Good afternoon, Prof. Gilberto Leung, Dr. the Honorable David Lam, Dr. Teresa Li, Dr. Law Chun Bon, Presidents and representatives from Sister Colleges, Fellows, Members, ladies and gentlemen,

Welcome you all to the conferment ceremony of our College this afternoon.

Congratulations:

Today is a very special day for our new Fellows. This is a very important milestone, and it signifies the beginning of the next phase of your career. May I take this opportunity to congratulate you, and on your behalf, thank your family, including your parents, your significant others and your children, for their unfailing support and encouragement; also thank your mentors, trainers and colleagues for their guidance; and thank the patients you have helped to manage, who have provided you with the training opportunity.

Chinese: 我代表香港病理學專科學院新院士多謝屋企人多年來嘅支持和包容，令他們現在可以成為香港病理學專科的新生力軍，為香港市民服務。希望你們在未來可以繼續支持他們，因為香港醫療界正面對許多的挑戰。

For those Members in the audience who have already passed our intermediate exam, I am sure it will be your turn very soon to come up here for your Fellowship conferment.

CME/CPD reminder and Genetic and Genomic Pathology:

To our new Fellows: Your future practice requires you to be competent in your field of practice. CME/CPD, continuing medical education/continuous professional development, is essential for your career development, and I believe Prof. Leung from the Academy will remind you again later.

Artificial Intelligence:

I am sure you have all heard a lot about Artificial Intelligence (A.I.) in the news recently, especially regarding the recent appointment of the CEO of OpenAI. A.I. has been a hot topic for some time, and its development has been remarkable, particularly in the field of medicine, including pathology. While this topic can easily fill an entire week of seminars and lectures, I will just highlight a few key points here:

The Academy and the College have released a policy on using Large Language Models (LLM) such as ChatGPT in written assignments of specialist trainees. I would like to emphasize that our College policy on Large Language Models does not impede their use. On the contrary, we believe it is crucial for our trainees to understand the advantages and limitations of these applications and the importance of proper declaration of their usage. By the way, to ensure the originality, I have chosen not to utilize ChatGPT to draft this speech.

A number of my friends have expressed concerns about the impact of A.I. on the field of pathology. However, as a pathologist, I believe that A.I. should be seen as an opportunity rather than a challenge. I draw a parallel to the discussions we had about the impact of genetic and genomic pathology in the past. Initially, there was a perception that molecular diagnosis could replace a significant portion of our work. But over time, we realized the pivotal role pathologists play in this field, especially in the era of personalized medicine. The College has now integrated Genetic and Genomic Pathology into various training programmes. Similarly, A.I. can enhance our efficiency and efficacy as pathologists, streamlining our diagnostic processes. It is therefore crucial for us to embrace this technology and update our knowledge on its utilization. A.I. will be a promising tool in the future.

Faculty Development and Position Paper:

Speaking of the future, I had the honour of participating in the Tripartite Medical Education Conference and the Strategic Planning Retreat on Education and Training 2023, organized by the Academy earlier this year. The purpose of these events was to formulate actions for the future of postgraduate medical education. The Academy has published a position paper that clearly outlines the recommendations made in various areas, including Faculty Development, training based on competency-based medical education, and the implementation of train-the-trainers and train-the-examiners programs.

In line with the Academy’s directions, our College has recently revised our > and the logbooks to incorporate the new concept of workplace-based assessment. This marks our College’s initial step towards implementing competency-based medical education, aligning with Academy’s future direction. Our College is also actively planning to contribute to the development of training programmes for the Shenzhen-HK Medical Specialist Training Centre, giving full support for the Academy’s effort to strengthen our collaboration with the Shenzhen medical profession.

International Liaison of Pathology Presidents (ILPP) 2023:

I would also like to highlight the International Liaison of Pathology Presidents (ILPP) meeting that our College hosted last month. ILPP is a platform for interaction and exchange of ideas among various overseas Pathology Presidents from around the world, including the United Kingdom, Ireland, Europe, Australia, Malaysia, United States, Canada and a representative from the World Health Organisation. During the two-day ILPP event, topics such as manpower shortage, professionalism and ethics, competency-based training, the adoption of digital pathology, the role of scientific officers and medical technologists, and so on, were all discussed. This ILPP has reaffirmed our unwavering commitment to maintaining international collaboration to advance the field of pathology, foster global sharing of ideas and knowledge, and collectively address the challenges and opportunities that lie ahead.

Young Fellows Chapter, Facebook and other Social Medias:

May I also take this opportunity to mention the Academy Young Fellows Chapter (YFC) and our College's active participation in it. I am honoured to have served as the first College representative in the Academy Young Fellows Chapter and as the inaugural Chairman of my College’s Young Fellows Chapter. I would like to express my sincere gratitude to Prof. Gilberto Leung and Prof. Lau Chak Sing for establishing the Academy YFC, which provides an excellent platform for Fellows from various sister Colleges to work and collaborate together.

To further engage our young fellows, we actively update our Facebook page. I invite you to come visit us on Facebook, give us “likes” and leave some comments. I have also created my personal Facebook and Instagram accounts, where you can learn more about my College responsibilities in the days to come.

About dinner:

This year, our College is finally able to host our Annual Dinner after the COVID-19 pandemic. However, due to a sister College hosting her AGM and Conferment Ceremony on the same day, some of our guests may not be able to attend. We encourage our College Fellows, Members and Associates to stay for the T.B. Teoh Foundation Lecture on “Everything Immunology, from diagnosis to treatment” by Prof. Lau Chak Sing, followed by the Annual Dinner. Let's enjoy this long-awaited event after its cancellation in the past few years due to COVID-19.

Lastly, I would like to express my gratitude to all those who have contributed to the preparation of this event. It has been a lot of hard work, as always. I wish everyone the best in the days to come.

Dr MAK Siu Ming

President

25 November 2023

Results of College Examination 2023 (Council Meeting 12 October 2023)

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Results of College Examination 2023 (Council Meeting 28 September 2023)

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Results of College Written Examination 2023 (Council Meeting 19 July 2023)

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Melioidosis: an urban outbreak in Hong Kong

Dr. Kristine LUK, Dr. May LEE and Dr. Wing Kin TO

Consultant Microbiologists, Department of Pathology, Princess Margaret Hospital, Hospital Authority

Volume 18, Issue 2, July 2023  (download full article in pdf)

Editorial note:

There was a significant upsurge of cases of melioidosis in Hong Kong in 2022, especially in the Kowloon region, raising public awareness to the condition. In this issue of the Topical Update, Drs. Kristine Luk, May Lee and WK To share their experience in investigating and managing the cases. We welcome any feedback or suggestion. Please direct them to Dr. Janice Lo (e-mail: janicelo@dh.gov.hk), Education Committee, The Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Volume 32, Issue 2 (click here to download the full pdf version)

Message from the President

It is with great pleasure that our College can finally host our Annual Dinner once again in 2023, following the return to normalcy in Hong Kong. I look forward to meeting all of you in the coming Annual General Meeting and the Conferment Ceremony. This year, we have offered the option of vegetarian dinner, and we have planned to introduce on-site download of event photos. We hope you will appreciate our new initiatives, and we shall review their popularity afterwards.

On top of organizing the regular College activities taking place each year, our College is particularly busy this year because of two special events.

The International Liaison of Pathology Presidents (ILPP) annual meeting was hosted by our College in Hong Kong on 30-31 October 2023. The last time the meeting was held in Hong Kong was back in 2010. Although it is demanding to be the host organization, it has provided us with an opportunity to introduce Hong Kong to the Presidents or representatives of other Pathology Colleges or organizations in the world.

Other than preparing for the meeting and the related social activities, we have also coordinated with the International Academy of Pathology, Hong Kong Division, to co-host an ILPP session during their Fall Scientific Meeting on 29 October 2023. The session was well received, and it covered hot topics such as artificial intelligence, digital pathology and globalization.

During the ILPP meeting, members shared the challenges and practice of their localities. It was a fruitful meeting for information sharing and network building. Hosting it in Hong Kong has also provided a precious opportunity for some of the younger Fellows to meet these international leaders in Pathology.

We were very lucky that the event went smoothly, and the weather has been on our side. I believe all of you will know how uncommon it is to see Victoria Harbour with a clear blue sky! The guests enjoyed the meeting and our hospitality, and I am very proud of our team in making this meeting a success. A big thank you to all those who have contributed to this exceptional event.

In addition to organizing the ILPP meeting, the other major task which our College has embarked on this year is the revision of the Regulations on Postgraduate Training and Examinations and the related logbooks. This labour-intensive task involves various Specialty Boards and the Training & Examinations Committee (TEC). The new version will be rolled out soon after endorsement of the regulations by the Education Committee of the Hong Kong Academy of Medicine. Detailed information will be promulgated by TEC in due course.

This year marks the end of my College Presidency. After nearly two decades in the College Council, I am happy to see many young and energetic successors ready to take up the future challenges of the College. These past few years have been exceptionally difficult for the medical profession and our College. I would like to express my sincere thanks to all Council members, College Secretariat and all those who have contributed to the College in many different ways.

Dr. CHAN Chak Lam, Alexander
President

November 2023

Volume 32, Issue 1 (click here to download the full pdf version)

Message from the President

Wish you all a Happy New Year of the Rabbit.

Our College hosted our Annual General Meeting (AGM) and the Conferment Ceremony successfully in 2022 despite the COVID-19 outbreak. Congratulations once again to all the new Members and Fellows. We are very fortunate that Dr. Michael BUCKLEY, our Honorary Fellow and the speaker of the TB Teoh Foundation Lecture, has attended the event in person despite the restriction related to the outbreak. Dr. Buckley’s enlightening lecture, “Exomes and Genomes: from Bedside to Bench-top", was very well received.

The 18th Trainee Presentation Session was also held on the same day of AGM. All the trainees who have participated in the session have done a great job, and congratulations to the winner Dr. Aden CHAN of Anatomical Pathology for his study “Combinations of Single Gene Biomarkers can Precisely Stratify 1,028 Adult Gliomas for Prognostication”. Thanks to Dr. Derek YAU, Dr. Christopher LAI and to all the judges for making the session a success.

The continuing medical education/continuous professional development (CME/CPD) 3-year cycle 2020-22 has just ended, and a new 3-year cycle (2023-25) has started. On behalf of the Education Committee, may I remind our new Fellows that CME/CPD is important in your career development and future practice. Failed compliance may result in suspension of your Academy’s Fellowship and your specialist registration status.

The coming year is a busy year for Training and Examinations Committee (TEC). TEC is currently engaging various Specialty Boards in the review of our training programmes. We are grateful for the advice we have received from our Academy’s Educationist, Dr. SO Hing Yu. Concurrent with the training programme review exercise, our College is also planning to contribute to the development of training programmes for the SZ-HK Medical Specialist Training Centre. This is in support of the Academy’s initiative to strengthen our collaboration with the Shenzhen medical profession.

These few years have been challenging times for all of us. Other than taking care of our patients, we also need to take good care of ourselves and those around us. Academy has taken various initiatives to promote the well being of doctors. Helpful material is available in the Academy’s website, and peer supporters (with some from our College) are ready to help. Our younger generation is encountering lots of challenges and uncertainties in life. As trainers and seniors, we should be supportive. On the other hand, I hope our younger members can also understand that the trainers are likewise facing unprecedented stress, but you should not hesitate to approach your seniors, colleagues or Academy’s peer supporters in case of need.

The multifactorial manpower shortage issue has great impact on our specialty and other medical professions. Our College will work closely with the Academy, the Hospital Authority, and the government, on exploring various options to tackle the matter. Meanwhile, we hope all new Fellows can stay longer in the public sector, to contribute to the training of our next generation of Pathologists.

Regarding government’s plan to explore empowering Chinese medicine practitioners (CMPs) to prescribe diagnostic imaging and laboratory tests for their patients, our College is working closely with our sister Colleges and the Academy to deliberate with the government and relevant parties. The Academy had a meeting with the Hong Kong Registered Chinese Medicine Practitioners Association on 16 January 2023, exploring the view of some CMPs.

Our College wishes to engage more young Fellows in College activity. After the establishment of the Academy‘s Young Fellows Chapter (YFC), we have set up our College’s own YFC. Some of our young Fellows are currently members of our College Council and some College committees. We treasure their input and contribution, and it is essential for succession planning. Our current YFC Chairman, Dr. Ivy CHENG, is also in charge of our College Facebook page. We hope to use this platform more often to share information, and your comment is most welcome. ( https://www.facebook.com/hkcpath/ )

The Academy’s Conferment Ceremony was held on 16 December 2022. Congratulations to our new Academy Fellows and to Dr. Lois CHOY, our College trainee in Chemical Pathology who won the gold medal of the Academy’s 2022 Best Original Research by Trainees (BORT) on that day.

International Pathology Day Workshop 2022 was held successfully on 17 December 2022. Dr. Rock LEUNG and his team have done an excellent job introducing our profession to over 200 secondary school students.

I represented our College to attend the Opening Ceremony of the New Territories (Shatin) Forensic Medicine Centre on 29 December 2022. The establishment of this new state-of-the-art facility is an important milestone for Forensic Pathology.

The year 2023 marks the 30th Anniversary of the Academy. The Tripartite Medical Education Conference on 14 January 2023 is the first of a series of celebration events, and Dr. MAK Siu Ming (Chairman of Training & Examinations Committee), Dr. Christopher LAI (Chairman of Education Committee) and I have attended the conference. Dr. MAK and I have also joined the Academy’s Strategic Planning Retreat on Education and Training on 4 March 2023, and it was a precious opportunity for experience-sharing amongst all Colleges and the Academy.

The next Academy’s anniversary celebration event will be Health for All, Move Forward Together on 19 March 2023. Our College has formed a competition team for the run, and some College members have also registered for the event.

The Academy has resumed the annual gathering with the media this year. I attended the HKAM Media Spring Tea Reception held on 3 February 2023. It was a good opportunity for the Academy to highlight the latest development of the Academy and promulgate the Academy’s view on some healthcare related policies and initiatives.

The next annual International Liaison of Pathology Presidents (ILPP) meeting has been planned to take place in Hong Kong in October 2023. Hong Kong was the hosting city in 2010. We look forward to hosting it once again after more than one decade.

With return to normalcy, I expect we shall be able to organise the long-overdue College annual dinner in 2023. I look forward to seeing you all.

Dr. CHAN Chak Lam, Alexander
President

March 2023

A Review on Complement Diagnostics

Volume 18, Issue 1, Jan 2023  (download full article in pdf)

Editorial note:

The complement system though commonly regarded as component of the innate immune system that protect our bodies from infection, it has increasingly evident that it has important roles in other immune surveillance and housekeeping functions, that it is involved in a wide and diverse range of clinical conditions. In this review, Dr Elaine Au provided an overview of the complement diagnostics and its clinical applications. We welcome any feedback or suggestions. Please direct them to Dr Elaine Au 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 Au Yuen Ling Elaine

Consultant, Division of Clinical Immunology, Department of Pathology, Queen Mary Hospital

The complement system not only as part of the innate immune system that contributes to the elimination of pathogens, and promote inflammation, it also modulates the adaptive immune response. Though its primary role is in host defense, it also serves an important role in clearance of apoptotic cells and immune complexes. Low or dysregulated activity in complement system has been described in a range of disease and pathological conditions.

The Complement system

The complement system comprises approximately 50 proteins, that are found in fluid phase or bound to cell surface (2). The central complement reaction involves the cleavage of C3 into C3b and C3a, which is promoted by the C3 convertase. Collectively, there are three activation pathways forming the C3 convertase. The classical pathway (CP) is triggered by the immune complexes, while the lectin pathway (LP) is triggered by the binding of mannan-binding lectin (MBL) or ficolins to carbohydrates or pathogen-associated molecular patterns. Both activation of CP and LP would lead to the formation of C4b2a as C3 convertase. On the other hand, in the alternative pathway (AP), there is a constant low-grade hydrolysis of C3, that binds factor B and cleaves factor D to generate a fluid phase C3 convertase, that is self- limited in healthy state. However, the AP will be activated and amplified through binding of the cleaved C3 to pathogens or altered tissues. Hence, AP helps to amplify complement activation initiated from CP and LP. The pathways converge in a common pathway to form the membrane attack complex (C5b-9). In addition, the cleavage of C3 and C5 generates C3a and C5 a, that act as anaphylatoxins, while the target bound C3 fragments (C3b, iC3b, C3d, g) facilitate phagocytosis.

The complement activation is delicately controlled by multiple soluble and membrane bound regulators. Factor H, C4b binding protein, the membrane proteins complement receptor 1 CR1 (CD35), decay acceleration factor (CD55), and membrane cofactor protein MCP (CD46), act as cofactors for plasma proteinase factor I, accelerating the decay of convertases. In addition, CD59 and C1 inhibitor regulate the C5b-9 complex and the C1 complex respectively.

Examples of complement diagnostics indications and associated disease conditions

A broad spectrum of clinical conditions is associated with complement deficiencies or its overactivation / dysregulation. The clinical consequences can be broadly categorized into three areas. 1) susceptibility to infection, 2) autoimmunity and 3) defects in controlling and limiting complement activation.

Infection susceptibility

In general, complement deficiencies are associated with increased risk of infections, especially encapsulated bacterial infections, most commonly Pneumococci, Hemophilus etc. In particular, individuals suffering from deficiencies in the terminal components (C5-C9) or properdin are susceptible to Neisseria infections. Hence, complement studies are indicated in the workup of young individuals suffering from recurrent infections (e.g. recurrent sinopulmonary infections, meningitis, etc), especially in recurrent infections caused by encapsulated bacteria. Nevertheless, primary component deficiency is rare, and most of these conditions are autosomal recessive (X-linked inheritance in properdin deficiency) (1).

Autoimmune diseases

Deficiency in early components of the CP, is frequently associated with lupus like autoimmune conditions. The associations range from 10% prevalence of lupus like conditions in C2 deficiency, to C1r/s (57% prevalence), C4 (75% prevalence) and C1q (90% prevalence) (2). These deficiencies can be confirmed in genetic studies and components measurement. Overall, primary deficiency is relatively uncommon. More often, lupus and other autoimmune immune complex diseases causes secondary complement components deficiency as consumption due to the immune complex activation. The component levels, e.g. C3 and C4 levels, are commonly employed in the workup and disease activity monitoring in these conditions. In some occasions, measuring autoantibodies, such as anti-C1q antibody in hypocomplementemic urticarial vasculitis syndrome (HUVS) and lupus, is useful for diagnosis and prognostication.

C3 nephropathy and Thrombotic microangiopathy (TMA)

Uncontrolled AP activation may result in a number of kidney diseases and systemic conditions. C3 glomerulopathy comprises C3 glumoerulonephritis (C3GN) and dense-deposit disease (DDD), is a pathological condition defined by predominant C3 accumulation, with absent or scantly immunoglobulin deposition. Atypical post infectious glomerulonephritis also falls in the continuum of C3 GN and DDD (3). In these conditions, underlying predisposition, be it genetic or acquired, may not be clinically evident until a triggering event, such as infection or pregnancy, that unfold the complement dysregulation. Besides genetic predisposition, presence of autoantibodies, e.g. C3 nephritic factor (C3 Nef), anti-factor H, have been observed in some patients. C3Nef are autoantibodies that bind to components of AP convertase, prolonging its functional half-life, leading to continuous C3 activation and consumption, with lowish CP and AP studies. Factor H has important role in the regulation of complement activation. In some patients, they are predisposed to the disease due to Factor H dysfunction caued by mutation or anti-Factor H. Useful workup for C3 nephropathy includes the complement pathways, components and activation products studies, testing for plasma cells disorders, determination of autoantibodies (C3 Nef, anti-factor H), along with gene panel (C3, CFH, CFI, CFB, CFHR1-5) testing (3).

aHUS is a primary TMA, that is characterized by uncontrolled AP activation, presenting with microangiopathic hemolytic anaemia, thrombocytopenia and acute renal failure. The dysregulated AP could be caused by mutations of complement regulators, most commonly factor H, and in around 6-10% of cases, by the presence of anti- factor H (4). Initial workup includes investigations to exclude other co-existing medical conditions associated with HUS or other forms of TMA. Similar to the workup of C3GN, checking the complement pathways, components and activation products, along with anti-factor H and genetic testing (C3, CFH, CGI, CFB, MCP, CFHR1-5, THBD, DGKE) are useful.

TMA leads to generalized endothelial dysfunction, that can progress to multiorgan injury. Apart from primary causes, some disease or medical conditions may predispose to TMA. In particular transplant associated TMA (TA-TMA) has been an important clinical entity, that carries high mortality and morbidity. Recent literature has shown that complement pathway dysregulation may play a role in the process. The pathogenesis in TA-TMA is complex, that multifactorial factors contribute to the endothelial injury and pathological process. Complications related to transplant, including GVHD or infections, may also stimulate the complement pathways. Complement blockage therapy, e.g. eculizumab, is useful in managing complex cases. After workup to exclude other potential differential diagnoses, risk assessment is important. Although not all patients with TA-TMA will have elevated sC5b-9, patients with elevation are at increased risk of death from TA-TMA (5). Hence, the activation product measurement has been used as risk stratification for consideration of complement blockade therapy (4,6).

Paroxysmal Noctural Hemoglobuinuria (PNH)

PNH is a rare acquired disorder, that patients suffered from hemolysis with acute exacerbations, leading to anaemia, bone marrow failure and increased risk of thrombosis. PNH arises from an expanded clonal proliferation of hematopoietic cells with somatic mutations of the X chromosomal gene PIG-A. Lack of PIG-A resulted in inability to bind GPI-anchored proteins, including the membrane bound complement regulators, DAF and CD59. As a result, cells having the mutation are susceptible to complement mediated intravascular haemolysis. Assessing the surface expression of CD55 and CD59 is helpful for the diagnosis.

Inherited and Acquired C1 inhibitor deficiency

Hereditary angioedema (HAE) and acquired angioedema (AAE), are rare diseases caused by C1 inhibitor deficiency. As a result, unregulated bradykinin formation leads to angioedema. HAE is an autosomal dominant condition, with majority of cases suffered from reduced concentration (Type I) or less commonly, reduced function (Type II), of C1 inhibitor. Some patients may have similar clinical presentations as HAE cases, but as an acquired condition due to the presence of autoantibodies against C1 inhibitor. These patients usually presented at an older age, and may have underlying hematological malignancies or autoimmune conditions as predisposition. The diagnosis of HAE is based on C1 inhibitor and C4 measurement. It is important to include both antigenic and functional assays for C1 inhibitor, since around 15% of cases may have normal or elevated dysfunctional C1 inhibitor protein (Type II). Furthermore, serum C1q concentrations can be used to differentiate HAE from acquired angioedema (AAE) as the latter is characterized by decreased C1q antigen concentration and autoantibodies against C1-INH. Genetic analysis for SERPING1 variants status may also help in the workup.

Monitoring of Complement Regulatory Drugs

In recent years, drugs targeting complement activation has been in clinical use. Eculizumab is the first approved complement inhibitor, that it is a humanized monoclonal antibody that hinder C5 proteolytic activation, inhibit the generation of C5a and the initiation of the membrane attack complex C5b-9, through its binding to the C5. Eculizumab is approved in the treatment of PNH, aHUS and refractory myasthenia gravis. Complement studies, such as CH50/ AH 50, and activation products (sC5b-9), have been employed in the treatment monitoring (7). In some specialized laboratory, C5 function may also be tested. The best time to monitor the therapy is at trough, immediately before the next dose. With effective drug treatment, CH50/AH50 and C5 function will be low. The activation products will also be suppressed.

Complement assays

The assays used in complement assessment can be broadly divided into 1) screening assays of total functional complement activity, 2) quantification of individual components, 3) quantitation of activation products 4) detection of autoantibodies against the complement components 5) assessing cell surface expression or tissue deposition of complement proteins/ breakdown products, 6) genetic assays.

Apart from the rare primary component deficiency, complement is associated in a number of disease conditions (such as infections, sepsis, malignancy, immune complex diseases, etc) by activation via different pathways. When a component is activated in vivo, the component is taken up by receptors on leukocytes or Kupffer cells. This results in secondary deficiency as consumption. Note that in complement studies, some assays are sensitive to in-vitro activation. Consumption can also be an artifact from heat labile nature of the complement proteins combined with delayed freezing of specimen after sample collection. Overall, the specificity of single complement test is low. Assessing several markers of the pathways and careful interpretation of results as a whole, is useful. In some situations, complementary use of genetic tests may help in cases suspecting primary in nature.

Since EDTA is able to inhibit complement activation in vitro, it is commonly used for quantification of complement components, in particular for activation products. Since heparin and citrate are insufficient inhibitors of complement activation, these are not suitable. Serum, on the other hand, is used for complement function and autoantibodies assessment. Plasma and serum received for complement assays should be separated within 2 hours from collection and frozen at -70 degree Celsius (4). Careful attention to the pre analytical steps and storage is crucial in complement studies.

Screening assays for total functional complement activity

The main indication for total complement function screen is to detect complement deficiencies. Such deficiencies can be genetic (primary), acquired (secondary, e.g. to consumption after pathway activation), or as a consequence of treatment. These tests reflect the total amount of active complement component present in a freshly sampled serum, and reflect the potential of the serum sample to achieve full activation in vitro after addition of activator. The traditional assays used are CH50 and AH50, based on studying the lysis of antibody sensitized sheep erythrocytes (CH50 for the CP activity) and the lysis of untreated rabbit erythrocytes (AH50 for the AP activity). The lysis of erythrocytes correlates with the formation of the terminal membrane attack complex downstream of the pathways’ activation. The results are usually expressed as reciprocal dilutions of the sample required to produce 50% lysis. Besides the traditional assays, a variety of modified methods based on the hemolytic assay were done in different centers. The functional screen can also be tested by measuring the deposition of activation products (ELISA detecting C9 neoepitope generated in terminal complex formation) upon activation of the serum with immobilized complement activating substances on a microtiter plate. Targeted molecules for each pathway are coated in wells of the microtiter plates; Ig M for CP, mannan /acetylated bovine serum albumin for LP and LPS for AP. (8)

In general, the pathway screens may provide some hint to the underlying disease process. Absent/low AH50 with normal CH50 suggests alternative pathway component deficiency, while absent/low CH50 with normal AH 50 suggests early classical pathway components (C1, C2, C4) deficiency. Absent/low results in both AH50 and CH50 suggests a deficiency affecting common components (C3, C5, C6, C7, C8, C9) shared in both pathways or complement consumption. Further investigations, including quantitation of individual components, would be helpful. In the settings of multiple components deficiency, consumptive depletion is likely.

Quantitation of individual components

In cases where the screening assays indicating a complement deficiency, quantitation of individual components and interpreting the results as a profile is useful to further delineate the affected pathways and pathogenesis.

Measurement of complement components is commonly done by immunoprecipitation assays with polyclonal antibodies against the protein of choice, e.g. nephelometry and turbidimetry. Other assays, such as gel precipitation assays or enzyme immunoassays were also used. Overall, these assays are relatively robust, however, do not provide information on the conformation or activation status in vivo.

Quantitation of activation products

Abnormal total complement functional screen could be due to primary deficiency or deficiency secondary to consumptive loss. Measurement of individual components level is not able to distinguish between primary from secondary loss. On the other hand, in vivo complement activation in acute phase reaction may not always lead to low components measurement despite ongoing consumption. Hence, quantitation of activation products would be helpful in the assessment of complement activation. Among the activation products available for measurement, detection of the soluble form of the terminal complement complex (sC5b-9), is the most promising screen for complement activation. The terminal complex reflects the activation to the final stage of the three pathways. Moreover, sC5b-9 has a relatively long in vivo half-life (60 mins), compared to other activation products, and is more stable with respect to in vitro activation compared to early components fragments (1,4). Overall, these activation markers can be rapidly produced by complement activation in vitro, therefore, proper sample collection and handling is important.

Autoantibodies against complement components

Autoantibodies to complement components have been linked to a number of disease conditions. The pathogenesis is often caused by the dysregulation of complement activation, as in the case of C3NeF and anti-Factor H. Occasionally, it may be affecting non-complement pathway, as in the case of anti-C1 inhibitor related angioedema, that it is due to inefficient inhibition of the kallikrein-kinin system and bradykinin release (4).

Most often, these autoantibodies could be detected by enzyme immunoassays. Functional assays were also helpful in the assessment. For example, in C3 Nef detection, a hemolytic assay that utilizes unsensitized sheep erythrocytes, or assay detecting fluid-phase C3 conversion after incubation of patient serum with normal serum at 37degree Celsius, were commonly used for the C3 Nef activity detection (9).

Assessing cell surface expression or tissue deposition of complement proteins/ breakdown products Measuring complement components and activation products directly on cell surface provides valuable information for the workup. For example, examining the deposition of various complement components in the glomeruli and peritubular capillary is useful for glomerulopathies assessment. Furthermore, studying the expression of membrane bound regulators is also helpful in some conditions, such as the use of flow cytometry assessment of CD55 and CD59 on blood cells in the diagnosis of PNH.

Genetic assays

With the advances in molecular diagnostics, complementary use of molecular diagnostics with traditional assays, has been increasingly employed in cases suspecting primary deficiency of complement factors or regulators. For example, gene panels study has been recommended in the workup of aHUS and C3 glomerulonephritis (3, 10-11).

Conclusion

With the vast and constantly growing knowledge in various disease process, along with expanding indications and emerging treatment options in complement mediated disorders, the application of complement diagnostics has been broadened and not limited to diagnosing rare primary genetic entities only. However, many of these assays remains highly subspecialized with limited availability, lack of standardization and complex interpretations. Careful standardization and close international collaborations and experience sharing, would be important for both the laboratory development and clinical applications in the field.

 

References

1. Kirschfink M, Mollnes TE. Modern complement analysis. Clin Diagn Lab Immunol. 2003 Nov;10(6):982-9.
2. Pickering MC, Botto M, Taylor PR, Lachmann PJ, Walport MJ. Systemic lupus erythematosus, complement deficiency, and apoptosis. Adv Immunol. 2000;76:227-324.
3. Angioi A, Fervenza FC, Sethi S, Zhang Y, Smith RJ, Murray D, Van Praet J, Pani A, De Vriese AS. Diagnosis of complement alternative pathway disorders. Kidney Int. 2016 Feb;89(2):278-88.
4. Ekdahl KN, Persson B, Mohlin C, Sandholm K, Skattum L, Nilsson B. Interpretation of Serological Complement Biomarkers in Disease. Front Immunol. 2018 Oct 24;9:2237.
5. Jodele S, Davies SM, Lane A, Khoury J, Dandoy C, Goebel J, Myers K, Grimley M, Bleesing J, El-Bietar J, Wallace G, Chima RS, Paff Z, Laskin BL. Diagnostic and risk criteria for HSCT-associated thrombotic microangiopathy: a study in children and young adults. Blood. 2014 Jul 24;124(4):645-53.
6. Jodele S, Dandoy CE, Lane A, Laskin BL, Teusink-Cross A, Myers KC, Wallace G, Nelson A, Bleesing J, Chima RS, Hirsch R, Ryan TD, Benoit S, Mizuno K, Warren M, Davies SM. Complement blockade for TA-TMA: lessons learned from a large pediatric cohort treated with eculizumab. Blood. 2020 Mar 26;135(13):1049-1057.
7. Ricklin D, Barratt-Due A, Mollnes TE. Complement in clinical medicine: Clinical trials, case reports and therapy monitoring. Mol Immunol. 2017 Sep;89:10-21.
8. Mollnes TE, Lea T, Frøland SS, Harboe M. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex. Scand J Immunol. 1985 Aug;22(2):197-202.
9. Nilsson B, Ekdahl KN. Complement diagnostics: concepts, indications, and practical guidelines. Clin Dev Immunol. 2012;2012:962702.
10. Goodship TH, Cook HT, Fakhouri F, Fervenza FC, Frémeaux-Bacchi V, Kavanagh D, Nester CM, Noris M, Pickering MC, Rodríguez de Córdoba S, Roumenina LT, Sethi S, Smith RJ; Conference Participants. Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference. Kidney Int. 2017 Mar;91(3):539-551.
11. Loirat C, Fakhouri F, Ariceta G, Besbas N, Bitzan M, Bjerre A, Coppo R, Emma F, Johnson S, Karpman D, Landau D, Langman CB, Lapeyraque AL, Licht C, Nester C, Pecoraro C, Riedl M, van de Kar NC, Van de Walle J, Vivarelli M, Frémeaux-Bacchi V; HUS International. An international consensus approach to the management of atypical hemolytic uremic syndrome in children. Pediatr Nephrol. 2016 Jan;31(1):15-39.