February 2014
Proteinuria
measurement and
urine-based markers
for preeclampsia
diagnosis
Technology Opportunity Assessment
M A IL ING A D DR E S S
PO Box 900922
Seattle, WA 98109
USA
A D DR E SS
2201 Westlake Avenue
Suite 200
Seattle, WA, USA
TEL: 206.285.3500
Prepared for the Merck for Mothers
Program
FAX: 206.285.6619
www.path.org
Copyright © 2014, PATH. All rights reserved.
Cover photo credit: PATH/Evelyn Hockstein.
The activities discussed in this publication were supported by funding from Merck through its Merck for
Mothers Program. The content of this publication is solely the responsibility of the author(s) and does not
represent the official views of Merck.
Table of contents
Statement of need............................................................................................................................. 1
Technology solutions landscape ...................................................................................................... 2
Candidate methods for the determination of proteinuria ................................................................. 3
Gold standard: total protein determination by 24-hour urine collection .................................. 3
Visually read dipstick (protein only) ....................................................................................... 3
Visually read ratiometric dipstick ............................................................................................ 4
Machine dipstick readers (ratiometric) .................................................................................... 6
Point-of-care non-dipstick readers (ratiometric) ...................................................................... 7
Specific urine-based candidate biomarkers for preeclampsia .......................................................... 8
Biomarker of misfolded protein: Congo Red dye assay .......................................................... 8
Tamm-Horsfall protein urine biomarker .................................................................................. 9
SERPINA1 urine protein biomarker ........................................................................................ 9
C5b-9 urine protein biomarker............................................................................................... 10
Investment opportunity .................................................................................................................. 10
References ...................................................................................................................................... 13
i
Statement of need
Preeclampsia (PE) is a life-threatening disorder that only occurs during pregnancy, childbirth, and the
postpartum period and is characterized by high blood pressure (hypertension) and protein in the urine
(proteinuria). Convulsions (fits) with signs of PE indicate eclampsia, although occasionally convulsions
occur in the absence of hypertension with proteinuria. Preeclampsia and eclampsia (PE/E) are among the
leading causes of maternal death and disability worldwide. The World Health Organization (WHO)
estimates that PE/E accounts for at least 16% of maternal deaths in settings with limited resources that
lack the skilled providers and facilities required for prevention, identification, and management of the
condition. 1 In most countries, PE/E rank second only to hemorrhage as specific, direct causes of maternal
death. The risk of PE/E varies greatly depending on where a woman lives; the risk that a woman in a lowresource country will die of PE/E is approximately 300 times greater than that for a woman in a highresource country. 2
Positive maternal and perinatal outcomes for women with PE/E depend on how soon the condition is
identified and how quickly the woman has access to the treatment package. * Prevention for PE/E has
focused on antenatal screening for high blood pressure and proteinuria as part of focused antenatal care
(ANC). † The presence of proteinuria (total protein excretion exceeding 300 mg/24 hours) 3 changes the
diagnosis from gestational hypertension to PE, and detection of proteinuria is key for making a diagnosis
of PE. In practice, however, the number of women accessing ANC remains far below recommended
visitation levels. ANC utilization is around 68% in low- and middle-income countries compared to 98%
in high-resource settings. 4 In fact more than half of all births occur at home, and many pregnant women
receive no prenatal care.5 Furthermore, the urgency to quickly get treatment is even more acute for
pregnant women in rural and even peri-urban areas who do not have easy access to well-equipped clinics
with trained staff. In areas with little or no prenatal care, case fatality rates are close to 3,000 per 10,000
births (30%), and most of the deaths appear to occur among “un-booked” women—women who were not
seen for any pregnancy-related medical care until the first seizure. These women, if they receive any care
at all, are often brought to a hospital in a coma after multiple seizures, and approximately 30% to 50% or
more of these women die.5
The “gold standard” for measuring proteinuria requires a 24-hour urine collection to directly determine
the daily total protein or albumin excretion. Such collections (typically during a minimum two-night
hospital stay) are resource intensive and require refrigeration of the urine during collection and
*
The treatment package includes in-patient monitoring, anticonvulsive and antihypertensive therapy, optimal timing
of childbirth, and skilled attendance at birth.
†
Focused ANC is evidence-based, goal-directed care that is tailored to the gestational age of pregnancy and individualized to each woman. It emphasizes quality of visits over quantity of visits and is conducted by a skilled health
care provider. Goals of focused ANC include early detection and treatment of complications, prevention of problems, birth preparedness/complication readiness, and the promotion of healthy practices to help ensure a positive
health outcome for the woman and her baby. Focused ANC is provided through a women-centered approach that
values the dignity and value of each woman and her family.
1
laboratory-based analysis. In fact, the number of 24-hour collections considered incomplete during studies
of pregnant women approaches 50%, more than double those in studies of nonpregnant populations. 6
Further complicating this procedure is the fact that the laboratory-based assays require trained staff in a
laboratory, typically a central laboratory at a major hospital. As a result, timed collections are rarely
practical if greater reach is sought outside formal clinics and urban settings.
There is, therefore, a great need for low-cost devices to measure proteinuria that meet or approach
WHO’s ASSURED criteria 7 (Affordable, Sensitive, Specific, User-friendly, Rapid and robust,
Equipment-free and Deliverable to end-users). Like protein-only dipsticks, proteinuria screening devices
must remain affordable, widely available, and functional at the most peripheral points of care. To impact
morbidity and mortality in PE/E, the urinalysis device should not only improve accuracy of proteinuria
measurements but should also increase reach to the community level by simplifying, to the greatest extent
possible, how proteinuria is measured. Increased access to tests that meet WHO’s ASSURED criteria and
are affordable would greatly improve the identification of women with PE and increase their opportunity
to access treatment packages, ultimately increasing the likelihood of survival for mothers with PE and
their infants.
Technology solutions landscape
This landscape analysis covers biomarkers and methods for their determination that are candidates for PE
screening. These methods are split into those designed for the determination of proteinuria (total protein
or albumin determination—see Section I) and those for the determination of urine biomarkers that are
more specifically correlated with PE (see Section II).
Although the measurement of proteinuria is part of the formal diagnostic criteria of PE, it is by itself a
less than perfect predictor of either maternal or fetal complications in women with PE. 8 Studies have
shown that 10% of women with clinical and/or histological manifestations of PE have no proteinuria, and
20% of women with eclampsia do not have significant proteinuria prior to their seizure.9,10 To further
complicate the diagnosis of PE based on proteinuria, primary renal disease and renal disease secondary to
systemic disorders, such as diabetes or chronic hypertension, are also usually characterized by proteinuria
and may first present in pregnancy. In addition, each of the current methods for proteinuria screening
possesses shortfalls which make use in low-resource settings (LRS) challenging. These challenges are
detailed in Appendix A which delineates a total of 16 technologies, the majority of which are
commercially available and a few of which are in development or in the conceptual stage. All 16
technologies are grouped by assay categories for comparative purposes. Each technology varies in
accuracy, cost, simplicity, and feasibility. These technologies are also described below with brief
summary statements under each technology heading.
2
Candidate methods for the determination of proteinuria
Urine consists of two primary groups of proteins. Albumin is the majority of plasma proteins that cross
the filtration barrier and is the predominant urinary protein in most proteinuric kidney diseases.
Uromodulin, also known as Tamm-Horfall protein (THP), is the major component of the nonplasma
urinary protein and originates in the renal tubules or urinary tract. Other minor subgroups are
immunoglobulins, low-molecular weight proteins, and light chains.11 In general, methods for the
determination of proteinuria measure the levels of total protein or its major constituent total albumin.
Specific urine biomarker assays, however, measure the concentration of a specific protein related to PE.
1. Gold standard: total protein determination by 24-hour urine collection
Timed urine collection is the gold standard and is very accurate for detecting and measuring proteinuria.
However, these assays require central laboratory services, and collection is highly prone to errors.
As mentioned above, the “gold standard” for measuring proteinuria requires a 24-hour urine collection to
directly determine the daily total protein or albumin excretion. More recently 2-, 8- and 12-hour
collections have been validated.12,13 Such collections (typically during a minimum two-night hospital
stay) are resource intensive and require refrigeration of the urine during collection and laboratory-based
analysis. The gold standard is measuring both protein and creatinine, which is typically done using the
benzethonium chloride assay (BCA) and Bradford protein assay for analysis. These are chemical
laboratory tests, and not widely available in LRS. In fact, the number of 24-hour collections considered
incomplete during studies of pregnant women approach 50%, more than double those in studies of
nonpregnant populations.6 The inherent variability associated with urinary retention and, therefore, timing
errors has even lead to the recommendation that pregnant women be hydrated and positioned in lateral
recumbency for 45 minutes or one hour before starting and before completing 24-hour collection. 14 As a
result, timed collections, while useful in the context of clinical studies and as a reference during
establishment of alternative PE/E screening methods, are not usually practical as primary screening
methods in LRS.
2. Visually read dipstick (protein only)
These tests are of mediocre accuracy for the determination of proteinuria and of only marginal usefulness
for PE screening, but they are widely available, and inexpensive.
The visually read urinary dipstick for protein is a semiquantitative colorimetric test that primarily detects
albumin (see Figure 1). It is widely used and inexpensive, costing less than US$0.10 per strip test.
Significant proteinuria, however, cannot be accurately detected or is excluded at a protein concentration
of 20‒30 mg/dL (the 1+ threshold, see Table 1) with point-of-care urine dipstick analysis. The threshold
for proteinuria with urine dipsticks is, therefore, around 100 mg/dL (2+ threshold). 15
3
Figure 1: Example of a visually read dipstick (protein only) and diagrammatic view of a dipstick.
Medi-TestCombi 2
Macherey-Nagel
Glucose pad or “feature”
Protein pad or “feature”
Medi-Test Combi 2: a two-parameter test (protein and glucose) used in point-of-care settings (UNICEF).
© 2014 by MACHEREY-NAGEL GmbH & Co. KG
Additional challenges to the urinary
Table 1: Protein dipstick grading.
protein-only dipsticks include poor
Approximate Amount*
correlation with protein analysis in 24Designation
Concentration
Daily
hour urine samples, inaccuracy due to
Trace
5 mg/dL to 20 mg/dL
the hour-to-hour variability of protein
1+
30 mg/dL
< 0.5 g/day
excretion, inaccuracy if the urine is
2+
100 mg/dL
0.5 g/day to 1 g/day
dilute or concentrated or has an
3+
300 mg/dL
1 g/day to 2 g/day
abnormal pH, dependency on observer
4+
> 2,000 mg/dL
> 2 g/day
interpretation, contamination by
bacteria or blood or vaginal secretions, *The diagnostic criterion for protein levels in PE is proteinuria of at least 300 mg
of protein in a 24-hour urine collection.
the relative broad specificity of
tetraphenol blue for all proteins, and the susceptibility of dipsticks to deterioration from humidity and
temperature. As a result of these issues, evaluations of protein-only dipsticks have shown them to have a
mediocre clinical sensitivity of less than 68% for proteinuria detection.16 A prospective st udy found that
the false positivity for proteinuria (truly negative samples resulting as test positive) ranged from 7% at the
3+ level to 71% at the 1+ proteinuria level, while false-negative (truly positive samples resulting as test
negative) rates were 7% for “nil” and 14% for “trace” proteinuria.8 Dipstick proteinuria was, however,
significantly more likely to be correct (true positive/true negative when compared with 24-hour
collection) if diastolic blood pressure was elevated > 90 mmHg (p = 0.032) and in the absence of
ketonuria (p = 0.001). 17
3. Visually read ratiometric dipstick
These tests have increased accuracy but are limited in availability and are expensive for LRS.
The concentration of protein in the urine may be compared to the creatinine level in a spot urine sample.
Protein:creatinine (Pr:Cr) uses urine creatinine as a surrogate marker to correct for variations in daily
urine volume. This is used in some institutions instead of a timed urine collection. Studies have
4
demonstrated that measurement of Pr:Cr in a spot urine sample accurately reflects 24-hour urine
collection for detection and quantification of protein in the urine and is simpler, faster, and a more useful
method for the diagnosis of significant proteinuria.18,19 In addition, there is potential for use as a
point-of-care test in facilities that do not have reliable electricity or a laboratory.
Multistix® PRO strips
The Multistix® PRO strips (Siemens), specifically the Multistix® PRO 10LS is one of two urinalysis
products on the market providing Pr:Cr. ‡ Among the ten pads (“features”), three pads are used for protein
and creatinine ratios: (1) the protein-low pad, specific for protein concentrations from trace to 15 mg/dL,
(2) the protein-high pad ranging from 30 mg/dL to greater than 2,000 mg/dL, and (3) a pad for
establishing urinary creatinine levels (from 10 mg/dL to 300 mg/dL). In an evaluation of 881 freshly
voided urine samples, the Multistix® PRO 10LS demonstrated a sensitivity and specificity to proteinuria
(when ratioing the Pr:Cr values) around 75% and 96.5%, respectively. 20 The study, however, did not
examine the correlation to PE. Currently at a cost of US$7.50 a strip, the Multistix® PRO is not
economically feasible for LRS.
Defeatured Pr:Cr dipstick (LifeAssay Diagnostics, South Africa)—in development
A “simplified” ratiometric dipstick for greater ease of evaluation is projected to have equal accuracy to
commercially available Pr:Cr dipsticks, while being comparatively inexpensive.
Currently available ratiometric dipsticks are economically unfeasible for use in LRS. PATH has identified
a manufacturer in South Africa—LifeAssay Diagnostics—developing a Pr:Cr strip test for economical
use in LRS. LifeAssay Diagnostics is an
Figure 2: Example image: lateral flow assay in
International Organization for Standardization cassette format (in conceptual development by PATH).
(ISO)-compliant manufacturer of diagnostics
for a variety of diseases in strip test format or
lateral flow assays in cassette format (see
Figure 2). Unlike the Multistix® PRO, which
includes seven other analyte pads, LifeAssay
can produce a simplified dipstick having two
or three pads only for protein and creatinine
analytes. Early cost estimates suggest that the
product will be between US$0.05–US$0.15
per strip. The LifeAssay Pr:Cr dipstick could
Photo: PATH/Allison Golden.
be included in an upcoming major clinical
study, Pre-eclampsia Integrated Estimate of RiSk or miniPIERS (part of a larger trial known as PREeclampsia-Eclampsia Monitoring, Prevention and Treatment or PRE-EMPT) in 2014. Verification testing
of the simplified dipstick prototype is being planned for early 2014 to verify the dipsticks prior to formal
‡
Multistix PRO and all associated marks are trademarks of Siemens Healthcare Diagnostics Inc. Additional information is available at: http://usa.healthcare.siemens.com/point-of-care/urinalysis/urine-test-strips-multistix-pro.
5
use in the clinical study. If verification is successful, the LifeAssay dipstick could be part of the clinical
trial’s routine screening methods of proteinuria. The clinical trial, with a recruitment of nearly 2,100
women, is planned for Brazil, Fiji, Pakistan, South Africa, and Uganda. The data will be correlated to
maternal and neonatal outcomes. Successful validation, as measured by improved performance and ease
of use, will be convincing evidence for implementation as a replacement to the common protein-only
dipstick in current pregnancy care algorithms. Furthermore, as a low-cost, highly portable solution, the
LifeAssay ratiometric dipstick could potentially impact task-shifting to community health care workers
and enable reaching more isolated pregnant women in rural areas.
Reagent-limited dipstick—in conceptual development by PATH
This conceptual dipstick assay would improve accuracy and reproducibility through a binary call of test
results.
PATH has conceptualized a dipstick assay where the need for evaluation of subtle color changes is
eliminated. Such a test would contain reagent-limited detection pads wherein graded levels of protein and
creatinine are laid out together on a single dipstick. Each pad, whether for protein or creatinine, would
measure specific threshold concentrations and thereby result in a binary change of indication color.
Instead of a subjective evaluation of color shading, the user would observe a single color change that
would correspond to a set threshold concentration. Observation of successive color levels indicating
increasing protein and creatinine concentration would then result in the proteinuria status. This concept
may help reduce the inherent variation arising from interpretation of colorimetric changes and thus
stabilize the range of accuracy values associated with ratiometric dipsticks.
4. Machine dipstick readers (ratiometric)
These test results have only a modest increase in accuracy but an advantage in data connectivity.
Clinitek® and Aution® PocketChem dipstick readers
Figure 3: Clinitek® Status + dipstick reader.
Sophisticated methods to read dipsticks, such as an
automated dipstick reader (Clinitek® [Siemens],
Germany; and Aution® [ARKRAY], Japan §—see
Figure 3), may increase precision of evaluating
colorimetric changes but require reliable electricity,
trained staff, and expensive equipment and are,
therefore, not suitable for most LRS. In general
dipstick readers demonstrate a modest improvement
in accuracy.15,21 While most studies are based on
dipstick evaluations by trained clinical staff in ideal
conditions of a laboratory, a reader study might be
Clinitek® photo courtesy of Siemens Healthcare Diagnostics.
©2014 Siemens Healthcare Diagnostics Inc.
§
Aution and all associated marks are trademarks of ARKRAY, Inc. Additional information is available at:
http://www.arkray.co.jp/english/products/urinalysis.html.
6
of greater benefit in variable point-of-care conditions with field care workers with variable training levels,
resulting in a greater consistency and accuracy of results. Another advantage of dipstick readers is
immediate connectivity to health management systems through the Internet. For rural locations, even
peri-urban locations, the connectivity would provide a link to care with physicians or for ready referral of
patient data in care management.
uChek™ urine Analyzer
The uChek™ urine analyzer, made by Biosense Technologies **, is a promising reader adapted for use in
LRS. The highly portable kit is complete with an illuminator box (Cuboid), a dipstick holding tray (Color
Mat), mobile phone, and dipsticks. The Cuboid and the Color Mat provides a stable platform for
photometric data collection by supporting consistent lighting and motion stabilization, hence a greater
precision of results determination. Data gathering, analysis, and storage programs are already uploaded in
the cell phone software. Furthermore, results can be transmitted by wireless networks to clinical services
or health management systems unlike other readers requiring hard line/computing infrastructure. While
the uChek™ system is less expensive than comparable readers, the cost of the system, US$300, remains
high for LRS.
5. Point-of-care non-dipstick readers (ratiometric)
These non-dipstick readers deliver high accuracy, but with higher user training requirements and at
higher cost.
At the most sophisticated end of the point-of-care technology spectrum are all-in-one analyzers (DCA
Vantage® [Siemens], US; and Afinion™ systems [Alere, Inc./Axis-ShieldPoC AS], UK) capable of
performing chemical assays utilizing antibody-specific binding of proteins. A small sample of urine,
typically less than
Figure 4: Afinion™ AS100 analyzer and cartridge.
5 microliters, is placed
in a disposable cassette
(Figure 4) that contains
anhydrous-reactive
chemicals.
Upon rehydration with
the urine sample and
insertion into the
analyzer, the analyzer
evaluates the resulting
color change or
Permission to use Afinion image granted from Alere. © 2014 Alere. All rights reserved. The Alere
Logo, Alere, and Afinion are trademarks of the Alere group of companies.
**
uChek is a trademark of Biosense Technologies Private Limited. Additional information is available at:
https://sites.google.com/site/ucheksystem/how-it-works.
7
immuno-turbidity change. Despite the capability of high performance for detecting proteinuria and
increased high throughput, the cost of even the smallest-capacity analyzers, US$2,500, far exceeds
feasibility at the community level, making them more appropriate for high throughput at established
medical institutions.
Specific urine-based candidate biomarkers for preeclampsia
Due to inherent lack of sensitivity and specificity of screening for PE by testing for proteinuria, a wide
variety of biomarkers have been proposed for diagnosing women with PE and identifying women at high
risk of developing PE. Research to uncover biomarkers for PE/E is rich and chiefly targeted to discovery
of signature markers in blood †† (to be discussed in greater detail in a separate landscape). If proven
effective, dipsticks and other ready-to-use methods to detect blood-based biomarkers could be developed
for use at point of care.
Urinary biomarkers, however, present an advantage in that urine samples are more easily obtainable, pain
free, in sufficient volume, and are neither infectious nor a biohazard in disposal. The payoff would be
high if a urine biomarker or a set of biomarkers could diagnose and predict PE/E or screen for the onset of
PE/E. Some biomarkers in urine may be present at high concentrations. They could likely be detected
with simple diagnostic rapid strip tests. This translation into lateral flow cassette format (see Figure 3
above) is not only a proven diagnostic platform but also a reliable, economically feasible format for lowresource countries. While some of these biomarkers appear promising, they remain investigational.
Several urine biomarkers are described below.
1. Biomarker of misfolded protein: Congo Red dye assay
Early study results using this assay show promising results for prediction for PE.
Protein research work conducted by Irina Buhimschi’s laboratory (while at Yale University) on urine
from women with severe PE identified the unique occurrence of aggregated fragments as a possible
consequence of misfolded proteins. 22 Congo Red is an azo dye which can non-reversibly intercalate
within misfolded protein complexes. 23 The Buhimschi team is evaluating the use of the dye as dot blots
on non-specialized, plain paper 24 as a means of a low-cost, usable method in LRS. In a 2009 abstract,25
Dr. Buhimschi’s group applied the Congo Red Dot (CRD) test, as well as blood-based biomarkers, serum
indicators, sFlt1/P1GF, and protein to creatinine ratios, to 347 pregnant women across five groups: 1) a
normotensive control group, 2) a chronic hypertension group, 3) a gestational hypertension group, 4) a
mild PE group, and 5) a group of women with severe PE. CRD test results were correlated to an outcome
††
The UNDP/UNFPA/WHO/World Bank Special Program of Research, Development and Research Training in Human Reproduction (HRP), in
collaboration with the Perinatal Research Branch of the National Institute of Child Health and Development, is currently conducting a multicenter
study to test whether changes in serum and urinary angiogenic proteins can be used as an effective method for identifying women at high risk of
developing PE.
8
of preterm delivery as a consequence of PE. Plots attached to the short abstract appear to show 1) CRD
can differentiate PE from non-PE hypertensive conditions, 2) an indication between a positive CRD test
and preterm delivery, and 3) marked higher performance in accuracy over sFlt1/P1GF and protein to
creatinine ratios. The Buhimschi group has received a 2013 Saving Lives at Birth award (USAID) to
expand work on the CRD.
2. Tamm-Horsfall protein urine biomarker
While in conceptual stage, this biomarker shows promise for prediction of PE, potentially higher
performance metrics, and adaptability to a lateral flow assay.
Abnormal proteinuria is classified into three categories of generalized origins: glomerular, tubular, and
overflow. About 40% of urinary proteins are glomerular in origin and the rest are tubular,14 mainly
composed of THP. THP, also known as uromodulin, is a naturally occurring glycoprotein produced by the
long tubes of the Henle’s loop. THP is naturally excreted. While the exact purpose of the protein is
unknown, two roles are hypothesized: 1) as an inhibitor of crystallization of calcium and 2) as a potent
immune suppressant in pregnant women. 26 In 1989, researchers 27 characterized a differential rate of
excretion between pregnant women, nonpregnant women, and pregnant women with PE. According to the
paper, a significant drop in THP excretion was found in pregnant women with PE. While normal
pregnancy is typified by an increase in THP excretion, greater than 51 mg/g creatinine, nonpregnant
women had a median THP excretion rate of 20 mg/g creatinine and hypertensive pregnant women with
PE exhibited a low THP excretion rate of 9 mg/g creatinine.
Using proteomics in a study of urine protein, THP was one of a few protein groups characterized in the
increase of renal disease and hypertension.28 David Carty (University of Glasgow) used conventional
capillary electrophoresis and mass spectrometry to longitudinally examine urine samples from pregnant
women from gestational weeks 12 to 16, 20, and 28. From comparison to nonpregnant controls, the
authors were able to develop a model of 50 urine biomarkers which increased markedly across the
sampling time points for urine samples taken from women who later developed PE. The 50 biomarkers
were identified as protein fragments of fibrinogen alpha chain, collagen alpha chain, and THP. In another
study, the investigators have identified one fragment of THP, near the promoter region of UMOD gene,
which is associated with hypertension independent of renal function.29 As a result of that work, the
authors speculate that THP, “as a key player in renal disease and hypertension, could point toward
subclinical vascular and renal damage in the early stage of preeclampsia that are indicated by differential
urinary expression of THP fragments and warrant further investigation.”
Anti-THP antibodies are already commercially available, and yet there is a surprisingly low number of
studies further investigating THP in pregnant women. A focused investigation in how THP levels vary
with pregnancy would inform the possible development of a lateral flow assay.
3. SERPINA1 urine protein biomarker
9
Also in the conceptual stage, this biomarker shows promise for prediction of PE, potentially higher
performance metrics, and adaptability to a lateral flow assay.
Dr. Buhimschi and her colleagues also published a proteomic analysis of urine from pregnant women
using mass spectrometry of a specialized technology, surface-enhanced laser desorption/ionization
(SELDI) technology. Interestingly their analysis uncovered a characteristic fingerprint of SERPINA1
protein (SERine Protease-inhibitor A1) and albumin protein fragments.30 A comparison of proteomic
profiling versus blood-based sFLt-1 to PIGF ratio ‡‡ or urine protein to creatinine ratio showed a much
higher accuracy for predicting PE and correlated to a surrogate outcome of mandated delivery. Since
SERPINA1 has been tied to pathophysiology of liver damage and encephalopathy, the authors
hypothesize that the supramolecular aggregates of misfolded SERPINA1 may be a result of abnormal
liver and neurologic changes which arise with PE.
4. C5b-9 urine protein biomarker
Potentially the most clinically sensitive and specific urine biomarker for PE to date.
C5b-9 plays an important role in inflammatory and cell-killing processes. Recently, in December 2013,
Burwick, et al. (Brigham and Women’s Hospital, Harvard University) associated the excretion of C5b-9
complement factor to 96% of cases with severe PE, 12% of controls with chronic hypertension, and 8% of
healthy controls. 31 Complement activation is part of the systemic inflammatory response to the fetus. In
order to protect itself from the maternal immune response, the fetus produces complement regulatory
proteins, inhibiting the complement activation cascade. The group hypothesizes that the excess presence
of complement factors in plasma during severe PE abets kidney injury, leading to increased concentration
in urine excretion. While all the complement factors were found to be excreted in increased amounts in
severe PE, urinary C5b-9 stands out dramatically in distinguishing severe PE from chronic hypertension.
As with other urine biomarkers, C5b-9 warrants further investigation in proof-of -concept studies,
validation, and feasibility. These development activities would be geared to translation for use in LRS as
a lateral flow assay.
Investment opportunity
PATH recommends the following opportunities for near-, intermediate-, and long-term investments to
create improved tools for proteinuria screening and/or PE diagnosis:
1. Near term (1-3 years): Validate and commercialize LifeAssay’s defeatured Pr:Cr dipstick
Despite the shortcomings of ratiometric dipstick tests, investment in the validation of the LifeAssay
dipstick could enable commercialization and scale-up of a near-term, affordable test for LRS.
‡‡
Soluble fms-like tyrosine kinase 1 (sFLt-1) and placental growth factor (PIGF) are both angiogenic proteins
identified as blood-based biomarkers for PE.
10
Specific activities could include:
•
Funding the inclusion of the LifeAssay test in the highly strategic mini-PIERS multi-country clinical
study planned for 2014. A funding gap to cover the costs for study supplies and labor, especially for
the use of gold-standard comparator tests alongside the LifeAssay test remains. LifeAssay has already
agreed to provide its tests at virtually no cost for this study.
•
Commissioning a research study to include user assessment, monitoring, and evaluation (in parallel to
PIERS). This study could be designed and prepared right after the PIERS data are available.
•
Supporting global and in-country advocacy with public-sector stakeholders for adding this test to
existing guidelines, government tenders, etc. (once the data are available).
•
Supporting marketing activities to public-sector customers by providing in-kind marketing expertise.
The LifeAssay defeatured Pr:Cr dipstick could be used with Biosense’s uChek™ reader to further
increase accuracy of interpreting the test, and results could be linked to mobile-based data management
systems. However, the many system changes required of a bundled product offering and the overall
implications for additional equipment, distribution, and training should not be underestimated. These
additional implications and trade-offs were clear when putting the LifeAssay defeatured Pr:Cr dipstick
combined with the uChek™ reader through the Strategic Prioritization tool (where it scored lower than
the LifeAssay dipstick alone).
2. Intermediate term (3-5 years): Build the next-generation Pr:Cr dipstick to improve
performance and usability
As a next-generation dipstick, PATH recommends advancing the defeatured Pr:Cr dipstick concept
further by developing a reagent-limited, dual dipstick for both protein and creatinine with thresholdlimited indicator fields. This will make it easier to accurately interpret results. A one- to three-year
development window from proof of concept to clinically evaluated prototype is anticipated.
Specific activities could include:
•
Conducting proof-of-principle research and development for reagent-limited, dual dipstick.
•
Evaluating reagent-limited, dual dipstick with clinical samples.
•
Commissioning a research study to include user assessment, monitoring, and evaluation.
•
Supporting global and in-country advocacy with public-sector stakeholders for adding this test to
existing guidelines, government tenders, etc.
3. Long term (4-7 years): Develop rapid assays for PE-specific urine biomarkers on a lateral flow
assay platform.
While further clinical studies are needed, there is the potential that PE-specific urine assays could be
developed by creating lateral flow-based assays for one or several of the biomarkers listed above. A
biobank, managed by Jim Roberts at the University of Pittsburgh, of over 2,000 well-characterized urine
11
samples from pregnant women could be available for validation of a biomarker assay. Translation into a
lateral flow assay, followed by verification and field validation, would be a longer-term project and could
be performed by PATH.
Specific activities could include:
•
Conducting proof-of-principle research and development, laboratory validation of rapid strip test, and
field validation of rapid strip test.
•
In parallel, conducting longitudinal clinical studies with biomarkers to confirm timing of their clinical
predictive validity.
•
Creating a commercialization strategy, including finding a commercialization partner.
•
Facilitating market development.
•
Supporting global and in-country advocacy with public-sector stakeholders for adding this test to
existing guidelines, government tenders, etc.
12
References
1. Khan KS, Wojdyla D, Say L, et al. WHO analysis of causes of maternal death: a systematic review.
The Lancet. 2006;367(9516):1066–1074.
2. Engender Health. Balancing the Scales: Expanding Treatment for Pregnant Women with LifeThreatening Hypertensive Conditions in Developing Countries, a Report on Barriers and Solutions to
Treat Preeclampsia & Eclampsia. New York: EngenderHealth; 2007. Available at:
http://www.engenderhealth.org/files/pubs/maternal-health/EngenderHealth-Eclampsia-Report.pdf.
3. Higby K, Suiter CR, Phelps JY, Siler-Khodr T, Langer O. Normal values of urinary albumin and total
protein excretion during pregnancy. American Journal of Obstetrics and Gynecology. 1994;171(4):984.
4. Firoz, T. Pre-eclampsia in low and middle income countries. Best Practice and Research Clinical
Obstetrics and Gynaecology. 2011;25(4):537–548.
5. Goldenberg RL, McClure EM, Macguire ER, Kamath BD, Jobe AH. Lessons for low-income regions
following the reduction in hypertension-related maternal mortality in high-income countries.
International Journal of Gynecology and Obstetrics. 2011;113(2):91–95.
6. Gaspari F, Perico N, Remuzzi G. Timed urine collections are not needed to measure urine protein
excretion in clinical practice. American Journal of Kidney Diseases. 2006;47:1–7.
7. Peeling RW, Holmes KK, Mabey D, Ronald A. Rapid tests for sexually transmitted infections (STIs):
the way forward. Sexually Transmitted Infections. 2006;82(Suppl 5): v1–v6.
8. Thangaratinam S, Coomarasamy A, O’Mahony F, et al. Estimation of proteinuria as a predictor of
complications of pre-eclampsia: a systematic review. BMC Medicine. 2009;7:10. Available at
http://www.biomedcentral.com/1741-7015/7/10.
9. Thornton CE, Makris A, Ogle RF, Tooher JM, Hennessy A. Role of proteinuria in defining preeclampsia: clinical outcomes for women and babies. Clinical and Experimental Pharmacology and
Physiology. 2010;37(4):466‒470.
10. Sibai BM. Eclampsia. VI. Maternal-perinatal outcome in 254 consecutive cases. American Journal of
Obstetrics and Gynecology. 1990;163(3):1049‒1054.
11. Viswanathan G, Upadhyay A. Assessment of Proteinuria. Advances in Chronic Kidney Disease.
2011;18(4):243–248.
12. Hofmeyr GJ, Belfort M. Proteinuria as a predictor of complications of pre-eclampsia. BMC Medicine.
2009;7:11. Available at http://www.biomedcentral.com/1741-7015/7/11.
13. Khazardoost S, Maryamnoorzadeh, Abdollahi A, Shafaat M. Comparison of 8-h urine protein and
random urinary protein-to-creatinine ratio with 24-h urine protein in pregnancy. Journal of MaternalFetal and Neonatal Medicine. 2012;25(2):138–140.
14. Lindheimer M, Katner D. Interpreting abnormal proteinuria in pregnancy: the need for a more
pathophysiological approach. Obstetrics and Gynecology. 2010;115(2 Pt 1):365–375.
15. Waugh JJ, Clark TJ, Divakaran TG, Khan KS, Kilby MD. Accuracy of urinalysis dipstick techniques
in predicting significant proteinuria in pregnancy. Obstetrics and Gynecology. 2004 Apr;103(4):769–777.
16. Gangaram R, Ojwang PJ, Moodley J, Maharaj D. The accuracy of urine dipsticks as a screening test
for proteinuria in hypertensive disorders of pregnancy. Hypertension in Pregnancy. 2005;24(2):117–123.
13
17. Phelan LK, Brown MA, Davis GK, et al. A prospective study of the impact of automated dipstick
urinalysis on the diagnosis of preeclampsia. Hypertension in Pregnancy. 2004;23(2):135–142.
18. UK Department of Health. Proteinuria: Detection and Quantitation in Adults Using ACR—
Information for GPs. London: Department of Health; 2009. Available at:
http://webarchive.nationalarchives.gov.uk/20130107105354/http://www.dh.gov.uk/prod_consum_dh/grou
ps/dh_digitalassets/documents/digitalasset/dh_096063.pdf.
19. Côté AM, Brown MA, Lam E, et al. Diagnostic accuracy of urinary spot protein:creatinine ratio for
proteinuria in hypertensive women: systemic review. BMJ. 2008;336 7651:1003–1006.
20. Croal, BL. Evaluation of the Bayer Multistix PRO 10LS point-of-care urine test. Point of Care.
2003;2(2):144–148.
21. Peele, JD, Gadsden RH, Crews R. Semi-automated vs. visual reading of urinanlysis dipsticks. Clinical
Chemistry. 1977;23(12):2242–2246.
22. Buhimschi I, Guomao Z, Baumbusch MA, Janciauskiene S, Buhimschi, CS. Preeclampsia is a disease
characterized by specific supramolecular aggregates of misfolded proteins and congophilia. American
Journal of Obstetrics and Gynecology. 2008:199(6) Supplement 1:S78.
23. Page on Congo Red Dot Test. Yale Scientific website. Available at
http://www.yalescientific.org/2010/12/congo-red-dot-test-a-groundbreaking-method-to-diagnosepreeclampsia. Accessed November 5, 2013.
24. Page on Congo Red Dot Test. United States Agency for International Development website. Available
at: http://www.usaid.gov/news-information/frontlines/open-development-developmentdefense/pinpointing-preeclampsia-simple-red. Accessed November 5, 2013.
25. Buhimschi I, Funai E, Zhao G, et al. Assessment of global protein misfolding load by urine “Congo
Red Dot” test for diagnosis and prediction of outcome in women with preeclampsia (PE). American
Journal of Obstetrics and Gynecology. 2009;201:12–13.
26. Liu Y, El-Achkar TM, Wu XR. Tamm-Horsfall protein regulates circulating and renal cytokines by
affecting glomerular filtration rate and acting as a urinary cytokine trap. Journal of Biological Chemistry.
2012;287(20):16365–16378.
27. Nesselhut T, Rath W, Grunow E, et al. The relationship between urinary Tamm-Horsfall glycoprotein
excretion and urinary activity of glycosidases in normal pregnancy and pre-eclampsia. European Journal
of Obstetrics & Gynecology and Reproductive Biology. 1993;48(1):23–31.
28. Carty DM, Siwy J, Brennand JE, et al. Urinary proteomics for prediction of preeclampsia.
Hypertension. 2011;57(3):561–569.
29. Padmanabhan S, Melander O, Johnson T, et al. Genome-wide association study of blood pressure
extremes identifies variant near UMOD associated with hypertension. PLoS Genetics. 2010;6(10):
e1001177.
30. Buhimschi, IA, Zhao G, Funai EF, et al. Proteomic profiling of urine identifies specific fragments of
SERPINA1 and albumin as biomarkers of preeclampsia. American Journal of Obstetrics and Gynecology.
2008:199(5):551.e1–551.e16.
31. Burwick RM, Fichorova RN, Dawood HY, Yamamoto HS, Feinberg BB. Urinary excretion of C5b-9
in severe preeclampsia: tipping the balance of complement activation in pregnancy. Hypertension.
2013;62(6):1040‒1045.
14