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Re-Phew! Consulting – Undergraduate Thesis Example
Comparing Renal Biomarkers and their Utility to
Monitor the Mainstay of Immunosuppression Therapy
Name
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Dissertation submitted in fulfilment
for the award ……………………………………
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2
Declaration
This work has not previously been accepted in substance for any degree and is not
currently submitted in candidature for any degree.
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Date.................................................................
STATEMENT 1
This dissertation is the result of my own investigations, except where otherwise stated.
Other sources are acknowledged by explicit references.
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STATEMENT 2
I hereby give consent for my thesis, if accepted, to be available for photocopying and for
inter-library loan, and for the title and summary to be made available to outside
organisations.
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Contents
A. Acknowledgments
B. Abstract
C. Abbreviations
1. Aims
2. Introduction – Chronic Kidney Disease (CKD)
3. Prevalence of Chronic Kidney Disease (CKD) in the UK
4. Target Patients in the UK
5. Renal Guidelines in the UK
6. Preventing Chronic Kidney Disease (CKD)
7. Reasons for Late Identification
8. Lack of Data on Early Detection
9. Impact/ Consequences of Late Identification
10. How to Detect/ Diagnose Chronic Kidney Disease (CKD)
11. Advantages for Early Detection
12. Clinical Testing
13. Urine Tests to Investigate Chronic Kidney Disease (CKD)
14. Blood Tests to Investigate Chronic Kidney Disease (CKD)
15. Why is Renal Point of Care Testing (POCT) Important?
16. POCT Methodology
17. What are the different Biomarkers for Renal Function?
18. Renal Transplant Immunobiology
19. Monitoring the Mainstay of Immunosuppression Therapy (IST)
20. Critique of Creatinine Methods
21. Critique of Proteinuria, ACR, Cystatin C, and NGAL Methods
22. Immunosuppression (IST) in Paediatric Post-Transplant Patients
23. Discussion
24. Further Research & Interventions
25. Conclusion
26. Summary Points
27. What this work adds
28. References
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A. Acknowledgements
I would like to thank my supervisor, who has been absolutely amazing during this
period. Thank you for your time in supervision.
I would like to dedicate this research to all my professional colleagues. I still have
far to go and much to learn. Thank you for the inspiration.
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B. Abstract
Background
Chronic Kidney Disease (CKD) is on the rise in all ethnicities. Evidence also highlights that this
disease negatively impacts quality of life and places an enormous financial implications on the
health care system for the provision of care to patients. Thus, it is of utmost importance to
devise strategies that prevent CKD and delay progressive loss of renal function in the wider
population.
Aim
This thesis aims to provide an overall perspective on Chronic Kidney Disease (CKD) as it
currently stands within the United Kingdom (UK) and evaluate the utility of renal biomarkers to
monitor nephrotoxicity and efficacy of immunosuppression therapy post renal transplantation.
Discussion
More emphasis is being placed on the prevention and early detection for CKD. Individuals who
are at high risk of renal failure need better ‘preparation’ and prompt early referral to secondary
care is a must in order to allow the best prognosis. In addition, the utility of biomarkers for
monitoring post-transplant immunosuppression therapy (IST) has to become more specific in
post-transplant IST monitoring. Neutrophil Gelatinase-Associated Lipocalin is becoming more
apparent in this respect.
Conclusion
Biomedical scientists now have a more active and larger role to play with respect to monitoring.
Whilst there have been guidelines on the screening and monitoring of anticoagulation-based
medication, there perhaps needs to be more collaborative guidelines to monitor mainstay IST
post-transplant patients receive. Specialists from both clinical and laboratory practice need to
come together to further inform which biomarkers would be specific for monitoring IST titres.
Keywords
Chronic Kidney Disease, Creatinine, Cystatin C, Immunosuppression Therapy, Proteinuria,
Neutrophil Gelatinase-Associated Lipocalin, Point of Care Testing
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C. Abbreviations
ACR Albumin Creatinine Ratio
CrCl Creatinine Clearance
CKD Chronic Kidney Disease
C-G Cockcroft-Gault Formula
CsA Cyclosporine
eGFR Estimated Glomerular Filtration Rate
ESRD End-Stage Renal Disease
GFR Glomerular Filtration Rate
GP General Practitioner
HLA Human Leukocyte Antigen
HD Haemodialysis
IST Immunosuppression Therapy
MHC Major Histocompatibility Complex
MDRD Modification of Diet in Renal Disease
NGAL Neutrophil Gelatinase-Associated Lipocalin
NSAIDs Non-Steroidal Anti-Inflammatory Drugs
NHS National Health Service
NSF National Service Framework
POCT Point of Care Testing
RRT Renal Replacement Therapy
SCr Serum Creatinine
QOL Quality of Life
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1. Aim
This thesis aims to provide an overall perspective on Chronic Kidney Disease (CKD) as it
currently stands within the United Kingdom (UK). Additionally this work seeks to evaluate
the utility of renal biomarkers to monitor the efficacy of immunosuppressive therapy
following renal transplantation.
2. Introduction - Chronic Kidney Disease (CKD)
Chronic Kidney Disease (CKD) is a long-term condition and has been described as the
gradual, and usually permanent, loss of kidney function over time. Early in the disease
process, people with CKD often experience no symptoms and CKD has, for a long time, been
an under-diagnosed condition (1). Even in the absence of symptoms, CKD appears to add
significantly to the burden of cardiovascular disease (CVD) and death (1).
In the 1970s renal diseases glomerulonephritis and pyelonephritis were the most prevalent
causes for enrolment into Renal Replacement Therapy (RRT) programmes. The prevalence of
these diseases diminished and diabetes is now increasingly one of the major causes of CKD,
(predominantly type-II) in addition to renal vascular diseases such as hypertension and
atherosclerosis. Several reasons have been put forward to explain this change including age,
ethnicity, increased Body Mass Index (2). Currently in the National Health Service (NHS),
provides diagnostic tests, medication and on-going treatments for renal failure. Together with
Haemodialysis (HD) treatment costs are around £20,000 - £25,000 per patient per year (The
National Service Framework for Renal Services (NSF) (3-4). renal function is close to the
level where dialysis is required, that is when not much can be expected of conservative
kidney protective treatments (4). Renal function is determined by degree of Glomerular
Filtration Rate (GFR)/ estimated GFR (eGFR). Table 1 provides definition of the five stages
of CKD by GFR (see below).
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Table 1: Stages of CKD
CKD Stage Definition
Stage 1 Kidney damage with normal or raised GFR
(≥ 90 ml/ min/ 1.73m2)
Stage 2 Kidney damage with normal or raised GFR
(60-89 ml/ min/ 1.73m2)
Stage 3 Moderately impaired GFR
(30-59 ml/ min/ 1.73m2)
Stage 4 Severely impaired GFR
(15-29 ml/ min/ 1.73m2)
Stage 5 End Stage Renal Failure or GFR
(< 15 ml/ min/ 1.73m2)
Table adapted from (1)
Proteinuria is an important parameter when considering CKD in a wider community.
Proteinuria originates from the kidney and occurs as a result of injury to either the glomerulus
or the renal tubule or both. In the UK, it is relatively common in the general population with
reported point prevalence of up to 8% but the prevalence falls to around 2% on repeated
testing. Chronic glomerular injury resulting in proteinuria may be secondary to prolonged
duration of diabetes or hypertension. A tubular origin of proteinuria may be associated with
inflammation of renal tubules triggered by prescribed drugs or ingested toxins. In the absence
of obvious clues to the cause of persistent proteinuria on history or clinical examination it is
worthwhile reviewing the patient's prescribed drugs to identify any potentially nephrotoxic
agents, e.g. Non-Steroidal Anti-Inflammatory Drugs (or NSAIDs).
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3. Prevalence of Chronic Kidney Disease in the UK
Over the last few years, collaborative efforts, enabled by a common definition of CKD, have
provided a description of the epidemiology, natural history, and outcomes of this disease and
have improved our understanding of its pathophysiology. There is increased recognition that
CKD is encountered in multiple settings and in all age groups, and that its course and
outcomes are influenced by the severity and duration of the causative event. The effect of
CKD on an individual patient and the resulting societal burden that ensues from the long-term
effects of the disease is attracting increasing scrutiny. There is evidence of marked variation
in the management of CKD due to a lack of awareness and an absence of standards for
prevention, early recognition, and intervention. These emerging data point to an urgent need
for a global effort to highlight that CKD is preventable, its course is modifiable and its
treatment can improve outcomes (5).
Kearns et al. (2013) inform that the identification of the prevalence of CKD relies on
opportunistic testing, and there is evidence that not everyone with CKD is being identified
through the Pay for Performance (P4P) scheme (where service providers under this
arrangement are rewarded for meeting pre-established targets for delivery of healthcare
services). Data from the Health Survey for England quote a national prevalence of 6%; the
corresponding estimate from the P4P scheme is 4.3%. Because of this difference, modelled
estimates of the prevalence of CKD are required to support case-finding for CKD.
Traditionally, P4P schemes have enabled General Practitioners (GPs) to identify and screen
target areas where there is a recognised under-detection of CKD. This has improved existing
methods for identifying individuals at risk of CKD such as testing based on currently
recommended risk factors (6). Quality-Adjusted Life Year (QALY) measures have also been
put in place for CKD and are used in cost-utility analysis to calculate the ratio of cost to
QALYs saved for a particular health care intervention (6).
One of the issues regarding accuracy in prevalence of CKD in the UK is that health
professionals in primary and secondary healthcare have different definitions of CKD and its
specific stage. Some use the Modification of Diet in Renal Disease (MDRD) to estimate GFR
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(eGFR) and some will use ClCr while others will look at other clinical parameters before
progressing to look for renal insufficiency. Because there is no overall UK consensus when to
detect CKD, prevalence may be exaggerated. However early laboratory analysis has a
positive effect on referral rates in a large adult population (7).
The National Institute for Health and Care Excellence 2013 guideline recommends Point of
Care Testing (POCT) in individuals at higher risk for CKD (8). These include individuals
with diabetes, hypertension, cardiovascular disease, connective tissue disorders, a family
history of renal disease and those prescribed potentially nephrotoxic drugs. Patients with
sudden onset of lower limb oedema and associated proteinuria should have a serum albumin
level measured to exclude nephrotic syndrome. Renal tract ultrasound will measure kidney
size, and detect scarring associated with chronic pyelonephritis or prior renal stone disease
which can cause proteinuria (8). Proteinuria prevalence is also an issue in long-term
management of patients with established CKD because this can also affect their albumin-
creatinine ratio more generally and it is understood that plasma albumin does decrease over
time (9-12). Analysis of individuals with unidentified CKD suggests that their risk profile
may be different to patients with identified CKD; this is an area that requires further research
(6).
4. Target Patients in the UK
Primary care records have been audited of patients aged 50-75 years who have either
hypertension or diabetes, and are therefore considered to be at high risk of developing renal
insufficiency (13). Kissmeyer et al. (1999) conduct research to identify if patients had their
blood pressure measured and urine tested for protein within 12 months, and SCr measured
within 24 months. Case notes and computer records in 12 general practices from inner and
greater London were examined retrospectively; the study revealed a total of 16,855 patients
were aged 50-75 years and informed that there is a high prevalence of chronic renal
insufficiency in hypertensive and diabetic patients (13). Renal failure as a complication of
diabetes is 10 times greater in South Asians than in Caucasians (14).
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Ellis and Cairns (2001) investigate hospital biochemistry databases for each individual's most
recent serum creatinine (SCr). Individuals with no result recorded in the previous year were
then invited for POCT: 189/365 (51.8%) attended. Data was collected on 821 of a total
potential population of 997 (15). Taking 120 mmol/l as the upper limit of normal, the overall
prevalence of renal disease in this population was 8.4%; 6.1% in the hypertensive patients,
12.6% in the diabetics and 16.9% in those with both. Significant proteinuria was present in
3.9% of the total population; 2.2% of hypertensive patients, 8.3% of diabetics and 3.9% of
those with both (15). At POCT, 44.5% of individuals had inadequately controlled blood
pressure. Renal impairment is common in high-risk population and POCT for renal disease is
simple, safe and gives a high yield of positive results (15).
As patients with renal failure owing to type II diabetes, hypertension or generalized CVD
have in most cases never experienced acute symptoms indicative of renal disease, such as
haematuria, severe hypertension or oedema (as patients with glomerular or interstitial
diseases), POCT programmes have to be designed to detect them at an earlier phase (16).
One study has found that Body Mass Index (BMI) is associated with an increased risk of the
development of CKD in men in the general population. The maintenance of optimal body
weight may reduce the risk of CKD, (17). Evidence suggest strategies to control serum uric
acid levels, fasting plasma glucose and proteinuria measurements are equally important in
detecting individuals at high risk for developing End-Stage Renal Disease (ESRD), (17).
Although prospective studies are needed, findings indicate that obesity, including metabolic
syndrome, is a potential treatable cause of CKD. Rigorous efforts should be made to optimize
weight control and reduce the risk of CKD and ESRD by a judicious combination of diet,
exercise, and psychological therapies (17).
5. Renal Guidelines in the UK
In the UK, the NSF for Renal Services provides specific guideline and framework of how
specific care packages should be delivered for patients who are newly diagnosed and long-
term renal patients, respectively. The NSF for Renal Services has two parts; Part I (Jan 2004)
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outlines Dialysis and Transplantation and Part II (Feb 2005) refers to CKD Acute Renal
Failure and End of Life Care (2-3).
Until recently, in the UK no agreed definition of CKD existed. SCr was commonly reported
as a surrogate marker of filtration. The absolute upper limit of ‘normal’ SCr value varies
between laboratories and is a sensitive biomarker (1). The number of patients taken into renal
replacement therapy (RRT) programmes has gradually increased over the last decade. This
may partly be due to improvements in dialysis techniques and a better availability of these
programmes. However, the pattern of the cause of end-stage renal failure has been changing
over time (18-19).
In (2006), the Kidney Disease: Improving Global Outcomes (KDIGO) initiative made
commendations that all countries should have a targeted-POCT program for CKD, focusing
on individuals known to have diabetes, hypertension and CVD (23). Research highlights that
measuring albumin creatinine ratio (ACR) (preferably on a first void morning specimen)
implementing POCT in all at-risk individuals is key (20-23).
6. Preventing Chronic Kidney Disease (CKD)
Given lack of specific early detection data, POCT middle-aged and older individuals for
proteinuria and treating some with ACEIs is, at best, a promising primary prevention strategy
for preventing CKD. A large population-based cohort study, with nested trial of ACEIs is still
required to evaluate whether this model of POCT for renal disease does more harm than good
(21).
In the case of a younger population and with respect to CKD and monitoring post-transplant
IST, dose modifications should be considered when prescribing therapeutic agents with a
known nephrotoxicity to adults. The lower age group subsets of the paediatric population
may be particularly sensitive to certain excipients that are not entirely inert and may have
side effects. Particularly, some excipients which can be used on adults may be toxic in
youngsters because of their immature and rapidly changing metabolic & elimination system
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(22). The salt of the active ingredient and the chemical nature of the preparation must be
carefully considered to avoid administration of excessive amounts of electrolytes (23).
Liquid preparations more often contains excipients like preservatives of which concentration
should be at the minimum level if not possible to eliminate. When preservatives are required,
the concentration should be at the minimum level and a thorough justification for the choice
of the preservative should be provided. Formulation used for chronic conditions may cause
repeated cumulative exposure to excipients. Therefore, the acceptable daily intake and safety
limits of excipients for youngsters must be checked, particularly for renal impaired
youngsters (lower renal elimination, kidney malfunction).
7. Reasons for Late Identification
Diabetes Mellitus and/or hypertension cause renal disease in up to 40% of patients requiring
dialysis. These patients are presumably being monitored by internists, endocrinologists or
cardiologists, and many referrals come from these physicians; other patients may be referred
by general practitioners. Data regarding disease status at the time of referral are also limited.
Substantial CVD and risk factors are evident at the time of referral. Most of the literature
describes data for those starting dialysis (i.e. late referral) rather than a broader spectrum of
all patients with renal insufficiency referred to nephrologists. Reasons for late referral include
insensitivity of current POCT tools (24).
Haemodialysis (HD) centres in the UK are overwhelmed by the increasing numbers of
patients accepted in for RRT. GP clinics are inundated with minor ailment complaints and
have a selection of patients who are at risk of renal failure. This is partly due to poor drug and
patient monitoring, but more because the lack of making sure the patient is self-aware,
especially diabetics and hypertensive recipients (24). There is growing awareness of a need
not only to identify patients with CKD at an earlier stage in the disease process, but also to
initiate treatment strategies earlier, in order to delay both progression of CKD and co-morbid
diseases and to define the optimal time required to prepare CKD patients for RRT. These
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three strategies are linked, and rely on appropriate identification of patients at risk of renal
disease (24).
De Jong et al (2006) investigate the current scale of the problem in a large region in England,
identifying the prior health care, patient characteristics, referral pattern, and outcomes of
those accepted onto RRT (16). A quarter of patients are referred for specialist nephrology
treatment at a very late stage, within 1 month of RRT. They are less likely to receive
interventions that could alter the progression of CRF or reduce its associated co-morbidity,
have a worse clinical state at the start of RRT, longer hospitalization and poorer survival.
These differences were much less marked for those referred within 1-4 months of starting
RRT, although this is an insufficient time to prepare for RRT. Further research is needed to
determine the missed opportunities for more proactive diagnosis and management of CKD
and also the management of immunosuppression therapy post-transplant (16, 25).
8. Lack of Data on Early Detection
The reasons for the under diagnosis (or lack of data on early detection of CKD) and under-
treatment of CKD still seems to be an on-going issue (26). Appropriate preventative
treatment and therapeutic interventions have to be implemented. POCT programmes should
1) promote early detection of CKD and co-morbid conditions; 2) have more concise use of
appropriate outcome measures to clarify patient care; 3) implement strategies to delay/
prevent disease progression or threat complications; 4) adequate preparation for timely
initiation of RRT (26).
Jurkovitz et al (2002) conduct a cross-sectional survey by way of a voluntary POCT of
relatives of patients with CKD in 10 communities in one South Eastern state of the USA.
Age; race; sex; family and personal history of CKD hypertension, and diabetes; height;
weight and blood pressure were determined. Proteinuria and random blood glucose and SCr
were also measured. Awareness of CKD is less than expected among relatives considering
the high prevalence of CKD in this population. Because data is still required in this
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community, POCT individuals would help form initial fundamental data and potentially
identify more individuals with early CKD (27).
In research by Jurkovitz et al (2002), individuals were screened for kidney disease risk
factors including blood pressure, blood glucose level, SCr level, haemoglobin level,
microalbuminuria, haematuria, pyuria, body mass index, and estimated glomerular filtration
rate (eGFR). The aim of this research was 1) To highlight the importance of early detection in
individuals with hypertension or diabetes from a first-order relative with hypertension,
diabetes, or CKD and 2) where data has been previous insufficient or robust (28). The team
summarized that targeted POCT is effective in identifying individuals with previously
unidentified or poorly controlled CKD risk factors, as well as individuals with a moderately
decreased eGFR (28).
Perico et al (2005) explore CKD in middle-income and low income countries, where the use
of RRT is scarce or non-existent. The team sought to demonstrate that in emerging countries
the best strategies against renal disease are prevention and early detection (29). Individuals
were instructed to void a clean urine specimen, and a dipstick test was performed. Individuals
with positive urinalysis were enrolled in a follow-up program with subsequent laboratory and
clinical checks (29). The researchers highlighted that mass POCT of the population for renal
disease is feasible in developing countries and can provide useful information on frequency
of renal diseases and this is all the more important in enhancing data on early detection (29).
Where lack of data on early detection is present, the use of long-term treatment of low dose
of Enalapril safely prevents increase in arterial blood pressure and progressively reduces
haematocrit and proteinuria. Aside from its scientific value, this particular investigation can
be taken as an example of how, (by rationalizing resources and investing in research
programmes), CKD progression and cardiovascular risk may eventually improve, which
ultimately should translate into less demand for HD, and thus provide alternatives to costly
RRT (29).
Assessment of albumin and/or protein excretion in urine is a key step in the early detection
and appropriate management of CKD. The approach to testing for albuminuria/proteinuria in
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the community is variable and often suboptimal. It is hampered by a variety of factors, some
include 1) variation in laboratory measurement; 2) lack of standard reference materials and
testing procedures; 3) variable definitions and units of reporting; 4) conflicting
recommendations and practices regarding who to test; and 4) uncertainty over when and how
testing is most appropriately done (19).
9. Impact/ Consequences of Late Identification
Nearly a quarter of patients are referred for specialist nephrology treatment at a very late
stage, within 1 month of RRT. They are less likely to receive interventions that could alter the
progression of CRF or reduce its associated co-morbidity, have a worse clinical state at the
start of RRT, longer hospitalization and poorer survival. These differences were much less
marked for those referred within 1-4 months of starting RRT, although this is an insufficient
time to prepare for RRT. Further research is needed to determine the missed opportunities for
more proactive diagnosis, (25). The consequences of late referrals include increased
morbidity, mortality, and resource utilization. There is also an impact on patients' quality of
life and missed opportunities for pre-emptive transplantation. Late referral also limits
therapeutic options, and these limitations have consequences on long-term outcomes once
patients are on dialysis (16), (18).
10. How to Detect/ Diagnose Chronic Kidney Disease (CKD)
Appropriate preventative treatment and therapeutic interventions have to be implemented.
Specific programmes should promote early detection of CKD and co-morbid conditions; use
of appropriate outcome measures to clarify patient care; implementation of strategies to delay
disease progression and/ or prevent or threat complications; adequate preparation for timely
initiation of RRT (26).
It is important to appreciate that eGFR and Cockcroft-Gault (C-G) formula use creatinine for
screening basis. Duncan et al (2001) conduct a research to estimate the prevalence of patients
who have significantly reduced GFR as calculated by the C-G formula. This study included
2781 outpatients referred by community physicians to an urban laboratory network for SCr
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measurement. GFR was estimated using the C-G formula. Patients were grouped according to
the concordance of SCr level abnormalities (abnormal >130 µmol/l) with significantly
abnormal C-G values (abnormal <50 ml/min). The C-G value of < or 50 ml/min was chosen
to reflect substantial renal impairment in all age groups (30). This study demonstrated that the
substantial prevalence of significantly abnormal renal function among patients identified by
laboratories as having normal-range SCr, including calculated estimates of GFR in routine
laboratory reporting may help to facilitate the early identification of patients with renal
impairment (30).
Abouchacra et al (2012) conduct research to also evaluate the diagnostic performance of
Neutrophil Gelatinase-Associated Lipocalin (NGAL) versus Cystatin C and eGFR using
CKD Epidemiology Collaboration (CKD-EPI - equation developed in an effort to create a
formula more precise than the Modification of Diet in Renal Disease (MDRD) calculation
formula, especially when actual GFR is > 60 mL/min per 1.73 m2), MDRD and Cystatin C in
post-renal transplant recipients and non-transplant CKD patients. This team identified
significant associations between NGAL, SCr and Cystatin C and eGFR (31), however the
implantation of GFR/ eGFR with Modification of Diet in Renal Disease (MDRD) calculation
can still be effective in detecting CKD and potentially in the use of Neoral IST monitoring
(32).
Several papers also describe the laboratory methods to measure albuminuria (albumin in the
urine), the way urine samples could be collected, the definitions for an abnormally elevated
albuminuria, and the way in which a population screened using POCT on albuminuria might
be organized (16), (18). Individuals with elevated levels of urinary albumin are at increased
risk for RRT and accelerated loss of renal function. Early detection of urine protein to slow
progression of CKD and decrease mortality is not cost-effective unless selectively directed
toward high-risk groups (older persons and persons with hypertension) or conducted at an
infrequent interval of 10 years, (16), (18). POCT for albuminuria identifies patients at
increased risk for progressive CKD, 40 – 50 % of who were previously undiagnosed or
untreated (16, 18).
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11. Advantages for Early Detection
Some conditions are self-limiting, while others may progress so slowly that there is sufficient
renal function for the person to live out their normal lifespan. Even when the kidneys begin to
fail, careful control of diet and blood pressure, and timely intervention to prevent
complications, can enable a person to survive in good health for many years. To support more
effective methods of monitoring CKD, it would be interesting for biomedical scientists to
prompt wider service discussions and identify if it would be feasible for the implementation
of a POCT programme in a community environment in addition to a Medicine Use Review
(MUR) service. CKD must represent a significant public health problem, be characterized by
a clear natural history with a detectable asymptomatic period, outcomes should be improved
by early treatment, and acceptable POCT should be more widely available. Health systems
must provide adequate and appropriate follow-up medical care for individuals with newly
detected CKD as well as post-transplanted patients in community (20).
Patients who decide on the spur of the moment to have a health check can be seen without
delay – it may be several days before there is an appointment at a local surgery. Patients may
also require further guidance, to be informed about self-care, and so perhaps the community
pharmacy setting should be explored for POCT and early detection? The community
pharmacy is an open environment and community pharmacists could provide this service,
perhaps more conveniently. Commissioning Enhanced Services under the NPC (2005), such
as POCT programmes can also compliment the on GP services (20).
Implementing Enhanced POCT services that encourage suspect or high risk patients to be
screened in community pharmacy would be another step forward in 1) early detection and
preventing CKD in suspect/ high risk patients, 2) potentially releasing workload of GPs and
3) help in early detection to prevent late referrals, 4) tighten control of medication with the
implementation of an MUR service, 5) strengthen collaborations in primary care and 6)
strengthen renal care according to the NSF for Renal Services (3), (4). Ultimately early
detection and proactive care/ POCT in pre-dialysis communities will not only improve
quality of life (QOL), but will also stand to save the NHS trust substantial funds (26).
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12. Clinical Testing
Biomedical scientists who work either in hospital or primary care and those with access to
patient records need to be familiar with test results and what they might mean. Not all
laboratory data are of direct relevance, but a general knowledge of these is recommended in
order to understand a patient’s medical background. Increasingly, Biomedical Scientists are
offering diagnostic POCT and therefore are able to interpret results and advise clinical teams,
appropriately (34).
POCT or test results can be expressed qualitatively, quantitatively or semi-quantitatively.
Haematology and biochemistry results are generally expresses quantitatively (i.e. as a set of
numbers) and microbiological results, for example tend to be semi-quantitative in that reports
will identify the micro-organisms present and describe their sensitivities to antibiotics (34). It
is important to be aware that reference values can range from one laboratory to another and
it’s always advisable to compare a result with the reference values in which a sample in
question has been sampled (or laboratory a test has been sent). POCT also offers greater
opportunities to explore monitoring immunosuppression treatment in renal post-transplant
recipients (33-36).
13. Urine Tests to Investigate Chronic Kidney Disease (CKD)
Urine is produced by the kidneys to remove soluble waste substances from the body. These
can be detected using dipstick methods (e.g. tests for pregnancy or diabetic ketoacidosis) or,
if more detailed information is required, urine can be sent from GP/ hospital settings to the
laboratory for analysis. Some substances can be measured in blood and urine. The main
advantage of tests on urine is that they are relatively non-invasive compared with blood tests.
However, if the urine specimen is collected incorrectly, this may affect results (35).
If a patient screens positive for Microalbuminuria, the test should be repeated at least twice
more. If one of the three tests taken is positive, then the patient is considered ‘normal’ and
can be screened again in a year. If two out of three are positive, it is considered that the
patient has Microalbuminuria. The ACR is measured at several visits. The current guidelines
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for confirmed Microalbuminuria are to start an inhibitor of the rennin-angiotensin system,
control glucose levels, control ABP, instigate aspirin and take measures to control
cardiovascular risks and other microvascular complications (36).
Creatinine is the principal endogenous marker that is used to measure GFR. Creatinine
clearance (CrCl) measurement, calculated from timed urine collection (24-hour urine) and
SCr, can result in overestimation of GFR due to tubular secretion of creatinine and problems
of accurate urine collection. Estimating GFR based on SCr and additionally correcting for
variables such as age, gender, racial origin and body weight can be more reliable than 24-
hour urinary CrCl (1).
At risk patients could be detected for progressive CKD and cardiovascular disease by POCT
for albuminuria. With this approach, there is the possibility of being able to detect patients
with stages 1 and 2 CKD, who cannot be detected by POCT only for GFR. Various review
papers have described the laboratory methods to measure albuminuria, the way urine samples
could be collected, the definitions for an abnormally elevated albuminuria, and the way in
which a population POCT on albuminuria might be organized (16), (18).
It would be feasible to screen patients in the community who are at increased risk of
developing CKD using a urinalysis to retrieve Albumin-Creatinine Ratio (ACR) would be
advantageous and cost-effective. Alongside a POCT in the wider community settings (e.g.
community pharmacy), more public health awareness and self-care campaigns are
compulsory in order that patients acknowledge renal disease and how it is associated with
primary diseases such as obesity, diabetes and hypertension.
14. Blood Tests to Investigate Chronic Kidney Disease (CKD)
Creatinine is a breakdown product of muscle. This means creatinine can be used as a measure
of glomerular filtration rate (GFR), and therefore renal function. Creatinine levels vary
according to an individual’s size and muscle mass. Urea can also be routinely used to
evaluate renal function. Estimated GFR (eGFR)/ Creatinine are still choice biomarkers to test
in suspect/ high risk renal patients, since traditionally they are also parameters used to
21
monitor renal function in long-term renal patients (and monitor IST), however some of
today’s tests – both at the POCT level and in laboratory measure the ratio between albumin
and creatinine (ACR) concentration. Creatinine concentration is affected by a wide range of
factors, including age, sex, ethnicity and body mass. Its benefit is greatly overshadowed by
the potential for misinterpretation it introduces. Measuring albumin alone is perhaps simpler
and more cost- effective and sufficient. The cut-offs for albumin are the same for men and
women of any age and ethnicity, and albumin concentration is not affected by muscle mass,
so long as the patient hasn’t consumed abnormal amounts of liquid, albumin concentration
alone offers the same sensitivity as albumin/creatinine – at less than half the cost. If
Biomedical scientist implement a POCT programmes, then there are pharmaceuticals offering
POCT kits that can support the analysis of a number of clinical parameters, as highlighted by
(37- 41).
15. Why is Renal Point of Care Testing (POCT) Important?
Providers, policy-makers, and payers should view CKD as a major public health problem and
initiate innovative POCT programmes to address this growing patient population (42). The
cost-effectiveness of POCT in wider settings, such as community pharmacy need to be
demonstrated as a means of achieving reductions in CKD through randomized trials (43),
(44). All of the biomarkers highlighted above can be used for POCT, which provides an
advantage to Biomedical Scientists to develop new protocols for diagnostic and analysis
development. In addition, POCT technology has advanced and sensitivity for CKD is
becoming more important in wider community environments. Suspect/ high risk individuals
for CKD can now also be offered POCT to help monitor:
Microalbumin in the urine (Microalbuminuria)
Haematuria (Blood in the urine)
Proteinuria (Protein in the urine)
Hyperlipidaemia (High quantity of Low Density Lipoproteins in the Blood)
22
Point of Care tests are those that can be carried out in a few minutes. Biomedical scientists
are in an ideal position to perform such tests. Indeed, several already do so. Changes in
technology are making a range of POCT test kits available. POCT can be used to fulfil three
main objectives:
To assess the risk of disease
To screen for the presence of disease
To manage the disease
(45)
Various tests can be used to screen for the early signs of disease or to assess the risk of a
disease. For example, fasting glucose can be used to screen for Diabetes Mellitus and systolic
blood pressure and the ratio of total serum cholesterol to high density lipoprotein can be
measured to help assess the risk of Coronary Heart Disease (CHD) (45).
POCT offers an opportunity for Biomedical Scientists to enhance their practice and services.
Lipid profiles can be measured to check disease progression and how patients are responding
to cardiovascular drug therapy. Monitoring of international normalized ratio (INR) can be
used in the management of anticoagulant therapy and testing SCr could be used to monitor
renal function (45). The methods and equipment chosen should take into account the number
of tests likely to be performed each day, how results compare with those from local
laboratories and the availability of support from local laboratories. Other factors, such as
providing seating for patients who may feel unwell after a blood test, should also be
considered (45).
Detecting Microalbuminuria requires sensitive immunoassays, specific for albumin and it is
expressed by urine albumin: creatinine ratio (ACR). Even when a person has proteinuria,
tight control of blood pressure can reverse it to Microalbuminuria levels for at least 3-4 years,
after which there is progression back to proteinuria and eventually CKD. If a patient has
stable renal control, a urine sample is taken annually. A conventional dipstick is used for a
protein assay and a Microalbuminuria assay is carried out on the sample to determine the
23
urine ACR; the patient’s SCr and eGFR can also be established (36). POCT can also be
implemented to:
Test for Haematuria (Blood in the urine)
Test for Proteinuria (Protein in the urine)
Test for Hyperlipidaemia (High quantity of Low Density Lipoproteins in the Blood)
Test Estimated Glomerular Filtration Rate (or EGFR)/ GFR.
Test for Cystatin C
Test Ferritin (Iron) Levels
Test Potassium, Sodium, and Calcium titres
(45)
16. POCT Methodology
A number of POCT methodologies have become available to provide rapid results. Post-
transplant renal patients, for example, regularly have their white blood cell counts checked,
particularly prior to each course of immunosuppression treatments. These are presently
performed in a hospital laboratory. If services were more widely available in primary care for
patients to have their immunosuppression titres measured, this can potentially save patients
an unnecessary trip to the hospital, should their results be abnormal. Several POCT white
blood cell counters have been used including: (1) The Chempaq XBC (Chempaq A/S,
Denmark), which measures haemoglobin concentration (red blood cells are lysed), leukocyte
counts and a 3-part differential (lymphocytes, monocytes and granulocytes, concentrations
and % of total) (46). Reagents are contained in a disposable cassette and measurements can
be taken from a finger stick or venous sample. Results are available in 3 minutes (2) The
HemoCue White Blood Cell (WBC) instrument (HemoCue AB, Angelholm, Sweden) is a
battery-run device utilising a disposable cuvette pre-loaded with reagent, along with an image
detector and an LCD display (47). The device measures white blood cells only, red blood
cells are lysed. Measurements require10μl (1 to 2 drops) (of capillary or venous blood and are
completed in 2 minutes. (3) The pocH-100i haematology analyser (Sysmex Corporation,
24
Kobe, Japan) provides a full blood count and a 3-part differential leukocyte count (49).
Neutrophils are reported separately (48). Readings take 2-3 minutes, per-sample. In
development: Point-of-care microfluidic single cell impedance cytometer, which performs
white blood cell differential count (T-lymphocytes, monocytes and neutrophils) based on
inherent electrical properties of cells (49).
Five hundred routine blood samples from a hospital were tested in parallel by the HemoCue
WBC compared to a reference analyser and data showed that the HemoCue WBC was
reliable for white blood cell counts within the analytical range of 0.4-30 x 109/l, except in
samples with high numbers of normoblasts or reticulocytes (e.g. in sickle cell anaemia or
thalassaemia major) (47). A total of 95% of the samples were within the acceptable
performance limit of 8-10% of the correct measurements as required by the United Kingdom
National External Quality Assessment Service. In patients exhibiting symptoms of infection,
a low or normal total WBC is usually associated with viral illness (50).
In a study by Rao et al (2008) a comparison of the Chempaq XBC analyser was made
between different locations (intensive care unit, emergency room, in-patient wards, primary
care, paediatric and obstetrics/ gynaecology clinics) and laboratory measurements showed
good correlation at all the locations for WBC, haemoglobin, granulocytes and lymphocytes (r
= 0.92–0.96), but not for monocytes (r = 0.88) (46).
Research by Osei-Bimpong et al (2009) conducted an evaluation of the pocH-100i
haematology analyser compared to conventional methods. The results demonstrated good
correlations for neutrophils (r2= 0.996) and lymphocytes (r2= 0.999), but less for the
“mixed” population of cells (r2= 0.611) (47). Brigg et al (2003) compared an impedance
cytometer with standard laboratory haematology analysis and identified good overall
correlations (95%) (49); the covariance (CV) factors between the results obtained from the
impedance cytometer and the reference device were: lymphocytes (95%), granulocytes (97%)
and monocytes (88%). The simplicity of impedance cytometry has potential application for
affordable POCT, but is still under development. No present studies on the use of POCT
counters for patients undergoing IST have been identified (49).
25
Currently very limited evidence exists on the cost-effectiveness and economic impact of
POCT for WBC count. Casey and Pichichero (2009) investigate the economic consequence
of POCT for C-reactive protein (CRP) and WBC count in patients with acute infections (51).
The study was undertaken in Japan and found that immediate testing led to a 30% reduction
in the cost of oral and parenteral antibiotics, although these savings were largely offset by the
prescription of new antiviral drugs in the immediate testing group. It also led to a non-
significant reduction in staff time and additional laboratory use. POCT for WBC needs to be
evaluated to assess whether it potentially delivers a cost-effective alternative to standard care
for a group of patients such as those receiving IST and those with acute infections. POCT
services for increased WBCs may provide better coordination of care in patients prescribed
immunosuppression post-transplant, potentially improving patient outcomes and cost
effectiveness. POCT services to measure white cell counts either alone or in combination
with inflammatory markers may also help reduce acute allograft rejection episodes.
17. What are the different Biomarkers for Renal Function?
A biomarker is defined as ‘a characteristic that is objectively measured and evaluated as an
indicator of normal biological processes, pathogenic processes, or pharmacologic responses
to a therapeutic intervention (21).
Biomarkers of kidney damage are affected by several factors including age. Certain
populations (children, older adults) may require special consideration as the normal
laboratory ranges are elevated. In paediatric patients, SCr reach adult levels by adolescence.
Moreover these patients are at high risk of developing drug induced nephrotoxicity. In
particular increased prevalence of accompanying diseases among older individuals in turn
increases their susceptibility to kidney diseases. Moreover the use of multiple concomitant
medicine use affects kidney function. Therefore increased risk of nephrotoxicity requires
advanced POCT in older adults (51). The different biomarkers for renal function include:
Cystatin C
Estimated Glomerular Filtration Rate (eGFR)
26
Glomerular Filtration Rate (GFR)
Neutrophil Gelatinase-Associated Lipocalin (NGAL)
Serum Creatinine (SCr)
Cystatin C or Cystatin 3 (formerly gamma trace, post-gamma-globulin or neuroendocrine
basic polypeptide) is a protein encoded by the CST3 gene, is mainly used as a biomarker of
kidney function. Recently, it has been studied for its role in predicting new-onset or
deteriorating CVD. If GFR declines, the blood titre of Cystatin C increases. Serum titres of
Cystatin C are a more precise test of renal function (as represented by eGFR/ GFR) than SCr
titre. Cystatin C titres are less dependent on age, sex, race and muscle mass compared to
creatinine. Cystatin C measurements alone have not been shown to be superior to formula-
adjusted estimations of renal function. Cystatin C can be analysed in a random sample of
serum using immunoassays (31).
Cystatin C may be a better predictor of GFR than SCr in patients with CKD and patients on
mainstream IST post-transplant, since as opposed to the latter, in addition to being a good
marker of filtration, it is not affected by age, gender confounders. It appears that in renal
post-transplant recipients, NGAL correlates well with Cystatin C and eGFR, most strongly
with Cystatin-based formula. Though this suggests potential use of NGAL as a screening test,
its weaker diagnostic performance raises some concern about its clinical usefulness. Larger
studies are needed to explore this further (31).
The renal biomarkers NGAL and Cystatin C are now emerging as potentially useful
indicators of GFR with the latter proposed as a gold standard. NGAL is a 25-kDa protein
bound to gelatinase originally identified from neutrophils, which is also expressed at very low
levels in several tissues including the kidneys and is released systemically in response to
renal epithelial damage or produced locally in renal tubules. NGAL undergoes glomerular
filtration followed by tubular uptake and is one of the earliest and most strongly induced
proteins in the kidney after ischemic or nephrotoxic injury in animals. NGAL can be easily
detected in the blood and urine post-transplant, hence is emerging as a major predictive
27
biomarker with potential use in risk stratification of eGFR, CKD progression and
Immunosuppression Therapy (IST) monitoring/ transplant rejection (31).
18. Renal Transplant Immunobiology
Only a kidney donated to a recipient by an identical twin will be the same genetically. In all
other cases, the organ will be recognised by the immune system of the new host as being
foreign, and an immune response will attempt to destroy it. The risk of this is reduced by (1)
Ensuring that the genetic make-up of the donor and recipient is closely matched (using
human leukocyte antigen (HLA) tissue typing) and (2) Suppressing the immune system of the
recipient with drugs for as long as the grafted kidney functions is important (28).
In man, the Major Histocompatibility Complex (MHC) is known as the HLA locus. HLA
antigens vary between individuals, making perfect matching extremely difficult.
Unfortunately, these antigens are also the strongest inducers of T-lymphocyte-mediated
kidney rejection (48). HLA tissue typing has been shown to reduce the frequency of rejection
episodes, thus achieving a more successful outcome (28).
Knowledge of the interaction of antigens with T-cells in the rejection process is essential to
understanding how immunosuppressive drugs work. Donor HLA molecules are presented via
antigen-presenting cells to resting T-cells that have receptors for that specific antigen. This
recognition process activates the T-cells, which produce and secrete cytokines (e.g.,
interleukins) and express cell-surface receptors to them (e.g., interleukin-2 receptors). The
lymphocyte colony recognises the donor HLA antigen, and differentiates and proliferates
under the influence of interleukin-2 (IL-2), which has been called T-cell growth factor.
Cytotoxic T-cells bind directly to donor cells and lyse them. Other T-cell sub-sets produce
more cytokines (e.g., IL-4 and interferon-γ) which lead to B-lymphocyte involvement,
antibody production, complement fixation and macrophage infiltration (50). The result is
destruction of graft tissue, which impairs the ability of the transplanted kidney to function.
Previous exposure of a recipient to other HLA antigens (e.g., from blood transfusions,
previous transplants or pregnancy) increases the likelihood of rejection. The risk can be
28
quantified using the panel reactive antibody (PRA) test, in which the higher the percentage
score the greater the recipient’s sensitivity. If the PRA score is greater than 85 per cent, the
potential recipient is considered to be highly sensitised and might require additional or
stronger immunosuppression (28).
19. Monitoring the Mainstay of Immunosuppression Therapy (IST)
The choice of mainstay immunosuppression medication currently available in the UK is listed
below. The maintenance regime is tailored to each individual patient.
Calcineurin inhibitors: Cyclosporine (CsA) – Neoral®, Sandimmun® (both
Novartis, UK), Deximune® (Dexcel, UK), Capimune® (Mylan, UK); Tacrolimus
(FK506) – Prograf®, Advagraf®, Modigraf® (all Astellas, UK), Adoport®
(Sandoz, UK), Tacni® (TEVA, UK), Vivadex® (Dexcel, UK), Mylan Tacrolimus
(Mylan, UK)
Antimetabolites: Azathioprine; Mycophenolate Mofetil (MMF) –CellCept®
(Roche, UK), along with several generic brands; Mycophenolate sodium (MMS) –
Myfortic® (Novartis, UK)
Corticosteroids: Prednisolone (non-branded)
Mammalian target of Rapamycin (mTOR) inhibitor: Sirolimus – Rapamune®
(Wyeth, UK) (mTOR is a protein related to cell growth)
(30)
Interleukin-2 Receptor Antibodies/ Monoclonal Antibodies
Basiliximab and Daclizumab are two of the first Interleukin-2 Receptor Antibodies (IL-2)/
Monoclonal Antibodies (MAbs) drugs that were licensed in the UK for use at the time of
renal transplant surgery. These MAbs are traditionally administered before surgery and they
exert their immunosuppressant effect only over the first few weeks post-transplant. Both are
licensed for use in combination with CsA and steroids, which then form the base-line therapy.
Both MAbs work by binding specifically to part of the IL-2 receptor that is only expressed on
29
activated T-lymphocytes. Inactivating IL-2 helps to prevent proliferation of antigen-
stimulated T-cells (30).
Calcineurin Inhibitors
Cyclosporine (CsA) and Tacrolimus (FK506) are calcineurin inhibitors that work early in T-
cell activation. T-cell activation involves a series of cascades, and the enzyme calcineurin is
one of the rate-limiting points. Calcineurin is the target of both CsA and FK506 complexes,
which inhibit it and prevent transcription of the genes encoding interleukin-2 (IL-2) and other
cytokines that cause early T-cell activation. CsA binds to an immunophilin (a cytoplasmic
protein) called cyclophilin A (30).
FK506 binds to another class of immunophilins known as FK binding proteins (FKBP),
specifically FKBP-12. CsA, FK506 and Sirolimus are metabolised in the liver via the
cytochrome P450 pathway (specifically the cytochrome P450 3A4 isoenzyme). Doses of CsA
used vary according to local practice. However, a typical initial oral dose would be 4mg/kg
twice daily. Doses are adjusted according to pre-dose (trough) whole-blood concentrations.
After six months or so, target ranges are reduced to aim for lower-dose maintenance therapy.
CsA levels are measured using an Enzyme Multiplied Immunoassay (EMIT) assay. Typical
target levels would be:
● 0–6 months, 150–300µg/l
● Over 6 months, 75–150µg/l
(30)
The major adverse effects of CsA and FK506 are nephrotoxicity, hirsutism, hyperlipidaemia,
glucose intolerance, hypertension, tremor, gingival hyperplasia, and hyperuricaemia.
Although CsA nephrotoxicity is largely dose-dependent, chronic toxicity also occurs,
necessitating the drug’s withdrawal (and possible change to Sirolimus or Mycophenolate-
based regimens) to attenuate the decline in renal function (30).
Most renal centres start with an oral dose of 0.1mg/kg twice daily for FK506, either for use as
primary immunosuppression or as rescue therapy (30). Doses are adjusted according to
30
trough blood levels and, after a few months, target ranges are reduced to aim for lower dose
maintenance therapy. Table 2 above provides Indications for Immunosuppression Laboratory
Testing, however FK506 typical desired whole blood trough levels would be:
● 0–6 months, 10–15 µg/l
● After 6 months, 5–10 µg/l
(30)
EMIT is a common method for qualitative and quantitative determination of
immunosuppression post-transplant. First introduced by Syva Company in 1973, it is the first
"homogeneous immunoassay" to be widely used commercially (52). The most widely used
applications for EMIT are for therapeutic drug monitoring (serum) and as a primary screen
for abused drugs or their metabolites in urine.
Like all immunoassays, EMIT uses antibodies that are specifically designed to bind the
molecule(s) of interest (analyte) without binding to other substances in the sample. Its unique
feature is the ability to detect this binding without resorting to a cumbersome separation of
the bound component. This is accomplished by including (in the mixture of antibodies and
sample) an enzyme that is attached to the analyte being tested for. Antibodies that do not
become bound to the sample drug bind instead to this analyte-bound enzyme. The analyte-
bound enzyme is designed so that when antibodies bind to its analyte portion, the enzyme is
deactivated (52). Two general examples are here given: (1) if a high amount of sample
analyte is present; this sample analyte will bind a large portion of antibodies leaving a large
portion of the analyte-bound enzymes free in solution. Much substrate will be converted by
high concentration of free enzyme. (2) If a low concentration of sample analyte is present,
this small concentration of sample analyte will bind only a small portion of the antibodies,
leaving a large portion of the antibodies to bind the analyte-bound enzymes and deactivate
them. In this case, much of the substrate will not be converted. Essentially, EMIT involves:
Taking a sample of post-transplant patient’s urine or serum containing
immunosuppression with a solution containing a known concentration of antibody and
enzyme substrate
31
After a short period (usually less than a minute) to allow binding, a known
concentration of conjugate is added
Measure the concentration in appearance by colour or fluorescence/ immuno
fluorescence
Determine immunosuppression efficacy or toxicity by comparing known
concentrations of the drug
(52)
Because EMIT assays are so sensitive, low or borderline EMIT results are sometimes
difficult to confirm by less sensitive procedures such as Thin-Layer Chromatography (TLC)
and, sometimes, gas chromatography (GC) and High-Performance Liquid Chromatography
(HPLC). Some centres are now performing C2 monitoring, (i.e., measuring blood
concentrations two hours post-dose). C2 is thought to predict more accurately individual
patient absorption than traditional trough monitoring, and results in a reduced incidence of
acute rejection episodes and acute renal dysfunction (52). CsA is 50 percent bound to
erythrocytes, 10 per cent to leucocytes and 30–40 per cent to plasma proteins. Only 1–6
percent exists in a free state (53), with 80–90 per cent bound to lipoproteins in plasma (54).
CsA is extensively metabolised in the liver and bowel under the influence of cytochrome
P450 3A4, so pharmacokinetic interactions are common. It is primarily eliminated by biliary
excretion with a median half-life of 6–8 hours (54). Table 2 below provides a number of
assays for Immunosuppression Laboratory Testing.
Table 2: Indications for Immunosuppression Laboratory Testing
Test Name Recommended Use Limitations Follow Up
Cyclosporine A by
Tandem Mass
Spectrometry
Method: Quantitative Liquid
Chromatography-
Tandem Mass
Spectrometry
Optimize dosage;
monitor compliance
Results from
different
methodologies
(mass
spectrometry
versus
immunoassay)
cannot be used
interchangeably.
Generally,
immunoassay
32
methods have
been reported to
have a positive
bias in results
when compared
to mass
spectrometry
due to antibody
cross-reactivity
Cyclosporine A, 2-
Hour Post Dose (C2)
by Tandem Mass
Spectrometry
Method: Quantitative Liquid
Chromatography-
Tandem Mass
Spectrometry
Optimize dosage;
monitor compliance
Everolimus by Tandem
Mass Spectrometry
Method:
Quantitative Liquid
Chromatography-
Tandem Mass
Spectrometry
Therapeutic
monitoring for
individuals
taking
Everolimus
Trough
concentrations
should be
assessed ~2
weeks after
commencing
treatment
Analytical
sensitivity –
limit of
detection is 2.0
ng/mL
Interferences
from commonly
used drugs and
associated
metabolites have
not been
observed
Results from
different
methodologies
(mass
spectrometry
versus
immunoassay)
cannot be used
interchangeably.
Generally,
immunoassay
methods have
been reported to
have a positive
bias in results
when compared
to mass
spectrometry
due to antibody
cross-reactivity
Mercaptopurine
Quantitative, Serum or
Plasma
Method:
Quantitative High
Performance Liquid
Chromatography
(HPLC) -Tandem Mass
Spectrometry
Monitor compliance Measures
concentration of
parent drug
only; risk of
toxicity
associated with
TPMT
deficiency
should be
evaluated by
TPMT, RBC
33
Mycophenolic
Acid (MMF)
Method:
High Performance
Liquid
Chromatography
(HPLC)
Optimize dosage;
monitor compliance
In vitro
conversion of
parent drug to
mycophenolic
acid can occur if
specimens are
collected shortly
after IV
administration
and could
contribute to
falsely elevated
concentrations
of mycophenolic
acid.
Therapeutic
ranges and toxic
thresholds are
not well
established
Sirolimus by Tandem
Mass Spectrometry
Method: Quantitative High
Performance Liquid
Chromatography
(HPLC) -Tandem Mass
Spectrometry
Optimize dosage;
monitor compliance
Results from
different
methodologies
(mass
spectrometry
versus
immunoassay)
cannot be used
interchangeably
Generally,
immunoassay
methods have
been reported to
have a positive
bias in results
when compared
to mass
spectrometry
due to antibody
cross-reactivity
Tacrolimus by Tandem
Mass Spectrometry
Method:
Quantitative Liquid
Chromatography-
Tandem Mass
Spectrometry
Optimize dosage;
monitor compliance
Results from
different
methodologies
(mass
spectrometry
versus
immunoassay)
cannot be used
interchangeably.
Generally,
immunoassay
methods have
been reported to
have a positive
34
bias in results
when compared
to mass
spectrometry
due to antibody
cross-reactivity
Thiopurine
Methyltransferase
(TPMT), RBC
Method: Enzymatic/Quantitative
Liquid
Chromatography-
Tandem Mass
Spectrometry
Detect risk for severe
myelosuppression with
standard dosing of
thiopurine drugs
Individualize dosing of
thiopurine drugs
Does not replace
clinical
monitoring
Genotype
cannot be
inferred from
TPMT activity
(phenotype)
TPMT inhibitors
may contribute
to false-low test
results
TPMT activity
should be
assessed prior to
treatment with
thiopurine drugs
Blood
transfusion
within 30 days
will reflect
donor status
Lymphocyte
Transplantation CD3
Method: Quantitative Flow
Cytometry
Monitor
immunosuppressive
therapy with OKT3; test
verifies CD3 antigen
removal
For testing of
immunocompromised
patients, order
Lymphocyte Subset
Panel 4 – T-Cell
Subsets Percent and
Absolute
Lymphocyte
Transplantation Profile
Method: Quantitative Flow
Cytometry
Monitor
immunosuppressive
therapy with anti-
lymphocyte drugs such
as OKT3 or ATG (anti-
thymocyte globulin)
Components include
CD2 percent and
absolute, CD3 percent
and antibodies, CD4
percent, CD8 percent,
CD4;CD8 ratio, CD19
percent
35
Table adapted from (53)
ImmuKnow is an immune cell function assay that detects cell-mediated immunity in an
immunosuppressed population. The assay detects cell mediated immunity by measuring the
concentration of ATP from CD4 cells following stimulation. ImmuKnow technology
combines cell stimulation, cell selection, and quantification of metabolic markers (Adenosine
Triphosphate - ATP) to measure cell-mediated immunity. ImmuKnow measures early
response to stimulation by detecting intracellular ATP synthesis in CD4 cells selected from
blood by monoclonal antibody-coated magnetic beads. The amount of ATP present in
stimulated blood specimens is a measure of lymphocyte activity. Since the CD4 lymphocytes
orchestrate cell-mediated immunity responses through immunoregulatory signaling, the
measurement of CD4 activation reflects the degree of immune function. The limit of ATP
detection of ImmuKnow is 1ng/ml (53).
Results of the ImmuKnow assay should be used in conjunction with clinical presentation,
medical history, and other clinical indicators when establishing the immune status of a
patient. This is a qualitative assay; therefore, the result does not quantify the level of IST.
Specimen will be rejected if greater than 30 hours old and needs to be other than
heparinized whole blood collected in a sodium heparin tube (53).
20. Critique of Creatinine Methods
Inulin was considered as the gold standard to measure GFR. Inulin is freely filtrated through
a semi-permeable membrane which is a strong argument for the absence of binding to
protein. Nevertheless, there are limitations to its use in daily practice. Because it’s relatively
high molecular weight, the molecule is relatively viscous and does not rapidly reach its
volume of distribution (55). Therefore, only methods using urinary clearance with constant
infusion rate seem accurate for this biomarker. Moreover, most of the methods (except the
enzymatic ones) are prone to interferences with glucose measurement and this can be a
limiting factor when measuring GFR in post-transplant renal patients (55).
36
Iohexol is a non-ionic contrast product, and was mainly used for myelography (a type of
radiological examination that uses a contrast medium to detect pathology of the spinal cord,
including the location of a spinal cord injury, cysts, and tumours). Iohexol’s molecular weight
is 821 Da. Iohexol is chronologically the last biomarker proposed for measuring GFR. It can
be used in all patients (except in patient with true allergy to contrast product). Its
measurement by HPLC was probably one of the most precise compared to other cold method
(inulin and iothalamate) (55). Iohexol is the less expensive biomarker and the cost of HPLC
is also low. More important, it must be underlined that an external quality control does exist
for Iohexol measurement. It can be concluded that the inter-laboratory Coefficient of
Variation for Iohexol measurement is very low (less than 5%) (55).
Due to its low value, using SCr provides an accurate measure in the assessment of renal
function and in the monitoring of IST post-transplant (56). The determination of of SCr or
urine is carried out by Jaffe’s reaction where creatinine reacts with Piciric acid and alkaline
medium. The alkanity is provided by sodium hydroxide (NaOH) (56). The most important
creatinine measurement range for detecting silent kidney disease and IST monitoring is
between 85 and 150μmol/L, corresponding to eGFR of approximately 60 mL/min/ 1.73m2
(but does also depend on age, sex, ethnicity and an isotope dilution-mass spectrometry
(IDMS)-based calibration (57).
Peake and Whiting (2006) summarize in an analytical commentary the measurement of SCr
and the work of the Laboratory Working Group of the National Kidney Disease Education
Program (NKDEP), USA (57). The NKDEP has published detailed recommendations for
improving the measurement of SCr (57). To assess the performance of current SCr methods,
31 serum samples covering a wide range of creatinine concentrations, plus a number of
controls were distributed to six laboratories in South Australia and Victoria using a total of
nine different instrument/method combinations. Each participating laboratory agreed to assay
the samples exactly as recommended by the manufacturer on two successive days.
Calibration materials for each method were run as “unknowns” as part of this protocol to
ensure that results reflected the manufacturer recommended calibration (and offsets). Target
37
creatinine concentrations were assigned by assaying ethanol extracts of samples in
quadruplicate using validated isotope dilution-mass spectrometry (IDMS) method based on
tandem Liquid chromatography–mass spectrometry (LCMS) (57). Table 3 summarizes details
of the eight routine creatinine methods and instruments are as follows (Jaffe assays unless
indicated otherwise).
Table 3: Routine Creatinine Methods and Instruments
Instruments Methods
1. Roche Hitachi 917 (H917)
[Roche Diagnostics]
• Uses a simple two-part reagent system with
Sodium Hydroxide (NaOH) in the first
reagent (rate-blanking) and picric acid in the
second. Results are corrected with an
“average” 26.5μmol/L offset for non-
creatinine chromogens
• Has a limited ability to correct for icteric
samples. IDMS calibration reference
intervals have been published for both this
assay and the Roche enzymatic assay.39,40 It
has been reported that enzymatic and Jaffe
creatinine results compare reasonably well
for blood donor samples and for pre- and
post-dialysis samples from CKD patients
(b) Enzymatic creatinine assay
• Widely accepted as one of the most
accurate routine methods available at present
and data indicates that this method produces
results for patient samples that agree closely
with IDMS
38
2. Roche Integra 700
[Roche Diagnostics]
• A simple assay system as for H917 (uses
offset of -18μmol/L), but lack of rate-
blanking results in interference exceeding
10% at relatively low concentrations of
bilirubin (85μmol/L)
3. Bayer Advia 2400
[Bayer HealthCare Diagnostics]
• A rate-blanked assay having the largest
recommended offset for non-creatinine
chromogens for all assays discussed here (-
35.4μmol/L)
4. Abbott 8200
[Abbott Diagnostics]
Rate-blanking and offsets are not officially
recommended and were not used with the
current method, although Abbott have
indicated that they plan to release an IDMS
aligned method by quarter 1, 2007
5. Beckman LX20
[Beckman Coulter]
• The STAT (cup) assay has an excellent
formulation to minimize protein, bilirubin
and glucose interference. Other than the
LCMS and Roche enzymatic assays, it is the
only Jaffe assay in this group that recovered
the weighed-in concentration (442μmol/L) of
an aqueous creatinine standard
6. Olympus 5400
[Olympus Diagnostic Systems]
• An offset of -18μmol/L is recommended for
the Olympus method in Australia and it
contains unstated additive to reduce bilirubin
interference.
• The package insert correctly indicates that
absorption of CO2 can alter the calibration of
39
the assay and this is an important
consideration for other assays because of the
high pH of all Jaffe assays
7. Dimension RxL
[Dade Behring]
• The only assay of this group using
ferricyanide to eliminate bilirubin
interference
• Calibration materials containing at least 30
g/L of protein are recommended, mainly to
partially compensate for protein interference
• No offsets are used
8. Vitros 950
[Ortho Clinical Diagnostics]
• A dry chemistry system using an enzymatic
reagent sequence discussed previously.
• Bilirubin and haemoglobin interferences are
minimized by retention in the spreading layer
Table adapted from (57)
21. Critique of Proteinuria, ACR, Cystatin C, and NGAL Methods
Shlipak et al (2011) identified that preoperative serum Cystatin C concentrations were
superior to SCr for the prediction of perioperative kidney injury (36). Preoperative urinary
ACR is also predictive of perioperative kidney injury in adults, but not in children (36-38).
Van et al (2009) highlight the current issues in using proteinuria as a biomarker for
measuring creatinine. There are key clinical issues that need to be addressed before a
unifying global guideline for proteinuria and IST post-transplant monitoring can be
developed; lack of data being one of those concerns (19).
In adults, post-operative urine interleukin-18 (IL-8) (IL-18, another novel biomarker) and
plasma NGAL levels peak within six hours of intensive care unit (ICU) arrival, whereas SCr
increases did not occur before 24–72 hours (36-38). Shlipak et al (2011) study higher urine
IL-18 and plasma NGAL levels were also associated with longer lengths of stay in ICU and
40
hospital and higher risk of dialysis or death, but urine NGAL was not predictive of kidney
injury or associated with adverse clinical outcomes. In the paediatric sub-study, urine NGAL
and urine IL-18, but not plasma NGAL, performed similarly to plasma NGAL and urine IL-
18 in the adult study for the prediction of kidney injury and adverse clinical outcomes (36).
In addition, it should be pointed out that robust functional measurements of GFR will also
continue to be important for IST monitoring and dosing. An improved platform to detect
different sources of NGAL in urine has been suggested and this may be a helpful tool to also
monitor pathophysiological changes for AKI development.
In contrast to creatinine, Cystatin C satisfies many features of an ideal filtration biomarker. In
some studies Cystatin C outperformed creatinine in detecting minor reductions in GFR (58),
(59). Furthermore, Shlipak et al (2006) identify that Cystatin C predicted mortality in
outpatients with apparently normal kidney function (59). The practical use of Cystatin C as a
biomarker of kidney function and/ or IST monitoring in post-transplant renal patients has not,
however, been thoroughly investigated. More research needs to take place following the work
of Abouchacra et al (31).
If novel biomarkers can be proven superior for these purposes, they may even replace SCr (or
Cystatin C) changes and urine output as our primary clinical tools to diagnose kidney injury
and monitor response to therapy. Currently however, in those countries where novel renal
biomarkers are available for clinical use, their function to inform assessments on differential
diagnosis and prognostic assessment and associated management decisions (triage, RRT
initiation, etc.) is probably where initial use would be appropriate (21). It is also unlikely that
biomedical scientists and clinical specialists will form an overall consensus that a single
biomarker will be able to provide an adequate monitoring of IST. A panel-specific selection
of biomarkers may be necessary to monitor titres between toxicity and efficacy (21).
41
22. Immunosuppression (IST) in Paediatric Post-Transplant Patients
In the case of paediatric post-transplant population, dose modifications should be considered
when prescribing IST with a known nephrotoxicity to adults. The lower age group subsets of
the paediatric population may be particularly sensitive to certain excipients that are not
entirely inert and may have side effects. Particularly, some excipients which can be used on
adults and older children may be toxic in neonates because of their immature and rapidly
changing metabolic & elimination system (61-62). The salt of the active ingredient and the
chemical nature of the preparation must be carefully considered to avoid administration of
excessive amounts of electrolytes (61-62).
In paediatrics, determination of CsA by HPLC is perhaps more specific; drug efficacy and
monitoring can be separated and quantified. However, this method is longer and requires
experienced biomedical scientists. The advantages of immunoenzymologic reactions are their
rapidity and automation. Different monoclonal antibodies (MAbs) also show a low cross-
reactivity with CsA metabolites. The fluorescence polarization immunoassay and the EMIT
for immunosuppression monitoring in the paediatric post-transplant population require an
initial manual pre-treatment step consisting of precipitation of erythrocytes and proteins by
methanol and centrifugation. Then, a second immunoenzymologic step is performed in the
equipment (52). This manual pre-treatment imposes frequent calibrations because of poor
reagent stability.
Liquid IST preparations more often contain excipients like preservatives of which
concentration should be at the minimum level if not possible to eliminate. When
preservatives are required, IST concentration should be at the minimum level and a thorough
justification for the choice of the immunosuppression should be provided. Formulation used
for chronic conditions may cause repeated cumulative exposure to excipients. Therefore, the
acceptable daily intake and safety limits of excipients for children must be examined,
particularly for children with renal impairment (lower renal elimination, kidney
malfunction) (61-62).
42
23. Discussion
Since implementation of the two National Service Renal Framework for Renal Services (2),
(3); September 2005 and May 2007 progress reports were also published (63), (64). The May
2007 report provides an update on prevention and early detection, new facilities, improving
services, and the standards and quality requirements of the Renal Service Framework.
Additional guides referring to Renal Service Framework include: The Multi-Professional
Criteria for Monitoring Implementation of the National Service Framework, British Renal
Society and Kidney Health (65), The Evidence Base for the National Service Framework for
Renal Services Modules One and Two: Part one - Dialysis and transplantation, October 2006
(66), Organs for Transplants, Organ Transplant Taskforce, January 2008 (67), Modernizing
Services for Renal Patients, Department of Health, July 2005 (68) and more recently, Kidney
Health: Delivering Excellence, A Kidney Health Report has been published (69) highlighting
the importance of screening with respect to Acute Kidney Injury (AKI). All guides are
prompting the importance of wider services being available for suspect/ high risk individuals
for CKD to be screened and in addition placing prominence on comparing renal biomarkers
and their utility to monitor mainstay of IST. More emphasis is being placed on the
prevention, and early detection of CKD to minimize progression to renal failure; patients who
are at high risk of renal failure need better ‘preparation’ and prompt early referral to
secondary care is a must in order to allow the best prognosis. At the same time, the
‘mentality’ of renal failure must change. Renal disease should not be viewed as a secondary
illness. Individuals can have renal failure without knowing (42).
C2 monitoring has debatable value. There are difficulties of obtaining samples at 2hrs +/- 15
minutes. C2 is used as it is considered to be a more accurate reflection of area under the curve
(AUC), which reflects the total exposure of the body to CsA from one dose and this affects
both absorption and elimination. Luminex Technology is now increasingly becoming
advanced. Luminex technology is built on proven, existing technology—flow cytometry,
microspheres, lasers, digital signal processing and traditional chemistry—that have been
combined in a unique way. Featuring a flexible, open-architecture design, Luminex
43
technology can be configured to perform a wide variety of bioassays quickly, cost-effectively
and accurately (70).
Luminex colour-coded beads, called microspheres are divided into 100 distinct sets. Each
bead set can be coated with a reagent specific to a particular bioassay, allowing the capture
and detection of specific analytes from a sample. Within the Luminex compact analyser,
lasers excite the internal dyes that identify each microsphere particle, and also any reporter
dye captured during the assay. Many readings are made on each bead set, further validating
the results. In this way, Luminex technology allows multiplexing of up to 100 unique assays
within a single sample, both rapidly and precisely. The Luminex system is a flexible analyser
based on the principles of flow cytometry that is designed to meet the needs of biomarkers
any size (70).
24. Further Research & Interventions
This work briefly highlights the number of biomarkers that can be used to monitor IST in
ensuring post-transplant patients receive adequate doses of their medication allowing
prolonged transplant function. There needs to be further investigations on which biomarkers
are most helpful specifically to monitor IST efficacy and this can only really be achieved
through proposed longitudinal studies or Randomized Controlled Trials (RCTs).
In 2004, an International Society of Nephrology (ISN) Consensus Workshop on Prevention
of Progression of Renal Disease recommended that individuals with diabetes and
hypertension have regular POCT for the potential development of CKD (71). In emphasis,
POCT is of equal prominence with respect to 1) identifying the most ‘suitable’ biomarker for
screening, 2) how patients on IST should be monitored through a POCT service in
community and 3) how POCT services should be implemented more widely. There should
otherwise be further research to potentially compare POCT kits available for the sensitivity of
some of the biomarkers.
Biomedical Scientists can also play a crucial role in identifying whether a regular patient on
IST is at risk of particular drug toxicity; this is important where preservation of renal function
44
is concerned. It is also important that any potential hepatoxicity/ nephrotoxicity is monitored.
Interestingly, Lab Tests Online-UK is a website designed to help the public understand the
many tests the clinical laboratory provides to the healthcare community’ (72) and a ‘new’
mobile application supports the rising increase in internet usage in the UK – 85% of adults
have used the internet, 68% are using it every day and 70% have used it to find out more
about their medical conditions. With the UK having one of the highest penetrations of smart
phones in the world at 58%, patients are already going online to expand their understanding
of their healthcare. The Lab Tests Online-UK app has also been added to the NHS Choices
Health App library, providing NHS endorsement for members of the public searching for
tools to support their healthcare (72).
Whilst there have been guidelines on the screening and monitoring of anticoagulation-based
medication (73), there perhaps needs to be more up to date clinical guidelines for monitoring
mainstay IST post-transplant patients receive. Interestingly guidelines highlight how POCT
services have expanded and a number of environments where this could be implemented to
serve wider communities (74-86). Suggested next steps include:
More studies of the accuracy and utility of POCT for white blood cell counters in the
primary care setting
Feasibility/ pilot studies in patients who are prescribed IST are required
Comparisons of white cell counts with POCT for other inflammatory markers for
patients are required to include effects on prescribing, patient satisfaction,
consultation rates, complications (e.g. hospital admission, delayed diagnosis) and cost
effectiveness.
(74-86)
Indeed, specialists from both clinical and laboratory practice need to come together to inform
further which biomarker would be more specific for CKD POCT and monitoring post-
transplant IST titres.
45
25. Conclusion
To conclude, biomedical scientists now have a more active and larger role to play with
respect to monitoring IST in post-transplanted individuals; NGAL is becoming more apparent
in this respect. Prospectively their expertise may be called upon where an in-depth insight on
specific renal biomarkers and sensitivity of laboratory assays are concerned. POCT offers
new advances in clinical care and testing; there are various costs to consider, but tighter
collaborations between laboratory staff and clinical teams who provide front-line care for
patients in CKD would be advantageous, this includes more teaching and continuous
professional development (CPD) opportunities where specialists from different teams come
together and gain more insight into what analytes/ biomarkers make better POCT and
diagnostic procedures. A thorough collaboration between professions in primary and
secondary care would be prodigious.
26. Summary Points
Robust POCT longitudinal data is required in a pre-CKD population to identify number of
Quality-Adjusted Life Years (QALYs) would be advantageous
Future Biomedical Scientists (with appropriate advanced training) may be eligible to
conduct POCT and this could include monitoring IST in post-transplanted patients
POCT may become routine in future practice for the purpose of identifying/ monitoring
hepatotoxicity/ nephrotoxicity and efficacy in post-transplant patients screening a number of
specific analytes according to GP/ specialist requests. This means that POCT kits will have
to become more sensitive for marker/s of intrigue
Biomedical scientists may be able to help develop more advanced POCT services with
specialists and GPs in response to identified needs in wider community
46
27. What this work adds
Identification of accurate biomarkers may be a key to improving outcomes in IST
monitoring
SCr is an index of glomerular filtration and is therefore not an ideal biomarker to monitor
titres of IST
The ideal biomarker for monitoring IST should provide timely results, have high accuracy,
and allow assessment of any signs of hepatoxicity/ nephrotoxicity, in addition to treatment
efficacy
Measurement of renal biomarkers should be affordable and reproducible
Novel biomarkers of early CKD and IST monitoring are being developed and evaluated, but
their exact role relating to POCT feasibility remains unproven
Further research should evaluate the role of novel biomarkers in multi-centre clinical
intervention studies throughout the patient pathway
Future studies require better integration between laboratory scientists and clinicians
Biomedical scientists need to carefully assess and coordinate between laboratory practices
and clinical teams to ensure patients are not being over-prescribed medication thus ensuring
efficacy and safety of treatment
47
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