive preclinical studies.9


    Table 1

     

    Early T-cell precursor ALL (ETP-ALL)


    ETP-ALL is recently characterized subtype of T-ALL. It constitutes around 12% of childhood ALL and 7.4% of adult ALL.11Genetic profiling showed ETP cells are similar to that of haemopoietic stem cells and myeloid progenitor cells.4This subgroup of ALL is characterized by the unique immunophenotype: cytoplasmic CD3+, surface CD3-, CD1a-, CD2-, CD5 dim (<75% positive), CD7 and positive for one or more stem cell and/or myeloid markers including HLA-DR, CD13, CD33, CD34, or CD117.5

    While activating mutation of NOTCH1 is a common mutation found in ALL and it account for 50% of cases of childhood ALL, this mutation is less common in ETP-ALL.3ETP-ALL commonly have mutations in FLT3, DNMT3A, IDH1, IDH2, etc.11

    ETP-ALL carries a poor prognosis with inferior overall survival when treated with standard chemotherapy regimen comparing with other subtypes of T-ALL.11This subgroup of T-ALL represented a distinct subtype with unique genetic profile and poor prognosis. 

     

    MRD in adult ALL


    Minimal residual disease (MRD) describe the very low level of disease burden which cannot be detected by morphology. Measurement of MRD not only pick up a submicroscopic level of disease but also can monitor the disease kinetic during the treatment process of haematological malignancies.10

    The following techniques can be used to detect MRD: 

    1. Multiparametric flow cytometry to detect leukaemia-associated immunophenotype (LAIP)
      By using a 4-color or 6-color panel of antibodies, we can identify LAIP in 90% of ALL caes.10Flow cytometry is a quick method and the result of MRD can be generated in a short period of time for clinical decision. The sensitivity of MRD detection by this method is 0.01%. However, in order to define the positive MRD, we need 10-40 cluster of cells and thus higher number of cells are required for assessment which may be difficult for reassessment samples after intensive chemotherapy.7In addition, antigenic shift is commonly occurred in leukaemic cells and normal cells during the therapy. The use of monoclonal antibodies, e.g anti-CD19, anti-CD22 for treatment of ALL will affect the gating strategy used to identify the leukaemic cells.10

    2. Detecting leukaemia-specific fusion transcript by PCR technique
      Quantitative reverse-transcriptase PCR can be employed to detect the amount of leukaemia-specific fusion transcript. The sensitivity is higher compared with flow cytometry (10-4to 10-6).7The test is relatively easy to be performed in standardized diagnostic laboratory. However, only 30-40% of cases of ALL carry leukaemia-specific fusion transcript and thus limited the eligibility of MRD detection by this method. Moreover, the interpretation is challenging for RNA-based test in those cases will poor RNA quality.

    3. Quantitative PCR for immunoglobulin (IG)-T cell receptor (TCR) gene targets
      Quantitative PCR is employed to detect the specific sequence of rearranged IG gene or TCR gene in the sample. The sensitivity of this method is 10-4to 10-5and this method can be applied to all cases of ALL. However, this method of MRD detection requires prior characterization of IG or TCR gene rearrangement by sequencing and designs patient-specific primers for each case for subsequent MRD detection. Extensive standardization and experience are needed for the laboratory to set up this test, which limit the use of this method of MRD detection in diagnostic laboratory. Moreover, the clonal evolution in leukaemic blasts during treatment can make the original rearranged sequence to be lost and thus generate a false negative result. Also, the non-specific primer annealing occurs during the process of marrow regenerative may yield false positive result for the test.7

     

    Application of MRD in treatment of adult ALL


    MRD-guided therapy had been gained extensive experience in childhood ALL.8The study group of German Multicenter Study Group for Adult ALL (GMALL) had conducted largest study for the role of MRD in adult Ph-negative ALL. They showed that molecular remission is the only parameters significantly affect the remission duration and survival.6Patients with positive MRD after induction therapy achieved better overall survival after receiving haemopoietic stem cell transplant. Early achievement of MRD negativity after induction chemotherapy is associated with good outcome for adult ALL.10Study showed that MRD level correlates with post-transplant outcome.13Another group found that haemopoietic stem cell transplant benefits the patients with positive MRD at week 6.1These findings may prompt reconsideration of the indications of haemopoietic stem cell transplant for adult patients with ALL, especially those patients achieve MRD negativity after treatment. 

     

    Concluding landmark


    The prognosis of acute lymphoblastic leukaemia in young adolescent and adult is poor. The recent discovery of new subtype of acute lymphoblastic leukaemia with characterization of genetic lesions make a breakthrough of understanding of disease biology. Precise disease prognostication can be made. Targeted therapies are being developed for treating those patients. Clinical trials are conducting for evaluating the targeted therapies in those new subtypes of acute lymphoblastic leukaemia. Moreover, the application of MRD monitoring and MRD-adapted therapy in adult ALL can further stratified the patients and select the appropriate candidates of haemopoietic stem cell transplant in order to reduce transplant-related mortality and morbidity. The advances in understanding of molecular mechanism and disease biology of ALL help to improve the risk stratification, rapid development of targeted therapies and hopefully improve the prognosis in young adolescent and adult patients. 

     

     

    Reference

     

    1. Beldjord K., Chevret S., Asnafi V., Huguet F., Boulland ML., Leguay T., Thomas X., Cayuela JM., Grardel N., Chalandon Y., Boissel N., Schaefer B., Delabesse E., Cavé H., Chevallier P., Buzyn A., Fest T., Reman O., Vernant JP., Lhéritier V., Béné MC., Lafage M., Macintyre E., Ifrah N., Dombret H; Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL). (2014) Oncogenetics and minimal residual disease are independent outcome predictors in adult patients with acute lymphoblastic leukemia. Blood, 2014,12;123(24):3739-49.
    2. Bradford J. Siegele., Valentina Nardi. (2018). Laboratory testing in BCR-ABL1-like (Philadelphia-like) B-lymphoblastic leukemia/lymphoma. Am J Hematol, 2018(93):971–977.
    3. Chao Gao., Shu-Guang Liu., Rui-Dong Zhang., Wei-Jing Li., Xiao-Xi Zhao., Lei Cui., Min-Yuan Wu., Hu-Yong Zheng and Zhi-Gang Li. (2014) NOTCH1 mutations are associated with favourable long-term prognosis in paediatric T-cell acute lymphoblastic leukaemia: a retrospective study of patients treated on BCH-2003 and CCLG-2008 protocol in China. Br J Haematol, 2014, 166(2):221-8. doi: 10.1111/bjh.12866
    4. Elaine Coustan-Smith., Charles G Mullighan., Mihaela Onciu., Frederick G Behm., Susana C Raimondi., Deqing Pei., Cheng Cheng., Xiaoping Su., Jeff rey E Rubnitz., Giuseppe Basso., Andrea Biondi., Ching-Hon Pui., James R Downing., Dario Campana. (2009) Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia.Lancet Oncol, 2009(10):147–56
    5. Elizabeth A. Raetz and David T. Teachey. (2016) T-cell acute lymphoblastic leukemia. Am Soc Haemaetolo Educ Program, 2016(1), 580-588
    6. Gökbuget N., Kneba M., Raff T., Trautmann H., Bartram CR., Arnold R., Fietkau R., Freund M., Ganser A., Ludwig WD., Maschmeyer G., Rieder H., Schwartz S., Serve H., Thiel E., Brüggemann M., Hoelzer D; German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia. (2012) Adult patients with acute lymphoblastic leukemia and molecular failure display a poor prognosis and are candidates for stem cell transplantation and targeted therapies. Blood, 2012,120(9):1868-76.
    7. Jacques J. M. van Dongen., Vincent H. J. van der Velden., Monika Br¨uggemann and Alberto Orfao. (2015) Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies.Blood, 2015;125(26):3996-4009
    8. Marianne Ifversen., Dominik Turkiewicz., Hanne V. Marquart., Jacek Winiarski., Jochen Buechner., Karin Mellgren., Johan Arvidson., Jelena Rascon., Lenne-Triin Ko¨rgvee., Hans O. Madsen., Jonas Abrahamsson., Bendik Lund., Olafur G. Jonsson., Carsten Heilmann., Mats Heyman., Kjeld Schmiegelow., Kim Vettenranta. (2019) Low burden of minimal residual disease prior to transplantation in children with very high risk acute lymphoblastic leukaemia: The NOPHO ALL2008 experience. British Journal of Haematology, 2019(184): 982–993
    9. Maude SL, Tasian SK, Vincent T, et al. (2012) Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia. Blood, 2012;120(17):3510-3518.
    10. Monika Bru¨ ggemann and Michaela Kotrova. (2017) Minimal residual disease in adult ALL: technical aspects and implications for correct clinical interpretation. Hematology Am Soc Hematol Educ Program.2017(1):13-21. doi: 10.1182/asheducation-2017.1.13.
    11. Nitin Jain., Audrey V. Lamb., Susan O’Brien., Farhad Ravandi., Marina Konopleva., Elias Jabbour., Zhuang Zuo., Jeffrey Jorgensen., Pei Lin., Sherry Pierce., Deborah Thomas., Michael Rytting., Gautam Borthakur., Tapan Kadia., Jorge Cortes., Hagop M. Kantarjian., Joseph D. Khoury. (2016) Early T-cell precursor acute lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) in adolescents and adults: a high-risk subtype. Blood, 2016;127(15):1863-1869
    12. Paul, S., Kantarjian, H., & Jabbour, E. J. (2016). Adult Acute Lymphoblastic Leukemia. Mayo Clin Proc, 91(11), 1645-1666. doi:10.1016/j.mayocp.2016.09.010
    13. Renato Bassan., Monika Brüggemann., Hoihen Radcliffe., Elizabeth Hartfield., Georg Kreuzbauer., Sally Wetten. (2019) A Systematic Literature Review And Meta-Analysis Of Minimal Residual Disease As A Prognostic Indicator In Adult B-Cell Acute Lymphoblastic Leukemia. Haematologica,2019 Mar 19. pii: haematol.2018.201053. doi: 0.3324/haematol.2018.201053.
    14. Roberts KG. (2018) Genetics and prognosis of ALL in children vs adults. Hematology Am Soc Hematol Educ Program. 2018(1):137-145. doi: 10.1182/asheducation-2018.1.137.
    15. Sergej Konoplev., Xinyan Lu., Marina Konopleva., Nitin Jain., Juan Ouyang., Maitrayee Goswami., Kathryn G. Roberts., Marc Valentine., Charles G. Mullighan., Carlos Bueso-Ramos., Patrick A. Zweidler-McKay., Jeffrey L. Jorgensen and Sa A. Wang. (2017) CRLF2-Positive B-Cell Acute Lymphoblastic Leukemia in Adult Patients: A Single-Institution Experience. Am J Clin Pathol, 2017(147):357-363
    16. Stadt UZ., Escherich G., Indenbirken D., Alawi M., Adao M., Horstmann MA. (2016) Rapid Capture Next-Generation Sequencing in Clinical Diagnostics of Kinase Pathway Aberrations in B-Cell Precursor ALL. Pediatr Blood Cancer, 2016;63(7):1283-6. doi: 10.1002/pbc.25975
    17. Tasian, S. K., Loh, M. L., & Hunger, S. P. (2017). Philadelphia chromosome-like acute lymphoblastic leukemia. Blood, 130(19), 2064-2072. doi:10.1182/blood-2017-06-743252
    18. Terwilliger, T., & Abdul-Hay, M. (2017). Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer J, 7(6), e577. doi:10.1038/bcj.2017.53
    19. Thai Hoa Tran and Mignon L. Loh. (2016). Ph-like acute lymphoblastic leukaemia. Haematology Am Soc Haemaetolo Educ Program, 2016(1), 561-566.
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    Results of College Written Exam (Council Meeting 16 July 2019)

    Wed, 2019-07-17 18:42

    Results of College Written Exam in Chemical Pathology & Clinical Microbiology and Infection 2019
     

    EXAM NO.

    RESULT

    E19213

    PASS

    E19214

    PASS

    E19216

    PASS

    E19217

    PASS


    Candidates who failed in written exam cannot proceed further in Fellowship Assessment in 2019.
    Passed candidates can proceed further in Membership or Fellowship Assessment in 2019.

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    Year 2019

    Thu, 2019-05-30 18:25

    Volume 28, Issue 2 (click here to download the full pdf version)

    Message from the President

    Dear Fellows and Members,

    In this issue of College Newsletter, I have to bring very sad news to all Members and Fellows. Our Past President, Dr. Michael Suen passed away peacefully on 6 January 2020 after battling against cancer for over 2 years. Dr. Suen was a leader with traditional Chinese wisdom, an excellent practising pathologist, a resourceful and encouraging mentor, a good friend with whom you can share your feelings, a caring husband and father. He was a great role model for our community, College, trainees, colleagues and his family members. We will never forget his smiling face and warm words of encouragement, in particular, during times of difficulty. May Dr. Suen rest in peace.

    On the other hand, there is a series of good news for our College in the past year. The breaking good news is the award of Professor Dennis Lo, Li Ka Shing Professor of Chemical Pathology, who was admitted as Honorary Fellow of The Hong Kong Academy of Medicine in December 2019 in recognition of his contribution to the field of cell-free plasma DNA for non-invasive prenatal and cancer diagnostics.

    The second good news is the award of Distinguished Young Fellows 2019 to Dr. Elaine Au, Consultant Immunologist and Dr. David Christopher Lung, Consultant Microbiologist. They received the award from Professor CS Lau, Academy President, in September 2019.

    The third good news is the successful Membership Examination and Fellowship Assessment for candidates in Anatomical Pathology, Chemical Pathology, Clinical Microbiology and Infection, Forensic Pathology as well as Haematology in August and September 2019.

    The fourth good news is the successful completion of the First Fellow Assessment for candidates in Genetic and Genomic Pathology in October 2019. The College Examinations could not have been successfully conducted without the help of our External Examiners, Chief Examiners and Local Examiners. During the past months of social unrest, some of our External Examiners were not able to come to Hong Kong physically. Tele-communication was adopted to be the alternative option by the College and Academy. Thank you for the hard work and contributions from all examiners.

    The fifth good news would be the award of The DS Nelson Trainee Oral Prize to Dr. Timothy Cheng, at the ‘Pathology Update 2019’ Conference in Melbourne. The title of his presentation was ‘Noninvasive Detection of Bladder Cancer by Shallow-Depth Genome-Wide Bisulfite Sequencing of Urinary Cell-Free DNA for Methylation and Copy Number Profiling’.

    In this issue, the Topic Update was on “Recent Advances in Acute Lymphoblastic Leukaemia “ by Dr. Albert Sin, and the Out of the White Coat Interviewee was Dr. Clarence Lam, Consultant Haematologist.

    The International Pathology Day Pre-Workshop was conducted to induce medical students to explore various disciplines of pathology in November 2019. Again due to social unrest, the International Pathology Day was postponed to a Saturday before Christmas when it was successfully conducted at the Pathology Teaching Laboratory of The Chinese University of Hong Kong, Prince of Wales Hospital.

    Finally, let me wish all of you a Prosperous Chinese New Year of the Rat!


    Dr. CHAN Ho Ming
    President

    Jan 2020

    Volume 28, Issue 1 (click here to download the full pdf version)

    Message from the President

    This is the first issue of our College Newsletter in 2019. In 2018, College was busy with conducting the activities associated with the new training programme in Genetic and Genomic Pathology including the second open forum and First Fellow application. We are now processing numerous applications. An interview will be conducted in October 2019 to finalise the eligibility.

    In 2018, the Hong Kong Academy of Medicine was also celebrating her 25th Anniversary with a series of fascinating events including the Congress of Medicine and Gala Dinner with an impressive drummer performance at the opening. Our Fellow, Prof. YUEN Kwok Yung was the Sir David Todd Orator in 2018. He presented his journey to becoming a renowned microbiologist with multiple showcases of Sherlock Holmes-style investigations for our community. In early 2019, the Academy also conducted a visit to Sichuan for our Young Fellows, so as to increase their understanding of the development of specialist care in Mainland China. Dr. MAK Siu Ming and Dr. CHENG Shui Ying, Ivy, represented our College to participate in this visit.

    In March 2019, the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPA-QAP) held one of their 30th Anniversary Quality Symposia in the Pao Yue Kong Auditorium at the Hong Kong Academy of Medicine Jockey Club Building. Local and overseas speakers came to give talks and opinions on how quality can be exercised as well as the future of quality in pathology. What an enjoyable experience-sharing opportunity!

    In the recent issue of Topical Update, Dr. CHONG Yeow Kuan, Calvin reviewed the technological development of biochemical genetics on three major categories of inherited metabolic diseases. With the introduction of expanded newborn screening using mass spectrometry, there is increasing awareness of these conditions in the community.

    Last but not the least, Dr. CHAN Sheung Wai, Gavin will share his concept of modern mortuary development, with a life paradigm shift from just being a place for the dead to a decent environment of bereavement for the family.

    I would like to thank all the above Fellows who have contributed so much to the build a positive image of pathologists. Hope you enjoy this issue of Pathologue!


    Dr. CHAN Ho Ming
    President

    May 2019
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    Results of College Supplementary Autopsy Exam 2018 (Council Meeting 19 March 2019)

    Tue, 2019-03-19 21:09

    Results of College Supplementary Autopsy Exam 2018 (Council Meeting 19 March 2019)

     

    Exam No.

    Result

    ES18101

    PASS

     

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    Biochemical genetics in the expanded newborn screening era

    Thu, 2018-12-27 16:05
    topical_update_v14i1

    Biochemical genetics in the expanded newborn screening era

     

    Volume 14, Issue 1 January 2019  (download full article in pdf)

     

    Editorial note

    In this topical update, Dr. Calvin Chong reviews and updates on the technological development of biochemical genetics for the three major classes of metabolic disorders, namely aminoacidopathies, organic acidurias and fatty acid oxidation defects. These conditions have increasing local awareness, in particular with the introduction of universal expanded newborn screening. With a rising clinical demand for confirmatory tests in biochemical genetics, the ways to achieve better analytical quality and capacity were discussed. We welcome any feedback or suggestions. Please direct them to Dr. Sammy Chen (email: 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.

    Fig 0

    Dr. Calvin Yeow-Kuan Chong
    Department of Pathology, Princess Margaret Hospital


    Introduction


    Biochemical genetics refers to the diagnosis of genetic disorders with biochemical markers. Sir Archibald Garrod first described the biochemical features of alkaptonuria in 19021, and is often named as the founding father of biochemical genetics. Throughout the past century, the practice of biochemical genetics has evolved from spot chemical tests towards the use of chromatography and mass spectrometry2,3. The recent implementation of the pilot study of expanded newborn screening means, on one hand, disorders are diagnosed earlier, and patients with such conditions would fare better, and on the other hand, this represents an increase in the use of confirmatory tests in biochemical genetics which is most acutely felt at the major chemical pathology laboratories.

    The screening panel of the government-initiated pilot study included 8 aminoacidopathies, 7 organic acidurias, 6 fatty acid oxidation defects, and 3 other disorders4. Apart from the three disorders which required separate measurements, all three other major classes of disorders (viz. the aminoacidopathies, organic acidurias, and fatty acid oxidation defects) are screened by the use of tandem mass spectrometric measurement of succinylacetone, amino acids and acylcarnitines, all done within a period of around two minutes.

    Table 1 lists the confirmatory markers and tests for the screened conditions 5,6: as can be seen, plasma amino acids, urine organic acids, and plasma acylcarnitines represents the main bulk work as confirmatory tests for these conditions. The present article aims therefore to explore the contemporary techniques available for these three major confirmatory tests in biochemical genetics and attempts to identify the approaches a laboratory may seek in order to cater for the increased demands.

    Fig t

    Table 1. Confirmatory markers and tests for conditions covered in the government-initiated pilot study of expanded newborn screening. Specialized tests not discussed in the present article are italicized.

    Amino acids


    Quantitative analysis of amino acids in plasma is the first-line confirmatory test for most aminoacidopathies. Amino acids are characterized by the presence of primary amine and carboxylic acid groups in one molecule, though some imino acids, namely proline and hydroxyproline, containing an imino (a functional group containing carbon-nitrogen double bond) as well as a carboxylic acid groups are also considered under the umbrella term amino acids in medical parlance and this use of terminology is adopted in the present article.

    The analytical difficulties for amino acids are obvious when one examine their chemical structures: they are amphoteric, very small (glycine has a molecular mass of 75.067 g/mol), and most of them do not possess conjugated double bonds which gives absorbance in the ultraviolet spectra7. The first and second characteristics make separation difficult when reverse phase chromatography is used, whereas the last two give rise to problems in mass spectrometric detection and ultraviolet detection respectively. There are three major methods commonly used in clinical laboratories: amino acid analysers, high performance liquid chromatography (HPLC) with pre-column derivatization, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Irrespective of the methods employed, protein precipitation is necessary before analysis.

    Amino acid analysers

    Amino acid analysers (AAAs) are standalone machines that employ cation-exchange chromatography using a lithium buffer system, with post-column derivatization with ninhydrin (at some 120-135 degree Celsius) and monitoring at two wavelengths8. It is considered the gold standard method for amino acid analysis. This method overcomes the difficulty in separation of amino acids by utilizing ion-exchange chromatography which relies on the charge of a molecule at a particular pH rather than the hydrophobicity and steric interactions of a molecule and that of detection by formation of purple complexes which absorb strongly at 570 nm for primary amino acids, and yellow complexes which absorb strongly at 440 nm for proline and hydroxyproline9,10. When compared with other methods, AAAs provides higher degree of automation and require less expertise from the laboratory; the major issues with AAAs are the lengthy analytical run (120 minutes is common), the cost of the analytical instruments and the proprietary nature of the reagents. Analytical interference in AAAs are rare but do occur with dipeptides, which occur in prolidase deficiency8,11, and aspartylglucosamine, which occur in aspartylglucosaminuria8, can be spotted by the 570/440 absorbance ratio.

    HPLC and LC-MS/MS methods

    HPLC coupled with pre-column derivatization is used in the author’s laboratory for PAA determination. The method relied on the pre-column (immediately before injection) derivatization of amino acids by orthophthalaldehyde (OPA) and 3-mercaptopropionic acid (MPA), followed by reversed phase chromatography and ultraviolet detection12-14. In the presence of a thiol reagent, OPA forms a fluorescent derivative with primary amino acids with peak absorbance/excitation at 340 nm and emission wavelength at 450 nm7,8. The major drawback of this method is the inability to detect proline (and hydroxyproline) as OPA does not react with imino acids.

    A newer derivatization reagent, AccQ-Tag (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate), which converts both primary and secondary amino acids into stable fluorescent derivatives, has been used with ultra-high performance liquid chromatography with ultraviolet detection15. This derivatization reagent has the advantages of covering both primary and secondary amino acids and as the derivatization products are stable, advanced autosamplers which can pipet reagent from one vial to another is not required16. Complete chemistry kit-sets that use this derivatization reagent is commercially available17.

    Liquid chromatography coupled with tandem mass spectrometry is also used locally for PAA determination. As these methods are often based on reversed phase chromatography, despite the use of mass spectrometric detector, derivatization is still often employed7. A method based on the proprietary derivatization reagent AccQ-Tag has been recently described in the literature with a run-time of 6 minutes18. Isotopic internal standards are required for LC-MS/MS-based methods and it is important to note that the impracticality of having isotopic internal standards for each and every analyte would mean that the robustness of the assay is considerably weaker than optical-based methods19.

    Compared with methods based on optical detection, LC-MS/MS methods had a shorter analytical runtime and better specificity. This is counterweighed by the higher capital and maintenance cost of acquiring an LC-MS/MS (and not to mention the cost of having a backup LC-MS/MS system), as well as the technical expertise necessary to operate and troubleshoot an LC-MS/MS.

    Choice of method and the future

    The question is different depending on the volume of testing as well as equipment availability. For laboratories that has existing HPLC-DAD equipment employing OPA derivatization there may be little incentive in adopting a new procedure: there is probably little competition for HPLC analyser time, and the inability to detect proline may not be a major issue as hyperprolinaemia type I is benign whereas hyperprolinaemia type II is classically diagnosed by the presence of N-(Pyrrole-2-carboxyl)-glycine in urine organic acids20.

    On the other hand, for a laboratory seeking to provide amino acids analysis for the first time, the use of stable derivatization reagent such as AccQ-Tag mentioned above has the advantage of not requiring higher-end liquid chromatographs and the availability of commercial kits means that development time is reduced. For a laboratory anticipating a high workload, a dedicated liquid chromatograph with UV detection appears to be the simplest solution as it is robust and inexpensive. On the other hand, for laboratories with lower service demand, mass spectrometric detection may in fact be more feasible as the notion of spare LC-MS/MS capacity means that the major downside of LC-MS/MS detection, viz capital cost and technical expertise, are sunk cost to the laboratory.

    Organic acids

    Organic acidurias are diagnosed most commonly by urine organic acid analysis with gas chromatography-mass spectrometry (GC-MS)21, and this technique is used by many local hospitals. Though the term organic acids refers to organic compounds with a carboxylic acid group, a broader spectrum of compounds, such as uracil and xanthine, are detected in practice. Urine organic analysis is usually qualitative though quantitative analyses of some compounds (e.g. orotic acid, methylmalonic acid) are often offered.

    GC-MS based urine organic acid analysis

    For GC-MS analysis of urine organic acids, a creatinine-corrected amount of urine is subject to liquid-liquid extraction with ethyl acetate after acidification using hydrochloric acid, the organic extract is then dried and derivatized by N,O-bis-tri(methylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)22. The resultant product is injected to the GC-MS operating in scan mode. For reliable detection of ketones, oximation with hydroxylamine hydrochloride can be performed prior to acidic extraction23.

    An alternative way of performing urine organic acid analysis is to bypass the extraction step. As no acidic extraction step is done, a limited panel of amino acids, purine, pyrimidines, and mono- and di-saccharides can be detected in the same analytical run. This method has been in-use in the author’s laboratory in the past 2 decades24,25, and allows the detection of a much wider range of metabolites in urine. With no extraction, the chromatograms are extremely complex due to co-elution of analytes and therefore this approach requires expertise in post-analytical data processing to be viable as a routine method.

    The interpretation of GC-MS data for urine organic acid analysis is commonly based on examination of the chromatogram (Figure 1), followed by a combination of peak integration in the total ion chromatogram followed by library searching and examination of extracted ion chromatogram at particular retention times for analytes which gives lower responses (Figure 2). The raw analyte response is then compared to locally-established age-specific reference intervals which allow clinical interpretation23.


    Fig 1

    Figure 1. Total ion chromatogram of a urine sample from a patient with malonic acidemia circulated in the ERNDIM qualitative organic acid program in 2016. The two abnormal peaks are indicated by asterisks: the first peak represents malonic acid (di-TMS derivative) and the second peak represents methylmalonic acid (di-TMS derivative).


    Fig 2

    Figure 2. Extracted ion chromatogram showing the quantifier ions (m/z 375, for aconitic acid in red; and m/z 254, for orotic acid, in black) and qualifier ions (m/z 285, for aconitic acid in green; and m/z 357, for orotic acid, in blue). They are co-eluting in many GC-MS based urine organic acid assays.


    LC-MS based urine organic acid analysis

    In the recent years, liquid chromatography-mass spectrometry-based methods have been published for analysis of urine organic acids. The benefit of liquid chromatography-mass spectrometry is clear: there is no need for the cumbersome derivatization step, and heat-labile analytes can be detected26,27. The major drawbacks of LC-MS based methods are the poorer separation of analytes and higher susceptibility towards ion suppression, as electrospray ionization is much less robust against matrix effects compared to electron ionization as used in GC-MS based methods28. The difficulty of developing an in-house LC-MS based method for urine organic acids lies in the procurement of a practically endless list of organic acid standards to establish the retention time and multiple reaction monitoring ratios. At the time of writing, there is at least one commercial kit that has been made available (Zivak Organic Acids LC-MS/MS analysis kit), but this commercial kit did not utilize isotopic internal standards even for critical analytes (such as hexanoylglycine and orotic acid) and one may wish to validate extensively the robustness of such assays against matrix effects.


    Choice of method and the future

    Out of the three assays discussed in the present article, urine organic acid analysis represents the most difficult of the three to establish in a laboratory. There is first the difficulty in establishing age-specific reference intervals, and then difficulty in acquiring a large number of chemical standards. A good starting point would be obtaining the bi-level quality controls for urine organic acid and special assays for urine from the ERNDIM network which consist of a number of critical analytes. As for choice of internal standards, while traditional choices such as tropic acid and pentadecanoic acid are often employed, deuterated internal standards covering critical analytes are much more available nowadays (e.g. methylmalonic acid-d3 and orotic acid-1,3-15N2) and their addition may improve quantitation of these critical analytes, the former being commonly quantified and the latter being prone to analytical errors29.

    For laboratories with existing GC-MS based urine organic acid assay, the question is probably whether to adopt LC-MS based solution. The belief that organic aciduria always results in sky-high level of abnormal metabolites in urine is flawed: the low-excretor phenotype of glutaric aciduria type I can serve as the classical example30. The difficulty may be mitigated somewhat if acylcarnitines and acylglycines are measured by the LC-MS based assays as glutarylcarnitine has been shown to be informative even for low-excretor GA I patients31. Overall, it remains to be seen whether LC-MS based organic acid analysis could stand alone replacing, rather than complementing, GC-MS based assays.


    Free carnitine and acylcarnitines


    Quantitative analysis of free carnitine and acylcarnitines in plasma represents the first-line confirmatory test for fatty acid oxidation disorders. Analysis of free carnitine with calculation of fraction of excretion can be helpful in the diagnosis of carnitine uptake defect, as is measurement of urine glutarylcarnitine for the diagnosis of glutaric aciduria type I as discussed above31. Carnitine is a quaternary ammonium compound with a carboxylic acid group, as well as a hydroxyl group with which acyl groups are attached to form acylcarnitines; as such, it forms zwitterions under physiological pH. Derivatization to esters by incubation with an alcohol (typically butanol) has been employed32 though locally underivatized analysis at acidic pH is commonly used33.

    For underivatized analysis, plasma is first diluted with a mixture of isotopic internal standards, and acidified acetonitrile is slowly added to precipitate plasma proteins. The sample is vortexed, centrifuged, followed by evaporation of the supernatant to dryness and reconstituted for analysis by positive electrospray ionization-tandem mass spectrometry with or without liquid chromatography separation. Data can be collected in multiple reaction monitoring (MRM) mode, or in precursor ion scan with accumulation of ions, commonly known as multichannel acquisition (MCA) mode34 (Figure 3). The American College of Medical Genetics Guideline, published in 2008, suggested the use of precursor ion scan as it permitted the evaluation of the whole acylcarnitine profile, as well as the detection of drug artefacts, interfering compounds and assessment of derivatization35.

    Fig 3

    Figure 3. Cumulative precursor scan mass spectrum for a blood-spot sample distributed in the ERNDIM Qualitative Acylcarnitine Program in 2014. In this specimen, elevated signals at m/z ratio 260 (C6-carnitine), 288 (C8-carnitine), and 314 (C10:1-carnitine) can be seen. The pattern would be compatible with MCAD deficiency. (Mass spectra courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)


    Derivatization and chromatographic separation

    In the analysis of carnitine and acylcarnitines, butyl-ester derivatization enhances the formation of positively-charged ion by reacting with carboxylic groups, and causes mass separation of dicarboxylic-carnitines and hydroxy-acylcarnitines (e.g. C4DC-carnitine and C5OH-carnitine), which are isobaric when underivatized36. Derivatization is typically performed at highly acidic conditions (e.g. 3N hydrochloric acid at 65°C for 15 minutes)35. On the other hand, chromatographic separation allows the separate determination of individual isomeric constituents (e.g. C4DC-carnitines include succinylcarnitine and methylmalonylcarnitine; and C5OH-carnitines include 3-hydroxyisovalerylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine) (Figure 4). With meticulous chromatographic separation, most biologically relevant isomeric species could be separately quantified37.


    Choice of method and the future

    The question for the laboratory is, first, whether to employ the derivatization procedure. The advantage of derivatization is the mass separation of hydroxylacylcarnitines and carnitine derivatives of dicarboxylic acids; the problems associated with derivatization are the partial hydrolysis of acylcarnitines because of the high temperature and strongly acidic condition employed33. The other considerations are whether to employ chromatography and if so, how extensive should it be: it would then be a delicate balance between through-put, diagnostic specificity, and analyser-time that is available. With the improvement of separation capability of liquid chromatographs and sensitivity of modern mass spectrometers, it is suggested that a short UHPLC program combined with both scheduled MRM and precursor ion scan function would be a good compromise.

    Fig 4

    Figure 4. SRM chromatogram of m/z 262>85 showing chromatographic separation of different species of isobaric (C4DC/C5OH) acylcarnitines (viz. succinylcarnitine at 4.68 minutes, two diastereomeric peaks of methylmalonylcarnitine at 5.2 and 5.35 minutes, and 3-hydroxyisovalerylcarnitine at 5.82 minutes) could be individually identified and quantified with liquid chromatography-tandem mass spectrometry without derivatization. (a) sample with normal amounts of succinylcarnitine; (b) sample with abnormal amounts of methylmalonylcarnitines (Chromatograms courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)


     

    Conclusions


    The three assays discussed above represent the bulk of workload for most metabolic laboratories. The planned implementation of universal expanded newborn screening in Hong Kong means that the demand would increase, and the phenotype will be less defined, as tests are requested for patients who may not yet present with features of an inborn error and importantly, patients with only borderline elevation of analytes.

    From a Bayesian point of view, this change in pre-test probability would mean that, if the analytical quality and interpretative capacity remains the same (which affect the likelihood ratio of positive results), the post-test probability would suffer from a negative impact. The quest for the metabolic bench of any major pathology laboratory is then to improve both throughput and quality of analysis at the same time, an impossible task, as the Duke of Norfolk wrote in 1538, “a man can not have his cake and eat his cake”38. The technology improvements as reviewed in the present article may aid in the analytical quality but the quest for improved interpretative capacity remains on the training and education of our present and coming generations of pathologists.



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    Abbreviations:
    Plasma amino acids (PAA):
    Arginine (Arg), Citrulline (Cit), Homocystine (Hcy), Isoleucine (Ileu), Leucine (Leu), Methionine (Met), Ornithine (Orn), Phenylalanine (Phe), Threonine (Thr), Tyrosine (Tyr), Valine (Val).

    Urine organic acids (UOA):
    ethylmalonic acid (EMA), glutaric acid (GA), 2-hydroxyglutaric acid (2-OHGA), 3-hydroxyglutaric acid (3-OHGA), 3-methylglutaric acid (3-MGA), 3-methylglutaconic acid (3-MGCA), hexanoylglycine (HG), 3-hydroxyisovaleric acid (3-OHIVA), Isovalerylglycine (IVG), 3-methylcrotonylglycine (3-MCG), Methylcitric acid (MCA), Methylmalonic acid (MMA), 3-hydroxypropionic acid (3-OHPA), propionylglycine (PG), phenylpropionylglycine (PPG), Tiglylglycine (TG).

    Plasma acylcarnitines (PAC):
    Free carnitine (C0).

    Others:
    6-pyruvoyl-tetrahydropterin synthase (PTPS), 3-hydroxy-3-methylglutaryl CoA (HMG-CoA), Carnitine-acylcarnitine translocase (CACT), Carnitine palmitoyltransferase II (CPT-II), Medium-chain acyl-CoA dehydrogenase (MCAD), Very long-chain acyl-coA dehydrogenase (VLCAD).
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