The Cytovale IntelliSep test is a semi-quantitative test that assesses cellular host response via deformability cytometry of leukocyte biophysical properties and is intended for use in conjunction with clinical assessments and laboratory findings to aid in the early detection of sepsis with organ dysfunction manifesting within the first 3 days after testing. It is indicated for use in adult patients with signs and symptoms of infection who present to the Emergency Department. The test is performed on an EDTA anticoagulated whole blood sample.

The IntelliSep test generates an IntelliSep Index value that falls within one of three discrete interpretation bands based on the probability of sepsis with organ dysfunction manifesting within the first three days after testing. The IntelliSep test represents the probability of the clinical syndrome of sepsis and is intended to be used alongside other clinical information and clinical judgement. It does not identify the causative agent of infection and should not be used as the sole basis to determine the presence of sepsis. The IntelliSep test is intended for in vitro diagnostic use.

Device Description

The Cytovale IntelliSep test is a short turn-around time (STAT) test, producing results in 10 minutes or less, to aid in the early identification of patients at risk for having or developing sepsis within three (3) days of testing. It assesses the state of immune activation in patients with clinical suspicion of infection who present in the Emergency Department (ED).

The IntelliSep test is run on the Cytovale System, a laboratory benchtop analyzer depicted in Figure 1. The Cytovale System is a closed system benchtop analyzer, comprised of three modules: Sample Preparation Module, Cell Imaging Module, and Imaging Analysis Module.

To run a test, the laboratory operator transfers 100 µL of whole blood into the sample preparation tube which is then placed into the Cytovale System. The system automatically lyses red blood cells, and washes the purified leukocytes in a diluent, producing a total volume of approximately 1mL of prepared sample, which the operator then transfers to the IntelliSep cartridge for analysis on the Cytovale System.

A microfluidic deformability cytometry technique is used to measure the biophysical properties of thousands of individual leukocytes in rapid succession. These properties have been shown to differ in quiescent white blood cell populations when compared to those in septic patients, enabling for rapid assessment of the host response and the likelihood of having or developing sepsis. Based on these measurements, the test provides a single score, the IntelliSep Index (ISI), ranging from 0.1-10.0, stratified into three discrete interpretation bands (Band 1, Band 2, Band 3) of sepsis likelihood.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the Cytovale IntelliSep test, based on the provided document:

Acceptance Criteria and Device Performance

The document does not explicitly state pre-defined quantitative acceptance criteria in a pass/fail format for clinical performance endpoints (e.g., a specific PPV or likelihood ratio threshold). Instead, it focuses on demonstrating a clear relationship between the IntelliSep Index (ISI) and the likelihood of sepsis, and the statistical distinctness of the interpretation bands. For analytical performance, criteria are generally established for standard deviations within precision studies and confidence intervals for reproducibility and carry-over.

Here's a table summarizing the reported device performance, categorized by the relevant studies:

Clinical Performance

Implicit Acceptance Criterion: A clear relationship between ISI and the increasing likelihood of sepsis, and statistically distinct interpretation bands (non-overlapping 80% confidence intervals between bands).

Performance Metric	IntelliSep Interpretation Band 1 (Lower Likelihood)	IntelliSep Interpretation Band 2 (Moderate Likelihood)	IntelliSep Interpretation Band 3 (Higher Likelihood)
Sepsis Predictive Value (Sepsis-3, Forced Adjudication)	11.1% (8.6%, 14.1% CI)	28.1% (23.5%, 33.2% CI)	49.4% (44.0%, 54.7% CI)
Sepsis Likelihood Ratio (Sepsis-3, Forced Adjudication)	0.35	1.08	2.69
Sepsis Predictive Value (Sepsis-2, Forced Adjudication)	28.0% (24.0%, 32.4% CI)	59.5% (53.9%, 64.9% CI)	72.9% (67.8%, 77.6% CI)
Sepsis Likelihood Ratio (Sepsis-2, Forced Adjudication)	0.38	1.44	2.64
Sepsis Predictive Value (Severe Sepsis-2, Forced Adjudication)	15.9% (12.7%, 19.6% CI)	43.9% (38.4%, 49.5% CI)	62.6% (57.2%, 67.7% CI)
Sepsis Likelihood Ratio (Severe Sepsis-2, Forced Adjudication)	0.31	1.28	2.74

Note: The clinical study explicitly states: "It is important to note that the algorithm for calculating the ISI ranges for the interpretation bands were defined based on prior sepsis-focused studies. No information from patients in this study influenced the calculations or interpretation bands of the ISI." and "Irrespective of the comparator scheme chosen, the probability of sepsis in the three ISI IntelliSep test Interpretation Bands was statistically distinct, defined as non-overlapping 80% confidence intervals between the bands." This suggests the demonstration of statistically distinct bands with appropriate clinical trends was the primary clinical performance criterion.

Analytical Performance

Performance Metric	Stated Acceptance Criteria	Reported Performance
Within-Laboratory Precision (Total, across all samples)	(Implicitly, standard deviations demonstrating acceptable variability)	Standard Deviation: 0.48 for 144 tests across 3 sites (range from 0.26 to 0.96 for individual samples)
Reproducibility (Total)	(Implicitly, standard deviations demonstrating acceptable variability)	Standard Deviation: 0.57 (combining within-lab 0.48 and across sites 0.30)
Reagents Lot-to-Lot Reproducibility	The 90% CI Lower and Upper Bounds are within (-1.0, 1.0) units of ISI	QC Level 1: (-0.12, 0.24) - PassQC Level 2: (-0.21, 0.19) - Pass
Cartridge Lot-to-Lot Reproducibility	The 90% CI Lower and Upper Bounds are within (-1.0, 1.0) units of ISI	Donors: (-0.68, 0.26) - Pass
Interfering Substances	No impact on results observed (up to listed concentrations)	Bilirubin (Unconjugated/Conjugated), Triglycerides, Hemolysate, Hemoglobin: No impact observed on ISI.
Sample Carry-Over	No sample carry-over effect found	Total ISI Difference (LL-HL): 0.03Total ISI Difference (HH-LH): 0.02 (No effect found)
Onboard Stability of Reagents	Stable for up to 30 days	Stable for up to 30 days when stored onboard the Cytovale System.
Sample Stability (K2-EDTA)	May be used for up to 5 hours after blood draw	May be used for up to 5 hours after blood draw.

Study Details

Sample sizes used for the test set and the data provenance:
- Test Set Sample Size: 572 evaluable subjects were included in the primary analyses for clinical performance.
- Data Provenance: The study was a "blinded, prospective, observational, multicenter cohort study" conducted at five hospitals at four geographically dispersed sites in the United States. The subjects were "representative of the adult population (≥ 18 years of age) typical of those presenting to EDs across the United States."
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- Number of Experts: A multi-tiered adjudication process was used:
  - Initially, two independent adjudicators reviewed each case.
  - If there was disagreement, a third adjudicator was involved in a consensus meeting.
- Qualifications of Experts: Physicians with board certification in Medical or Surgical Critical Care (CC), Infectious Diseases (ID), Emergency Medicine (EM), or Internal Medicine (IM) or related fields, from different institutions. They were not participating as an Investigator in the Study.
Adjudication method (e.g. 2+1, 3+1, none) for the test set:
- The adjudication method was 2+1 (two independent adjudicators, with a third used for arbitration in case of disagreement).
- Outcomes were categorized as 'unanimous' (both initial adjudicators agreed), 'consensus' (third adjudicator facilitated agreement), or 'forced' (majority two out of three if consensus couldn't be reached). The performance data is presented for all three schemes (forced, consensus, unanimous).
If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- No, an MRMC comparative effectiveness study was not explicitly stated or performed. The study evaluated the IntelliSep test as an aid in conjunction with clinical assessments and laboratory findings, but it did not directly compare human reader performance with and without AI assistance (i.e., IntelliSep test results). The test provides a quantitative index and interpretation bands, not an image for interpretation by human readers.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- Yes, a standalone performance evaluation was done. The IntelliSep test generates an "IntelliSep Index" (ISI) automatically from the measured biophysical properties of leukocytes. The clinical performance section directly assesses the predictive values and likelihood ratios of this automatically generated ISI, independent of real-time human interpretation during the study (treating physicians were unaware of study enrollment and IntelliSep test results). The test is intended for use in conjunction with clinical assessments, but its performance metrics are derived from the algorithm's output itself.
The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
- The ground truth was established through retrospective physician adjudication based on consensus definitions for Sepsis-2 and Sepsis-3. This involved a multi-tiered process by qualified physicians:
  - Collection of detailed patient information (demographics, labs, vitals, medical history, hospital encounter, infectious disease, medications, discharge disposition, SOFA scores, clinical impression).
  - Compilation into a Case Report Summary (CRS).
  - Review by independent adjudicators with arbitration for discordant results.
- It's a form of expert consensus based on clinical data.
The sample size for the training set:
- The document states: "It is important to note that the algorithm for calculating the ISI ranges for the interpretation bands were defined based on prior sepsis-focused studies. No information from patients in this study influenced the calculations or interpretation bands of the ISI."
- This implies the training of the algorithm and definition of interpretation bands occurred prior to and separate from this specific clinical validation study. The sample size for the training set is not provided in this document.
How the ground truth for the training set was established:
- Similar to the point above, the document only mentions that the ISI algorithm and interpretation bands were "defined based on prior sepsis-focused studies."
- The method for establishing ground truth for those prior training studies is not detailed in this document.

Summary

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December 20, 2022

Cytovale Inc. Juliet Carrara Exec. Vice President of Regulatory & Clinical Affairs and Quality Assurance 150 Executive Park Blvd, Suite 4100 San Francisco, California 94134

Re: K220991

Trade/Device Name: IntelliSep test Regulation Number: 21 CFR 866.3215 Regulation Name: Device To Detect And Measure Non-Microbial Analyte(S) In Human Clinical Specimens To Aid In Assessment Of Patients With Suspected Sepsis Regulatory Class: Class II Product Code: OUT Dated: March 31, 2022 Received: April 4, 2022

Dear Juliet Carrara:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

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statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

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Noel Gerald Branch Chief Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K220991

Device Name IntelliSep test

Indications for Use (Describe)

The IntelliSep test generates an IntelliSep Index value that falls within one of three discrete interpretation bands based on the probability of sepsis with organ dysfunction manifesting within the first three days after test represents the probability of the clinical syndrome of sepsis and is intended to be used alongside other clinical information and clinical judgement. It does not identify the causative agent of infection and should not be used as to determine the presence of sepsis. The IntelliSep test is intended for in vitro diagnostic use.

Type of Use (Select one or both, as applicable)
-------------------------------------------------

Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

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Name:	Cytovale, Inc.
Address:	150 Executive Park Blvd, Suite 4100, San Francisco, CA 94134
Telephone:	1-415-417-2188
Contact Person:	Juliet Carrara
Email Address:	juliet.carrara@cytovale.com

1 510(k) Owner / Submitter Information Name

2 Date Summary was Prepared December 19, 2022

3 Device Name and Classification

Trade Name:	IntelliSep test
Instrument Name:	Cytovale System
Classification:	Class II (Special Controls)
Classification Name:	Device to detect and measure non-microbial analyte(s) in human clinical
	specimens to aid in assessment of patients with suspected sepsis (21 CFR
	866.3215)
Product Code:	QUT
Panel:	83 - Division of Microbiology Devices

4 Predicate Device Information

Predicate Product	510(k)Number	Date ofClearance	Classification	Regulation	ClassificationProduct Code
VIDAS B·R·A·H·M·SPCT (PCT)	K162827	February 23, 2017	Class II(Special Controls)	866.3215	PRI

Table 1: Predicate device information

5 Device Description

The IntelliSep test is run on the Cytovale System, a laboratory benchtop analyzer depicted in Figure

Image /page/3/Figure/13 description: The image shows a Cytovale system, which includes several modules. There is a Sample Preparation Module (SPM), a Cell Imaging Module (CIM), and an Imaging Analysis Module (IAM). The image also shows Cytovale reagents, as well as a printer and barcode scanner.

Figure 1: The Cytorale System is a closed system benchtop andyzer, comprised of three modules: Sample Preparation Module, Cell Imaging Module, and Imaging Analysis Moduled are the Cytorale System Reagent (Cytovale Reagent Kit, Cytovale Diluent, Cytovale Cleanse), Barcode Scanner and Printer (not shown: IntelliSep Cartridge, Sample Preparation Tube),

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The IntelliSep test requires use of the materials described in Table 2.

IntelliSep test Materials & Equipment	Description
Cytovale System:(Required and supplied)• Sample Preparation Module (SPM)• Cell Imaging Module (CIM)• Imaging Analysis Module (IAM)	A closed system benchtop analyzer as shown in Figure 1. Itis comprised of three modules: Sample Preparation Module,Cell Imaging Module and Imaging Analysis Module.
Barcode scanner(Optional and supplied)	Scanner used to automatically enter sample and materialidentifiers.
Printer(Optional and supplied)	Printer used to print a hard copy of results.
Sample Preparation Tube(Required and supplied)	A single-use, commercial off-the-shelf test tube used forsample preparation.
IntelliSep Cartridge(Required and supplied)	Closed system test cartridge which presents the sample fortesting in the Cell Imaging Module.
Cytovale Reagent Kit(Required and supplied)	Nonbiological salt solutions (Reagent A Lysis and ReagentB Quench), for use with the Sample Preparation Module tolyse red blood cells and quench the lysis reaction.
Cytovale Diluent and Cleanse Reagents(Required and supplied)	The Diluent solution is used in the Sample PreparationModule to suspend white blood cells for analysis. TheCleanse is a rinsing solution for the SPM.
IntelliSep Quality Control Kit(Quality controls are required but notsupplied)	A two-level Quality Control set, derived from stabilizedwhole blood.Note: Per 42 CFR 493.1256, quality controls are required for theIntelliSep test; however, at their discretion, laboratory directorsmay develop their own quality control materials or order theIntelliSep Quality Control Kit.

Table 2: Materials and equipment required for the IntelliSep test

6 Intended Use

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dysfunction manifesting within the first 3 days after testing. It is indicated for use in adult patients with signs and symptoms of infection who present to the Emergency Department. The test is performed on an EDTA anticoagulated whole blood sample.

7 Indication for Use

See "Intended Use"

8 Overall Comparison Between Subiect Device and Predicate

The indications for use for both the subject and predicate devices are comparable, as they are both intended for use in conjunction with other laboratory findings and clinical assessments to aid in the risk assessment of sepsis in acute patient populations. The indications for use for the subject and predicate devices differ in the type of analytical method used to detect their specific intended assay markers and in the specific subpopulations of acute patients being assessed (e.g., Emergency Dept vs ICU).

Identification and analysis of non-microbial host immune response analytes is the scientific foundation for both the subject and predicate devices. For the predicate device, quantification of procalcitonin is performed to assess the inflammatory response to bacterial infection. The subject device instead quantifies changes in cellular biophysical properties of leukocytes (immune cells) that occur as part of the inflammatory response to infection. While the intended uses of the predicate and subject device are similar, the predicate device measures a released biomarker and the subject device directly evaluates cellular morphology.

Both the predicate and the subject device aid in the identification of sepsis in conjunction with other laboratory findings and clinical assessments. None of the differences constitute a new intended use for the subject device.

	Candidate Device	Predicate Device
Item	Cytovale System and IntelliSep test	VIDAS B·R·A·H·M·S PCT(K162827)
Intended Use /Indications forUse	The Cytovale IntelliSep test is a semi-quantitative test that assesses cellular host response via deformability cytometry of leukocyte biophysical properties and is intended for use in conjunction with clinical assessments and laboratory findings to aid in the early detection of sepsis with organ dysfunction manifesting within the first 3 days after testing. It is indicated for use in adult patients with signs and symptoms of infection	VIDAS B·R·A·H·M·S PCT is an automated test for use on the instruments of the VIDAS family for the determination of human procalcitonin in human serum or plasma (lithium heparinate) using the ELFA (Enzyme-Linked Fluorescent Assay) technique.Used in conjunction with other laboratory findings and clinical assessments, VIDAS
Item	Candidate Device	Predicate Device
	Cytovale System and IntelliSep testwho present to the Emergency Department.The test is performed on an EDTAanticoagulated whole blood sample.The IntelliSep test generates an IntelliSepIndex value that falls within one of threediscrete interpretation bands based on theprobability of sepsis with organ dysfunctionmanifesting within the first three days aftertesting. The IntelliSep test represents theprobability of the clinical syndrome of sepsisand is intended to be used alongside otherclinical information and clinical judgement. Itdoes not identify the causative agent ofinfection and should not be used as the solebasis to determine the presence of sepsis. TheIntelliSep test is intended for in vitrodiagnostic use.	VIDAS B·R·A·H·M·S PCT(K162827)B·R·A·H·M·S PCT is intended for use asfollows:• to aid in the risk assessment ofcritically ill patients on their first dayof ICU admission for progression tosevere sepsis and septic shock, to aidin assessing the cumulative 28-dayrisk of all-cause mortality for patientsdiagnosed with severe sepsis or septicshock in the ICU or when obtained inthe emergency department or othermedical wards prior to ICUadmission, using a change in PCTlevel over time,• to aid in decision making onantibiotic therapy for patients withsuspected or confirmed lowerrespiratory tract infections (LRTI)defined as community-acquiredpneumonia (CAP), acute bronchitis,and acute exacerbation of chronicobstructive pulmonary disease(AECOPD) – in an inpatient settingor an emergency department,• to aid in decision making onantibiotic discontinuation for patientswith suspected or confirmed sepsis.
Site of Use	To be performed by trained laboratorypersonnel in a clinical or hospital laboratorysetting	To be performed by a trained operator in aprofessional setting, such as a hospital centrallaboratory
Specimen Type	Human venous whole blood (K2 EDTA)	Human serum, plasma (lithium heparinate)
SpecimenStability	Five (5) hours after draw at ambientconditions	The sera or plasma separated from the clot canbe stored at 2-8°C in stoppered tubes for up to48 hours; if longer storage is required, freezeat -25 ± 6°C. Six-month storage of frozensamples does not affect the quality of results.Three freeze/thaw cycles were validated
SpecimenProcessing	1. Automated closed system (SamplePreparation Module) which only operates withproprietary reagents.2. The operator transfers the processed sampleinto the IntelliSep Cartridge which is insertedinto the Cell Imaging Module for analysis.	Instruments of the VIDAS family: VIDAS,miniVIDAS or VIDAS 3, using conjugate andsolid phase antibodies and custom reagents
Analyte(s)	Leukocyte biophysical properties	Procalcitonin (PCT)
Item	Candidate Device	Predicate Device
AssayPrinciple/Method	Microfluidic deformability cytometry	Quantitative immunofluorescent assay, basedon Sandwich immunoassay principles, whichmeasures a specific fluorescence signature(fluorescence (ELFA) of 4-methyl-umbelliferyl measured at 450 nm) that isproportional to the antigen (PCT)concentration.
Controls	IntelliSep Quality Control Kit including twolevels of controls derived from stabilizedwhole blood:• Level 1 Control• Level 2 Control	Two levels of antigen concentration. Each vialcontains lyophilized recombinant PCT inTRIS NaCl buffer (pH 7.3) and preservatives.
InstrumentPlatform	Cytovale System	Instruments of the VIDAS family: VIDAS,miniVIDAS or VIDAS 3
Result Output	The IntelliSep Index (ISI, range 0.1 to 10.0)that falls within one of three discreteinterpretation bands (Band 1, Band 2, Band 3)based on likelihood of sepsis within three (3)days of testing	Calculated estimate of concentration ofcirculating PCT, in units of ng/mL
Reagent Stability	1. In original shipping containers unopened atambient temperature (15-30°C): up to thestated expiration date;2. After opening, onboard at ambienttemperature (15-30°C): 30 days	1. In original shipping containers unopened at2-8°C: up to the stated expiration date (12months);2. After opening, onboard at 2-8 °C: 29 days
Limitations	• Using a different Sample PreparationTube than the one provided may lead toerroneous results.• Smeared blood on the side of the SamplePreparation Tube may cause the test tofail. Remove blood smears from the sidesof the sample preparation tube using thespecified lint-free foam swab. Using adifferent swab other than the one requiredmay lead to no result returned.• $100 \mu L \pm 5 \mu L$ of specimen volume isneeded for sample preparation. Anerroneous result may occur if the volumeof the sample transferred to the SamplePreparation Tube is different from therequired volume.• Use only EDTA anticoagulated wholeblood within 5 hours of collection.• Predictive values (estimated probabilitiesof sepsis) are dependent on prevalence ofdisease and likelihood ratios measured for	VIDAS B·R·A·H·M·S PCT (PCT) is notindicated to be used as a standalone diagnosticassay and should be used in conjunction withclinical signs and symptoms of infection andother diagnostic evidence.Decisions regarding antibiotic therapy shouldNOT be based solely on procalcitoninconcentrations.PCT results should always be interpreted inthe context of the clinical status of the patientand other laboratory results. Changes in PCTlevels for the prediction of mortality, andoverall mortality, are strongly dependent onmany factors, including pre-existing patientrisk factors and clinical course.The need to continue ICU care at Day 4 andother covariates (e.g., age and SOFA score)
	Candidate Device	Predicate Device
Item	Cytovale System and IntelliSep test	VIDAS B·R·A·H·M·S PCT(K162827)
	the clinical trial population as reported inthe Clinical Performance Summary. Usersshould establish or verify that theseparameter values are appropriate for thepatient population being tested.The clinical performance has not beenestablished in the following populations:Patients below 18 years of age. Patients with a history of a hematologicmalignancy (any leukemia, lymphoma, ormyeloma), myelodysplastic syndrome, ormyeloproliferative disorder Patients who have undergone ahematopoietic stem cell transplant or anysolid organ transplant Patients receiving a cytotoxicchemotherapeutic agent in the past 3months Patients who are residents or patients of ahospital-based skilled nursing facility Patients who received systemiccorticosteroids were not excluded fromthe clinical study. However, the study wasnot powered to evaluate the performanceof the ISI specifically in this populationand as such clinical performance has notbeen established in this population. Patients with pre-existing end stage renaldisease (ESRD) who undergohemodialysis were not excluded from theclinical study. However, the study was notpowered to evaluate the performance ofthe ISI specifically in this population andas such clinical performance has not beenestablished in this population. This test was not evaluated for sequentialmonitoring of patients, or for use inpatients past the initial ED encounter. The clinical study was not adequatelypowered to evaluate differences indemographics and subpopulations,therefore results should be interpreted inconjunction with clinical assessments andother laboratory findings.	are also significant predictors of 28-daycumulative mortality risk.Certain patient characteristics, such as severityof renal failure or insufficiency, may influenceprocalcitonin values and should be consideredas potentially confounding clinical factorswhen interpreting PCT values.Increased PCT levels may be observed insevere illness such as polytrauma, burns,major surgery, prolonged or cardiogenicshock.PCT levels may not be elevated in patientsinfected by certain atypical pathogens, such asChlamydophila pneumoniae and Mycoplasmapneumoniae.The safety and performance of PCT-guidedtherapy for individuals younger than age 17years, pregnant women, immunocompromisedindividuals or those on immunomodulatoryagents, was not formally analyzed in thesupportive clinical trials.

Information related to this comparison is tabulated in Table 3.

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Table 3: Comparison between subject device and predicate device

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9 Performance Testing

Clinical Performance Summary 9.1

Cytovale conducted a blinded, prospective, observational, multicenter cohort study at five hospitals at four geographically dispersed sites comprised of both academic and community hospital Emergency Departments (EDs) in the United States. The study cohort was representative of the adult population (≥ 18 years of age) typical of those presenting to EDs across the United States with signs or suspicion of infection (defined as meeting two or more Systemic Inflammatory Response Syndrome (SIRS) criteria where one must be aberration of temperature or white blood cell count OR an order placed for cultures of blood, sputum, urine, or sterile body fluids).

599 subjects were enrolled in the study and 572 evaluable subjects were included in the primary analyses. As shown in Table 4, subjects spanned across ranges of age (18 to 103 years of age; 33% of subjects ≥ 65), race (62% white, 30% Black or African American, 2% Asian, and 6% other), ethnicity (6% Hispanic), and sex (44% female). They encompassed those with common comorbidities, including but not limited to diabetes, hypertension, chronic kidney disease, cancer, and obesity. Infection sources affecting or originating from all major organ systems (i.e., respiratory, genitourinary, skin, gastrointestinal, cardiovascular system, central nervous system, and bone) were represented in the study. Additionally, because this study was conducted during the COVID-19 pandemic, 9% of the subjects enrolled in the study were SARS-CoV-2 positive.

The IntelliSep test was performed on an EDTA anticoagulated whole blood sample obtained within 4 hours of the first recorded vital sign (triage). Subsequently, subjects were followed with retrospective chart review for outcome information. Subjects received standard of care and treating physicians were unaware of study enrollment and IntelliSep test results.

Population Characteristic		Total	Sepsis Status(Sepsis-3 Forced)
		(N = 572)	Positive(N = 152)	Negative(N = 420)
	Median (Q1 - Q3)	56.0 (40.0 - 68.0)	63.0 (46.0 - 73.0)	53.00 (37.0 - 66.0)
Age	Subjects ≥ 65N (%)	187 (32.69%)	69 (45.39%)	118 (28.10%)
Sex	Female	250 (43.71%)	57 (37.50%)	193 (45.95%)
N (%)	Male	322 (56.29%)	95 (62.50%)	227 (54.05%)
Ethnicity	Hispanic	35 (6.12%)	5 (3.29%)	30 (7.14%)
N (%)	Non-Hispanic	527 (92.13%)	145 (95.39%)	382 (90.95%)
	Not Provided	10 (1.75%)	2 (1.32%)	8 (1.90%)
	American Indian orAlaska Native	8 (1.40%)	1 (0.66%)	7 (1.67%)
	Asian	10 (1.75%)	0 (0.00%)	10 (2.38%)
RaceN (%)	Black or AfricanAmerican	172 (30.07%)	42 (27.63%)	130 (30.95%)
	Native Hawaiian or OtherPacific Islander	6 (1.05%)	2 (1.32%)	4 (0.95%)
	White	356 (62.24%)	101 (66.45%)	255 (60.71%)
	Other	20 (3.50%)	6 (3.95%)	14 (3.33%)
Source of	Bone / Joint	19 (6.64%)	11 (7.24%)	8 (5.97%)
infection in	Cardiovascular System	20 (6.99%)	13 (8.55%)	7 (5.22%)
Subjects	Central Nervous System	13 (4.55%)	12 (7.89%)	1 (0.75%)
Adjudicated asInfected, multiple	Gastrointestinal /Abdominal	32 (11.19%)	17 (11.18%)	15 (11.19%)

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Population Characteristic		Total(N = 572)	Sepsis Status(Sepsis-3 Forced)
			Positive(N = 152)	Negative(N = 420)
sources includedper subject(N, % of infected)	Respiratory System	125 (43.71%)	81 (53.29%)	44 (32.84%)
	Skin	57 (19.93%)	21 (13.82%)	36 (26.87%)
	Urine & Urine System /Genitourinary	76 (26.57%)	49 (32.24%)	27 (20.15%)
	Other or Unknown	24 (8.39%)	11 (7.24%)	13 (9.70%)
SARS-CoV-2TestingN (%)	Number Tested	391 (68.36%)	128 (84.21%)	263 (62.62%)
	Number Positive (ofTested)	54 (13.81%)	37 (28.91%)	17 (6.46%)
	Number Positive (ofTotal)	54 (9.44%)	37 (24.34%)	17 (4.05%)

Table 4: Study population characteristics per sepsis-3 consensus definition in the forced adjudication scheme)

The performance of the IntelliSep test was evaluated by comparison to non-reference retrospective physician adjudication. As detailed below, all subjects were adjudicated by two qualified physicians following the consensus definitions for Sepsis-2 and Sepsis-3. Discordant results were arbitrated through a committee meeting that included a third physician. The adjudication process determined each case to be 'Sepsis' with 'unanimous', 'consensus' or 'forced' determinations for both Sepsis-3 consensus definitions. No cases were excluded due to inability to arbitrate. All study personnel, including study adjudicators. were blinded to the IntelliSep test results.

A multi-tiered adjudication process was utilized to standardize and reduce the inherent subjectivity and variability of using a non-reference comparator. Approximately 30 days after the subject was enrolled, a chart review, using a structured case report form (CRF), was performed. All sites were required to extract the same information to enable adjudication based on the case report form. The information captured included demographics, laboratory results, vital signs, past medical history, hospital encounter information, infectious disease information, medications, and discharge disposition. Additionally, objective evidence of infection (present at the time of ED presentation) and organ dysfunction (manifesting within 3 days following ED presentation) using the Sequential (sepsis-related) Organ Failure Assessment (SOFA) score was captured. Furthermore, a clinical impression was prepared by designated study-trained physicians that were not involved in the subject's care.

Information obtained from the Demographic and Clinical Case Report Forms, Objective Evaluation, and the clinical impression was then compiled into a case report summary (CRS) and sent to two independent adjudicators. If the two adjudicators agreed in their determination, the case was considered 'unanimous' adjudication. If there was disagreement, the CRS was sent to a third adjudicator and a consensus meeting was held. If all could agree, the case fell under 'consensus' or if following discussion consensus could not be reached, a 'forced' (majority two out of three) adjudication determination was reached. The study independent adjudication committee comprised of physicians with board certification in Medical or Surgical Critical Care (CC), Infectious Diseases (ID), Emergency Medicine (EM) or Internal Medicine (IM) or related fields, from different institutions who were not participating as an Investigator in the Study. Individual cases were assigned to the adjudicators by a third-party vendor, without involvement from Cytovale. Adjudicators were blinded to the identity of the Site Investigators and Medical Monitors.

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The prevalence of sepsis, using the forced Sepsis-3 consensus definition adjudication scheme, was 26.6%. Results across all approaches to analyzing retrospective physician adjudication results were consistent, i.e., a clear relationship was observed between IntelliSep Index (IST) and the increasing likelihood of having or developing sepsis within 3 days across (Figure 2). Irrespective of the comparator scheme chosen, the probability of sepsis in the three ISI IntelliSep test Interpretation Bands was statistically distinct, defined as non-overlapping 80% confidence intervals between the bands (Table 5 through Table 7). It is important to note that the algorithm for calculating the ISI ranges for the interpretation bands were defined based on prior sepsisfocused studies. No information from patients in this study influenced the calculations or interpretation bands of the ISI.

Image /page/11/Figure/3 description: The image contains nine scatter plots arranged in a 3x3 grid, each displaying the "Sepsis Predictive Value" on the y-axis against three "Bands" on the x-axis. The plots are grouped into three rows labeled (A), (B), and (C), representing different Sepsis conditions: "Sepsis-3", "Sepsis-2 (Sepsis)", and "Sepsis-2 (Severe Sepsis)", respectively. Within each row, the plots illustrate different adjudication schemes: "Forced Adjudication Scheme", "Consensus Adjudication Scheme", and "Unanimous Adjudication Scheme". Each data point on the scatter plots represents the sepsis predictive value for a specific band, with error bars indicating the variability or uncertainty associated with each value.

Figure 2: Plots of sepsis predictive value in each band across all approaches to analyzing retrospective physician adjudication results: (A) Sepsis-3 consensus definition, (B) Sepsis 2 consensus definition, (B) Severe Sepsis-2 consensus definition, (C) Severe Sepsis-2 consensus definition, for forced, consensus, and unanimous adjudication schemes.

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Sepsis-3 consensus definition, forced adjudication scheme (n=572)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value(80% CI)	Sepsis LikelihoodRatio
Band 1	28	224	11.1% (8.6%, 14.1%)	0.35
Band 2	45	115	28.1% (23.5%, 33.2%)	1.08
Band 3	79	81	49.4% (44.0%, 54.7%)	2.69
			Sepsis Prevalence
Total	152	420	26.6%	NA
Sepsis-3 consensus definition, consensus adjudication scheme (n=570)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value(80% CI)	Sepsis LikelihoodRatio
Band 1	28	222	11.2% (8.7%, 14.2%)	0.35
Band 2	45	115	28.1% (23.5%, 33.2%)	1.08
Band 3	79	81	49.4% (44.0%, 54.7%)	2.68
			Sepsis Prevalence
Total	152	418	26.7%	NA
Sepsis-3 consensus definition, unanimous adjudication scheme (n=450)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value(80% CI)	Sepsis LikelihoodRatio
Band 1	20	186	9.7% (7.1%, 12.9%)	0.31
Band 2	34	86	28.3% (23.0%, 34.3%)	1.14
Band 3	62	62	50.0% (43.9%, 56.1%)	2.88
			Sepsis Prevalence
Total	116	334	25.8%	NA

Table 5: Distribution of subjects, sepsis prevalence, and sepsis likelihood ratios in the three ISI IntelliSep test Interpretation Bands for Sepsis-3 consensus definition for forced, consensus, and unanimous adjudication schemes.

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Sepsis-2 (sepsis) consensus definition, forced adjudication scheme (n=517)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value(80% CI)	Sepsis LikelihoodRatio
Band 1	60	154	28.0% (24.0%, 32.4%)	0.38
Band 2	88	60	59.5% (53.9%, 64.9%)	1.44
Band 3	113	42	72.9% (67.8%, 77.6%)	2.64
			Sepsis Prevalence
Total	261	256	50.5%	NA
		Sepsis-2 (sepsis) consensus definition, consensus adjudication scheme (n=516)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value(80% CI)	Sepsis LikelihoodRatio
Band 1	60	153	28.2% (24.2%, 32.5%)	0.38
Band 2	88	60	59.5% (53.9%, 64.9%)	1.43
Band 3	113	42	72.9% (67.8%, 77.6%)	2.63
			Sepsis Prevalence
Total	261	255	50.6%	NA
	Sepsis-2 (sepsis) consensus definition, unanimous adjudication scheme (n=411)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive ValuePrevalence (80% CI)	Sepsis LikelihoodRatio
Band 1	37	143	20.6% (16.7%, 24.9%)	0.34
Band 2	58	54	51.8% (45.3%, 58.2%)	1.41
Band 3	83	36	69.8% (63.7%, 75.3%)	3.02
			Sepsis Prevalence
Total	178	233	43.3%	NA

Table 6: Distribution of subjects, sepsis prevalence, and sepsis likelihood ratios in the three ISI IntelliSep test Interpretation Bands for sepsis per Sepsis-2 consensus definition for forced, consensus, and unanimous adjudication schemes.

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Sepsis-2 (severe sepsis) consensus definition, forced adjudication scheme (n=517)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value (80% CI)	Sepsis Likelihood Ratio
Band 1	34	180	15.9% (12.7%, 19.6%)	0.31
Band 2	65	83	43.9% (38.4%, 49.5%)	1.28
Band 3	97	58	62.6% (57.2%, 67.7%)	2.74
			Sepsis Prevalence
Total	196	321	37.9%	NA
Sepsis-2 (severe sepsis) consensus definition, consensus adjudication scheme (n=516)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value (80% CI)	Sepsis Likelihood Ratio
Band 1	34	179	16.0% (12.8%, 19.7%)	0.31
Band 2	65	83	43.9% (38.4%, 49.5%)	1.28
Band 3	97	58	62.6% (57.2%, 67.7%)	2.73
			Sepsis Prevalence
Total	196	320	38.0%	NA
Sepsis-2 (severe sepsis) consensus definition, unanimous adjudication scheme (n=411)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value (80% CI)	Sepsis Likelihood Ratio
Band 1	20	160	11.1% (8.2%, 14.7%)	0.25
Band 2	43	69	38.4% (32.3%, 44.8%)	1.23
Band 3	75	44	63.0% (56.8%, 68.9%)	3.37
			Sepsis Prevalence
Total	138	273	33.6%	NA

Table 7: Distribution of subjects, sepsis prevalence, and sepsis likelihood ratios in the three ISI Interpreation Bands for severe sepsis per Sepsis-2 consensus definition for forced, consensus, and unanimous adjudication schemes.

The IntelliSep ISI risk stratification score trended upwards with the adjudication determination of sepsis independent of demographic groups (age, sex, race, and ethnicity; Table 8). Additionally, trends were observed between increasing ISI values across the Interpretation bands and hospital care metrics (e.g., hospital admission, ICU admission and transfer, hospital length of stay, and administration of antibiotics; Table 9). It is important to note, that the study was not designed nor powered to evaluate endpoints with regards to severity of illness or hospital care metrics (e.g., mortality, severity of illness scores, hospital admission and length

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of stay, etc.) and any significant trends or correlations observed between these metrics and the ISI and its Interpretation bands are only observational.

Additionally, subjects who have received systemic corticosteroids and subjects with preexisting end stage renal disease (ESRD) who undergo hemodialysis were not excluded from the clinical study, however, the study was not powered to evaluate the performance of the ISI specifically in these subpopulations.

Population Characteristic		IntelliSep test Results
		Band 1(N = 252)	Band 2(N = 160)	Band 3(N = 160)
Age	Median (Q1 – Q3)	54.0 (39.5 - 67.0)	56.0 (39.0 - 68.0)	58.0 (41.5 - 71.0)
	Subjects ≥ 65, N (%)	77 (30.56%)	54 (33.75%)	56 (35.00%)
Sex, N (%)	Female	106 (42.06%)	68 (42.50%)	76 (47.50%)
	Male	146 (57.94%)	92 (57.50%)	84 (52.50%)
Ethnicity, N (%)	Hispanic	20 (7.94%)	8 (5.00%)	7 (4.38%)
	Non-Hispanic	227 (90.08%)	148 (92.50%)	152 (95.00%)
	Not Provided	5 (1.98%)	4 (2.50%)	1 (0.62%)
Race (%)	American Indian or Alaska Native	2 (0.79%)	3 (1.88%)	3 (1.88%)
	Asian	7 (2.78%)	2 (1.25%)	1 (0.62%)
	Black or African American	76 (30.16%)	47 (29.38%)	49 (30.62%)
	Native Hawaiian or Other Pacific Islander	3 (1.19%)	0 (0.00%)	3 (1.88%)
	White	154 (61.11%)	104 (65.00%)	98 (61.25%)
	Other	10 (3.97%)	4 (2.50%)	6 (3.75%)

Table 8: Study population demographic characteristics per IntelliSep test interpretation bands.

Population Characteristic			IntelliSep test Results
		Total(N = 572)	Band 1(N = 252)	Band 2(N = 160)	Band 3(N = 160)
All-Cause	3-day	3 (0.52%)	1 (0.40%)	0 (0.00%)	2 (1.25%)
Cumulative In-Hospital MortalityN (%)	7-day	9 (1.57%)	3 (1.19%)	1 (0.62%)	5 (3.12%)
	30-day	24 (4.20%)	6 (2.38%)	7 (4.38%)	11 (6.88%)
Admitted to Hospital, N (%)		360 (62.94%)	123 (48.81%)	107 (66.88%)	130 (81.25%)
Admitted to ICU, N (%)		71 (12.41%)	20 (7.94%)	23 (14.38%)	28 (17.50%)
Transferred to ICU within 3 Days, N(%)		25 (4.37%)	1 (0.40%)	15 (9.38%)	9 (5.62%)
Length of stayMedian (Q1-Q3)	Alive at 30 days	3.0 (0.0 - 7.0)	2.0 (0.0 - 4.0)	4.0 (0.0 - 8.0)	5.0 (3.0 - 9.0)
	Deceased in-hospital within30 days	8.5 (6.5 - 14.5)	9.0 (5.5 - 12.0)	9.0 (8.5 - 12.5)	8.0 (5.0 - 16.5)
ICU Length ofstayMedian (Q1 - Q3)	Alive at 30 days	0.0 (0.0 - 0.0)	0.0 (0.0 - 0.0)	0.0 (0.0 - 0.0)	0.0 (0.0 - 0.0)
	Deceased in-hospital within30 days	6.5 (2.0 - 12.5)	5.5 (0.0 - 11.5)	8.0 (7.0 - 11.0)	5.0 (2.0 - 10.0)
AntibioticsPrescribed	Yes	301 (52.62%)	99 (39.29%)	81 (50.62%)	121 (75.62%)
	No	267 (46.68%)	152 (60.32%)	77 (48.12%)	38 (23.75%)
N (%)	Unknown	4 (0.70%)	1 (0.40%)	2 (1.25%)	1 (0.62%)

Table 9: Study population severity of illness and hospital course characteristics per IntelliSep test interpretation bands. Note: In the calculation of APACHE II scores, for whom a lab value was not collected per standard of care, the value was considered normal.

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9.2 Descriptive (Non-Powered) Analyses

In addition to examining study endpoints, descriptive non-powered analyses were performed to explore test performance by demographic subgroups. These non-powered descriptive analyses evaluated the association of IntelliSep Index (expressed in terms of its Interpretation Bands) with the predictive value of sepsis (an adjudicated non-reference method) using the Sepsis-3 consensus standard definition, for demographic subgroups split by age and sex.

The association was examined by the non-overlap of the 80% confidence intervals around point estimates for the probability of sepsis in Band 1 and Band 3 These descriptive analyses were performed using a forced adjudication scheme with evaluable subjects and are shown in Table 10 and Figure 3 (age, <65 and ≥65 years) and Table 11 and Figure 4 (sex, male and female).

Age < 65, Sepsis-3 consensus definition, forced adjudication scheme (n=385)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value (80% CI)
Band 1	17	158	9.7% (6.9%, 13.2%)
Band 2	25	81	23.6% (18.3%, 29.7%)
Band 3	41	63	39.4% (33.0%, 46.2%)
Age ≥ 65, Sepsis-3 consensus definition, forced adjudication scheme (n=187)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value (80% CI)
Band 1	11	66	14.3% (9.3%, 20.8%)
Band 2	20	34	37.0% (28.2%, 46.8%)
Band 3	38	18	67.9% (58.6%, 76.1%)

Table 10: Descriptive (Non-Powered) Analysis: Results for Sepsis-3 Forced by Age Group

Image /page/16/Figure/7 description: The image contains two plots comparing sepsis predictive values for different age groups using the Sepsis-3 Forced Adjudication Scheme. The left plot represents individuals strictly under 65 years of age, while the right plot represents individuals over 65 years of age. Both plots show sepsis predictive values for three bands, with corresponding sample sizes (n) indicated for each band. The sepsis predictive value appears to increase from Band 1 to Band 3 in both age groups, with the over 65 age group showing a higher predictive value in Band 3.

Figure 3:Plot of sepsis predictive value (% of subjects adjudicated as septic) in each band for Sepsis-3 Forced by Age Group

Females, Sepsis-3 consensus definition, forced adjudication scheme (n=250)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value (80% CI)
Band 1	9	97	8.5% (5.2%, 13.1%)
Band 2	19	49	27.9% (20.8%, 36.1%)
Band 3	29	47	38.2% (30.7%, 46.1%)
Males, Sepsis-3 consensus definition, forced adjudication scheme (n=322)
IntelliSep Result	Adjudicated +	Adjudicated -	Sepsis Predictive Value (80% CI)
Band 1	19	127	13.0% (9.5%, 17.3%)
Band 2	26	66	28.3% (22.1%, 35.1%)
Band 3	50	34	59.5% (52.0%, 66.7%)

Table 11: Descriptive (Non-Powered) Analysis: Results for Sepsis-3 Forced by Sex

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Image /page/17/Figure/2 description: The image contains two plots comparing the sepsis predictive value for females and males using the Sepsis-3 Forced Adjudication Scheme. The y-axis represents the sepsis predictive value, ranging from 0.0 to 1.0, while the x-axis represents three bands with different sample sizes for each gender. For females, the sepsis predictive values for Band 1 (n=106), Band 2 (n=68), and Band 3 (n=76) are approximately 0.1, 0.3, and 0.4, respectively. For males, the sepsis predictive values for Band 1 (n=146), Band 2 (n=92), and Band 3 (n=84) are approximately 0.15, 0.3, and 0.6, respectively.

Figure 4: Plot of sepsis predictive value (% of subjects adjudicated as septic) in each band for Sepsis-3 Forced by Sex

9.3 Analytical Performance

9.3.1 Analytical Sensitivity

Analytical sensitivity (limit of detection, limit of blank, and limit of quantitation) performance testing is not applicable to the IntelliSep test.

9.3.2 Precision and Repeatability / Reproducibility

9.3.2.1 Within-Laboratory Precision (Repeatability)

The within-laboratory precision for the IntelliSep test was measured according to CLSI EP05-A3. Testing was conducted at 3 sites. At each site, 4 donor samples covering the range of ISI were tested. For each sample, 2 operators performed runs on 2 instruments in triplicates, for a total of 144 tests.

Number of tests	Components of within-laboratory precision			Total Within-laboratory Precision
	Repeatability(within run)	Between Operators	Between Instruments
	Standard Deviation	Standard Deviation	Standard Deviation	Standard Deviation
144	0.44	0.10	0.16	0.48

Table 12: Summary of results, Within-Laboratory Precision

The following tables expand upon the total within-laboratory precision values described above, showing the within-laboratory precision values per site.

Within Laboratory Precision, Site 1
Sample	N	Mean ISI	Repeatability(within run)Standard Deviation	BetweenOperatorsStandard Deviation	BetweenInstrumentStandard Deviation	Within-LaboratoryPrecisionStandard Deviation
#1	12	3.98	0.17	0.02	0.20	0.26
#2	12	4.01	0.80	0.53	0.00	0.96
#3	12	4.90	0.25	0.06	0.00	0.26
#4	12	7.13	0.45	0.04	0.11	0.46

Table 13: Within-Laboratory Precision results, Site 1

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Within Laboratory Precision, Site 2
Sample	N	Mean ISI	Repeatability(within run)Standard Deviation	BetweenOperatorsStandard Deviation	BetweenInstrumentStandard Deviation	Within-LaboratoryPrecisionStandard Deviation
#5	12	3.24	0.41	0.00	0.40	0.57
#6	12	4.88	0.42	0.34	0.22	0.58
#7	12	5.10	0.29	0.00	0.31	0.42
#8	12	6.08	0.31	0.00	0.00	0.31

Table 14: Within-Laboratory Precision results, Site 2

Within Laboratory Precision, Site 3
Sample	N	Mean ISI	Repeatability (within run)Standard Deviation	Between OperatorsStandard Deviation	Between InstrumentStandard Deviation	Within-Laboratory PrecisionStandard Deviation
#9	12	2.56	0.37	0.09	0.16	0.42
#10	12	4.68	0.37	0.18	0.18	0.45
#11	12	5.53	0.36	0.00	0.10	0.37
#12	12	6.41	0.48	0.22	0.00	0.53

Table 15: Within-Laboratory Precision results, Site 3

A supplemental within-laboratory precision study was performed at one site with 4 donor samples, spanning the high end of the ISI range with scores between 7.3-10.0 ISI. For each sample, 2 operators performed runs on 2 instruments in triplicates, and repeated the testing at least one hour after the initial testing for a total of 96 tests.

Components of within-laboratory precision across samples, Supplemental Study
Sample	N	Repeatability(within run)Standard Deviation	Between-RunStandard Deviation	Between-OperatorStandard Deviation	Between-InstrumentStandard Deviation	Within-laboratoryPrecisionStandard Deviation
#13	24	0.29	0.07	0.00	0.00	0.30
#14	24	0.43	0.34	0.00	0.00	0.55
#15	24	0.37	0.56	0.00	0.15	0.69
#16	24	0.39	0.21	0.14	0.07	0.47

Table 16: Supplemental Within-Laboratory Precision study results

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9.3.2.2 Reproducibility

Total reproducibility was calculated by adding within-laboratory precision and Across Sites reproducibility. The Across Site reproducibility was measured according to CLSI EP05-A3 using IntelliSep Quality Control Kit samples. Testing was conducted at 3 sites using the same lots of control material. At each site, testing was performed for 40 non-consecutive days for both Quality Control levels, for a total of 240 tests.

Sample	Number of tests	Repeatability	Across Sites
		Standard Deviation	Standard Deviation
QC Level 1	120	0.66	0.34
QC Level 2	120	0.49	0.25
Both levels	240	0.59	0.30

Table 17: Summary of results, Reproducibility

Combining the within-lab precision (0.48 units) with the Across Sites reproducibility (0.30 units) resulted in a total reproducibility of 0.57 units.

Reagents Lot-to-Lot Reproducibility 9.3.2.3

The Cytovale reagents (consisting of the Cytovale Reagent Kit, Diluent and Cleanse solutions) lot-to-lot reproducibility was tested according to CLSI EP26-A using IntelliSep Quality Control samples.

Testing was performed on two lots of reagents for each of the three types listed above. For each lot of reagents, each IntelliSep Quality Control level (L1 and L2) was tested five times over three days, resulting in 60 IntelliSep tests. An equivalence test examined if the Cytovale reagents sets have an absolute mean difference of less than 1.0 ISI unit.

Sample	Reagent Set #1 mean ISI minus Reagent Set #2mean ISI (Cytovale Cat. No. CV-REA-001)	Acceptance criteriaThe 90% CI Lower andUpper Bounds are within (-1.0, 1.0) units of ISI
	Mean		90% CI
QC Level 1	0.06	(-0.12, 0.24)	Pass
QC Level 2	-0.01	(-0.21, 0.19)	Pass

Table 18: Summary of results, Reagents Lot-to-Lot Reproducibility

Cartridge Lot-to-Lot Reproducibility 9.3.2.4

The IntelliSep Cartridge lot-to-lot reproducibility was tested according to CLSI EP26-A using donor samples.

Two lots of IntelliSep Cartridges were evaluated during this reproducibility study, where three donor samples across three IntelliSep Index ranges (Low, Moderate and High) were evaluated. Five replicates of each sample were tested, resulting in 30 IntelliSep tests.

Sample	Cartridge Lot #1 mean ISI minusCartridge Lot #2 mean ISI		Acceptance criteriaThe 90% CI Lower andUpper Bounds are within (-1.0, 1.0) units of ISI
	Mean	90% CI
Donors	-0.47	(-0.68, 0.26)	Pass

Table 19: Summary of results, Cartridge Lot-to-Lot Reproducibility

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9.3.3 Analytical Measuring Range

Nonclinical analytical sensitivity, linearity, and detection limit(s)/cutoff testing is inapplicable to the IntelliSep test. Clinical sensitivity, clinical specificity, and clinical cut-off information were evaluated as part of the clinical validation study (reference Section 9.1).

9.3.4 Interference

Interfering Substances 9.3.4.1

The effect of the following endogenous substances and pharmaceutical compounds on assay performance was tested according to CLSI EP07. Interferences were tested up to the listed concentrations and no impact on results was observed. Cross-reactivity evaluations are inapplicable to the IntelliSep test.

Substance	Concentration	Control GroupMean ISI	Test GroupMean ISI	MeanDifference
Unconjugated Bilirubin	40 mg/dL	3.4	3.8	-0.4
Conjugated Bilirubin	40 mg/dL	2.8	3.0	-0.2
Triglycerides	1500 mg/dL	2.5	2.7	-0.2
Hemolysate	1000 mg/dL	2.7	2.7	0.0
Hemoglobin	1000 mg/dL	3.3	3.3	0.0

Table 20: Summary of results, Interfering Substances testing

9.3.4.2 Sample Carry-Over

The study to evaluate sample carry-over was based on the principles of CLSI EP10-A3-AMD and measured the impact to the ISI due to carryover between two samples whose cells have different biophysical properties.

The study was conducted over 5 days at 1 site with 1 instrument and 1 operator. Two levels from the IntelliSep Quality Control Kit (L1 and L2) were evaluated using eleven replicates of L1 ("L" or "Low") and ten replicates of L2 ("H" or "High") cycled between each other. On each test day, a specified carryover sample order test set was performed to determine the impact on ISI of an L2 sample preceding an L1 sample ("HL"), and an L1 sample preceding an L2 sample ("LH"). No sample carry-over effect was found as shown in the following table.

Day	LL MeanISI	HL MeanISI	LL - HLDifference (ISI)	HH MeanISI	LH MeanISI	HH - LHDifference (ISI)
Day 1	2.46	2.22	0.24	7.28	7.40	-0.12
Day 2	2.14	2.20	-0.06	7.00	7.24	-0.24
Day 3	2.36	2.32	0.04	7.14	6.90	0.24
Day 4	2.34	2.42	-0.08	7.18	6.90	0.28
Day 5	1.98	2.00	-0.02	7.02	7.08	-0.06
Total	2.26	2.23	0.03	7.12	7.10	0.02

Table 21: Summary of results, Sample Carry-Over

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9.3.5 Stability

Onboard Stability of Cytovale Reagents 9.3.5.1

The stability of the three Cytovale reagents (Cytovale Reagent Kit, Diluent and Cleanse) was tested according to CLSI EP25-A using IntelliSep Quality Control Kit samples. All three Cytovale reagents are stable for up to 30 days when stored onboard the Cytovale System.

9.3.5.2 Sample Stability

The stability of K2-EDTA anti-coagulated whole blood samples was tested according to CLSI EP25-A using 20 donor samples spanning the range of ISI values. The K2-EDTA sample may be used for the IntelliSep test for up to 5 hours after blood draw.

9.4 Healthy Reference Range

The reference range for the IntelliSep test was measured according to CLSI EP28-A3C. In a population of 243 self-reported healthy individuals (49% Female), the 95% Reference Range for the IntelliSep Index was calculated as 1.0-4.6 ISI.

Population	ISI Reference Range(2.5%-97.5%)	ISI Lower Reference Limit(90% CI)	ISI Upper Reference Limit(90% CI)
Female	0.9-4.7	0.7-1.1	4.3-4.9
Male	0.7-4.7	0.4-1.2	4.1-4.7
All	1.0-4.6	0.9-1.2	4.2-5.0

Table 22: Summary of results, Healthy Reference Range

10 Proposed Labeling

The labeling is sufficient and it meets the requirements of 21 CFR Parts 801 and 809, as applicable, and the special controls for this device under 21 CFR 866.3215.

11 Conclusions

The submitted information in this premarket notification is complete and supports a substantial equivalence determination.

Regulation Number and Section

§ 866.3215 Device to detect and measure non-microbial analyte(s) in human clinical specimens to aid in assessment of patients with suspected sepsis.

(a)
Identification. A device to detect and measure non-microbial analyte(s) in human clinical specimens to aid in assessment of patients with suspected sepsis is identified as an in vitro device intended for the detection and qualitative and/or quantitative measurement of one or more non-microbial analytes in human clinical specimens to aid in the assessment of patients with suspected sepsis when used in conjunction with clinical signs and symptoms and other clinical and laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the device's detailed Indications for Use statement describing what the device detects and measures, the results provided to the user, whether the measure is qualitative and/or quantitative, the clinical indications for which the test is to be used, and the specific population(s) for which the device use is intended.
(2) Premarket notification submissions must include detailed documentation of the device description, including (as applicable), all device components, software, ancillary reagents required but not provided, explanation of the device principle and methodology, and for molecular devices include detailed documentation of the primer/probe sequence, design, and rationale for sequence selection.
(3) Premarket notification submissions must include detailed documentation of applicable analytical studies, such as, analytical sensitivity (Limit of Detection, Limit of Blank, and Limit of Quantitation), precision, reproducibility, analytical measuring range, interference, cross-reactivity, and specimen stability.
(4) Premarket notification submissions must include detailed documentation of a prospective clinical study or, if appropriate, results from an equivalent sample set. This detailed documentation must include the following information:
(i) Results must demonstrate adequate device performance relative to a well-accepted comparator.
(ii) Clinical sample results must demonstrate consistency of device output throughout the device measuring range likely to be encountered in the Intended Use population.
(iii) Clinical study documentation must include the original study protocol (including predefined statistical analysis plan), study report documenting support for the Indications for Use(s), and results of all statistical analyses.
(5) Premarket notification submissions must include evaluation of the level of the non-microbial analyte in asymptomatic patients with demographic characteristics (
e.g., age, racial, ethnic, and gender distribution) similar to the Intended Use population.(6) As part of the risk management activities performed under 21 CFR 820.30 design controls, you must document an appropriate end user device training program that will be offered as part of your efforts to mitigate the risk of failure to correctly operate the instrument.
(7) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling, and a detailed explanation of the interpretation of the limitations of the samples (
e.g., collected on day of diagnosis) must be included in the device's 21 CFR 809.10(b)(10) compliant labeling.