2019 College Annual Report and Year Book 2018-19

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

Annual Report 2019

Year Book 2018-19 2019

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

Fellowship Assessment:

Membership Examination:

Recent Advance in Acute Lymphoblastic Leukaemia

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

Editorial note

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

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

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.

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

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.

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

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

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.

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 spectrometry^2,3. 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.

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

Organic acids

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 decades^24,25, 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.

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).

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 detected^26,27. 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

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)

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.

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

Reference

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3. Schulze A, Lindner M, Kohlmüller D, Olgemöller K, Mayatepek E, Hoffmann GF. Expanded newborn screening for inborn errors of metabolism by electrospray ionization-tandem mass spectrometry: results, outcome, and implications. Pediatrics. 2003;111(6):1399–406.

4. Mak, CM. Newborn Screening: Past, Present and the Future. Topical Update, The Hong Kong College of Pathologists. 2016;11(2):1–10.

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6. Saudubray J-M, Berghe G van den, Walter JH. Inborn Metabolic Diseases. Springer-Verlag Berlin Heidelberg; 2012.

7. Kaspar H, Dettmer K, Gronwald W, Oefner PJ. Advances in amino acid analysis. Anal Bioanal Chem. 2009 Jan;393(2):445–52.

8. Duran M. Amino Acids. In: Blau N, Duran M, Gibson KM, editors. Laboratory Guide to the Methods in Biochemical Genetics [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited 2018 Nov 24]. p. 53–89. Available here.

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10. Cunico RL, Schlabach T. Comparison of ninhydrin and o-phthalaldehyde post-column detection techniques for high-performance liquid chromatography of free amino acids. Journal of Chromatography A. 1983;266:461–70.

11. Ferreira CR, Cusmano-Ozog K. Spurious Elevation of Multiple Urine Amino Acids by Ion-Exchange Chromatography in Patients with Prolidase Deficiency. JIMD Rep. 2016 Apr 12;31:45–9.

12. Fürst P, Pollack L, Graser TA, Godel H, Stehle P. Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials. Journal of Chromatography A. 1990;499:557–69.

13. Schuster R. Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications. 1988;431:271–84.

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Application for First Fellow in Genetic and Genomic Pathology is now opened and please download the application form here.

Results of College Exam 2018 (Council Meeting 27 September 2018)

Fellowship Assessment:

Membership Examination:

Results of College Written Exam in Chemical Pathology, Clinical Microbiology & Infection, and Haematology 2018
 

Candidates who failed in written exam cannot proceed further in Fellowship Assessment in 2018.
Passed candidates can proceed further in Membership or Fellowship Assessment in 2018.