Diagnostic Utility of a Novel Point-of-Care Test of Calprotectin for Revision Total
Conditions: Prosthetic Joint Infection
Sponsor: The Cleveland Clinic

 
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Diagnostic Utility of a Novel Point-of-Care Test of Calprotectin for Revision Total Knee 
Arthroplasty 
 
 
 
 
 
NCT: NCT03694925 
 
Date: 3/17/20  
 
 
 
 
STUDY PROTOCOL 
 
PI- Carlos A Higuera, MD 
 
Department of Orthopaedic Surgery 
Section of Adult Reconstruction 
Cleveland Clinic Foundation 
 
 
 
 
 
Correspondence: 
Alison Klika 
Clinical Research Coordinator 
Cleveland Clinic, Cleveland, OH 
Department of Orthopaedic Surgery - A4-516 
Office: 216-444-4954 
FAX: 216-445-6255 
Email: klikaa@ccf.org 
 
 
 
 
 
 

 
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Table of Contents 
 
 
 
I. 
Synopsis…………………………………………………………………………………3 
II. 
Background……………………………………………………………………………...4 
III. 
Rationale………………………………………………………………………………...5 
IV. 
Hypothesis………………………………………………………………………………6 
V. 
Aims………………………………………………………………………………….....6 
VI. 
Study Setting……………………………………………………………………………6 
VII. 
Eligibility Criteria……………………………………………………………………...6 
VIII. 
Study Procedure………………………………………………………………………...7 
IX. 
Data Collection…………………………………………………………………………8 
X. 
Analysis…………………………………………………………………………………9 
XI. 
Data Safety……………………………………………………………………………..10 
XII. 
References……………………………………………………………………………...11 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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I. 
Synopsis 
 
Title 
Diagnostic Utility of a Novel Point-of-Care Test of Calprotectin for 
Revision Total Knee Arthroplasty 
Sponsor 
Orthogenics 
Sites 
Cleveland Clinic Main Campus (Cleveland, OH) and Cleveland 
Clinic Florida (Weston, FL) 
Objectives 
To investigate whether there are quantifiable differences in the level 
of calprotectin in the synovial fluid that allow separation of different 
modes of joint implant failure (e.g. infected, aseptic loosening).  A 
subset of primary TKA patients (with history of OA) will be 
included as a baseline. 
Design 
Prospective clinical trial involving patients undergoing primary or 
revision TKA. 
Test Product 
Calprotectin POC test is a lateral flow device that detects 
calprotectin, a marker of inflammation, in the synovial fluid.  
Number of 
Patients 
N=30 primary TKA 
N=120 revision TKA (of which, n=70 aseptic and n=50 septic) 
Procedure 
Synovial fluid will be collected pre-operatively or intra-operatively 
from patients meeting the inclusion/exclusion criteria, and will be 
tested for infection using the Calprotectin POC test, which will be 
validated against then CALPRO Calprotectin ELISA test. 
Statistical 
Methods 
Diagnostic parameters of Calprotectin POC test will be assessed 
using the MSIS criteria as the gold standard.  
 
 
 
 
 
 
 
 
 
 
 
 

 
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II. 
Background 
 
Total knee arthroplasty (TKA) is considered to be a very successful procedure which has 
changed treatment of severe degenerative joint disease, improving the quality of life of 
millions[1,2].  However, infection of these implants, called periprosthetic joint infection (PJI), is 
a serious complication of arthroplasty[3].  PJI can lead to additional surgeries, revision, fusion, 
and other adverse events. It is important to accurately diagnose PJI, because its management 
differs from that of other causes of arthroplasty failure. 
 
 
Unfortunately, no single test or clinical sign is currently available to diagnose infection. The 
classic signs of infection such as redness, warmth, swelling and pain can result from a variety of 
septic and aseptic etiologies. Additionally, some cases of PJI, especially those caused by indolent 
organisms, are often associated with relatively little redness, warmth, swelling and pain.  Though 
rare, the only clinical sign that has been strongly associated with PJI is a sinus tract leading from 
the skin to the joint. Although clinical signs and symptoms should alert a clinician to the 
possibility of infection, there is general agreement in the field that they are not useful in making 
a definitive diagnosis. Therefore, a combination of laboratory testing techniques has guided the 
diagnostic decision-making related to PJI[4–6].  
 
Recognizing the limitations of any one test for diagnosing PJI, the Musculoskeletal Infection 
Society (MSIS) has generated a criteria-based definition of PJI for diagnosing PJI[6]. This 
criteria-based definition of PJI places emphasis on culture techniques that identify pathogens.  
However, because the diagnostic sensitivity of cultures is about 70%[7,8], and because tissue 
culture results from intraoperative samples are only available after surgery, the MSIS definition 
also includes serological and synovial inflammatory markers that can be used to diagnose PJI. A 
major disadvantage of the MSIS criteria-based definition of PJI is that two of the essential 
criteria, tissue culture and histological analysis, are only available after surgery.  In cases where 
the surgical samples are critical for the diagnosis of infection, the physician may need to adjust 
his or her treatment post operatively for the patient based on the treatment provided.  The 
availability of a preoperative test that correlates well with the MSIS criteria could improve 
patient care.  Although the currently available pre-operative tests have been shown to be useful, 
these tests often require laboratory expertise, and the results may not be available in a timely 
fashion. Therefore, there has been growing interest in point of care (POC) test for the diagnosis 
of infection. 
 
Currently, some of the POC tests available include the alpha defensin and leucocyte esterase 
(LE) tests. Both these test measure markers of infection present in the synovial fluid. The LE 
test, though cheap and easy to perform, is easily affected by the presence of blood in the synovial 
fluid[9,10]. Although centrifugation of the bloody samples might improve the accuracy of the LE 
test, it is cumbersome, and not easily available. As a result, a large number of bloody samples are 

 
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unable to be analyzed by the LE test. The alpha-defensin lateral flow device is another promising 
POC test which has sensitivity of about 70%[11].  Although the clinical results of this are 
promising, they are not as good as previous studies using alpha-defensin levels measured in a 
laboratory[11]. Moreover, there are concerns that the alpha defensing test will yield false 
positive results in cases of metal on metal revisions[12]. Therefore, better tests are required. 
 
 
III. Rationale 
 
Calprotectin is a biomarker closely associated with leucocytes in general, and is present in high 
volumes in neutrophil cells[13–15] . Calprotectin is also produced by infiltrating monocytes and 
macrophages, where calprotectin is released upon phagocytosis[16].In neutrophils, calprotectin is 
stored intracellularly and are released upon activation of the cell. The determination of number 
of neutrophils and proportion of neutrophils out of total number of inflammatory cells is a 
diagnostic strategy commonly used in diagnosis of infection[6,17]. Upon encounter with a 
pathogen, neutrophils have several strategies to fight infections , and produce high-levels of 
calprotectin [13,18]. Activation of neutrophils, and release of calprotectin, can be for any reason 
causing activation of the complement system and aseptic Inflammatory responses[13]. Moreover, 
Calprotectin is a danger associated molecular patterns (DAMP) signal influencing the 
inflammatory responses[14]. The level of activated neutrophils in PJI provide basis of the 
presence of calprotectin in the synovial fluid of PJI patients, and thus, for calprotectin as a 
potential biomarker for PJI. Calprotectin-levels in the synovial fluid do not merely reflect the 
level of leucocytes and neutrophils present in the synovial fluid, but levels are correlated to the 
WBC content. Calprotectin is likely to reflect the number of activated cells and surpass the 
diagnostic accuracy of total WBC counts and neutrophil percentage for PJI diagnosis. 
 
Wouthuyzen-Bakker et al. [19] demonstrated that a level of calprotectin of 50 mg/L in the 
synovial fluid has very good diagnostic accuracy for PJI, supported by area under the curve 
values of more than 0.9. This break point produced a negative predictive value (NPV) of 95% for 
all 42 patients in the study. In a subgroup analysis for patients with chronic PJI, a NPV of 97% 
was observed[19]. The excellent NPV may assist in the orthopaedic clinic to rule out the 
presence of infection and consider diagnostic alternatives for aseptic loosening and pain revision 
of the joint patient. A rapid and accurate distinction between these two causes is important as PJI 
and aseptic loosening are managed differently with regards to surgical Intervention and follow-
up.  
 
Point of Care Test diagnostics by lateral flow devices provides reliable test results within 
minutes of sample collection. Currently, Calprotectin can be detected by such lateral flow 
devices (developed by Orthogenics, Tromsø, Norway). The speed and ease of use of this test 
allows for diagnosis at patient’s bed side. These tests can be applied in the physician's office, 

 
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operating room, an ambulance, the home, the field, or in the hospital. As the results are timely 
they allow rapid diagnostic and identifies treatment alternatives for the patient. This technology 
empowers clinicians to make decisions at the "point-of-care", and can have significant impact on 
health care delivery and ability to address challenges of health disparities. However, it is 
important to validate the diagnostic utility of calprotectin POC in a diverse set of patients 
undergoing revision arthroplasty. 
 
 
IV. Hypothesis 
 
The study hypothesis is that the use of Calprotectin POC test will help differentiate between septic 
and aseptic failure following TKA. 
 
 
V. Aims 
 
Compare levels of calprotectin in these different patient groups: 
 septic revisions v aseptic loosening 
 aseptic loosening v other non-infected failure modes 
 primary knees stratified by OA grade  (or simply for baseline comparison?) 
 
 
VI. 
Study Setting 
 
This will be a prospective clinical trial. Patients undergoing total knee arthroplasty will be 
eligible to participate in this study. The clinical trial will be conducted with samples 
collected at a Cleveland Clinic Main Campus (Cleveland, OH) and Cleveland Clinic 
Florida (Weston, FL) with high volumes of revision surgery. The goal is to enroll n=30 
primary TKA patients, n=70 aseptic revision TKA patients, and n=50 septic revision TKA 
patients.   
 
 
VII. 
Eligibility Criteria 
 
 
Subject Inclusion Criteria 
 Subject is ≥18 years of age. 
 Patient with a diagnosis of OA (for primary TKA only) 
 Subject has had no recent injections or surgeries of the joint (within past 6 weeks). 
 Subject has or will have all of the medical tests required to allow MSIS classification 
(revision cases only) 

 
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 Subject signs informed consent form. English Speaking 
 
 
Subject Exclusion Criteria 
 MSIS criteria labs no older than 60 days (revision cases only) 
 Results are not available for medical tests required to perform MSIS classification (revision 
cases only) 
 Sample was obtained via lavage 
 Quantity not sufficient (at least 1 ml required). 
 
VIII. Study Procedure 
 
Synovial fluid samples will be collected from the affected joint either during pre-operative work-
up or intraoperatively. Sample will be transported to testing site. Once the sample is received by 
the laboratory or research personnel, fluid will be first aliquoted for use in the physician-ordered 
diagnostic laboratory testing. The remainder of the specimen will be used for Calprotectin testing 
using the testing protocol described by Orthogenics. The specimen for the MSIS criteria labs 
need not be collected at the same time as specimen used for the Calprotectin POC test. In brief, 
the Calprotectin POC testing technology utilizes the natural occurring liquid movement through 
membranes, and by combining this with lines of specific antibodies and nanoparticles (gold or 
latex), biomarker detection in the fluid can be achieved. When Calprotectin is present in the 
fluid, a test line will form together with a control line that demonstrate the validity of the test. 
The test will be read as: green (no inflammation), yellow (moderate inflammation) or red 
(massive inflammation). This will then be validated by comparison against the CALPRO 
Calprotectin ELISA test. ELISA samples will be diluted 20x for a primary TKA sample and 
1000x for a revision TKA sample.  
 
 
The septic revision cases will be defined using MSIS criteria. All non-infected revision patients 
are considered as aseptic cases. MSIS criteria-defined PJI diagnosis will be determined by an 
independent three-physician adjudication panel with expertise in infection who have access to all 
the necessary patient data for clinical diagnosis (eg, all MSIS criteria and patient history).  The 
adjudication panel will be blinded to the results of the Calprotectin POC test. There are two 
major criteria, and five minor criteria in the MSIS definition of PJI.  The existence of one 
positive major criterion is sufficient for the diagnosis of PJI.  Similarly, the existence of any 
three of the five positive minor criteria is considered sufficient for the diagnosis of PJI.  Table 1 
presents the MSIS criteria based definition of PJI. 
 
Table 1: MSIS Workgroup Standard Definition for PJI [6] 
One of the following must be met for diagnosis of PJI. 

 
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1.  A sinus tract communicating with the prosthesis (major criteria);    
  A pathogen is isolated by culture from two separate tissue or fluid samples obtained from 
the affected prosthetic joint (major criteria); 
3.  Three of the following five minor criteria exist: 
      a. Elevated ESR and CRP (ESR≥30mm/hr; CRP≥10mg/L) 
      b. Elevated synovial fluid WBC count (≥ 3000) 
      c. Elevated synovial fluid neutrophil percentage (≥80%) 
      d. Isolation of a microorganism in one periprosthetic tissue or fluid culture 
Greater than 5 neutrophils per high-powered field in 5 high-power fields observed from 
histological analysis of periprosthetic tissue at 400 times magnification 
 
All samples will be classified based on the MSIS criteria into positive (infected) or negative (not 
infected). The MSIS criteria will be considered as the gold-standard for diagnosis. 
 
The interpretation of Calprotectin POC test will be made in a quantitative way by classifying all 
patients into a positive or a negative test. A positive test will be the presence of yellow or red 
line. This is expected to improve the sensitivity as Calprotectin POC test is intended to use as a 
screening tool. However, additional analysis will be performed for different thresholds. 
 
The aseptic revision cases will be identified first by gross loosening on xray. If no loosening 
evident, a bone scan may  be performed. 
 
 
IX. Stability Experiment  
Stability Experiment Protocol 
Stability 
Definition 
The length of time an analyte can be stored at a specific condition without a 
significant change in value and the temperature a specimen with the analyte can be 
stored at a specific condition without a significant change in value. This is an 
important quality indicator for sample acceptability and result interpretation after 
specimen storage.   
Experiment 
Overview 
Four temperature conditions will be tested (room temperature, refrigerated, frozen 
and freeze/thaw cycles) over a designated period of time on at least three (3) 
calprotectin  levels, spanning the device range of 14-300 mg/L. Each specimen will 
be tested in triplicate for each condition and at baseline and calprotectin will be 
measured with the Lyfstone Rapid Test.  
Experiment 
Details 
1. Create at least three samples with concentrations in the Low, Mid and High 
ranges of the calprotectin test.  These samples will be prepared using one or 
more samples with high calprotectin concentrations.  The high sample(s) will be 
leftover from de-identified patient(s) enrolled in his study after the test is 

 
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performed per the study protocol.  The high sample (s) will be diluted as 
needed to reach the target concentrations using never frozen leftover synovial 
fluid from the clinical laboratory as the diluent.  The use of leftover de-identified 
synovial fluids was approved by the section head of the laboratory section. Up 
to 40 mLs synovial fluid will be used.  
2. The following conditions will be tested for each sample in triplicate:  
a. Time Zero (T0), baseline result 
b. Room Temperature (RT), room temperature near 25°C, tested at two 
(2), four (4) and eight (8) hour intervals 
c. Refrigerated (REF) (2-8°C), tested at ten (10) and twenty-four (24) hour 
intervals 
d. Frozen (F) (-90 to -70°C) with two (2) freeze thaw cycles. Freeze thaw 
cycle defined as: Samples will be completely frozen then removed to 
RT and completely thawed. Upon sample being thawed, one set will be 
tested (1X) and the other sample will be placed back in the freezer to 
completely freeze. Sample will then be removed from freezer to RT to 
completely thaw and be tested for analyte (2X) 
3. Assay specimens for Calprotectin concentrations, obtain both qualitative and 
quantitative results, in triplicate following the IFU instructions. 
 
Notes:  All raw data must be saved. Record performance time of assay, stability 
condition of specimen, and qualitative and quantitative values documented.  
Residual synovial fluid specimens will be used as matrix for this experiment to dilute 
a known high value of calprotectin from already tested knee synovial fluid samples 
as described above. The standard calprotectin (64.25 mg/L CALPRO AS, Norway) 
may also be spiked as needed to obtain the target concentrations. 
 
Data 
 
Qualitative agreement: After storage, the qualitative result is in agreement 
with the baseline (T0) result for all samples.  
 
Quantitative agreement: Samples will be analyzed for % bias from baseline 
(T0) using the following equation 
 
%𝐵𝑖𝑎𝑠 
 
= (𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛 (𝑋𝑋) 𝑟𝑒𝑠𝑢𝑙𝑡−𝑇0 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛)
𝑇0 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛
𝑥100 
 

 
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X. Data Collection 
 
Data will be collected preoperatively, and during surgery or the hospital stay.  All data will be 
entered and maintained in REDCap, an electronic data capture tools hosted at Cleveland 
Clinic[20]. REDCap (Research Electronic Data Capture) is a secure, web-based application 
designed to support data capture for research studies, providing 1) an intuitive interface for 
validated data entry; 2) audit trails for tracking data manipulation and export procedures; 3) 
automated export procedures for seamless data downloads to common statistical packages; and 4) 
procedures for importing data from external sources.   
 
  
 
The following will be collected on all patients in the study: 
1. 
Demographics –MRN, Age, Gender, BMI, Race, Smoking status, Joint type, current anti-
inflammatory medications, history of inflammatory disease, ongoing antibiotic therapy, history of 
infectious disease 
2. 
Comorbidities – Charlson or Elixhauser cormorbidity index 
3. 
Surgical history- Previous joint replacement related surgeries, severe synovitis status 
4. 
Laboratory values- ESR/CRP, WBC, RBC, PMN%, LE(optional), Culture, Histology, 
hemolysis index, synovasure (alpha defensin) test when available,  
5. 
Imaging – Xrays and bone scans (if available) 
6. 
Calprotectin results (POC and ELISA result) 
 
 
Schedule of Events 
 
Evaluation 
Screening* 
Aspiration 
TKA 
Demographics 
X 
 
 
 
 
 
Samples are considered stable if the value is within ±30% bias; larger bias 
will be investigated to rule out experimental issues.  Due to the higher 
imprecision observed in previous experiments, this target may be revisited 
after obtaining the data.  
 
Analyze data using a statistical software. 
Notes 
This experiment represents the minimum verification necessary, and the medical 
director may require additional steps. An open conversation should be maintained 
during the entire verification. 
 

 
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Medical/Surgical history 
X 
 
 
Sample screening 
X 
X 
Introp data collection 
 
 
X 
Laboratory details 
Calprotectin ELISA and POC 
CRP and ESR 
Cell counts 
Cultures 
Histology 
Leukocyte esterase 
Hemolysis index  
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Radiographic details 
Xrays 
Bone scan (when appropriate) 
 
X 
X 
 
 
* from timepoint closest to surgery 
 
XI. Analysis 
 
 
A. 
Statistical analysis 
 
Descriptive statistics will be generated for demographics and medical history. Categorical 
variables will be summarized by frequency counts and percentages. Continuous variables will be 
summarized by number of observations (N), mean, standard deviation (SD), median, minimum 
and maximum, and 95% confidence intervals (CIs). 
 
For testing of diagnostic utility, MSIS criteria will be used at the gold-standard for PJI. The 
following diagnostic parameters will be evaluated: sensitivity, specificity, positive predictive 
value, negative predictive value and accuracy. Additionally, receiver operating characteristics 
curves will be plotted. Calprotectin test will be considered to be positive when there is at least 
moderate or massive inflammation (i.e. yellow or red). The diagnostic parameters will also be 
evaluated considering only massive inflammation (red) as positive. 
 
The Calprotectin test will be validated by comparison to the CALPRO Calprotectin ELISA test 
using established diagnostic values from Lyfstone.  
 
Subgroup analysis will be performed based on the infecting organism type, joint, and presence of 
inflammatory conditions. 
 
 

 
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XII. 
Data Safety 
The sample obtained from the patient will be stored at the testing site at -80°. At the end of 
the study, the samples will be transported to the site of principal investigator. Only members 
of the study team (i.e. the personnel listed on the IRB application) will have access to 
protected health information of patients included in this study. All data procurement in this 
research project will utilize the standard of care follow-up visits to collect study data. Only 
secure computers will be used on main campus, all will be password protected. Only 
investigators approved by the IRB will be allowed access to this data. The data will be 
maintained in the patient’s medical records, unaltered from its form prior to the study. 
 
XIII. 
Co-enrollment 
Patients from IRB 16-549 may be co-enrolled in this trial. As this trial is observational no special 
consideration need to be made for recruitment and enrollment. Patients will be identified 
independently using a search of scheduled cases on EPIC by the clinical research fellow who is 
running each individual study. Patients who are eligible for both studies will then be contacted by 
phone several days before surgery to go over the consent form and explain the trial for each study 
independently. Patients will also be sent a copy of the consent form for the given trial for their 
review via mail or email, based on their preference. Patients will then formally consented with all 
signatures necessary obtained on the day of surgery for each study independently using a consent 
form for each study.  
 
 
 
XIV. 
References 
 
 
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