Screening for cancer: are biomarkers of value?

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Screening for cancer: 

are biomarkers of value? 

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Editor’s letter 

3 

– February/March 2011 

Prostate cancer management: a continuing challenge 

Accounting for 

between 6 - 10 % of 

cancer deaths in men, 

prostate cancer (PCa) 

is the second most 

frequently diagnosed 

cancer in the West, 

with around one in six 

men in the developed 

world eventually being diagnosed 

with the disease. While older age 

and family history increase the risk 

for PCa, there are no established 

modifiable risk factors; reduction 

in the mortality rate is dependent 

on early diagnosis and timely treatment. 

Although a routine blood test 

revealing an elevated prostate-specific 

antigen (PSA) level may detect 

PCa before symptoms have developed, 

despite its name this marker 

is actually far from specific. It may 

be elevated in several benign conditions, 

with the result being unnecessary 

biopsies. In addition, a considerable 

proportion of men with low 

levels of PSA below the cut-off value 

of 4 ng/mL actually have prostate 

cancer. Various research groups have 

thus been looking for more effective 

methods of measuring PSA as well 

as for more specific and sensitive 

markers for diagnosis and prognosis 

of the disease. 

Over the past decade several groups 

from prestigious institutes have 

reported that the use of prostatespecific 

antigen velocity (PSAV), 

which involves the change in PSA 

level over a specified time interval, 

is a more effective strategy for risk 

prediction, diagnosis and prognosis 

of PCa. Even in cases with total PSA 

levels below the cut-off value, and 

certainly with values between 4 and 

10 ng/mL, an elevated PSAV above 

0.4 ng/mL/year, has been reported 

to be a strong predictor of prostate 

cancer risk. An elevated PSAV was 

found to be significantly more likely 

to result in aggressive disease and 

ultimately death from PCa. 

However a recent comprehensive 

US study, reported in the Journal of 

the National Cancer Institute and 

involving 5,000 men, found that 

even a rapid increase in PSA level 

was not necessarily linked to prostate 

cancer if the actual absolute 

level of the antigen was low. The 

researchers concluded that the 

PSA level varies naturally over 

time, so that men whose PSA level 

suddenly increases should have a 

repeat test rather than undergo a 

possibly unnecessary biopsy. 

However the controversy surrounding 

PSAV and its value in 

PCa screening can probably be 

explained by the fact that the many 

different PSA assays available are 

not interchangeable, nor are they 

necessarily referenced to the same 

laboratory standards. To give reliable 

results, longitudinal monitoring 

of PSA must use the same assay 

and standard each time a subject is 

tested. Such individual risk assessment 

based on the doubling time 

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of relevant markers, as well as the 

development of a panel of biomarkers 

incorporating PSA isoforms, 

will hopefully not only reduce 

unnecessary biopsies, but will have 

a significant impact on the overall 

prostate cancer mortality rate. 

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Contents 

FRONT COVER 

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FEATURES 

Weekly news updates on www.cli-online.com | February/March 2011 | Volume 35 



Also in this issue : 

Molecular forms Serum free light chain 

of PSA Pg. 14 analysis Pg. 18 

Pg.06 

Genetic biomarkers in 

colorectal cancer Pg. 26 

[6 - 8] Screening for cancer: are biomarkers of value? 

[10 - 12] Hybrid muliplex assays for the early detection 

of colorectal cancer 

Intuitively, the measurement of biomarkers for 

cancer screening has great appeal. However, 

those markers which have been most widely 

studied for cancer screening have been disappointing 

as regards reducing mortality from 

cancer, especially in asymptomatic populations. 

The real utility of biomarkers in screening looks 

more likely to be in high-risk populations, where 

the prevalence of cancer is high. One example 

of this is the use of human chorionic gonadotropin 

for screening for gestational trophoblastic 

neoplasia in patients who have had a previous 

diagnosis of a hydatidiform mole. 

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– February/March 2011 6 Tumour markers 



Intuitively, the measurement of biomarkers for cancer screening has 

great appeal. This article reviews the current status of the biomarkers 

most widely studied in cancer screening, but concludes that their use 

to date has been disappointing in reducing mortality from cancer, 

especially in asymptomatic populations. Their main utility in screening 

is likely to be in high-risk populations, where the prevalence of cancer 

is high. An example is the use of human chorionic gonadotropin 

for screening for gestational trophoblastic neoplasia in patients 

who have had a previous diagnosis of a hydatidiform mole. 

by Prof. Michael J Duffy 

Screening for cancer has great intuitive appeal, 

since it is widely believed that the early detection 

of malignancy followed by the initiation 

of treatment results in improved outcome. 

Consequently, population-based screening 

for different types of cancer is now available 

in many countries, including mammography 

for breast cancer in women over 50 years of 

age, the PAP smear for cervical cancer and 

either colonoscopy or faecal occult blood 

testing (FOBT) for colorectal cancer. 

Compared to procedures such as mammography, 

colonoscopy and smear testing, the 

measurement of biomarkers is considerably 

simpler and more convenient [1]. However, 

as previously pointed out by Dr Sturgeon [3], 

biomarkers generally have low sensitivities 

and low specificities for early malignancy. 

These disadvantages, when combined with 

the low prevalence of cancers in a healthy 

population, greatly limit the use of biomarkers 

in screening programmes, especially 

when used alone. Despite these disadvantages, 

several biomarkers have either undergone 

or are currently undergoing investigation 

for cancer screening [Table 1]. The aim 

of this article is to briefly review the current 

status of the biomarkers most widely 

studied for cancer screening. 

PSA in screening for 

prostate cancer 

Although ad hoc or opportunistic screening 

for prostate cancer is now common, the 

benefit of this is unclear. Ideally, prior to 

being used in general population screening, 

a new screening modality should be shown 

to reduce disease-specific mortality in a 

large prospective randomised trial. In 2009, 

preliminary results from two such trials on 

prostate cancer screening were published. 

One of these trials, the European Randomised 

Study of prostate Cancer (ERSPC) 

[4], was carried out in seven European 

countries, while the second, known as the 

Prostate, Lung, Colon and Ovary (PLCO) 

trial [5] was performed in the USA. After 

7-10 years of follow-up in this study, similar 

rates of death from prostate cancer were 

found in the screened and control arm. This 

trial however, had several flaws including a 

high frequency of PSA testing prior to the 

start of the trial and substantial contamination 

of the control group (i.e., subjects in the 

control group had high rates of PSA testing). 

Furthermore, there was a low rate of 

diagnostic biopsies in subjects with elevated 

PSA levels during the trial. 

Compared to the PLCO trial, the European 

study had a greater number of participating 

subjects, longer follow-up and less contamination 

of the control arm. Although 

Marker or test 

it showed that screening resulted in a 20% 

reduction in mortality, the authors calculated 

that 1410 men would have to be 

screened and 48 additional cases of prostate 

cancer would have to undergo treatment to 

prevent one death from prostate cancer [4]. 

With these conflicting findings, it is not 

surprising that published guidelines on 

prostate cancer screening differ in their 

recommendations as to whether or not 

asymptomatic men should undergo PSA 

testing for prostate cancer [6]. Although 

expert panels differ in their recommendations 

regarding PSA screening, most state 

that prior to any testing, men should be 

counselled regarding potential risks and 

benefits of screening and that a shared 

approach to decision making between 

doctor and patient should occur. 

Faecal Occult Blood Testing 

in screening for colorectal cancer 

In contrast to the role of PSA in prostate 

cancer screening, four large randomised 

prospective trials have all shown that 

screening apparently healthy subjects over 

50 years of age using FOBT reduces mortality 

from CRC [7]. Combined results from 

the four trials showed that the screening 

resulted in a 16% reduction in the relative 

risk of CRC mortality [7]. Following adjustment 

for those subjects that failed to attend 

screening, mortality reduction increased to 

25%. Combined results from the four trials 

however, failed to show a significant difference 

in all-cause mortality between the 

screened and control groups. This failure 

Malignancy 

Effect on reducing 

mortality 

FOBT Colorectal Yes 

PSA Prostate Unclear 

CA 125 Ovarian Unclear 

AFP Hepatocellular* Yes** 

VMA/HVA Neuroblastoma No 

Pepsinogen Stomach* Unclear 

HCG Trophoblastic*** Yes 

Table 1. Biomarkers that have undergone or are currently undergoing evaluation in screening for 

cancer. *Only in high-risk areas/high risk subjects. **Shown in a randomised trial to reduce mortality 

in high-risk subjects in China (2). ***In patients who have had a previous hydatidiform mole.

7 


to show a reduction in all-cause mortality 

is likely to be due the fact that mortality 

from CRC made a relatively low contribution 

to overall mortality. Nevertheless, 

based on the above results, pilot or regional 

CRC screening trials have now commenced 

in several countries and are under 

consideration in several others. 

The FOBT used in the above randomised trials 

was performed with the guaiac test which 

relies on the pseudo peroxidase activity of 

haem, either as intact haemoglobin or free 

haem. The guaiac FOBT (gFOBT) system 

however, has several limitations such as lack 

of specificity for human haemoglobin (certain 

foodstuffs and medications may interfere 

with the test) and relatively low clinical sensitivity 

and specificity for colorectal neoplasia 

[8]. In addition, it is difficult to automate, 

making it unsuitable for population-based 

screening [8]. 

In an attempt to overcome some of these 

limitations, the faecal immunochemical test 

(FIT) was introduced. This test specifically 

detects the globin component of haemoglobin 

in an immunochemical assay. Some 

of the main advantages of FITs over gFOBTs 

are summarised in Table 2. Because of these 

advantages, European Group on Tumor 

Markers expert panel recently recommended 

use of a FIT for the new centres embarking 

on FOBT screening for CRC [8]. 

AFP in screening for 

hepatocellular cancer 

The incidence of hepatocellular cancer 

(HCC) varies widely throughout the world, 

being highest in South-East Asia and Sub- 

Saharan Africa and relatively low in Europe 

and North America [9]. Its incidence in the 

West however, has increased in recent years, 

mainly due to infection with the hepatitis 

C virus. The most important risk factor for 

HCC is liver cirrhosis which most commonly 

results from infection with hepatitis 

B or C virus, or high intake of alcohol. Since 

groups at high risk of developing HCC can be 

identified, screening had been advocated for 

detecting early malignancy in these subjects. 

The biomarker most widely used in screening 

for HCC is AFP. However, as AFP lacks 

sensitivity for early disease, it is usually 

combined with abdominal ultrasound 

(US) in screening for HCC. Thus, the 

National Academy of Clinical Biochemistry 

(NACB) [10] recommends that both AFP 

and abdominal US be performed at sixmonthly 

intervals in patients at high risk 

of HCC, especially those with hepatitis B 

and hepatitis C-related liver cirrhosis. The 

NACB also state that “AFP concentrations 

that are >20 µg/L and increasing should 

prompt further investigation, even if US 

is negative” [10]. Similarly, the National 

Comprehensive Cancer Network (NCCN) 

recommends ultrasound and measurement 

of AFP every six months to screen 

high risk subjects for HCC. This organisation 

also recommends additional imaging, 

such as contrast CT if AFP levels are rising, 

or following identification of a liver mass 

nodule on ultrasound [11]. In contrast 

to the NACB and NCCN, the American 

Association for the Study of Liver Disease 

(AASLD) recommends that AFP should 

not be used in the surveillance of highrisk 

groups for HCC unless ultrasound 

is unavailable [12]. New guidelines form 

the AASLD however, do not include AFP 

determination in their recommendations 

for HCC screening (http://www.aasld.org/ 

practiceguidelines/Pages/NewUpdated- 

Guidelines.aspx). 

CA 125 in screening for 

ovarian cancer 

Compared to the cancers discussed above, 

ovarian cancer is relatively rare, with an 

incidence rate of approximately one in 70 

women [13]. Although relatively rare, ovarian 

cancer is the 4 th most common cause 

of cancer-related death in women and the 

most lethal gynaecological malignancy. 

The high death rate, at least in part, reflects 

the fact that most women with ovarian 

cancer are diagnosed at an advanced stage, 

i.e., 65-75% of women with ovarian cancers 

have stage III/IV disease at diagnosis. The 

5-year survival rate for these patients is 

only about 20%-30%. On the other hand, 

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FITs have better analytical sensitivity and specificity for human haemoglobin than 

guaiac-based tests. 

Some FITs can be automated, thus increasing throughput and reproducibility. 

Some FITs can be quantitated, enabling adjustment of sensitivity, specificity and 

positivity rates to meet local needs. 

Unlike the guaiac tests which may be affected by certain dietary components 

(e.g., red meat, vitamin C) and some medications (e.g., aspirin), FITs are free 

from interference by these factors. 

Table 2. Advantages of the faecal immunochemical test (FIT) over the guaiac faecal occult blood 

testing (gFOBT). Data summarised from [8]. 

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a 5-year survival rate of >90% can be 

achieved when disease is confined to the 

ovary. Unfortunately, only about 25% of 

ovarian cancers are detected at this early 

stage. This correlation between 5-year survival 

rates and stage at diagnosis suggests 

that screening and early detection may 

improve outcome. 

Because of its relatively low prevalence, a 

screening strategy for ovarian cancer must 

have an extremely high specificity to minimise 

the number of false-positive results. 

Based on a prevalence of 40 cases per 

100,000 women, it has been estimated that 

in order to achieve an acceptable positive 

predictive value (at least 10%), an ovarian 

cancer screening strategy should have a 

specificity of 99.6% [14]. 

The main screening tests undergoing evaluation 

for ovarian cancer are CA 125 and 

transvaginal ultrasound (TVS). Currently, 

two large prospective trials are evaluating 

these modalities in screening healthy 

women for ovarian cancer, namely the 

PLCO study in the US [15] and the United 

Kingdom Collaborative Trial of Ovarian 

Cancer Screening (UKCTOCS) [16]. 

Preliminary results from the UK trial are 

promising. Thus, of the 58 cancers detected 

with CA 125 and TVS, 28 (48%) were 

found to be either stage I or II. Sensitivity, 

specificity and PPV of the two tests for 

primary and tubal malignancies combined 

were respectively 89.4%, 99.8% and 35.1%. 

Currently, it is unclear whether screening 

with CA 125 and TVS reduces mortality 

from ovarian cancer [16]. Guidelines 

therefore recommend against the use of 

CA 125 either alone or in combination 

with TVS in screening for ovarian cancer 

in asymptomatic average-risk women outside 

the context of a randomised controlled 

trial [6,17]. 

HCG in screening for gestational 

tropohoblastic neoplasia 

Gestational trophoblastic neoplasia (GTN) 

is a rare malignancy that originates from 

placental tissue. Although most GTNs 

develop following a molar pregnancy, they 

can occur after any antecedent pregnancy. 

As previously pointed out [3,18], the use of 

human chorionic gonadotropin (HCG) to 

screen for GTN in patients diagnosed with 

a hydatidiform mole approaches the ideal 

use of a screening biomarker as: 

• HCG levels are increased in almost all 

patients with trophoblastic disease, 

• HCG is a highly sensitive marker for 

small volume trophoblastic disease, 

• The prevalence of malignant trophoblastic 

disease in women diagnosed with a previous 

hydatidiform mole is relatively high 

(3 to 15%) and 

• Effective chemotherapy is available for 

malignant trophoblastic disease. 

The combination of HCG measurements, 

organised follow-up and availability of 

effective chemotherapy means that GTN 

is one of a few human malignancies that is 

curable, even in advanced stages of the disease. 

Indeed, cure rates for this malignancy 

now approach 100%. An important practical 

point in measuring HCG in GTN is that the 

assay used should detect all the main forms 

of the protein, especially the beta subunit. 

Conclusion 

Although the measurement of biomarkers 

has great appeal for cancer screening, 

their use to date has been disappointing 

from the point of view of reducing mortality 

from cancer, especially in asymptomatic 

populations. Their main utility in screening 

is likely to be in high-risk populations, 

where the prevalence of cancer is high. A 

good example of this is the use of HCG in 

screening for GTN in patients who had a 

previous diagnosis of a hydatidiform mole. 

References 

1. Duffy MJ. J Int Fed Clin Chem Lab Med (JIFCC) 

2010;21:issue 1. 

2. Zhang B-H, Yang B-H, Tang ZY. J Cancer Clin 

Oncol 2004;130:417-422. 

3. Sturgeon C. A wide role for tumour markers in 

screening. Clin Lab Int April 2006. 

4. Schröder FH, Hugosson J, Roobol MJ, et al. N Engl J 

Med 2009;26;360:1320-8. 

5. Andriole GL, Crawford ED, Grubb RL 3rd, et al. N 

Engl J Med 2009;360:1310-9. 

6. Sturgeon CM, Duffy MJ, Stenman UK et al. Clin 

Chem 2008;54:e11-79. 

7. Hewitson P, Glasziou P, Irwig L, Towler B, Watson 

E. Cochrane Database of Systematic Reviews 2007, 

Issue 1, Art. No.: CD001216. DOI: 10.1002/14651858. 

CD001216.pub2. 

8. Duffy MJ, van Rossum LG, van Turenhout ST et al. 

Int J Cancer 2011;128:3-11. 

9. Parikh S, Hyman D. Am J Med 2007;120:194-202. 

10. Sturgeon CM, Duffy MJ, Hofmann BR et al. Clin 

Chem 2010;56:e1-48. 

11. National Comprehensive Cancer Network 

(NCCN) Clinical Practice Guidelines in Oncology, 

Hepatobiliary Cancers Version 2. 2010. http:// 

www.nccn.org/ (Accessed, 27 Jan, 2011). 

12. Bruix J, Sherman M. Hepatology 2005;42:1208-36. 

13. Clarke-Pearson DL. N Engl J Med 2009;361:170-176. 

14. Jacobs I, Bast RC. Human Reprod 1989;4:1-12. 

15. Buys SS, Partridge E, Greene MH et al. Am J Obstet 

Gynecol 2005;193:1630-1639. 

16. Menon U, Gentry-Maharaj A, Hallett R et al. Lancet 

Oncol 2009;10:327-40. 

17. Duffy MJ, Bonfrer JM, Kulpa J et al. Int J Gynecol 

Cancer 2005;15:679-691. 

18. Duffy MJ. Crit Rev Clin Lab Sci 2001;38:225-262. 

The author 

Michael J Duffy 

Dept of Pathology and Laboratory 

Medicine 

St Vincent’s University Hospital, Dublin 4, 

UCD School of Medicine and Medical Science, 

University College Dublin, Dublin 4, Ireland. 

Corresponding address: 

Professor M J Duffy 

Dept of Pathology and Laboratory 

Medicine 

St Vincent’s University Hospital 

Elm Park, Dublin 4, Ireland. 

Tel. +353-1-2094378 

e-mail: michael.j.duffy@ucd.ie 

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Hybrid multiplex assays for the early 

detection of colorectal cancer: 

a perspective 

The early detection of colorectal cancer (CRC) is one of the great challenges in the 

battle against this disease. This article illustrates the power of immunoproteomic 

analysis of the serum antibody repertoire to pinpoint tumour-associated antigens 

directly from patient serum samples. Accumulating evidence indicates that 

certain intestinal bacteria can also play an important signalling function in CRC 

diagnostics. As the immune response against infectious agents is in general more 

pronounced than the response to altered self-proteins from tumour cells, antigens 

from an infectious agent could be instrumental in the immunodiagnosis of this 

disease. Eventually hybrid multimarker assays could be developed with a diagnostic 

accuracy that meets the stringent criteria for CRC screening at the population level. 

by Dr Harold Tjalsma 

The need for early 

detection of CRC 

Sporadic cancers of the colon 

and rectum are the second 

most frequent malignancy in 

Western societies. Almost 1 

million cases occur annually 

worldwide, and nearly half 

a million patients die from 

CRC each year. About 45% 

of the cases are detected at 

an advanced stage, which is 

mainly because the flexibility of 

the colon and its content allows 

relatively large tumours to be 

asymptomatic for a long time. 

Treatment is then difficult or 

even impossible, whereas early 

stage CRC can be effectively 

cured by surgical removal of 

the diseased part of the colon 

[1,2]. Thus, the largest decrease 

in CRC mortality will probably 

not be achieved by better therapeutics, 

but by better tools for 

CRC diagnosis at an early stage. 

Importantly, CRC progresses 

very slowly; the diagnostic 

window of opportunity may 

be as long as one decade [Figure 

1]. Faecal occult blood tests 

(FOBT) are often used nowadays 

to pre-select high-risk 

individuals for further colonoscopic 

evaluation. Among the 

weaknesses of the FOBT is its 

limited sensitivity, which is 

estimated to be around 50% for 

tumours. Importantly, highrisk 

adenomas (polyps) are 

Figure 1. Temporal response to bacterial & tumour antigens during 

CRC development. 

only detected in about 10-20% 

of cases as these are not often 

accompanied by intestinal 

bleedings [3]. Nevertheless, it is 

generally believed that screening 

the elderly (>50y) population 

for CRC would decrease 

morbidity and mortality. Thus, 

a simple and accurate screening 

assay is the holy grail in CRC 

diagnostics. 

Antigens as 

molecular markers 

An ideal molecular marker for 

CRC should be one that is specifically 

produced by tumour 

cells, or is closely associated 

with diseased cells [4]. For 

clinical applications, blood is 

the body fluid of choice for biomarker 

assessment as homeostasis 

means its composition is 

normally stable. Immunoproteomic 

approaches take advantage 

of the fact that higher 

organisms have a sophisticated 

system to distinguish “normal” 

(self) from “abnormal” (nonself) 

proteins. In most cases, 

non-self proteins originate 

from invading fungi, parasites, 

bacteria and viruses, however, 

aberrant host proteins can 

also be recognised as non-self. 

In the latter case, the immune 

response is driven by abnormal 

expression of proteins or 

by molecular alterations, such 

as those resulting from mutations, 

misfolding, aberrant 

degradation, glycosylation 

or truncated frameshift peptides 

[4,5] that render these 

proteins immunogenic. 

For diagnostic purposes, several 

features of circulating antibodies 

are important: i) they 

reflect a molecular imprint of 

disease-related antigens from 

the entire human body; ii) 

although an antigen may be 

present only briefly, the corresponding 

antibody response 

is likely to be persistent; iii) 

the half-life of antibodies is 

about 15 days which minimises 

daily fluctuations; iv) antibodies 

are highly stable compared 

to many other serum proteins 

making serum-handling protocols 

less stringent; v) the amplification 

cascade controlled by 

the humoral immune system 

causes a surplus of circulating 

antibodies after appearance of 

the cognate (low-abundance) 

antigen. Circulating IgGs can 

be monitored in a bottomup 

ELISA setup, meaning 

that the reactive serum IgG is 

sandwiched between the antigen, 

which is immobilised 

in a microtitre plate, and the 

labelled anti-human IgG detection 

antibody. Antigen (micro) 

arrays allow the simultaneous, 

multiplexed measurement 

of serum antibodies against 

multiple antigens. In addition, 

fluid-phase assay systems 

based on antigens coupled to 

addressable beads have been 

developed [4,6,7]. The latter 

systems may be best suited 

for future implementation of 

multiplexed antigen assays in 

clinical laboratories.

11 


Colon tumour antigens 

Colon tumour antigens have 

been identified by different 

approaches, including blotting 

of colon tumour cell lines [8], 

phage display of a CRC cDNA 

library [9], and commercial 

protein microarrays [10]. In all 

these cases serum from CRC 

patients and controls is used 

to select those antigens that 

are specifically recognised by 

antibodies from the patient 

sera [4,6]. The sensitivity and 

specificity of single antigens for 

detection was promising, but 

still below the required standards 

for diagnostic purposes. A 

higher accuracy was obtained 

by using a panel of antigens 

reaching a sensitivity and specificity 

both of 92%. The specificity 

of this test could even be 

further increased to 96% by the 

inclusion of carcino embryonic 

antigen (CEA) as marker in this 

panel [9]. In addition to protein 

antigens, an immune response 

to aberrant glycosylated mucins 

has also been implicated as a 

potential marker for CRC [11]. 

Notably, however, the immune 

response against altered selfproteins 

from tumour cells is in 

general inferior to the response 

against antigens from an infectious 

agent. Therefore, the close 

interaction of colonic (tumour) 

cells with intestinal bacteria 

may provide new opportunities 

for finding antigenic 

biomarkers for CRC. 

The colon: a hybrid 

super organ 

The colonic epithelium is in 

constant contact with an enormous 

number of bacteria (also 

known as microbiota or microbiome 

) that largely outnumber 

the epithelial cells. It is estimated 

that the human microbiota 

is composed of around 10 14 

bacterial cells, comprising >10 3 

species [12,13]. This dense bacterial 

population is essential for 

digestion of food and control of 

intestinal epithelial homeostasis. 

Thus, human and bacterial 

cells together form a complex 

ecosystem that, as a whole, 

interactively performs various 

biological processes. In this way 

the colon can be regarded as a 

hybrid superorgan. It is important 

to know that inter-individual 

variations in microbiota 

composition are huge, whereas 

the intra-individual composition 

of intestinal microbiota 

is relatively stable throughout 

life [14,15]. Accumulating 

evidence also supports a 

relationship between certain 

mucosa-associated bacterial 

and intestinal diseases such as 

CRC. Two models for intestinal 

dysbiosis can be envisaged. 

First, disease-promoting bacteria 

that are part of the intrinsic 

microbiome of a certain individual 

induces epithelial damage. 

Second, a diseased state 

of the host epithelium, such as 

CRC, induces local microbiome 

dysbalance. Both models 

are discussed below. 

CRC-associated bacteria 

Several recent studies have 

provided mechanistic evidence 

for the involvement of 

gut bacteria in the development 

of CRC, which includes 

the production of DNA damaging 

superoxide radicals, 

genotoxins or toxic metabolites 

and the induction of cell 

proliferation and/or pro-carcinogenic 

pathways. As intestinal 

microbiomes differ from 

individual to individual, the 

intrinsic intestinal microbiota 

of a specific person may contain 

an unfavourable number 

of pathogenic bacteria. In the 

long term, their activities may 

compromise the activities of 

the health-promoting (commensal) 

bacterial population. 

For example, carcinogenic 

activities have been ascribed 

to certain Enterococcus faecalis, 

Escherichia coli and Bacteroides 

fragilis strains [16- 

20]. Monitoring the activity 

of this group of bacteria may 

thus be important for the risk 

assessment of CRC. 

On the other hand, the dramatic 

physiological and metabolic 

alterations that result from colon 

carcinogenesis itself disturbs the 

local intestinal microenvironment. 

This causes (local) shifts 

in microbiota composition as 

the altered tumour metabolites 

and intestinal physiology will 

recruit a bacterial population 

with a competitive advantage in 

this specific microenvironment. 

This is exemplified by infections 

of the opportunistic intestinal 

pathogen Streptococcus gallolyticus 

(aka Streptococcus bovis 

biotype I) that have been associated 

with CRC for many years 

[21,22]. This bacterial species 

can adhere to the collagens that 

become exposed to the intestinal 

lumen during the formation 

of polyps [23]; pre-malignant 

sites appear to be a preferred 

niche for this bacterial species 

by which it can gain access to 

the human body. Importantly, 

most S. gallolyticus infections 

remain subclinical and thus may 

occur much more often than 

is currently realised. A similar 

mechanism may explain the 

association between Clostridium 

septicum and CRC [24]. The latter 

group of bacteria may play 

an important signalling role in 

CRC diagnostics. 

Microbiotarelated 

antigens 

In our laboratory, we investigated 

whether the occurrence 

of subclinical S. gallolyticus 

infections could be 

helpful in CRC diagnostics. 

Using an immunocapture mass 



spectrometry approach, we observed that 

associated antigen responses were diagnostic 

for both CRC and polyp patients 

[25,26]. The presence of serum IgG against 

ribosomal protein (Rp) L7/L12 was monitored 

in two independent sample collections 

using an ELISA-based approach. 

This demonstrated that anti-RpL7/ 

L12 response was most pronounced in 

patients with early stage CRC and significantly 

different from that of healthy control 

subjects [27]. A drawback of the use 

of this antigen is its conservation in bacteria. 

Consequently, the cross reactivity of 

this assay results in a large overlap in anti- 

RpL7/L12 titres between CRC cases and 

controls. The specificity for CRC was only 

57% at a sensitivity of 75%. Accordingly, 

current activities include the identification 

of alternative S. gallolyticus antigens that 

are more specific for this bacterial species. 

We anticipate that a broader approach will 

yield more accurate assays that may be 

applied on the individual level. This view 

is corroborated by a recent study showing 

that an ELISA-based assay using a mixture 

of surface proteins from S. gallolyticus as 

potential antigens yielded a similar sensitivity 

of 73%, but an improved specificity 

of 83% [28]. The identification of diagnostic 

antigens from other intestinal bacteria 

is also in progress. These include antigens 

from species such as S. gallolyticus, which 

take advantage of a pre-malignant lesion 

to invade the human body and thereby 

cause an antigen response, but antigens 

will also be sought from pathogenic bacteria 

that have been implicated in CRC 

initiation as discussed previously [Figure 1]. 

FIigure 2. Hybrid antigen panels for the early 

diagnosis of CRC. 

Hybrid multimarker assays 

for CRC 

From a genetic perspective, CRC is a highly 

heterogeneous disease [29,30], which 

makes accurate diagnosis based on a single 

biomarker unlikely at the population level. 

On the contrary, multimarker assays are 

likely to provide diagnostic platforms that 

can be used to screen populations for CRC 

and select high-risk individuals or individuals 

with early stage disease for follow up. 

As illustrated in Figure 1, certain mucosaassociated 

bacteria may be involved in 

CRC initiation. Invasiveness of these pathogens 

or exposure to their antigens may 

elicit an IgG response that is valuable for 

CRC risk assessment in individuals. These 

individuals may not need a bowel examination 

immediately, but can be enrolled in a 

more strict monitoring programme. When 

an IgG response is detected against bacterial 

antigens that use polyps or tumours as 

a portal of infection, this may be an important 

sign of early stage disease, especially 

when IgGs against tumour antigens are also 

detected in the same subject [Figure 2]. It 

may be envisaged that immune responses 

against bacterial antigens decrease upon 

disease progression [27], whereas assays 

based purely on tumour antigens may 

leave early disease stages undetected. 

Thus a hybrid approach combines indirect 

bacterial markers that are associated 

with CRC risk and early stage disease with 

tumour markers that are diagnostic for the 

disease itself. 

Conclusions 

Hybrid antigen panels provide a promising 

new concept for CRC screening. However, 

much still needs to be learned about bacterial 

interference in CRC. It goes without 

saying that, when established, hybrid antigen 

panels would need a thorough clinical 

validation phase before decision algorithms 

based on these antigen panels can 

be developed. 

Acknowledgements 

The author would like to thank Annemarie Boleij, 

Rian Roelofs, Bas Dutilh, Wilbert Peters, Julian 

Marchesi and Ikuko Kato for inspiring discussions. 

The financial support from the Dutch Cancer Society 

(project KUN-2006-3591) and the Dutch Digestive 

Diseases Foundation (project WO10-53) for 

our work on innovative CRC diagnostics is warmly 

acknowledged. 

References 

1. Jass JR, Morson BC. J Clin Pathol 1987; 40: 

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2. Booth RA. Cancer Letters 2007; 249: 87-96. 

3. Simon J. N Engl J Med 1998; 338: 1151-1152. 

4. Tjalsma H. Exp Rev Proteomics 2010; 7: 879-895. 

5. Reuschenbach M, Kloor M, Morak M et al. 

Familial Cancer 2010; 9: 173-179. 

6. Tjalsma H, Schaeps RMJ, Swinkels DW. Proteomics 

Clin Appl 2008; 2: 167-180. 

7. Vignali DA. J Immunol Methods 2000; 243: 

243-255. 

8. He YJ, Mou ZR, Li WL et al. Int J of Colorecta Dis 

2009; 24: 1271-1279. 

9. Ran YL, Hu H, Zhou Z et al. Clin Cancer Res 2008; 

14: 2696-2700. 

10. Babel I, Barderas R, Diaz-Uriarte R et al. Mol Cell 

Proteomics 2009; 8: 2382-2395. 

11. Silk AW, Schoen RE, Potter DM et al. Mol Immunol 

2009; 47: 52-56. 

12. Dethlefsen L, Eckburg PB, Bik EM et al. Trends 

Ecol Evol 2006; 21: 517-523. 

13. Qin JJ, Li RQ, Raes J et al. Nature 2010; 464: 

59-U70. 

14. Green GL, Brostoff J, Hudspith B et al. J Appl 

Microbiol 2006; 100: 460-469. 

15. Costello EK, Lauber CL, Hamady M et al. Science 

2009; 326: 1694-1697. 

16. Toprak NU, Yagci A, Gulluoglu BM et al. Clin 

Microbiol Infect 2006; 12: 782-786. 

17. Wang XM, Allen TD, May RJ et al. Cancer Res 

2008; 68: 9909-9917. 

18. Wu SG, Rhee KJ, Albesiano E et al. Nature Medicine 

2009; 15: 1016-U1064. 

19. Cuevas-Ramos G, Petit CR, Marcq I et al. Proc 

Natl Acad Sci USA 2010; 107: 11537-11542. 

20. Lee SH, Hu LL, Gonzalez-Navajas J et al. Nature 

Medicine 2010; 16: 665-U665. 

21. Hausen HZ. Int J Cancer 2006; 119: XI-XII. 

22. Boleij A, Schaeps RMJ, Tjalsma H. J Clin Microbiol 

2009; 47: 516-516. 

23. Boleij A, Muytjens C, Bukhari S et al. J Infect Dis 

2011; in press. 

24. Wentling GK, Metzger PP, Dozois EJ et al. Dis 

Colon Rectum 2006; 49: 1223-1227. 

25. Tjalsma H, Scholler-Guinard M, Lasonder E et al. 

Int J Cancer 2006; 119: 2127-2135. 

26. Tjalsma H, Lasonder E, Scholler-Guinard M et al. 

Proteomics Clin Appl 2007; 1: 429-434. 

27. Boleij A, Roelofs R, Schaeps RM et al. Cancer 

2010;116: 4014-4022. 

28. Abdulamir AS, Hafidh RR, Mahdi LK et al. BMC 

Cancer 2009; 

29. Kaiser J. Science 2006; 313: 1370-1370. 

30. Sjoblom T, Jones S, Wood LD et al. Science 2006; 

314: 268-274. 

The author 

Dr Harold Tjalsma 

Department of Laboratory Medicine (830), 

Nijmegen Institute for Infection, Inflammation 

and Immunity (N4i) & Radboud University 

Centre for Oncology (RUCO) of the Radboud 

University Nijmegen Medical Centre. 

P.O. Box 9101, 6500 HB Nijmegen 

The Netherlands 

Tel. +31-24-3618947 

e-mail: h.tjalsma@labgk.umcn.nl

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Molecular forms of prostate specific 

antigen (PSA) in serum: clinical and 

analytical implications 

Prostate specific Antigen (PSA) is widely used as a disease biomarker 

for diagnosis and monitoring of prostate cancer (PCa). Numerous 

different immunoassays are available for the measurement of 

PSA and its subforms in serum. The assays can be referenced to 

different laboratory standards and are not interchangeable. Patients 

and physicians should be aware of which assay was used, and 

longitudinal monitoring should be performed with the same test. 

by Dr Katharina Braun, Dr David Ulmert and Dr Hans Lilja 

Prostate-specific antigen (PSA) is a kallikrein-related 

peptidase encoded by a five 

exon gene 7.1 kb (KLK3), one of fifteen 

genes clustered in a 280 kb locus on the long 

arm of chromosome 19 in the cytogenic 

region q13.3-4 [1]. KLK3 (encoding PSA) 

and KLK2 (encoding kallikrein-related 

peptidase 2 or hK2) share approximately 

80% amino acid sequence identity and the 

two proteins are produced and secreted at 

highly abundant levels by prostate epithelium 

although some expression can also 

be detected in certain other extra-prostatic 

tissues [2]. 

PSA is synthesised as a 261-amino-acid 

(aa) pre-pro precursor that is processed to a 

non-catalytic zymogen through removal of 

a ≈17-aa signal peptide upon transfer to the 

endoplasmic reticulum, whereas the short 

activation peptide must be released, e.g. by 

hK2, to convert the non-catalytic ≈244-aa 

zymogen to the mature 237-aa catalytic 

single-chain PSA [2]. 

Originally called gamma-seminoprotein, 

a seminal fluid protein was identified in 

1966 and characterised in 1971 by Hara et 

al [3]. The authors anticipated that the protein 

would be a potential marker for seminal 

fluid applicable in the field of forensic 

medicine. In 1979, PSA was purified from 

prostatic tissue, and was later found to be 

identical to gamma-seminoprotein [4]. Subsequently, 

several studies recognised PSA 

as a potential marker for PCa [5]. The first 

assay for PSA in serum was developed by 

Kuriyama et al [6] shortly after Papsidero 

and coworkers [5] identified PSA in blood. 

PSA is synthesised in normal prostate epithelium, 

benign prostate hyperplasia (BPH) 

and all stages of prostate adenocarcinoma. 

The concentration of PSA in seminal fluid 

is up to 10⁶ fold higher than in blood [7]. 

The median concentration of tPSA in blood 

is ≈0.7 ng/mL in healthy men at early middle 

age [8], whereas in advanced cancer the 

amount of PSA in the blood can increase up 

to 10,000 fold [7]. 

Although recent data from the large population-based 

randomised trials in Europe 

and the US have demonstrated that PSAbased 

prostate cancer screening can reduce 

mortality from prostate cancer by about half 

after fourteen years, these important benefits 

are tempered by considerable overdetection 

and consequential risks for overtreatment 

associated with current screening modalities 

[9]. Risk of prostate cancer diagnosis, 

metastasis and death from prostate cancer 

are very strongly associated with concentration 

of PSA in blood [8]. This strong rationale 

explains the widespread use of PSA as a 

key biomarker to assess disease risk, monitor 

therapeutic intervention and disease 

recurrence and as a key component in 

various prognostic models. 

Molecular forms of PSA in serum 

PSA added to blood in vitro exists in three 

forms: one fraction will occur complexed with 

inactivating protease inhibitors, one portion 

as non-complexed non-catalytic PSA, and a 

third as active PSA entrapped by macroglobulins 

[10]. However, the “total PSA” (tPSA) 

detected in clinical samples comprises the 

sum of the concentration of both free PSA 

and PSA complexed to protease inhibitor 

ACT [11]. Data from the original discovery 

and characterisation of the proportion of free 

PSA versus PSA-ACT complexes suggested a 

mean free-to-total PSA ratio of 22% (range 

7-50%) in patient’s serum samples [11]. Based 

on PSA-measurements at early middle age 

in a large, highly representative populationbased 

cohort of men, the median proportion 

of free-to-total PSA in blood has later been 

shown to be ≈33% (IQR 28%; 38%) [12]. 

Complexed PSA 

In the blood circulation, the majority of noncatalytic 

PSA is covalently complexed with 

the protease inihibitor α1-antichymotrypsin 

(ACT or SERPINA5). Active PSA can also 

be enveloped by α-macroglobulins such as 

α2-macroglobulin (A2M) and pregnancy 

zone protein (PZP) [10]. Unlike the interactions 

with ACT, the complex-formation 

with A2M or PZP does not inactivate PSA 

although it blocks catalytic PSA from access 

to protein substrates [10]. It is noteworthy 

that such macromolecules mask epitopes recognised 

by commercially available assays and 

thus stay undetected by these methods [1,11]. 

Since the original discovery in the early 1990s 

it has been carefully documented that the proportion 

of PSA-ACT is higher in men with 

PCa compared to men with BPH [6,10], that 

the free-to-total PSA ratio is an independent 

predictor of prostate cancer risk [9], and that 

the free-to-total PSA ratio enhances discrimination 

of men with BPH from those with evidence 

of PCa beyond that of total PSA alone 

[13]. A systematic review and meta-analysis of 

66 subsequent studies found that the free-tototal 

PSA ratio (“%fPSA”) enhanced the accuracy 

in predicting the diagnostic outcome of 

a prostate biopsy compared to that based on 

tPSA alone [14]. 

Free PSA and subforms 

The non-complexed, free PSA in blood 

is a mixture of different inactive forms 

circulating unattached to any plasma 

proteins. These inactive forms can be 

separated into two main fractions: single 

chain “intact” forms with or without 

truncated remainders of the short activation 

peptide, and forms that are inactive 

due to internal cleavages. The most

15 


studied forms of the latter subgroup are 

PSA with internal cleavages at Lys145- 

Lys146 (“nicked PSA”) or cleavages at 

Lys182-Ser183 (“BPSA”) [15]. 

While free PSA in men with BPH is correlated 

with a higher ratio of internally cleaved 

PSA, increased concentration of intact noncomplexed 

forms and truncated precursor 

forms of PSA are found in patients with 

presence of prostate cancer [16]. 

Serum PSA measurement 

More than 80 antibodies against PSA have 

been very carefully characterised based on 

their binding regions on the protein [17]. 

Because of the high degree of amino acid 

sequence identity between PSA and hK2 

(80% identity), many monoclonal antibodies 

(MAbs) against PSA cross-react with hK2 – 

also with identical binding affinity to each of 

these two highly similar proteins. 

Three distinct antigenic regions of PSA can 

be identified in reference to the ability to recognise 

free PSA, both free and complexed 

PSA, and the cross reactivity with hK2 [17]. 

Non-linear antigenic domains that are in 

close proximity to amino acids 86-91 are 

highly specific for free PSA. Epitopes specific 

for PSA without cross reactivity with hK2 are 

located at or close to amino acids 158-163. 

The shared epitopes between PSA and hK2 

are located close to amino acids 3-11, which 

are close to the identical amino-terminal end 

of both proteins. Knowledge of antibody specificity 

is important for selecting appropriate 

antibody pairs when designing immunoassay 

[17]. Numerous commercial immunoassays 

are available for the measurement of PSA 

and its subforms in serum. Specific assays 

for fPSA as well as dual assays for fPSA and 

tPSA, PSA-ACT assays and hK2 assays have 

been developed [18 - 21]. Additionally assays 

detecting different proPSA forms have been 

made available for use in research. 

There appears to be no access to any of the 

antigenic PSA epitopes subsequent to a stable 

complex that formed between PSA and 

α2 Macroglobulin (A2M), which makes 

measurement of PSA-A2M technically complicated 

and not clinically informative [22]. 

Alternatively, denaturation with sodium 

dodecyl sulphate at high pH can be used to 

disrupt the PSA-A2M complex and release 

PSA from this complex, which then can be 

detected with a conventional ELISA [23]. 

During the past two decades, multiple studies 

compared PSA values measured by commercially 

available immunoassays with - at least 

in part - inconsistent and conflicting results. 

Graves et al compared the polyclonal assay 

from Yang laboratories with the Hybritech 

two site monoclonal assay in 1990 using 

samples from a group of 27 patients, and 

found a two-fold difference in PSA-levels 

between the two assays [24]. Semjonow et al 

reported a correction factor of 0.94 to 2.35 

when comparing Beckman Coulter Access 

and Hybritech Tandem E assays in 1996 [25]. 

In contrast to these findings, Roehrborn et al, 

comparing three monoclonal based assays in 

a group of 86 patients (Hybritech Tandem E, 

Abbott ImX and Tosoh AIA 600) found no 

differences for total PSA [26]. Leewansangtong 

et al also reported a high correlation 

of PSA level ranges between the Hybritech 

Tandem E and Abbott AxSYM assay [27]. 

Figure 1 illustrates one of the novel duallabel 

monoclonal antibody tests designed 

to selectively measure both fPSA as well as 

simultaneously enabling detection of total 

PSA with equimolar detection of free PSA 

and PSA-ACT complex [12]. 

In 1994, the Second Stanford Conference 

on International Standardisation of International 

Standards proposed the use of a standard 

produced by Stamey et al. This standard 

calibrator is composed of 90% PSA-ACT 



and 10% fPSA, similar to the distribution 

found in the circulation of PCa patients [28]. 

This 90:10 PSA preparation was established 

as the World Health Organisation standard 

(WHO 96/670) [29]. PSA assays using the 

WHO 96/670 standard yield 20-25% lower 

PSA values than those using the Hybritech 

standards [30]. 

In 2004 Link et al compared the Beckman 

Coulter Access and Bayer Centaur system 

as well as the third generation DCP Immulite 

System, and found higher PSA values 

measured with Access than Centaur and 

similar results with the Centaur and Immulite 

systems [31]. Blijenberg et al compared 

the Hybritech Tandem E, Beckman Coulter 

Access, DCP Immulite, Roche Diagnositcs 

Elecsys and Defia Prostatus systems and 

showed similar measurements for total 

PSA but not for fPSA [32]. 

These findings were confirmed by two 

recent studies comparing equimolar assays 

calibrated to WHO standards. Kort et al 

compared tPSa, fPSa and cPSA in 70 samples 

in 6 different assays (Beckman Coulter 

Access, Abbott ARCHITECTS and Abbott 

AxSYM, Bayer Centaur, DPC Immulite 

2000, Roche Modular Analytics E170). 

Results showed variation in values for tPSA 

from 0.5 to 1.0µg/L and for fPSA from 0.12 

to 0.40µg/L. Overall results showed less 

diversity for tPSA than fPSA, but tPSA 

assays were still not interchangeable [33]. 

Stephan et al investigated the interchangeability 

of tPSA, fPSA and %fPSA between 

Beckman Coulter Access, DPC Immulite 

2000, Abbott AxSYM, Bayer Centaur and 

Roche Diagnositcs Elecsys assays and still 

found significant interassay variability. 

This may be due to the different epitope 

specificity of the antibodies used [34]. 

Figure 1. Design of immunoassay for simultaneous 

measurement of free, uncomplexed forms of PSA 

and total PSA. Monoclonal antibodies coated on 

plate as capture antibody for free and complexed 

forms in equimolar fashion (Mab1). Monoclonal 

antibodies to detect PSA-ACT and free PSA (Mab2) 

and monoclonal antibodies accessible for fPSA 

epitope only (Mab3), both measureable 

with fluorescence (27). 

Conclusion 

Since the introduction of WHO 96/670 

Standards and development of tPSA-assays 

designed to detect free PSA and PSA-ACT 

on an equimolar basis, inter-assay variability 

has decreased – in particular regarding 

tPSA values. Nevertheless results of commercially 

available tPSA assays are not yet 

interchangeable, not uniformly standardised, 

and with no widely accepted conversion 

factor to correct the accuracy. Large 

discrepancies in fPSA values may result in 

clinical misinterpretation as the decision to 

consider a prostate biopsy may be based on 

the ratio of fPSA to tPSA. 

Persisting discrepancies between assays 

result from a combination of the overall 

design, epitope specificity and affinity 

of capture and detector antibodies, use 

of monoclonal or polyclonal antibodies, 

cross-reactivity and non-specific interferences, 

as well as standardisation. Physicians 

should therefore be aware of which assay 

and standards have been used and note 

whether the same test is also being used for 

longitudinal monitoring of their patients. 

Acknowledgements 

Grant support: Swedish Cancer Society, Swedish 

Research Council (Medicine), The Tegger Foundation, 

Lund University Medical Faculty ALF grants, 

the National Cancer Institute [P50-CA92629], the 

Sidney Kimmel Center for Prostate and Urologic 

Cancers, David H. Koch through the Prostate 

Cancer Foundation, Fundación Federico SA, and 

German Association of Urology (DGU), Ferdinand 

Eisenberger research grant Competing interest 

declaration: Dr Hans Lilja holds patents for free 

PSA and hK2 assays. 

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28. Nurmikko P, Vaisanen V, Piironen T, et al. Clin Chem 2000; 

46:1610-1618 

18. Black MH, Grass CL, Leinonen J, Stenman UH, Diamandis 

EP. Clin Chem 1999; 45:347-354 

19. Zhu L, Leinonen J, Zhang WM, Finne P, Stenman UH. Clin 

Chem 2003; 49:97-103 

20. Bjork T, Piironen T, Pettersson K, et al. Urology 1996; 

48:882-888 

21. Piironen T, Lovgren J, Karp M, et al. Clin Chem 1996; 

42:1034-1041 

22. Lilja H, Haese A, Bjork T, et al. J Urol 1999;162:2029-2034; 

discussion 2034-2025 

23. Baumgart Y, Otto A, Schafer A, et al. Clin Chem 2005; 

51:84-92 

24. Graves HC, Wehner N, Stamey TA. J Urol 1990; 

144:1516-1522 

25. Semjonow A, Brandt B, Oberpenning F, Roth S, Hertle L. 

Prostate Suppl 1996; 7:3-16 

26. Roehrborn CG, Gregory A, McConnell JD, Sagalowsky AI, 

Wians FH, Jr. Urology 1996; 48:23-32 

27. Leewansangtong S, Goktas S, Lepoff R, Holthaus K, Crawford 

ED. Urology 1998; 52:467-469 

28. Prestigiacomo AF, Chen Z, Stamey TA. Scand J Clin Lab 

Invest Suppl 1995; 221:57-59 

29. Rafferty B, Rigsby P, Rose M, Stamey T, Gaines Das R. Clin 

Chem 2000; 46:1310-1317 

30. Stamey TA. Urol Clin North Am 1997;24:269-273 

31.Link RE, Shariat SF, Nguyen CV, et al. J Urol 2004;171: 

2234-2238 

32. Blijenberg BG, Yurdakul G, Van Zelst BD, et al. BJU Int 2001; 

88:545-550 

33. Kort SAR, Martens F, Vanpoucke H, van Duijnhoven HL, 

Blankenstein MA. Clinical Chemistry 2006;52:1568-1574 

34. Stephan C, Klaas M, Muller C, et al. Clin Chem 

2006;52:59-64 

The authors 

Katharina Braun 1,5 , David Ulmert 1,3,4 and 

Hans Lilja 1,2,3 

Departments of 1 Surgery (Urology), 2 Clinical 

Laboratories, and Medicine, Memorial Sloan- 

Kettering Cancer Center, New York, USA 

Departments of 3 Laboratory Medicine, and 

4 

Urology, Lund University, Skåne University 

Hospital, Malmö, Sweden 

5 

Department of Urology, Marienhospital 

Herne, University Bochum, Herne, Germany 

Corresponding author: 

Hans Lilja, MD, PhD. 

Memorial Sloan-Kettering Cancer Center 

Department of Clinical Laboratories, Urology, 

1275 York Avenue, Box 213, New York, NY 

10065, USA 

e-mail: liljah@mskcc.org

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Myeloma screening for the 21 st century 

The role of the serum free light chain assay* continues to evolve. 

Dr Jerry Katzmann, Laboratory Director Mayo Clinic, Rochester, 

USA addressed this topic from a laboratorian’s perspective at 

the recent 6 th International Symposium on Clinical Applications 

of Serum Free Light Chain Analysis. This thought-provoking 

presentation and what it means for the future is discussed. 

by Alison M. Levoguer and Richard G. Hughes 

Historical perspective: 

the oldest tumour marker 

Monoclonal Bence-Jones protein from myeloma 

patients, named after Dr Henry Bence 

Jones’ initial description, is the oldest tumour 

marker available. It is essential that both clinical 

chemists and clinical laboratory staff know 

how to recognise it. Such analysis is normally 

made by protein electrophoresis, which 

detects and quantifies an abnormal protein 

band (or M-spike), and immunofixation electrophoresis, 

which identifies heavy and light 

chain types. Together these techniques provide 

diagnostic and monitoring information 

but do not provide prognostic insights. 

Historically, in multiple myeloma (MM) 

the recommendation has always been to 

obtain a serum and urine sample from the 

patient, since the light chains produced by 

the tumour clone are excreted in the urine. 

The quantitative serum free light chain (FLC) 

assay has, since its availabilty in 2001, caused 

a phase shift in this idea. This can be elegantly 

demonstrated from data published in 

2001 [1] by plotting non – secretory multiple 

myeloma (NSMM) patients’ serum free light 

chain values on a logarithmic kappa/ lambda 

FLC dot plot. Doing so shows this approach 

to be an effective tool for diagnosing NSMM 

from serum tests alone [Figure 1]. Second, 

because it is quantitative, this test allows 

monitoring of the patient response to treatment. 

Subsequent studies have provided 

further insights into the analogous diseases 

of light chain deposition and have revolutionised 

the diagnosis of AL amyloidosis [2]. 

This is because such diseases are difficult to 

identify and quantitate by electrophoresis yet 

amenable to detection and quantification by 

serum free light chain assay. Since then, further 

studies have supported this initial work. 

Perhaps it is now time to reconsider the absolute 

need for undertaking urinalysis at initial 

diagnostic screening. 

Screening for plasma cell 

proliferative disorders (PCD) 

The current International Myeloma Working 

Group (IMWG) recommendation [3] for 

myeloma screening is a panel combination 

of three serum tests; protein electrophoresis, 

immunofixation electrophoresis and quantitative 

free light chain assay. There is little 

reason to perform urine studies as part of 

an initial diagnostic screening panel unless 

primary amyloidosis is suspected clinically. 

Once a plasma cell disorder (PCD) is diagnosed, 

urine studies should be carried out. 

This has an enormous impact on a clinical 

Figure 1. Serum FLC concentrations in patients with NSMM compared with normal individuals and 

patients with light chain multiple myeloma (LCMM). Reproduced from Serum Free Light Chain Analysis 

Sixth Edition, AR Bradwell 2010. 

laboratory. There is no need for two types 

of samples – pertinent since obtaining a 24 

hour urine collection is often difficult. A single 

serum sample allows the laboratory to 

carry out all IMWG screening panel tests. 

The sole reason for a urine sample request 

at screening is now for urine total protein 

assessment, its distribution and to quantitate 

any large M-spike if present. This small 

change in practice has a big potential impact. 

Dr Katzmann’s lab showed this in a study of 

1,877 newly diagnosed PCD patients where 

all serum and urine tests were performed [4]. 

In performing all the tests that can be carried 

out in the clinical chemistry laboratory, 

namely serum protein electrophoresis (PEL), 

serum immunofixation electrophoresis (IFE) 

and the serum free light chain assay as well as 

urine immunofixation electrophoresis (urine 

IFE) nearly all, but not every patient can be 

detected: whilst powerful tests, interaction 

and dialogue with clinical colleagues are 

still necessary to make a diagnosis in some 

cases. This is particularly true of light chain 

diseases (2% amyloid cases missed) and 

extramedullary forms of myeloma. 

Retaining free light chain testing but omitting 

urine studies does miss diagnosing a 

few more patients, but not cases of myeloma, 

Waldenström’s macroglobulinaemia or 

smoldering myeloma (SMM). Some MGUS 

patients (by definition a pre-malignant disorder, 

predominantly laboratory defined) 

and 1% more amyloidosis cases are indeed 

missed. Overall, adding serum free light 

chain testing increases sensitivity with only 

a small concomitant decrease from omitting 

urine studies. 

In conclusion, none of these assays works 

successfully on its own. Assays should be 

used in combination to provide an effective 

screening panel. As we look to future laboratory 

and clinical practice, an effective PCD 

screening panel does not require urinalysis 

unless primary amyloidosis is suspected and 

may not require serum IFE. Dr Katzmann 

suggests that this may be reflected in the next 

iteration of the IMWG Guidelines. 

Prognosis – benign 

versus malignant 

monoclonal gammopathies 

Patient prognosis is another area that 

impinges on laboratory practice. Since 

the free light chain assay has been found 

to be prognostic in every PCD studied 

so far, including the significant number 

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of pre-malignant MGUS and SMM cases 

that are diagnosed each year, it is important 

to have a strategy for managing 

“benign monoclonal gammopathies” versus 

“malignant monoclonal gammopathies”. 

A study of 1,384 MGUS cases from 

a South Eastern Minnesota, USA cohort 

demonstrated that 1% of MGUS patients 

progressed to myeloma per year, the size 

of the M-spike and heavy chain isotype 

being prognostic for progression [5]. This 

was extended to include a third factor – 

an abnormal serum free light chain ratio 

and a risk stratification model was developed 

that identified “very low risk MGUS 

patients” using low serum monoclonal 

protein levels, IgG isotype and a normal 

serum free light chain ratio [6]. These 

MGUS patients’ disease progression rate 

was 0.1% per year. Whilst not synonymous 

with a “normal case” it is consistent 

with placement at the benign end of 

the monoclonal gammopathy disease 

spectrum. Conversely, MGUS patients at 

very high risk are starting to be identified; 

the risk is not necessarily high enough at 

presentation to warrant immediate treatment, 

but these patients are most likely to 

exhibit the clinical symptoms and signs 

required (i.e. by CRAB criteria) for a 

diagnosis of myeloma in the future. 

11-12% of MGUS patients 

undetected by a simplified 

screening panel of serum PEL and 

FLC: should we be concerned? 

If MGUS patients’ lab tests are negative with 

serum protein electrophoresis and free light 

chain assay, with a small M-spike and a normal 

free light chain ratio, thus there is no, or 

just one adverse prognostic factor, they are 

placed in a low risk group. There is a very 

low probability of progressing to disease and 

it may be preferable that laboratorians and 

clinicians do not identify them. This avoids 

placing a psychological burden on patients 

and an economic strain on the healthcare 

system. More incumbent is to examine why 

recommendations for monitoring these individuals 

are what they are. Our patients’ lifelong 

interests are probably better served if 

Assay 

Coefficient of 

Variability 

Figure 2. Serum FLC terminology: involved, uninvolved 

and difference in free light chain derivation. 

their clinical symptoms and signs of disease 

are monitored in the community by their 

primary healthcare physician in the first 

instance, as opposed to extensive lab-based 

monitoring under the hospital out-patient 

care of a haematologist. 

Monitoring 

The free light chain immunoassay is recommended 

for monitoring of oligosecretory 

PCD (AL, NSMM, LCDD), that is, those 

patients who do not have a measurable 

serum or plasma M-spike and for documenting 

a response to therapy [6,7]. The recommendation 

for what a partial response (PR) 

is in these oligosecretory PCD patients has 

been set at a 50% reduction in the involved 

free light chain or dFLC [Figure 2]. 

Dr Katzmann presented newly generated 

data from stable patients addressing the total 

coefficient of variation (CV) of some of these 

screening panel assays i.e., the biological and 

analytical variation combined. This patient 

cohort was not receiving therapy, had a stable 

diagnosis over time and blood was sampled 

every few months [Figure 3]. These data 

show that serum M-spike quantitation has a 

CV of 6.1% implying that a minimal change 

is approximately 12.2% in M-spike (i.e. 2 

standard deviations) with confidence in a 

partial reduction at 50%. Less change could 

be due to chance alone or evolution of disease 

over time. Immunoglobulin quantitation has 

a similar CV. The urine M-spike, in sharp 

contrast, has a CV that is much higher at 

29.1%, in accordance with a minimal change 

Analytic CV 

Biologic CV 

Serum M-spike [SPE] 6.1% 5% 3.5% 

Serum Ig [nephelometry] 9.9% 5% 8.5% 

Urine M-spike [SPE] 29.1% 6% 28.5% 

iFLC 26.0% 6% 25.3% 

rFLC 38.1% 

Figure 3. Coefficients of variability of monoclonal protein measurements in stable patients. Personal 

communication, Dr. Katzmann, February 2011. Reproduced with permission. iFLC = involved FLC, 

rFLC = free light chain ratio. 

in a urine M-spike, which itself necessitates at 

least a 50% change to define it correctly and 

more than a 90% reduction to consider it a 

partial response. 

A long held assumption has been that this 

very high variability was due to the quirks 

associated with 24 hour urine collection. 

However, when Katzmann examined the 

CV for the free light chain assays these data 

[Figure 3] showed a very similar CV to that 

of urine M-spike quantitation. So for both 

assays, despite low analytical CVs [Figure 3, 

column 3], some biological CV common to 

both [Figure 3, column 4] leads to a higher 

overall coefficient of variation. This biological 

variation is related to the light chains 

themselves in a way that is currently not fully 

understood but is most likely related to the 

short half lives of the free light chains. Table 3 

shows that biological variability of the serum 

free light chains, measured in this study at 

25.3% as well as last year [8] is similar to that 

for the urine M-spike at 28.5%. With this in 

mind a review of the IMWG guidelines is 

timely. From these data there is no reason to 

think that serum free light chain analysis is 

inferior to urinalysis, but criteria should be 

used for sFLC assay changes that are comparable 

to those currently used for urine assays. 

Perhaps it is time for a 90% reduction in FLC 

to be considered a partial response. In conclusion 

we should both honour the past contribution 

made by Henry Bence Jones but also 

address the need for assays to become more 

stringent and standardised internationally. 

References 

1. Drayson M et al. Blood 2001; 97: 2900-2902. 

2. Lachmann HJ et al. Br J Haematol 2003; 122: 78-84. 

3. Dispenzieri A et al. Leukemia 2009; 23: 215-224. 

4. Katzmann JA et al. Clin Chem 2009: 55: 

1517-1522. 

5. Kyle RA et al. N Engl J Med 2002; 346: 564-569. 

6. Rajkumar SV et al. Blood 2005; 106: 812-817. 

7. Gertz MA et al. Am J Hematol 2005; 79: 

319-328. 

8. Snyder M et al. Blood 2009; 114: 1803a. 

Editors comments: clarifications to the IMWG 

criteria for coding CR and VGPR in patients in 

whom the only measurable disease is by serum 

FLC levels were published during the writing of 

this manuscript: 

S. Vincent Rajkumar, Blood. Prepublished online 

Feb 3 2011;doi:10.1182/blood-2010-10-299487. 

The authors 

Alison Levoguer, Scientific Affairs Manager 

Richard Hughes, Senior Research Scientist 

The Binding Site Group Limited, 

Birmingham, UK 


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Role of PSA isoforms in improving 

PSA specificity in asymptomatic men 

An overview of the European Randomised Study of Screening for 

Prostate Cancer (ERSPC) side studies indicates that biomarker panels 

would enhance accuracy of PCa screening in asymptomatic men [1]. 

by Dr Chris Bangma 

The diagnosis of prostate cancer (PCa) 

continues to challenge both clinicians 

and their laboratory colleagues. With 

the incidence of PCa rising progressively 

each year, it is now the most common 

form of malignancy amongst men 

in Europe. [2]. Prostate-specific antigen 

(PSA) has been the main driver for early 

detection of prostate cancer. Increasingly, 

over the last decade, we have seen how 

awareness of the PSA test has generated a 

storm of diagnostic activity, particularly in 

countries like the US, even in men with few 

or no symptoms. 

This tumour marker has attracted extraordinary 

scientific attention regarding its 

biochemical characteristics, epidemiological 

and clinical value, alongside huge 

commercial interest and increasing debate, 

even in the lay press [3, 4]. The level of 

PSA at initial screening is highly indicative 

of PCa being diagnosed later in life 

– however, approximately half of cancers 

detected in this way are found to be indolent 

[5]. It has always been recognised that 

many prostate cancer patients died with, 

rather than because of, their disease, but 

it was not foreseen that the impact of PSA 

screening of asymptomatic men would 

result in such a large number of indolent 

(slow growing) cancers being detected. 

The ERSPC believes that the benefits of 

screening would be significantly increased 

by the introduction of new biomarkers 

such as PSA isoforms [Figure 1], and 

Complexed PSA 

Total PSA 

Other 

inactive 

PSA 

Truncated 

proPSA 

Free PSA 

Figure 1. SA and PSA Isoforms – overview. 

Adapted by Chris Bangma from original table 

published in European Journal of Cancer, Oct 

2010 [1]. 

BPSA 

the subsequent development of a panel of 

screening tests. The additional analysis it 

carried out of candidate markers provides 

early encouragement. So far, the most 

promising results have been obtained from 

the analysis of free PSA, proPSA, nicked 

PSA and hK2 [6]. The availability of a 

panel of such markers could increase the 

specificity and sensitivity of screening for 

prostate cancer, enabling new strategies to 

be developed for monitoring and asessing 

patient risk. This would reduce unnecessary 

biopsies, with surgical intervention 

only offered to men at the highest risk. 

The first breakthrough in assessing the 

effectiveness of PSA testing on PCa mortality 

assessment came in March 2009 with the 

publication of the ERSPC study (involving 

an overall follow-up of up to 12 years) [7]. 

It reported an initial reduction of at least 

20% in mortality, which rose to approximately 

30% when adjustment was made for 

non-attendance of men enrolled in the 

study [8]. 

Solving the dilemma of 

overdiagnosis 

The likelihood of being diagnosed with prostate 

cancer in later life is highly dependent 

on the level of PSA at initial screening. PSA 

increases more quickly over time in men who 

get detectable cancer. However, the ERSPC 

study highlighted the serious limitations of 

current PSA testing – that of overdiagnosis. 

In order to save one life, 1410 men would 

have to be tested and 48 men diagnosed with 

prostate cancer. To achieve this, an unacceptably 

high number of biopsies would 

subsequently be found to be negative. 

Current clinical practice uses a serum PSA 

value of more than 4 ng/mL to indicate 

abnormality; this is likely to lead to a prostatic 

biopsy. The case is less clear cut with 

men with a serum PSA between 2-4ng/mL. 

After undergoing a biopsy, 20 -30% of these 

men will be seen to have PCa, and of those, 

approximately one third of these cancers 

are indolent and unlikely to cause death [9]. 

Many patients have therefore undergone an 

unnecessary, invasive procedure– at a cost 

to both the men themselves and the health 

service involved. Based on the annual incidence, 

it is estimated that the annual number 

of unnecessary biopsies is around 750,000 in 

the US alone [10, 11]. 

Throughout the duration of the study, the 

eight ERSPC countries involved used total 

PSA value as the sole indicator for biopsies. 

However, the study involved such a large 

sample (182,000 participants initially) that it 

was possible to carry out a number of additional 

country-specific studies specifically 

focusing on different candidate markers. 

Alongside evaluating changes in PSA concentrations 

over time (PSA velocity, PSA 

doubling), various PSA-isoforms, kallikreins 

and molecular markers were assessed retrospectively 

in different ERSPC cohorts of men 

both with and without prostate cancer. The 

objective was to see what additional information 

these candidate markers could add to 

the total PSA result hopefully enabling more 

selective PCa screening models to be developed 

for asymptomatic men. 

PSA is a kallikrein-like serine protease (also 

known as kallikrein-related peptidase 3), 

which is cleaved into smaller fragments as 

part of its biological profile. Family members 

include kallikrein-related peptidase 2 (hK2), 

which is genetically and structurally very 

similar to PSA and also primarily localised to 

the epithelial cells of the prostate. hK2 rapidly 

converts the inactive precursor form of PSA, 

proPSA, to active PSA and appears to be more 

strongly associated with prostate tumours 

than total PSA, being highly expressed in 

poorly differentiated cancer cells. 

Candidate markers — 

a brief glimpse at the possibilities 

From the side studies, the most promising 

potential biomarkers included % free PSA 

and free PSA forms (intact/nicked PSA 

and proPSA) and hK2 [6]. ProPSA is found 

in normal prostatic epithelium together 

with truncated forms. Mikolajczyk et al. 

identified a clinically important form, (-2) 

proPSA, containing 239 amino acids. This 

form is found in prostate cancer tissue as 

well as serum [12]. Indications are that the

23 


various precursor forms of PSA contribute 

unique predictive values. 

The use of free PSA in addition to total 

PSA has been shown to reduce the number 

of sextant negative biopsies at a PSA cutoff 

level of 3 ng/mL by 30 % — at a cost of 

missing 10 % of detectable cancers (which 

were found microscopically to be predominately 

well differentiated) [13]. 

The Swedish cohort demonstrated that 

the kallikrein markers would considerably 

improve the detection of pCa and the 

accuracy of predicting high-grade tumours 

(Gleason score ≥7 at biopsy). Using the panel 

of four kallikrein markers, at a biopsy-threshold 

of a 20% risk of prostate cancer, would 

have reduced the number of biopsies by 57% 

and missed only 31 out of 152 low-grade and 

3 out of 40 high-grade cancers [14]. 

Men with these high-grade tumours appeared 

to have significantly greater p2PSA/%fPSA 

ratios then those with tumours that were less 

aggressive or indolent. Adding more support 

for a multi-kallikrein panel, in the first round 

of a population screening study the Rotterdam 

section of the ERSPC showed how 

the panel was found to predict PCa in men 

with an elevated PSA. This would reduce 

the number of biopsies by 413 per 1000 

men while missing only 60 (out of 216) low 

grade cancers, and one out of 43 high-grade 

cancers [6]. 

Based on ERSPC retrospective analysis, a 

range of studies demonstrated that the implementation 

of %fPSA in population screening 

would have resulted in a considerable 

increase in specificity at the cost of a small 

loss of sensitivity. %fPSA was shown to be 

statistically significant in the PSA level range 

of 3 to 10ng/mL. In a study of almost 10,000 

Swedish patients, based on a total PSA ≥3ng/ 

mL combined with %fPSA ≤18% the cancer 

detection rate would have been 14% higher 

with a positive predictive value of 36% [15]. 

In a study involving 1726 patients, using a 

%fPSA cut-off of 16% or less as an indicator 

for biopsy would have reduced the number 

of false-positive results by 37%, at the cost of 

missing 11% of the detectable cancers [16]. 

A variety of free to total PSA ratios were also 

analysed prospectively in order to assess their 

relative sensitivity and specificity as indicators 

for biopsy [Table 1]. When PSA level 

was below 3ng/mL, a low %fPSA (


[20]. PCA3 was also added to the panel, 

but at a later stage. 

Call for pan-European bioBank to 

share data and speed validation 

Better strategies are needed for clinicians and 

their supporting laboratory teams seeking 

safe ways to avoid invasive treatment in men 

likely to have indolent cancers. If clinicians 

are to wait before sending certain patients 

with PSA levels < 10 ng/mL for biopsy, they 

must be sure that indolent tumours can be 

more accurately diagnosed, and that robust 

clinical tools are available to more precisely 

indicate the need for intervention. The lack 

of validation of novel genomic or proteomic 

markers in tissue, blood, or urine is one of 

the major restrictions. There are many examples 

of candidate markers reported in the 

literature, which are validated in subsets of 

ERSPC cohorts, but the potential contribution 

of each candidate marker can only be 

fully established by simultaneous assessment 

in a large population-based cohort with a 

follow up of five to 10 years. This is being 

delayed by the lack of adequate repositories, 

in particular for urine, as well as the labourintensive 

isolation of genomic material from 

cancer cells from prostatic biopsies. 

What is needed is a pan - European biorepository 

to allow for simultaneous retrospective 

assessment of markers in relation to final 

disease outcome. Ownership of the samples 

themselves would remain with the source, 

but by the establishment of a virtual computerised 

biobank, data, comparison and assessment 

could be more comprehensively shared 

and evaluated. What would be especially useful 

would be to hold and compare information 

on serial serum markers from patients 

who had needed little intervention, or in 

whom intervention had been avoided for several 

years e.g., in men in ‘watchful waiting’ or 

active surveillance programmes, or in those 

with benign prostatic hyperplasia (BPH). 

Reference 

Parameter for 

Biopsy 

As the ERSPC data matures, more is becoming 

known about the fate of men with and 

without prostate cancers. Because of the 

close follow-up involved, the ERSPC study 

can shed light on the predictive value of 

clinical and laboratory markers over time. 

Future screening appears to rest on the 

development of a panel of markers, alongside 

equations and individual risk assessments 

[21]. This combined information 

would enable diagnostic teams to determine 

both the risk of PCa and, if present, whether 

it would be aggressive or indolent. The role 

of PSA is likely to change. It could be incorporated 

into a more comprehensive, and 

more specific, combination of tests within 

a kallikrein panel, or used initially, but followed 

by a more personalised risk assessment 

invoving more specific markers. 

References 

1. Bangma CH et al. Eur J Cancer 2010; (46) 3109-3119. 

2. Quinn M et al. BJU Int. 2002; 90:162-73. 

3. De Angelis G et al. Rev Urol 2007; 9(3):113-23. 

4. Hernandez J et al. Cancer 2004; 101(5): 894-904. 

5. Draisma G et al. J Natl Cancer Inst 2003; 95(12): 868-78. 

6. Vickers AJ et al. Clin Cancer Res 2010; 16(12): 3232-9. 

7. Schröder FH et al. New Eng J of Med 2009; 360: 

1320-1328 

8. Roobol MJ et al. Eur Urol 2009; 56, Issue 4 584-591 

9. Roehl KA et al. J Urol 2002; 168: 922. 

10. Jemal A et al. Cancer J Clin 2009; 59(4): 225-49. 

11. Fadare O et al. Arch Pathol Lab Med 2004; 128(5): 

557-60. 

12. Mikolajczyk SD et al. Cancer Res 2001; 61(18): 

6958-63. 

13. Bangma CH et al. Urology 1995; 46 (6) 773-8.Bangma 

CH et al. Urology 1995; 46 (6):779-784. 

14. Vickers AJ et al. BMC Med 2008; 6:19. 

15. Becker C et al. J Urol 2003; 170(4 Pt 1):1169-74. 

16. Bangma CH et al. Urology 1995; 46(6):773-8. 

17. Finne P et al. Eur Urol 2008; 54(2): 362-70. 

18. Raaijmakers R et al. Eur Urol 2007; 52(5): 1358-64. 

19. Haese A et al. Prostate 2001; 49(2): 101-9. 

20. Roobol MJ et al. Urology 2004; 63(2): 309-13; 

discussion 313-5. 

PSA range 

Raaijmakers et al [22] Total PSA 2-3 

Finne et al [17] No biopsy < 3 

Recker et al [23] FT ratio 4 

Roobol et al [21] 

PSA-doubling time 

< 4 years, PSA 

velocities 

1-2 

Finding 

%fPSA predictive for 

aggressiveness, NOT 

for positive biopsy 

%fPSA < 15 strong 

predictor of later Pca 

Low cancer specificity 

of FT ratio 

No benefit of early 

rescreen 

No independent 

predictor for cancer 

Table 1. Results from ERSPC’s Prospective ERSPC Side Studies. Adapted by Chris Bangma from 

original table published in European Journal of Cancer, October 2010 [1]. 

21. Roobol MJ et al. Eur Urol 2010: 57 (1): 79-85. 

22. Raaijmakers R et al. J Urol 2004;171(6 Pt 1):2245-9. 

23. Recker F et al. J Urol 2001;166(3):851-5. 

24. Rietbergen JB et al. J Urol 1998;160(6 Pt 1):2121-5. 

The author 

Chris Bangma 

Director of the ERSPC and 

chairman & head of the department of 

urology Erasmus Medical Center, 

Rotterdam, The Netherlands 


Beckman Coulter’s novel marker 

and index improve probability 

of detecting PCa 

In Europe, Beckman Coulter, Inc., has recently 

introduced a new prostate disease marker, 

p2PSA, and the Prostate Health Index (phi) 

for the non-invasive identification of patients 

who are most likely to have a negative prostate 

biopsy.* 

The new marker, named Access Hybritech 

p2PSA, measures the [-2]proPSA molecule, 

an isoform of free PSA. Studies have demonstrated 

that when p2PSA measurements are 

combined with Access Hybritech PSA and free 

PSA measurements, the resulting index demonstrates 

a significant improvement in clinical 

specificity for prostate cancer detection, relative 

to PSA and % fPSA alone, in the PSA range 

2–10 ng/mL in men >50 years and with nonsuspicious 

digital rectal exam (DRE) findings 

[1]. The extra dimension offered by the Prostate 

Health Index (phi) means that the lab can 

provide the clinician with an automatic calculation 

from the index using the PSA, free PSA 

and p2PSA test results. The phi result gives the 

probability of prostate cancer. It does this by 

providing a significant increase in specificity 

over total, free PSA, or %fPSA while maintaining 

the accepted cancer detection rate. 

CLI reported in late 2009 on the forthcoming 

findings of the first prospective study 

looking at the effectiveness of p2PSA and phi 

and these have now been published in The 

Journal of Urology. They have confirmed that 

the Beckman Coulter phi score demonstrates 

significantly higher clinical specificity for 

prostate cancer detection relative to PSA and 

%fPSA alone in men in the PSA range 2-10 

ng/mL (using the Hybritech calibration of 

PSA). Clinical interpretive criteria have been 

developed for men in this entire range [2]. 

* Relates to Europe only 

1. Mikolajczyk SD, Marker KM, Millar LS et al. Cancer 

Res 2001; 61: 695 

2. Le BV, Griffin CR, Loeb S, Carvalha IGF, Kan D, 

Baumann NA, Catalona WJ. J Urol .2010; 183 Issue 

4 1355-1359

News in brief 

25 


Simple test for aggressive 

lung adenocarcinoma 

in never-smokers 

An inexpensive immunohistochemistry 

test can effectively 

identify a sub-group of neversmoking 

lung cancer patients 

whose tumours express a molecule 

associated with increased 

risk of disease progression 

or recurrence. Researchers at 

the Mayo Clinic, Rochester, 

USA, found that 8% - 12% of 

patients with lung adenocarcinoma 

who have never smoked 

carry tumours that express 

anaplastic lymphoma kinase 

(ALK). This subset of patients 

is at more than twice the risk of 

experiencing disease progression 

or recurrence within five 

years of initial diagnosis compared 

to never-smokers whose 

lung adenocarcinoma tumours 

are ALK-negative. The ability 

to test for ALK status in these 

patients might help in selecting 

the most appropriate therapies. 

http://tinyurl.com/6c8c5nz 

tumours had mutations in 

the epidermal growth factor 

receptor (EGFR) gene. All were 

being treated with the drug 

erlotinib, which acts on the 

EGFR molecule. The researchers 

set out to find genes that 

predicted which patients 

would have a lasting benefit, 

and which would not. To do 

this, they used NanoString, an 

integrated digital technology 

with high levels of precision 

and sensitivity that detects 

AD-CLSI_AD-LabMedica.qxd 25/02/2011 10:29 Pagina 1 

expression levels of hundreds 

of genes in a single reaction. 

They examined the expression 

levels of 48 different genes in 

43 patients with EGFR-mutant 

non-small cell lung cancer 

who were being treated with 

erlotinib, and found that AEG 

(astrocye elevated gene 1, also 

known as metadherin) was 

the strongest predictor of progression-free 

survival in these 

patients. Progression-free survival 

was 27 months for those 

with low AEG-1 expression, 

compared to 12 months for 

those with high expression. 

AEG-1 is a cancer-associated 

gene with multiple functions 

that contribute to several 

hallmarks of cancer including 

drug-resistance. For those 

patients in the high-risk group, 

clinical oncologists can be prepared 

for early progression 

and an appropriate alternative 

management strategy. 

http://tinyurl.com/5sxy5ev 

Oncogene AEG-1 

strongly predicts 

response to erlotinib 

treatment in 

EGFR-mutant 

lung cancer 

21 st International Congress of 

Clinical Chemistry and Laboratory Medicine 

19 th IFCC - EFCC European Congress of 


8 th Annual Meeting of the German Society of 


Researchers in the Spanish 

Lung Cancer Group (SLCG), 

in cooperation with Pangaea 

Biotech, in the USP Dexeus 

University Institute, Barcelona, 

Spain have identified a 

gene whose expression level 

strongly predicts how well certain 

lung cancer patients will 

respond to treatment with the 

drug erlotinib. The researchers 

studied 55 patients with nonsmall 

cell lung cancer, whose 

Berlin, Germany 

ICC Berlin - Internationales Congress Centrum 

15 - 19 May 2011 

MARK YOUR CALENDAR 

30 March 2011 

Deadline for reduced fee registration 

www.berlin2011.org

– February/March 2011 26 Molecular diagnostics 

Genetic biomarkers in colorectal cancer 

Colorectal cancer is at the leading edge of the emerging era of 

personalised oncology. Mutational analysis of the KRAS and BRAF 

oncogenes on tumour tissue is already in widespread clinical use 

to guide anti-epidermal growth factor receptor (EGFR) therapy, and 

additional promising molecular biomarkers are being evaluated. 

In this review, we detail both established and emerging genetic 

biomarkers of colorectal cancer that inform on prognosis and predict 

treatment responses, and highlight pre-analytic and analytic factors 

that impact on the accuracy of genetic testing in solid tumour tissue. 

by Dr Carlos J Suarez, Dr Eric Q. Konnick and Dr Colin Pritchard 

the current standard of care for CRC, and 

the topoisomerase-I inhibitor irinotecan. 

Most studies suggest that 5-FU offers little 

benefit to patients with MSI CRC; however, 

lack of complete agreement in the literature 

has limited the use of MSI testing to guide 

5-FU therapy [3]. Several studies with irinotecan 

have demonstrated increased survival 

in patients with MSI-associated CRC 

[10, 11], but the data is limited. Therefore, 

MSI testing in CRC is currently primarily 

used as a screening assay for Lynch syndrome, 

and much less frequently to guide 

prognosis or treatment [Table 1]. 

Colorectal cancer (CRC) is the third most 

common cancer and the fourth most 

common cause of cancer deaths worldwide, 

with 1.2 million cases and 609,000 

deaths reported in 2008 [1]. Advances in 

our understanding of the pathogenesis 

and evolution of CRC have demonstrated 

that molecular features that are not easily 

inferred from traditional morphology can 

predict both prognosis and response to 

treatment. To better characterise the molecular 

variants of colon cancer, biomarkers 

have been developed that assess the status 

of cellular pathways known to be associated 

with CRC prognosis and treatment 

response. Although many biomarkers have 

been investigated and proposed, the best 

validated and most clinically accepted 

biomarkers to date are those associated 

with microsatellite instability (MSI)/mismatch 

repair (MMR) system and the Epidermal 

Growth Factor Receptor (EGFR) 

signalling pathway. Here, we focus on established 

prognostic and predictive biomarkers 

related to the EGFR signalling pathway 

and MSI/MMR system, with a brief discussion 

of several promising new biomarkers 

[summarised in Table 1]. We conclude with 

Colorectal cancer: genetic biomarkers can inform 

on prognosis and predict treatment responses. 

a discussion of laboratory considerations 

that influence biomarker detection and 

interpretation of results. 

Microsatellite instability (MSI) and 

the Mismatch Repair System 

Microsatellites are short segments of repetitive 

DNA that are prone to errors during 

DNA replication [2]. To correct these errors 

and prevent deleterious mutations, all species 

have a well-conserved DNA mismatchrepair 

(MMR) system, which comprises a 

group of proteins including MLH1, MSH2, 

MSH3, MSH6, and PMS2 in humans [3]. 

Deficiencies in the DNA mismatch-repair 

system cause cumulative mutations in 

microsatellite sequences, leading to what is 

referred as microsatellite instability (MSI). 

Approximately 15% of all colorectal cancers 

present with MSI [4, 5], and within 

those cases about 3% are associated with 

Lynch syndrome [5, 6], and ~12% are due 

to sporadically acquired deficiencies in the 

MMR system [7, 8]. In Lynch syndrome, 

also known as hereditary non-polyposis 

colorectal cancer (HNPCC), the functional 

alterations in the MMR system are 

secondary to germline mutation in the 

MMR genes commonly affecting MLH1, 

MSH2 and MSH6. In contrast, the majority 

of sporadic MSI tumours are due to hypermethylation 

of the MLH1 promoter, resulting 

in gene silencing [9]. 

Both germline and sporadic CRC tumours 

with MSI are less prone to invasion, metastasis, 

and lymph nodes spread, and have a 

better prognosis than their microsatellite 

stable (MSS) counterpart [8]. In addition, 

some studies have shown an association 

between MSI and response to adjuvant 

chemotherapy with 5-fluorouricil (5-FU), 

The EGF signalling pathway 

The discovery of EGFR expression in 

malignant cells, and its potential role in 

pathogenesis of malignancy, led to the 

development of monoclonal antibodies 

that could be used as targeted anti-cancer 

therapy. It was quickly noted that not all 

CRC patients treated with the new anti- 

EGFR medications showed a demonstrable 

response, prompting a deeper investigation 

into the signalling pathways of EGFR. 

This lead to the realisation that mutations 

in CRC downstream of EFGR in the pathway 

allow for autonomous activation of the 

pathway, and subsequent activation of the 

mitogen activated kinase (MAPK) pathway, 

leading to signals which promote cell 

survival and growth. 

KRAS 

KRAS is a key downstream mediator of 

EGFR signalling that is mutated in ~40% of 

CRC. Activating mutations in KRAS codon 

12 and 13 are observed early in tumourigenesis, 

are conserved through cancer 

progression, and are mutually exclusive of 

BRAF mutations [12]. Mutations in KRAS 

codon 12 or 13 predict resistance to anti- 

EFGR therapy (cetuximab and panitumumab) 

[13-16], however, even patients 

with wild-type KRAS do not consistently 

respond to anti-EGFR therapy (~20-50%) 

[17]. Testing of neoplastic tissue is now the 

standard of care for CRC patients who are 

being considered for anti-EGFR therapy 

because of the near complete lack of drug 

response when KRAS is mutated. 

BRAF 

BRAF is a protein kinase downstream of 

KRAS in the EFGR signalling pathway that 

is mutated in 10-15% of CRC. BRAF mutations 

occur about 10-times more frequently 

in MSI tumours (~50%) compared to MSS

27 


CRC (~5%), and are mutually 

exclusive of KRAS mutations 

[12]. The majority of mutations 

are a single base pair substitution 

leading to a V600E change 

in the amino acid sequence. 

Patients with BRAF mutations 

have an apparent decreased 

overall survival compared 

to patients with wild-type 

BRAF when treated with anti- 

EFGR therapy [18]. CRC with 

mutated BRAF do not appear 

to respond to monoclonal 

antibody treatment, although 

larger studies are needed to 

confirm the results observed to 

date [19]. BRAF inhibitors are 

in various stages of development, 

and some are currently 

under evaluation in clinical trials, 

suggesting the possibility 

that BRAF mutational analysis 

may be used in the future to 

guide BRAF inhibitor therapy 

in CRC. 

Possible future 

biomarkers 

PI3K/PTEN 

The phosphatidylinositol 

3-kinase (PI3K) pathway is 

thought to be involved in modulating 

EFGR signalling and is 

mutated in up to 40% of CRC. 

Mutations in the PI3K pathway 

are not mutually exclusive 

of KRAS or BRAF mutations. 

Deregulated signalling in this 

pathway has been linked to 

either an activating mutation 

in the PIK3CA p110 subunit or 

inactivation of the PTEN phosphatase. 

Studies have suggested 

that alterations in the PI3K/ 

PTEN pathway lead to resistance 

to anti-EFGR treatment, 

although the association is not 

as definitive as observed in 

KRAS or BRAF [20, 21]. PI3K 

may also be a target for drug 

development, however, the 

utility in CRC may be impacted 

by the coexistence of PI3K and 

KRAS mutations [22]. 

Loss of 18q 

The loss of the long arm of chromosome 

18 (18q) is observed 

in ~70% of CRC, and is associated 

with a worse prognosis, 

however results have not been 

consistent to date [23]. Several 

genes thought to be involved in 

predisposing to CRC, including 

transforming growth-factor 

beta (TGFβ) pathway mediators 

such as SMAD4 are located 

in this region. There are ongoing 

clinical trials evaluating the 

predictive utility of 18q status. 

Topoisomerase I 

Increased protein expression 

of topoisomerase, based on 

IHC, has been correlated with 

an increased response to the 

topoisomerase inhibitor irinotecan, 

and a decrease in IHC 

signal correlated with a lack of 

additional benefit [24]. This 

is not surprising, given that 

irinotecan is a topoisomerase 

I inhibitor, but replication 

of these results is warranted 

before routine use of topoisomerase 

level determination 

to direct therapy decisions. 

Laboratory 

considerations 

Diagnosis of MSI and 

differentiation of sporadic 

CRC from Lynch 

Syndrome/HNPCC 

MSI can be suspected clinicopathologically 

in colon 

adenocarcinomas that are 

right-sided, with focal mucinous 

differentiation, high histologic 

grade, absence of “dirty 

necrosis”, prominent tumourinfiltrating 

lymphocytes, and a 

Crohn’s-like host response [25]. 

However, confirmation of the 

genetic alterations associated 

with MSI is necessary to make 

the diagnosis. 

Genetic diagnosis of MSI is 

primarily based on PCR amplification 

of specific microsatellite 

loci with fluorescent primers, 

and subsequent fragment 

analysis by capillary electrophoresis 

[26] . The length of 

target microsatellite loci in 

tumour and normal cells from 

the same patient are compared, 

and if differences are identified 

in more than 30% of the loci 

analysed, a diagnosis of MSI is 

given (also referred to as MSIhigh, 

or MSI-H). When the differences 

only involve 10-29% or 

less than 10% of the loci, these 

findings are diagnosed as MSIlow 

and MSS, respectively. For 

both prognostic and predictive 

purposes, MSI-low and MSS are 

usually considered equivalent. 

In 1997, an international consensus 

meeting [27] established 

a reference panel of 5 microsatellite 

loci (2 mononucleotide 

loci, BAT25 and BAT26, and 

3 dinucleotide loci, D5S346, 

D2S123 and D17S250) with 

high sensitivity and specificity 

for MSI, known as the Bethesda 

panel, and also established the 

aforementioned cut-off point 

to interpret results. Since then, 

other panels containing alternative 

loci, particularly mononucleotide 

loci that seem to 

be more specific for MSI than 

dinucleotide loci, have been 

developed. Currently, many 

molecular laboratories use 

panels of five mononucleotide 

loci including BAT25, BAT26, 

NR21, NR24 and MONO21. 

An alternative and complementary 

strategy for the diagnosis of 

MSI is immunohistochemistry 

(IHC) analysis of MMR proteins. 

Antibodies against 

MLH1, MSH2, MSH6 and 

PMS2 are used to identify the 

loss of expression of any of 

these MMR proteins. The performance 

of the IHC analysis 

has been estimated to be very 

similar to the MSI analysis with 

approximately >93% sensitivity 

and 100% specificity, although 

interobserver variability may 

be greater with IHC compared 

to MSI testing [28]. 

Once the diagnosis of MSI 

has been established, it is 

critical to determine whether 

the MMR deficiency is due 

to Lynch syndrome or sporadically 

acquired MSI for 

genetic counselling purposes. 

BRAF mutational analysis can 

help with this determination 

because ~50% of sporadic MSI 

tumours have BRAF mutations, 

whereas hereditary 

MSI tumours almost never 

have mutations in BRAF [12]. 

However, a definite diagnosis 

of Lynch syndrome requires 

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– February/March 2011 28 Molecular diagnostics 

germline sequencing and insertion/deletion 

analysis of the MMR genes. If the 

tumour has been previously analysed by 

immunohistochemistry, targeted germline 

sequencing and insertion/deletion 

analysis of the gene corresponding to the 

missing MMR protein can be performed. 

Pre-analytic and analytic 

considerations of molecular 

diagnostic methods 

Genetic testing of solid tissue specimens 

presents a unique challenge for the clinical 

laboratory. Samples vary widely in tissue 

quantity (e.g. fine needle biopsy vs. resection 

specimen), quality (e.g. fresh frozen 

vs. formalin-fixed for 72 hours), and heterogeneity 

of tumour and non-tumour 

tissue. Due to these factors, highly robust 

and sensitive methods that can detect a 

clinically significant variant present in a 

low proportion of the sample are needed. 

Traditional Sanger sequencing has a sensitivity 

of only ~15-20% to detect minor 

allele populations within a mixture, 

which can result in false negative results 

for KRAS testing [29]. Pyrosequencing 

and melting curve analysis generally have 

better sensitivity, at ~1-10% [29]. Newer 

methods that increase sensitivity further 

to ~0.1-1% include co-amplification at 

lower denaturation temperature (COLD)- 

PCR, and peptide nucleic acid (PNA) 

clamping [30, 31]. Such sensitive methods 

have been described in the literature for 

KRAS [30-32] and BRAF [32] mutation 

detection in CRC, and have been shown 

to improve diagnostic accuracy. Additional 

techniques which could allow ultrasensitive 

detection of somatic mutations 

include next-generation sequencing [33] 

and limiting-dilution-PCR [34]. Importantly, 

with the increased ability to detect 

minor mutant populations within the 

sample, it will be necessary to determine 

the clinically relevant threshold in addition 

to the absolute analytical threshold. 

Conclusions and 

recommendations 

Marked improvements in our understanding 

of CRC biology over the last few years 

have led to the discovery of several genetic 

biomarkers that guide treatment and 

establish prognosis of patients with CRC. 

Although many biomarkers have been 

studied, currently only KRAS, BRAF, and 

MSI are sufficiently validated to support 

routine clinical use. Cases in which MSI is 

suspected based on clinicopathologic data 

should be tested. If MSI is demonstrated, 

the distinction between Lynch syndrome 

and sporadic CRC is fundamental to determine 

the necessity of genetic counselling 

and close oncologic surveillance. Sporadic 

CRC cases should be evaluated for KRAS 

and possibly BRAF if anti-EGFR therapy is 

an option. In the future, testing the PI3K 

pathway genes PIK3CA and PTEN may 

help define the population which is most 

likely to benefit from the expensive anti- 

EGFR treatment. The performance of the 

molecular methods used for the above 

determinations needs to be considered 

and clearly communicated so that patients 

receive the most accurate and meaningful 

information, and do not over-interpret the 

laboratory data. 

As the era of molecular oncology and 

personalised medicine unfolds, many 

changes in the way cancer is currently 

diagnosed and treated are expected to 

occur. Laboratory directors and staff alike 

have the responsibility of keeping up with 

the pace of change to continue offering 

the best diagnostic testing to clinicians 

and patients. 

References 

1. Karsa LV et al. Best Pract Res Clin Gastroenterol 

2010; 24(4): 381-396. 

2. Boland CR et al. Gastroenterology 2010; 

138(6):2073-2087.e2073. 

3. Vilar E et al. Nat Rev Clin Oncol 2010; 7(3):153-162. 

Biomarker Freq. Detection Method Current Uses Possible Future Uses 

KRAS codon 12/13 

mutations 

40% DNA mutation testing Anti-EGFR resistance Prognosis 

BRAF V600E mutation 10% DNA mutation testing 

Microsatellite 

instability (MSI) 

15% PCR/sizing 

Anti-EGFR 

resistance,Lynch/ 

HNPCC diagnosis 

Lynch/HNPCC 

diagnosis 

PIK3CA mutations 20% DNA mutation testing None 

BRAF inhibitor sensitivity 

Prognosis,5-FU 

resistance,Irinotecan 

sensitivity 

Anti-EGFR resistance,PI3K 

inhibitor sensitivity 

PTEN loss 30% IHC None Anti-EGFR resistance 

18q loss 50% FISH, cytogenetics None Prognosis 

Topoisomerase 1 low 50% IHC None Irinotecan sensitivity 

Freq.: Frequency in colorectal cancer; 5-FU: 5-fluorouracil; IHC: Immunohistochemistry; FISH: 

Fluorescent in situ hybridization 

Table 1. Genetic Biomarkers in colorectal cancer. 

4. Aaltonen LA et al. N Engl J Med 1998; 338(21): 

1481-1487. 

5. Hampel H et al. N Engl J Med 2005; 

352(18):1851-1860. 

6. Hampel H et al. J Clin Oncol 2008; 26(35):5783-5788. 

7. Ward R et al. Gut 2001; 48(6):821-829. 

8. Popat S et al. J Clin Oncol 2005; 23(3):609-618. 

9. Kane MF et al. Cancer Res 1997; 57(5):808-811. 

10. Fallik D et al. Cancer Res 2003; 63(18):5738-5744. 

11. Bertagnolli MM et al. J Clin Oncol 2009; 27(11): 

1814-1821. 

12. Rajagopalan H et al. Nature 2002; 418(6901):934. 

13. Bokemeyer C et al. J Clin Oncol 2009; 

27(5):663-671. 

14. Benvenuti S et al. Cancer Res 2007; 

67(6):2643-2648. 

15. Van Cutsem E et al. J Clin Oncol 2007; 25(13): 

1658-1664. 

16. Amado RG et al. J Clin Oncol 2008; 

26(10):1626-1634. 

17. Normanno N et al. Nat Rev Clin Oncol 2009; 6(9): 

519-527. 

18. Roth AD et al. J Clin Oncol 2010; 28(3):466-474. 

19. Di Nicolantonio F et al. J Clin Oncol 2008; 26(35): 

5705-5712. 

20. Jhawer M et al. Cancer Res 2008; 68(6):1953-1961. 

21. Sartore-Bianchi A et al. Cancer Res 2009; 69(5): 

1851-1857. 

22. Janku F et al. Mol Cancer Ther 2011. 

23. Popat S et al. Eur J Cancer 2005; 41(14):2060-2070. 

24. Braun MS et al. J Clin Oncol 2008; 26(16):2690-2698. 

25. Greenson JK et al. Am J Surg Pathol 2003; 27(5): 

563-570. 

26. Laghi L et al. Oncogene 2008; 27(49):6313-6321. 

27. Boland CR et al. Cancer Res 1998; 58(22):5248-5257. 

28. Shia J. J Mol Diagn 2008; 10(4):293-300. 

29. Tsiatis AC et al. J Mol Diagn 2010; 12(4):425-432. 

30. Milbury CA et al. Clin Chem 2009; 

55(12):2130-2143. 

31. Pritchard CC et al. BMC Clin Pathol England 2010; 

10: 6. 

32. Mancini I et al. J Mol Diagn 2010; 12(5):705-711. 

33. Mardis ER et al. Hum Mol Genet 2009; 18(R2): 

R163-168. 

34. Pohl G et al. Expert Rev Mol Diagn 2004; 4(1):41-47 

The authors 

Carlos J Suarez, Eric Q. Konnick and Colin 

Pritchard 

Department of Laboratory Medicine 

University of Washington 

Seattle, WA 98195, USA 

The authors contributed equally to this work. 

Corresponding author: 

Colin C. Pritchard MD, PhD 

University of Washington 

Department of Laboratory Medicine 

Molecular Diagnostics Laboratory, Room 

NW-275 

Seattle, WA 98195-7110, USA 

Tel. +1 206 598 6131 

Fax +1 206 598 6189 

e-mail: cpritch@uw.edu.

Microbiology 

29 


Laboratory diagnosis of syphilis: 

indirect detection methods 

Due to diverse clinical manifestations which are shared with other 

treponemal and non-treponemal diseases, it is important to diagnose 

syphilis with the aid of appropriate and reliable laboratory tests. 

by Dr Meghna Patel 

Syphilis is a sexually transmitted disease 

caused by the spirochete, Treponema pallidum 

[1, 2]. It is a chronic disease with many 

very serious complications, especially during 

pregnancy, affecting the cardio-vascular 

and nervous systems in particular. It can also 

facilitate the transmission of HIV [2, 3]. During 

the course of the disease there are diverse 

clinical manifestations, which are indistinguishable 

from those of many other diseases 

[1, 4]. In recent years the incidence of syphilis 

has greatly increased in the United States and 

in Germany [1, 5]. Syphilis is easy to cure in 

the early stage, but can become disabling in 

the later stages and can ultimately be fatal [1]. 

The disease progresses through four stages: 

the primary, secondary, latent and tertiary 

stages. The stage of the disease at which the 

patient presents has implications for diagnosis 

and treatment [6]. 

There are several laboratory tests, which can be 

categorised into direct and indirect detection of 

T. pallidum, to diagnose syphilis at the various 

stages of the disease. This article will focus on 

indirect detection methods; with direct methods 

there are difficulties in cultivation and 

maintenance of T. pallidum in the laboratory, 

and in samples from patients with the latent 

and late stages of the disease, there is a paucity 

of spirochetes for direct observation. However 

a good number of indirect detection methods 

have been developed and are routinely used to 

diagnose syphilis. Broadly these are classified 

as non treponemal and treponemal tests. 

Non treponemal tests 

These tests are often referred to as non specific, 

reagin or cardiolipin tests, as they use 

standardised antigen containing cardiolipin, 

cholesterol and lecithin to detect the presence 

of IgG and IgM antibodies to lipoidal material 

released from host cells [4]. US CDCapproved 

standard tests include the Venereal 

Diseases Research Laboratory (VDRL) slide 

test, the Rapid Plasma Reagin (RPR) card 

test, the Unheated Serum Reagin (USR) test 

and the Toluidine Red Unheated Serum Test 

(TRUST) [6]. Serum is preferred over plasma 

for both treponemal and non treponemal 

tests, however plasma can also be used for the 

RPR test and the TRUST. The VDRL and USR 

tests are microflocculation tests that are read 

using a microscope. Even though the VDRL 

is the only test directly applicable for CSF testing, 

its utility is limited because the antigenic 

suspension is unstable and must be freshly 

prepared every day for reliable results, and 

the serum sample must be heat-inactivated. 

However, the USR uses a very stable antigenic 

suspension and an unheated serum sample, 

which facilitates use. Being macroscopic flocculation 

tests, a microscope is not required to 

perform the RPR and TRUST. The RPR uses a 

stabilised VDRL antigen containing charcoal 

particles to facilitate reading of the test. In the 

TRUST, charcoal is replaced by toluidine red 

dye that gives pink coloured floccules with 

positive samples. These tests can be used as 

both qualitative and semi-quantitative assays 

to determine the titre of the sample. Non 

treponemal tests are of great value for monitoring 

the course of the disease during and 

after treatment, and for diagnosis of reinfection 

and relapse. 

In spite of these advantages, non treponemal 

tests cannot be used alone for syphilis diagnosis 

as they have limited sensitivity, especially 

in primary and late latent syphilis patients, 

and the prozone phenomenon in secondary 

syphilis can result in false negatives. False positives 

can also occur due to cross reactivity in 

patients with hepatitis, chicken pox, infectious 

mononucleosis, measles, malaria and connective 

tissue disease, as well as in pregnancy [4]. 

Span Diagnostic’s non treponemal tests RPR 

and TRUST provide stable, aqueous ready to 

use reagents with standardised immunoreactivity. 

They offer simple, rapid and inexpensive 

test for syphilis and are even suitable for epidemiological 

and field studies. The company 

has further improved its non treponemal tests 

with the use of synthetic VDRL antigen, manufactured 

by US-FDA approved technology, 

licensed from the CDC. Synthetic VDRL antigen 

offers better stability compared to natural 

VDRL antigen. 

Treponemal tests 

These are sometimes referred to as specific 

tests because they use T. pallidum or its components 

as antigen/s. Treponemal tests are 

mainly used for confirming the results of 

non treponemal tests that have low specificity. 

They are also important for detecting 

very early, congenital or late cases of syphilis 

when non treponemal tests are negative. 

However the usefulness of treponemal tests 

is limited by their cost and complexity, as 

well as by persistent positive results even in 

patients who have been successfully treated. 

In treponemal tests, anti-treponemal antibodies 

are detected by techniques such as 

agglutination, Enzyme Immuno Assay (EIA), 

immunofluorescence, immunoblotting and 

immunochromatogrphy. 

Indirect Agglutination tests 

The Treponema Pallidum HaemAgglutination 

(TPHA) and Treponema Pallidum Particle 

Agglutination (TPPA) tests are examples 

of indirect agglutination tests. In the TPHA 

classically sheep RBCs are coated with sonicated 

T. pallidum preparations. In positive 

samples these are agglutinated by the anti 

treponemal antibodies present. The TPHA 

is a highly sensitive test in all stages of the 

disease except in early primary syphilis. It is 

also reasonably specific but there are some 

false positives, from the use of sheep RBCs, 

in patients suffering from infectious mononucleosis, 

leprosy, collagen disease and other 

miscellaneous conditions; false positives are 

rare in healthy individuals [4]. The use of 

chicken, turkey and more recently human ‘O’ 

RBCs has successfully reduced the number of 

false positive results, as has the use of coloured 

gelatine or polymer particles, which has also 

greatly decreased the complexity of the test 

[2]. The TPPA test is an appropriate substitute 

for the microhaemagglutination assay 

(MHA-TP), as it is as sensitive as the fluorescent 

treponemal antibody absorbed (FTA- 

ABS) test for primary syphilis and as useful as 

the RPR for screening blood donors [7]. Furthermore 

it has been reported that sensitivity 

has increased due to the improved IgM binding 

capacity of sensitised gel particles. 

Enzyme Immuno Assay (EIA) 

The indirect competitive capture EIA, detecting 

IgG and/or IgM treponemal antibodies, 

has been developed by numerous laboratories. 

The tests have similar or higher sensitivity and

– February/March 2011 30 Microbiology 

specificity compared with the FTA-ABS and 

TPPA [6]. In addition they can be automated, 

facilitating the objective reading of results. The 

reader can be linked to the laboratory computer, 

which reduces the variation and subjectivity 

of manual reading. However, EIAs based 

on treponemal antigens, like other treponemal 

tests, give positive results throughout life even 

after successful completion of therapy. The 

sensitivity of EIA is also no greater than the 

TPHA for primary syphilis. The CDC recommends 

that if the EIA is used for screening, an 

RPR test should be performed on all positive 

samples, and a second treponemal test, such as 

TPPA or FTA-ABS, should be used to confirm 

a positive result. 

Fluorescent Treponemal Antibody 

Absorption Test (FTA-Abs) 

This indirect fluorescent antibody test utilises 

fluorescent dye to detect antibodies against 

treponemal antigens. Serum or plasma is 

pretreated with absorbent to remove groupspecific 

treponemal antibodies. The FTA 

- Abs double staining test is a modified FTA- 

Abs which uses an additional counter stain 

to improve the reliability of the results. Even 

though it is highly sensitive, especially in early 

primary syphilis, it is less sensitive than other 

treponemal tests for detecting markers of 

past infection [2]. The specificity of FTA-Abs 

is also lower than in other treponemal tests. 

False positives occur in approximately 1-2% 

of the normal population, mainly in patients 

with immune haemolytic anaemia, some viral 

infections and in narcotic addicts [2]. Furthermore 

an effective, specialised and qualitycontrolled 

microscope is required for the test. 

Immunoblotting 

This technique detects antibodies to individual 

T. pallidum proteins. Antibodies reacting to 

some of the treponemal antigens such as 15 

kDa, 17 kDa, 44.5 kDa and 47 kDa are diagnostic 

for acquired syphilis [2]. Either IgG or IgM 

antibodies are detected, which is useful for a 

confirmatory test [4, 6]. Recently recombinant 

antigens, in place of electrophoretically fractionated 

proteins, have improved the test, but it 

cannot be performed in a small scale laboratory; 

the test is only available in selected laboratories. 

Rapid tests 

Rapid tests using treponemal antigen(s) 

coated on latex particles and nitrocellulose 

(ICT) strips are available for testing blood, 

serum or plasma. Such simple, rapid tests 

are more useful in the field, in resource-poor 

settings and in the physician’s office, as technical 

expertise and specialised instruments 

are not required. However rapid tests do not 

incorporate an internal Quality Control, so 

a periodic external Quality Control using 

laboratory-based tests is recommended [6]. 

One such test is Span Diagnostics’s Signal –Tp 

ver. 2.0, an Immunodot test utilising a mixture 

of purified recombinant, treponemal antigens 

(47 kDa, 17 kDa), which are among the 

major immunoreactive antigens in Western 

Blot analysis and have shown great promise 

in the development of a rapid diagnostic test 

for syphilis [3,4]. Another test, Span Diagnostics’s 

Crystal- Tp, is based on an immunochromatographic 

technique which detects all 

major classes of antibodies i.e. IgG, IgM, IgA 

to treponemal antigens. A mixture of purified 

recombinant antigens is used- 15 kDa, 47 kDa 

and 17 kDa- which are major immunoreactive 

antigens in the Western Blot technique [3,4]. 

These two tests have an inbuilt control dot/ 

line. They are 100% sensitive when compared 

with a confirmed positive serum sample panel 

for anti treponemal antibodies, and 100% specific 

when compared with the TPHA. 

Practical utility of serological tests 

In the laboratory diagnosis of syphilis, it is most 

important to detect the antibody response 

with a screening test and confirm positive 

results using a confirmatory test. An ideal 

screening test should be simple, readily available, 

cost-effective, rapid, user-friendly, suitable 

for resource-poor situations and easily 

adapted for use with a large number of specimens. 

Sensitivity should be high, whereas the 

confirmatory test should have both high sensitivity 

and specificity, even at the cost of ease, 

economy and simplicity, to rule out all false 

positives without missing any positive samples. 

Although this combination provides an 

excellent screen for all stages of syphilis except 

for very early primary infections, when the 

treponemal test will not be positive, the cost 

should be considered. In collaboration with 

CDC, Span Diagnostics has developed world’s 

first dual-test in a flow-through format, the 

Signal Spirolipin, which detects reagin (i.e. 

nonspecific) as well as anti treponemal (i.e. 

specific) antibodies against a mixture of 47 

kDa and 17 kDa antigens in a single test. This 

can be used for screening and confirmation 

of syphilis in almost all disease stages without 

any prozone effect or false positives, and 

can also be used to monitor treatment with 

high specificity and sensitivity. Promising test 

results have been found in neuro-syphilis, 

congenital syphilis and in cases of syphilis/ 

HIV coinfection. This test makes diagnosis 

quicker, more reliable, easier and more economical, 

without the need for specialised 

instruments and expertise. It is thus suitable 

for field and epidemiological studies. The Signal 

Spirolipin is a visual three dot assay with 

an inbuilt control dot to validate successful 

completion of the assay procedure; results are 

available in 10 minutes. 

References 

1. CDC fact sheet, syphilis, 2008. 

2. Nesteroff S. Serology, syphilis, RCPA Quality Programs Pty 

Limited, 2004. 

3. Egglestone SI and Turner AJL. Serological diagnosis of syphilis. 

Communicable disease and public health 2000; 3:158-62. 

4. Larsen SA, Steiner BM and Rudolph AH. Laboratory diagnosis 

and interpretation of tests for syphilis. Clinical Microbiology 

Reviews 1995; 8: 1-25. 

5. Muller I, Brade V et al. Is serological testing a reliable tool in 

laboratory diagnosis of syphilis? Meta-analysis of eight external 

Quality control surveys performed by German Infection 

Serology Proficiency testing program, Journal of Clinical 

Microbiology 2006; 44:1335-1341. 

6. Ratnam S. The Laboratory diagnosis of syphilis. The Canadian 

Journal of Infectious disease and medical Microbiology 2005; 

16:45-51. 

7. Pope V et al. Comparison of serodia Treponema plallidum 

particle agglutination Captia Syphilis-G and SpiroTek regain 

II test with standard test technique for diagnosis of syphilis, 

Journal of Clinical Microbiology, 2001;38:2543-2548. 

The author 

Dr M. Patel 

SPAN DIAGNOSTICS LTD. 

Udhna, Surat, 

INDIA 


Nova Biomedical increases 

manufacturing capability 

Nova Biomedical announced recently that, in 

response to rapid growth in its diabetes and 

whole blood point-of-care testing products 

business, it has purchased an additional 7,500 

square metres of manufacturing/warehouse 

facility in Billerica, MA, USA. According to 

Lou Borrelli, Nova Biomedical’s CFO, this 

additional state of the art manufacturing facility 

will ensure that the company’s manufacturing 

capabilities keep pace with the increasing 

demand for its StatStrip Hospital Glucose 

products as well as its Nova Max consumer 

diabetes products. 

One of the main drivers for Nova’s strong 

growth is the rapid adoption of its StatStrip 

Hospital Glucose Monitoring System. Since 

its inception just four years ago, StatStrip has 

become the fastest growing hospital glucose 

meter in the world. StatStrip uses a novel glucose 

test strip technology that measures haematocrit 

and other common interferences such 

as maltose, galactose, xylose, acetaminophen, 

ascorbic acid and oxygen, and eliminates erroneous 

glucose results caused by these interfering 

substances. StatStrip lab-like accuracy and 

freedom from interference has been validated 

in more than 50 published clinical studies 

worldwide, in a broad variety of critical care 

settings including ICU, Dialysis, NICU, OR, 

ED, and Tertiary Care. This remarkable rate of 

StatStrip publications by some of the world’s 

leading hospitals, is testimony to the importance 

of this technology breakthrough for bedside 

glucose testing and suggests the reason for 

its popularity.

Product News 

31 


Human molecular 

karyotyping panel 

A multiplexed assay kit for human molecular 

karyotyping, the nCounter human 

karyotype panel provides a cost-effective 

way to monitor cell line quality and carry 

out cytogenetics research. With this kit, 

researchers can accurately quantify chromosome 

number and detect aneuploidy 

– the existence of fewer or more than the 

normal two chromosomes in a diploid 

genome. These assays are ideal for monitoring 

human-derived samples and cell 

lines for chromosomal abnormalities, and 

features less hands-on time than other 

products. Additional applications include 

the screening of human samples for clinical 

research and non-human cell lines for 

human contamination as well as next-generation 

sequencing library quality control. 

NanoString Technologies, Inc 

Seattle, WA, USA 


Legionella pneumophila 

monoclonal antibodies 

Legionella pneumophila is a gram negative 

organism, found in the natural environment, 

which can cause an infection of 

the pulmonary alveolar macrophages, the 

condition also known as Legionnaire’s disease. 

Though there are 64 serogroups of 

Legionella, 70-90% of cases of pneumonia 

are caused by serogroup 1 of L. pneumophila. 

Two new monoclonal antibodies react 

with the lipopolysaccharide (LPS) of all the 

serogroup 1 strains tested (11 strains). These 

antibodies can form a pair for the detection 

of SG1 LPS from various samples. 

ViroStat, Inc 

Portland, ME, USA 


Agar for rapid MRSA screening 

The newly improved Brilliance MRSA 2 Agar 

chromogenic medium saves valuable time for 

infection control teams by allowing rapid and 

reliable screening for methicillin-resistant 

Staphylococcus aureus (MRSA). With accurate, 

easy-to-read results available in just 18 

hours, the agar enables prompt initiation 

of appropriate infection control measures 

to help minimise the opportunities for further 

transmission of MRSA. The enhanced 

formulation ensures even easier interpretation 

of results. New inhibitory components 

in the medium further reduce the growth 

of non-target organisms, while a novel pink 

counter stain ensures that any organisms 

that do grow are easily distinguished from 

the distinctive blue MRSA colonies. The agar 

minimises false-positive results, even in low 

MRSA prevalence settings. This saves time 

and resources by reducing the need to reincubate 

plates and minimising confirmatory 

testing. Convenient 

and extremely 

easy to use, the 

agar is suitable for 

inoculation direct 

from screening 

swabs, or it can be 

used to plate out an 

isolate or suspension. 

Thermo Fisher Scientific 

Basingstoke, Hants, UK 


Dimeric antigen for greater 

specificity in Borrelia diagnostics 

An innovative dimeric Outer surface 

Proctein C (OspC_ antigen provides over 

30% higher specificity than conventional 

OspC in the serological diagnosis of Lyme 

disease. Antibodies against OspC are formed 

in as many as 90% of persons infected with 

Borrelia and represent the most important 

marker for early-stage borreliosis. Recent 

research has revealed that the biologically 

active form of OspC is a homodimer. The 

recombinant OspC used in immunoassays is, 

however, typically produced in monomeric 

form. To compensate for its correspondingly 

low sensitivity it is often coated at high 

concentrations, leading to numerous unspecific 

reactions. Now recombinant OspC has, 

for the first time, been produced in dimeric 

form using state-of-the-art molecular biological 

methods. This dimeric antigen, OspC 

adv, has the same characteristics as the native 

dimeric protein. Its exceptionally high specificity 

minimises the risk of false-positive 

results, and it retains a high sensitivity. Moreover, 

in contrast to native protein which is 

difficult to isolate, 

this dimeric antigen 

can be produced 

using standardised 

methods, thus 

ensuring a highly 

consistent antigen 

quality. The antigen 

is now included in the Anti-Borrelia EURO- 

LINE-RN-AT immunoblot (IgG and IgM) 

from Euroimmun. This line blot system provides 

an extensive portfolio of diagnostically 

relevant antigens, including the important 

marker VlsE. Each antigen preparation is 

tailored to provide maximum sensitivity and 

specificity. The IgM blot features OspC adv 

from four different Borrelia species (afzelii, 

burgdorferi, garinii, spielmanii), encompassing 

all currently known human pathogenic 

species. The Anti-Borrelia immunoblot also 

includes immunoreactive membrane lipids 

— exclusive to this system — which further 

enhance the efficiency of the analysis, and is 

ideal for the second, confirmatory step of the 

recommended two-step strategy for serological 

Borrelia diagnostics. The system is also 

suitable for investigating CSF in the diagnosis 

of neuroborreliosis. The assay is easy to 

perform and evaluate, and the entire analysis 

can be automated using specially developed 

devices and software. 

EUROIMMUN AG 

Luebeck, Germany 


Targeted sequencing solution 

for FFPE samples 

An ultra-deep targeted sequencing system 

for fresh frozen and Formalin-Fixed Paraffin 

Embedded (FFPE) samples, the Deep- 

Seq FFPE solution is based on microdroplet-based 

single molecule PCR technology. 

Delivering high specificity, accurate quantification 

and unbiased allelic representation, 

it enables researchers to interrogate as many 

as 500 targets at up to 50,000-fold coverage 

across extensive collections of wellannotated 

clinical samples to discover rare 

cancer and other disease-specific mutations 

that represent as little as one percent of a 

heterogeneous sample. 

RainDance Technologies, Inc 

Lexington, MA, USA 


– February/March 2011 32 

Product News 

Incubator with O 2 

and N 2 

control 

The MCO-19M CO 2 

incubator provides 

extremely accurate control of 

the environment within the incubation 

chamber. CO 2 

concentration 

is measured using a dual infra red 

solid state sensor that is not affected 

by humidity or temperature changes 

when the incubator door is opened. In 

addition, the CO 2 

sensor remains calibrated 

continually without the need 

for timed recalibration procedures. 

O 2 

concentration is measured using a solid state zirconium sensor 

that is maintenance-free, thereby removing the need for constant 

recalibration. CO 2 

and O 2 

levels are controlled using a PID controller 

to ensure rapid recovery without excessive overshoot of the 

chosen gas-level set-points. A stable and uniform temperature is 

provided within the chamber using Sanyo’s Direct Heat Air Jacket 

system, which is PID controlled to ensure rapid recovery after a 

door opening. The even distribution of temperature throughout 

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23–26 May 2011 

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the chamber contributes to the shelf-to-shelf reproducibility of 

conditions. The interior of the chamber is manufactured from 

InCu saFe, a copper-enriched stainless steel antimicrobial alloy, 

which inhibits the growth of fungi, mycoplasma and bacteria. For 

critical applications the SafeCell UV system automatic decontamination 

systems can be incorporated; this optional UV Illuminated 

air duct automatically maintains contamination-free air 

and humidity within the chamber. For applications which require 

additional decontamination, the hydrogen peroxide decontamination 

system is extremely effective and fast: a complete cycle only 

takes 130 minutes. This system is also safe, since the H 2 

O 2 

vapour 

is contained within the incubator and is broken down to water and 

oxygen at the end of the cycle. The door interlock and alarm also 

ensure that the system is safe. 

Sanyo BiomedicaL 

Etten-Leur, The Netherlands 


Midkine ELISA 

Midkine (MK) — also 

known as neurite growth 

promoting factor 2 

(NEGF-2) — is a cytokine 

that is expressed during 

embryonic development. 

In healthy adults MK is 

expressed at only low levels. 

However, MK levels are found to be elevated in the blood and 

urine of patients with carcinomas including oesophageal, stomach, 

colon, pancreatic, thyroid, lung, urinary, hepatocellular, neuroblastoma, 

glioblastoma, and Wilm´s tumour. MK levels can be 

measured in the early stage of cancer development, making MK 

an optimal marker for screening and preventive healthcare. The 

determination of MK in blood even detects organ-located solid 

tumour malignancy. High MK levels are associated with poor 

prognosis in some types of cancer. The combination of MK detection 

together with established oncologic markers improves cancer 

diagnosis. The Human Midkine ELISA kit extends BioVendor’s 

portfolio of diagnostic kits for oncology. Suitable for serum and 

plasma samples, the 96 well-microtitre plate format is suitable for 

use with standard ELISA equipment or robotic systems. 

Biovendor 

Modrice, The Czech Republic 


Clinical chemistry analyser 

Designed for small- to mid-sized 

hospitals and laboratories, the 

BioMajesty 6010 is a very compact 

fully automatic chemical 

analyser which can handle up to 

1200 tests per hour. With 43 reagents 

and 84 sample positions, 

it provides the flexibility needed 

for routine use. Both photometric 

and immunoturbidimetric 

assays can be performed as well as Na, K and Cl electrolyte 

determination using indirect ISE methods. Special emphasis has 

been given to the software, which combines a high degree of user

Product News 

33 


friendliness with optimally secure results. 

An integrated on-board haemolysis function 

optimises HbA1c determination. In 

addition, only very small sample volumes 

are required, so it is ideal in paediatric and 

geriatric settings. The system has readyto-use 

reagents with excellent on-board 

stability; and reagent consumption is very 

low. 

DiaSys Diagnostic Systems 

GmbH 

Holzheim, Germany 


Xylene-free tissue 

processing 

Traditionally xylene 

has been used as 

a clearing agent in 


for many years, primarily 

because of 

its miscibility with 

both alcohol and 

wax, while very effectively clearing the tissue 

of alcohol. However, xylene also exposes 

lab personnel to hazardous fumes. Fortunately 

this is no longer an issue with Tissue- 

Tek Tissue-Clear, which is a non-toxic, nonflammable, 

odourless and biodegradable 

(thus environmentally-friendly), substitute 

for xylene. It is also compatible with a common 

mounting medium, and its biodegradable 

and non-hazardous properties allow 

for easy disposal (depending on permission 

by appropriate local authorities). Substituting 

xylene by Tissue-Tek Tissue-Clear in a 

traditional processing programme on a Tissue-Tek 

VIP 6 will not jeopardise specimen 

integrity or quality. Whether a laboratory 

chooses to use a long or short programme 

for processing, lab personnel no longer have 

to be subjected to hazardous xylene fumes. 

Sakura Finetek Europe B.V. 

Alphen Aan Den Rijn, The Netherlands 


NGAL test for diagnostic use 

NGAL is a novel biomarker for diagnosing 

acute kidney injury (AKI). The key advantage 

of NGAL is that it responds earlier than 

other renal status markers such as serum creatinine 

and shows a proportionate response 

to injury. NGAL therefore provides a new 

way to identify patients at risk of developing 

potentially severe acute kidney injury (AKI) 

24-72 hours before the problem would otherwise 

be detected. The NGAL Test from Bio- 

Porto, previously available for research use 

and successfully tested in selected hospitals in 

Europe, the USA and Southeast Asia, is now 

CE-marked and available for use in Europe 

for early diagnosis of acute kidney injury. It 

will soon be possible to obtain The NGAL 

Test for diagnostic use in approximately 40 

countries. Using only a few drops of plasma 

or urine the test provides results in just 10 

minutes and thus addresses the widespread 

demand for urgent NGAL determination. 

Designed to run on open channels of chemistry 

analysers from numerous manufacturers, 

most laboratories thus have a convenient 

and easy way to measure NGAL. 

BioPorto Diagnostics A/S 

Gentofte, Denmark 


Rivaroxaban calibrators and 

controls for monitoring therapy 

Rivaroxaban is a 

new oral anticoagulant, 

developed 

by Bayer Healthcare 

and sold under 

the trade name 

of Xarelto. It is a 

direct factor Xa 

inhibitor approved 

in more than 80 

countries for the 

prevention of VTE after hip or knee surgery. 

Rivaroxaban is expected to be approved in 

the near future for other indications such 

as stroke prevention in patients with atrial 

fibrillation, thus making it a very useful and 

important anticoagulant. The STA-Rivaroxaban 

Calibrator & Control solution is 

designed for the determination of rivaroxaban 

concentration. Including rivaroxaban 

calibrators and controls, this solution can 

be used in two methods for different clinical 

needs and laboratory requirements. These 

are respectively the PT-derived method 

(STA-Neoplastine CI Plus), a cost effective 

global test for screening purposes, and the 

Anti-Xa method (STA-Liquid Anti-Xa), 

which is insensitive to analytical and biological 

variables and has a wide working range 

for specific and more sensitive dosage determination. 

Results are expressed in ng/mL 

of rivaroxaban. Reagents are barcoded for 

optimal ease of handling and traceability, 

and methods can be fully automated on the 

STA line, with dedicated test setups. Results 

are available in a few minutes, sso the system 

is ideal for STAT sample requirements. 

Diagnostica Stago 

Asnières sur Seine, France 


Treponema pallidum IgG 

test system 

Syphilis is a bacterial infection that is 

usually sexually transmitted, but may 

also be passed from an infected mother 

to her unborn child. Syphilis is a curable 

sexually transmitted disease (STD) which, 

however if left untreated can eventually 

lead to irreversible damage to the heart 

and nervous system. Despite the existence 

of effective prevention measures, the 

disease remains a global problem with an 

estimated 12 million people infected each 

year. The ZEUS ELISA IgG test system 

allows the qualitative detection of specific 

human IgG class antibodies to Treponema 

pallidum in human sera. The presence of 

antibodies to T. pallidum specific antigen, 

in conjunction with non-treponemal 

laboratory tests and clinical findings, may 

aid in the diagnosis of syphilis infection. 

Together with the company’s AtheNA 

Multi-Lyte Treponema pallidum IgG Plus 

Test System, multiple testing solutions 

for the qualitative detection of specific 

human IgG class antibodies to T. pallidum 

in human sera are now available. 

Zeus scientific 

Raritan, NJ, USA 


PCR-ready human genomic 

DNA extraction 

It is now possible 

to extract and 

purify human 

genomic DNA 

from whole 

blood much 

faster than with 

a spin column 

for use in genetic 

testing, pharmacogenomics 

and 

forensic applications. 

Using an Eppendorf epMotion 

automated pipetting station, Akonni 

TruTip delivers with the simple push of 

a button inhibitor-free, PCR-ready DNA 

up to five times faster than ‘gold standard’ 

spin columns. Alternatively one to 

eight ultra-rapid manual extractions can 

be performed with TruTip and a Rainin 

EDP 3Plus single- or multi-channel 

pipette, in as few as four minutes. Yields 

and purity are comparable to those 

obtained using gold standard kits. 

Akonni Biosystems 

Frederick, MD, USA 


– February/March 2011 34 

Product News 

STAT immunoassays for cardiac 

biomarker testing 

Roche has introduced a complete battery of 

STAT immunoassays for cardiac biomarker 

testing on the cobas 6000 analyser series, the 

integrated system designed for diagnostic 

labs with medium testing volumes. With a 

nine-minute duration, the assays are faster 

than any other cardiac immunoassay tests 

currently available on an integrated platform 

and enable labs to deliver results to 

doctors treating cardiac patients in about 

half the time of standard Roche tests. The 

tests include troponin T, CK-MB, myoglobin 

and NT-proBNP, and are virtually 

equivalent to the company’s 18-minute tests 

in performance, precision and sensitivity. 

Roche diagnostics 

Mannheim, Germany 


Immunofluorescence 


A complete 

system for the 

management of 

autoimmune IF 

lab procedures, 

the i-mLS is an 

extremely userfriendly 

software 

that provides a high level of control of all 

aspects of autoimmune IF testing. Designed 

to be the ideal companion for the I-mLD 

microscope, the software enables users to 

capture and store microscope images and to 

create a customised image catalogue for use 

as a reference. The software also allows easy 

result reporting and the addition of comments 

from the analyst, which can be introduced 

either into the basic system or into 

a report format. In addition, the software 

enables the inclusion of additional information 

such as clinical comments and images 

of patient tests, the consultation of results 

by patient, test or session, as well as communication 

with the LIS. When connected 

to the i-Pro immunofluorescence processor, 

the i-mLS software can import current session 

information, read results at the microscope 

and then export data back to the i-Pro 

information on a new working session with 

new or repeat test request. The system also 

allows laboratory managers to maintain 

traceability of reagents used in all tests performed 

on the i-Pro and to remotely access 

stored results from their own office. 

BioSystems 

Barcelona, Spain 


Platelet disfunction test system 

The next-generation of Siemens’ best-in-class 

technology to detect inherited, acquired, and 

drug-induced platelet dysfunction, the new 

INNOVANCE PFA 200 system has userfriendly 

updates including a colour LCD 

touchscreen to access detailed patient and 

result information, a barcode reader for digital 

patient result tracking and LIS connection, 

and a USB port to archive stored data. The 

system also has multiple user-friendly software 

updates, including new software that 

supports closure curve illustrations for use in 

scientific studies. Results, which are avaiable 

in 4-8 minutes, allow rapid comprehensive 

bleeding assessment. The system carries out 

collagen/EPI and collagen/ADP tests and the 

company’s latest addition, the P2Y test for 

detection of platelet P2Y12-receptor blockades 

in patients. 

Siemens Medical Solutions 

Diagnostics 

Tarrytown, NY, USA 


Vitamin D control 

Now available throughout the European 

Union, Fujirebio Diagnostics’ Vitamin D 

Control contains both 25(OH) Vitamin D2 

and 25(OH) Vitamin D3, and is intended 

for use as a quantitative, assayed serum 

control. The control has excellent stability, 

value assignment across platforms and 

clinically relevant levels, enabling laboratories 

to monitor the performance of their 

vitamin D assays. 

Fujirebio diagnostics 

Malvern, PA, USA 


Calendar of events 

May 7-10, 2011 

21st ECCMID / 27th ICCs 

Milan, Italy 

Tel. +41 61 686 77 11 

Fax +41 61 686 77 88 

e-mail: eccmid@escmid.org 

www.eccmid-icc2011.org 

May 9-13, 2011 

4th International Congress of 

Myology 

Lille, France 

Tel. +33 4 78 176 276 

http://myology2011.org/ 

index_us.html 

May 13-15, 2011 

XIIth International Congress of 

Paediatric Laboratory Medicine 

(ICPLM) 


Tel. +39 0266802323 

e-mail: icplm2011@mzcongressi.com 

www.icplm2011.org/ 

May 15-19, 2011 

IFCC-Worldlab-Euromedlab 

Berlin 2011 Congress 


Tel. +39 02 66802323 

Fax +39 02 6686699 

e-mail: info@berlin2011.org 

www.berlin2011.org 

May 23-26, 2011 

FOCUS 2011 

Harrogate, UK 

Tel. +44 141 434 1500 

Fax +44 141 434 1519 

e-mail: focus2011@meetingmakers.co.uk 

www.focus-acb.org.uk 

May 24-27, 2011 

Hospitalar 2011 

São Paulo, Brazil 

www.hospitalar.com/ingles/ 

June 25-29, 2011 

10th World Congress on 

Inflammation 

Paris, France 

Tel. 33 1 53 85 82 71 

Fax 33 1 53 85 82 83 

Email: 

info@inflammation2011.com 

www.inflammation2011.com 

July 17-20, 2011 

6th IAS Conference on HIV 

Pathogenesis, Treatment and 

Prevention 

Rome, Italy 

www.ias2011.org 

July 24-28, 2011 

2011 AACC Annual Meeting 

Atlanta, GA, USA 

www.aacc.org/ 

events/2011am/ 

September 21-24, 2011 

14th Annual Meeting of the 

European Society for Clinical 

Virology (ESCV) 

Funchal, Madeira, Portugal 

www.escv.org/meetings/meetings.asp 

October 2-6, 2011 

12th International Congress of 

Therapeutic Drug Monitoring 

and Clinical Toxicology 

Stuttgart, Germany 

Tel. +49 711 8931 636 

Fax +49 711 8931 370 

e-mail: info@iatdmct2011.de 

www.iatdmct2011.de 

October 15 – 16, 2011 

The Lancet / ESCMID Conference 

on Healthcare-Associated 

Infections and Antimicrobial 

Resistance 

Beijing, China 

Tel. +86 21 61333077 

Fax +86 21 52980210 

e-mail: summitenquiry@elsevier.com 

October 24–26, 2011 

ESCMID Conference on 

Diagnosing Infectious Diseases: 

Future and Innovation 

Venice, Italy 

Tel. +39 041 52 62 530 

Fax +39 041 52 71 129 

e-mail: ico@icorganization.it 

www.escmid.org 

For more events see: 

www.cli-online.com/events/ 

Dates and descriptions of future 

events have been obtained from 

official industrial sources. CLi cannot 

be held responsible for errors, 

changes or cancellations.

Who can really help me grow? 

Only Siemens has the innovative solutions your lab needs 

to reach the top and the vision to keep you there. 

Planning for your future starts with choosing the right diagnostics partner today. Siemens provides comprehensive and 

customizable solutions so laboratorians and clinicians can improve productivity every day. And, with a 130-year tradition 

of innovation, you can trust Siemens to stay on the leading edge of emerging trends and technologies, so together we 

can set a new standard in patient care for years to come. www.siemens.com/diagnostics 

Answers for life. 

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© 2011 Siemens Healthcare Diagnostics. All rights reserved.

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