Ortho-Count Calibration Kit for ORTHO CYTORONABSOLUTE Laser Flow Cytometry System, contains a Calibrator Bead Suspension for absolute lymphocyte count calibration and Verifier Bead Suspensions (I, II and III) to verify accuracy of absolute count calibration. The Calibrator Bead Suspension is manufactured as known "events"/unit volume (i.e. the Calibration Number) in the form of microparticle beads suspended in a proprietary buffer. The size of the microparticles has been selected to emulate the range of size of leukocytes detected by the ORTHO CYTORONABSOLUTE under specified gating conditions. The Verifier Bead Suspensions (I, II and III), are composed of materials identical to the Calibrator Bead Suspension and collectively provide a range of particle bead concentrations. The target values chosen approximate values expected from patients with low (Verifier I), normal (Verifier II) and high (Verifier III) white blood cell counts, The Verifiers are used to verify accuracy of the absolute respectively. lymphocyte count calibration immediately following calibration and are also used as daily quality control verification that the ORTHO CYTORONABSOLUTE is in calibration. A properly calibrated instrument will provide absolute counts within + 10% of the labeled events/uL for each Verifier supsension. In the context of immune status monitoring, absolute cell count is defined as the number of cells per unit volume. The ORTHO CYTORONABSOLUTE uses precision stepper motors to deliver diluted sample to the instrument's flow cell at a constant rate. Since constant flow rate means movement of a fixed volume per unit time, analysis time determines the volume of sample analyzed. Calibration of the ORTHO CYTORONABSOLUTE consists of setting analysis time such that a volume corresponding to 1 uL of undiluted sample is examined. Ortho-Count Calibrator Bead Suspension, representing a known number of particles per unit volume (i.e. events/uL) at the same dilution as used for testing samples, is counted until its "Calibration Number" is reached; the instrument is set to this analysis time. In subsequent analyses events are to passage of the equivalent of 1 uL of undiluted sample the instrument flowcell. As a result of this calibration, sample results can be read in absolute count, i.e. "events"/uL.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Acceptance Criteria and Device Performance

The core acceptance criterion for the Ortho-Count Calibration Kit is that a properly calibrated ORTHO CYTORONABSOLUTE instrument will provide absolute counts within ±10% of the labeled events/uL for each Verifier suspension.

Acceptance Criteria	Reported Device Performance
Absolute counts within ±10% of labeled events/uL for each Verifier suspension.	Verifier I: Mean across sites = 1693, Std. Dev. = 55, % CV = 3.2% (Compared to a target value, which is not explicitly stated but implied to be within 10% of the mean)
	Verifier II: Mean across sites = 7748, Std. Dev. = 193, % CV = 2.5%
	Verifier III: Mean across sites = 15140, Std. Dev. = 409, % CV = 2.7%
No significant differences in absolute lymphocyte counts attributable to instrument or calibration method when compared to predicate device/method.	Table 1: Ratios of Mean Method 1/JT2, Method 2/JT2, and Method 2/Method 1 generally hover around 1.0, with mean standard deviations around 0.04-0.05, indicating no significant differences. For example, the mean ratio of Meth2/Meth1 is 0.99 with a standard deviation of 0.05.
Inter-laboratory reproducibility.	Table 2: Overall Inter-Lab % CV values for Verifiers I, II, and III are 3.2%, 2.5%, and 2.7% respectively, which are comparable to within-laboratory precision, indicating good inter-laboratory reproducibility.
Strong correlation between direct ORTHO CYTORONABSOLUTE counts and those calculated from combined hematology and other flow cytometer/reagent results.	Figure 2: Regression analysis yields a slope of 0.89, a y-intercept of -31 ± 13, and a correlation coefficient (r) = 0.96. This indicates a strong positive correlation.
Utility as quality control indicators over time (60 days).	Figure 3: Shows "tight agreement" in measurements made using each Verifier Bead Suspension over 60 days, confirming suitability as an accurate quality control measure.

Study Details

Sample size used for the test set and the data provenance:
- Table 1 (Comparison to Predicate Device): 10 donors. Data provenance not explicitly stated (e.g., country of origin), and it's not clear if it was retrospective or prospective.
- Table 2 (Inter-laboratory Reproducibility): Verifier Bead Suspensions (I, II, and III) were used across four different ORTHO CYTORONABSOLUTE instruments located in different regions of the United States. The frequency or total number of measurements for establishing "Mean" values for each verifier is not specified, but it's likely prospective for this component.
- Figure 1 (Consistency of Calibration/Cross-Calibration): 41 samples (whole blood panel). Provenance not explicitly stated beyond "different regions of the United States" for the instruments. The samples were handled "overnight at room temperature or shipped for next day delivery to each site," suggesting prospective collection for this study, though the source of the donors/patients is not specified.
- Figure 2 (Direct vs. Calculated Absolute Counts): Data points for CD19, CD3+CD8+, CD3+CD4+, and Total CD3+. The total number of individual samples is not specified, but the regression analysis implies a substantial number of data points for these subsets. Provenance not explicitly stated.
- Figure 3 (Quality Control Indicators): Counts/volume were measured on the three verifier suspensions twice per week over a period of 60 days for four ORTHO CYTORONABSOLUTE instruments.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- The document does not explicitly state the number or qualifications of experts used to establish ground truth for the test sets. Instead, the ground truth is established through:
  - Comparison to a predicate device (Coulter JT2 calibrated with S-CAL Kit) (for Table 1).
  - Labeled 'events'/uL for the Verifier Bead Suspensions themselves, which are manufactured values (for Table 2 and Figure 3).
  - "Calculated from combined hematology and other flow cytometer/reagent results" (for Figure 2's comparative ground truth).
Adjudication method (e.g. 2+1, 3+1, none) for the test set:
- No adjudication method involving experts is described. The comparisons are quantitative measurements against established methods or known values.
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 MRMC comparative effectiveness study involving human readers (as in interpretation of images or complex data) was conducted. This device is a calibration kit for an automated instrument, not an AI diagnostic tool requiring human interpretation. Therefore, the concept of "human readers improve with AI" is not applicable here.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- The study primarily focuses on the standalone performance of the calibration kit in ensuring the accuracy and reproducibility of the ORTHO CYTORONABSOLUTE instrument's absolute counting function. The ORTHO CYTORONABSOLUTE itself is an automated device, so its performance (when calibrated) is inherently "algorithm only" in terms of cell counting. The "human-in-the-loop" would be the technician operating the flow cytometer and applying the calibration kit.
The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
- Known Reference Values: For the Verifier Bead Suspensions, the ground truth is the "labeled events/uL," which are manufactured and accurately determined "events"/unit volume.
- Predicate Device/Method: For the comparison of absolute lymphocyte counts (Table 1), the ground truth is established by a "Coulter JT2 calibrated using S-CAL Kit" which is the predicate device/method.
- Combined Method: For Figure 2, the ground truth is established by "values calculated from separate flow cytometers and automated hematology analyzers."
The sample size for the training set:
- The document does not describe a "training set" in the context of machine learning or AI. This is a calibration kit, and its performance is evaluated against established methods and internal consistency, not through an iterative training process for an algorithm.
How the ground truth for the training set was established:
- As there is no described training set for an algorithm, this question is not applicable. The kit itself is manufactured with known characteristics (e.g., "known 'events'/unit volume" for the Calibrator Bead Suspension).

Summary

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ITEM 7.

807.87 (h) . A 510(k) SUMMARY AS DESCRIBED IN 807.92 OR A 510(k) STATEMENT AS DESCRIBED IN 807.93.

K935720

MAY - 9 1996

Ortho Diagnostic Systems Inc. Ortho CrionomaBSOLUTE " Laser Flow Cytometry System Page 56

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510 (k) SUMMARY

SUBMITTER : Ortho Diagnostic Systems Inc. CONTACT : Ms. Joanne Harris Tel.: (908) 218-8404 1001 U.S. Hwy 202 Raritan, NJ 08869-0606 Fax.: (908) 218-8168 DEVICE NAME: Ortho-Count Calibration Kit PREDICATE: S-CAL® Kit for ORTHO CYTORONABSOLUTE [K813375] Laser Flow Cytometry System Coulter Diagnostics

[864.8185 Calibrator for red cell and white cell counting}

DATE: November 29, 1993

DEVICE DESCRIPTION:

The Calibrator Bead Suspension is manufactured as known "events"/unit volume (i.e. the Calibration Number) in the form of microparticle beads suspended in a proprietary buffer. The size of the microparticles has been selected to emulate the range of size of leukocytes detected by the ORTHO CYTORONABSOLUTE under specified gating conditions.

The Verifier Bead Suspensions (I, II and III), are composed of materials
identical to the Calibrator Bead Suspension and collectively provide a range of particle bead concentrations. The target values chosen approximate values expected from patients with low (Verifier I), normal (Verifier II) and high (Verifier III) white blood cell counts, The Verifiers are used to verify accuracy of the absolute respectively. lymphocyte count calibration immediately following calibration and are also used as daily quality control verification that the ORTHO CYTORONABSOLUTE is in calibration. A properly calibrated instrument will provide absolute counts within + 10% of the labeled events/uL for each Verifier supsension.

In the context of immune status monitoring, absolute cell count is defined as the number of cells per unit volume. The ORTHO CYTORONABSOLUTE uses precision stepper motors to deliver diluted sample to the instrument's flow cell at a constant rate. Since constant flow rate means movement of a fixed volume per unit time, analysis time determines the volume of sample analyzed. Calibration of the ORTHO CYTORONABSOLUTE consists of setting analysis time such that a volume corresponding to 1 uL of undiluted sample is examined.

Ortho Diagnostic Systems Inc. Ortho-Count Calibration Kit for
ORTHO CYTORONABSOLUTE™ Laser Flow Cytometry System Page 57

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Ortho-Count Calibrator Bead Suspension, representing a known number of particles per unit volume (i.e. events/uL) at the same dilution as used for testing samples, is counted until its "Calibration Number" is reached; the instrument is set to this analysis time. In subsequent analyses events are to passage of the equivalent of 1 uL of undiluted sample the instrument flowcell. As a result of this calibration, sample results can be read in absolute count, i.e. "events"/uL.

INTENDED USE:

Ortho-Count Calibration Kit for ORTHO CYTORONABSOLUTE Laser Flow Cytometry System is intended to be used to calibrate and verify calibration of the absolute lymphocyte counting function of the ORTHO CYTORONABSOLUTE. This intended use is substantially equivalent to the intended use of S-CAL Kit, the predicate device, for lymphocyte counting. The intended use is different than that for S-CAL Kit in that Ortho-Count Calibration Kit may not be used to calibrate the ORTHO CYTORONABSOLUTE for parameters other than lymphocyte counting. This difference does not affect the safety or effectiveness of Ortho-Count Calibration Kit for its intended use since the ORTHO CYTORONABSOLUTE is not intended for all of the same uses as the Coulter Systems with which S-CAL Kit is intended to be used.

TECHNOLOGICAL CHARACTERISTICS:

Ortho-Count Calibration Kit and S-CAL Kit both contain components which are recognized by cell counters as leukocytes and are used to convert the electronic signal obtained from such instruments into accurate leukocyte count results expressed in clinical terms. Both kits consist of suspensions of particles, whose size, shape, concentration and other
characteristics have been accurately determined. Ortho-Count Calibration Kit employees microparticle beads, while S-CAL Kit uses fixed erythrocytes in suspension, to simulate leukocytes.

PERFORMANCE DATA:

Performance of an ORTHO CYTORONABSOLUTE calibrated using Ortho-Count Calibration Kit (Meth 2) was compared to performance of the same instrument calibrated using whole blood whose total lymphocyte count was determined with a Coulter JT2 calibrated using S-CAL Kit (Meth 1), and to performance of the Coulter JT2 calibrated with S-CAL Kit (JT2). As shown in Tabla.1. there were no significant differences in absolute lymphocyte counts that could be attributed to either the instrument or the method of calibration.

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TABLE 1

LYMPHOCYTE COUNTS FRON A HENATOLOGY ANALYZER VERSUS

LYMPHOCYTE COUNTS FRON ORTHO CYTORONABSOLUTE CALIBRATED BY

DIFFERENT METHODS

DonorNumber	MeanJT2	Std.Dev.	MeanMethod 1	Std.Dev	MeanMethod 2	Std.Dev	RatioMeth1JT2	RatioMeth2JT2	RatioMeth2Meth1
1	1983	75	1776	23	1940	90	0.90	0.98	1.09
2	2383	41	2189	94	2147	88	0.92	0.90	0.98
3	1767	137	1736	71	1668	93	0.98	0.94	0.96
4	2217	75	2076	81	2129	90	0.94	0.96	1.03
5	1800	47	1805	52	1807	110	1.00	1.00	1.00
6	2250	55	2265	29	2132	56	1.01	0.95	0.94
7	1600	122	1557	25	1476	98	0.97	0.92	0.95
8	2300	71	2323	138	2271	61	1.01	0.99	0.98
9	3820	164	3730	81	3855	94	0.98	1.01	1.03
10	2517	117	2481	73	2413	141	0.99	0.96	0.97
MeanStandard Deviation							0.970.04	0.960.04	0.990.05

Four ORTHO CYTORONABSOLUTE instruments located in different regions of the United States, were calibrated using Ortho-Count Calibration Kit to assess inter-laboratory reproducibility os evidenced by Ortho-Count Calibration Kit Verifier Bead Suspensions. Table 2 shows the accuracy and precision of the absolute counts at each site. Overall precision of the absolute counts between laboratories was equivalent to the within laboratory precision.

Table 2. Across Site Accuracy and Precision of Absolute Counts on Four Microparticle Calibrated CytoronAbsolutes

Location	SiteOne	SiteTwo	SiteThree	SiteFour	OverallInter-Lab
Verifier I
Mean	1736	1699	1669	1670	1693
Std. Dev.	61	43	43	44	55
% CV	3.5	2.5	2.6	2.6	3.2
Verifier II
Mean	7851	7726	7805	7614	7748
Std. Dev.	174	225	101	173	193
% CV	2.2	2.9	1.3	2.3	2.5
Verifier III
Mean	15434	15169	15209	14755	15140
Std. Dev.	257	492	225	288	409
% CV	1.7	3.2	1.5	2.0	2.7

Consistency of calibration of ORTHO CYTORONABSOLUTE instruments was demonstrated in a study comparing absolute lymphocyte counts obtained on a panel of donors at each of the four study sites.

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Mean absolute lymphocyte counts from eight determinations on each donor per site were plotted against the overall mean lymphocyte count for each donor across sites (Figure 1).

Image /page/4/Figure/1 description: Figure 1 shows the absolute lymphocyte counts measured on cross-calibrated ORTHO CYTORONABSOLUTE Instruments. The figure is a title for a scientific paper or presentation. The title is written in a clear and concise manner.

Image /page/4/Figure/2 description: This image is a scatter plot comparing the lymphocyte count at different sites with the mean lymphocyte count. The x-axis represents the mean lymphocyte count, ranging from 0 to 3500, while the y-axis represents the lymphocyte count at sites, ranging from 0 to 4000. The plot includes data points from four different sources: ODSI, Rush, UCLA, and USC, each represented by a different symbol. The data points generally show a positive correlation between the mean lymphocyte count and the lymphocyte count at sites.

Figure 1. Absolute lymphocyte counts were determined using cross-calibrated ORTHO CYTORONABSOLUTE instruments for a whole blood panel kept overnight at room temperature or shipped for next day delivery to each site. The average lymphocyte count obtained at each site is plotted versus the mean lymphocyte count across sites for each of 41 samples.

The determination of an absolute lymphocyte subset count requires both a lymphocyte count and the percentage of lymphocytes positive for the marker. By convention, these measurements have required separate flow cytometry and hematology instruments. A study was performed to demonstrate that when calibrated using Ortho-Count Calibration Kit it is possible to obtain equivalent absolute lymphocyte subset counts directly from the ORTHO CYTORONABSOLUTE. Figure 2 shows strong correlation between absolute count values determined directly on the ORTHO CYTORONABSOLUTE and those calculated from combined hematology and other flow cytometer/reagent results. Regression analysis of the data yields a slope of 0.89, a y-intercept of -31 t 13 and a correlation coefficient (r) = 0.96.

Ortho Diagnostic Systems Inc. Ortho-Count Calibration Kit for ORTHO CYTORONABSOLUTE Laser Flow Cytometry System Page 60

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Direct Determination of Absolute Lymphocyte Counts on the ORTHO Figure 2. CYTORONABSOLUTIE Versus Hematology Plus Other Flow Cytometer Calculations

Image /page/5/Figure/1 description: The image is a scatter plot comparing lymphocyte counts. The x-axis represents lymphocyte counts measured by hematology plus alternate flow cytometers, ranging from 0 to 3000. The y-axis represents lymphocyte counts measured on Cyloron Absolute, also ranging from 0 to 3000. The plot displays data points for CD19, CD3+CD8+, CD3+CD4+, and Total CD3+.

Absolute lymphocyte subset values were determined directly using a Figure 2. single ORTHO CYTORONABSOLUTE. These data are plotted versus absolute lymphocyte counts determined using separate flow cytometers and automated hematology analyzers. Absolute lymphocyte subsets plotted are: CD19, CD8+ cells that are also CD3+, CD4+ cells that are also CD3+ and total CD3+ cells

The utility of the Ortho-Count Calibrator Kit Verifier Bead Suspensions as quality control indicators of proper calibration of the ORTHO CYTORONABSOLUTE was demonstrated in a study in which counts/volume (absolute counts) were measured on the three verifier suspensions twice per week over a period of 60 days. Figure 3 shows the absolute counts obtained at each laboratory over the two months. The tight agreement in measurements made using each of the Verifier Bead Suspensions confirms the suitability of Ortho-Count Calibration Kit as an accurate quality control measure of the status of calibration of the ORTHO CYTORONABSOLUTE.

Ortho Diagnostic Systems Inc. Ortho-Count Calibration Kit for
ORTHO CYTORONABSOLUTE™ Laser Flow Cytometry System Page 61

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Ortho-Count Calibration Kit Verifier Bead Suspensions as Figure 3. Quality Control Indicators of Status of Calibration of the ORTHO CYTORONABSOLUTE

Image /page/6/Figure/1 description: The figure shows a scatter plot of verifier counts versus days post calibration. The y-axis shows the verifier counts in events per microliter, ranging from 0 to 16000. The x-axis shows the days post calibration, ranging from 0 to 60. There are three distinct clusters of data points, one around 14000, one around 8000, and one around 1000.

Four ORTHO CYTORONABSOLUTE instruments were calibrated using Ortho-Pigure 3. Count Calibration Kit. The absolute count of each Verifier Bead Suspension is plotted for each laboratory on each day throughout the study.

CONCLUBIONS :

1. Ortho-Count Calibration Kit is substantially equivalent to S-CAL Kit used to calibrate Coulter hematology instruments, for the intended use of calibration and verification of the calibration of the absolute lymphocyte counting function of the ORTHO CYTORONABSOLUTE.
1. Ortho-Count Calibration Kit functions as a suitable crosscalibration standard to ensure inter-laboratory reproducibility.
Ortho-Count Calibration Kit provides accurate calibration 3. and verification of calibration of the ORTHO CYTORONABSOLUTE, permitting direct determination of absolute lymphyocyte subset values equivalent to those obtained using values calculated from separatte flow cytometers and hematology analyzers.
Ortho-Count Calibration Kit provides reliable quality 4 . control of the calibration status of the ORTHO CYTORONABSOLUTE.

Regulation Number and Section

§ 864.5220 Automated differential cell counter.

(a)
Identification. An automated differential cell counter is a device used to identify one or more of the formed elements of the blood. The device may also have the capability to flag, count, or classify immature or abnormal hematopoietic cells of the blood, bone marrow, or other body fluids. These devices may combine an electronic particle counting method, optical method, or a flow cytometric method utilizing monoclonal CD (cluster designation) markers. The device includes accessory CD markers.(b)
Classification. Class II (special controls). The special control for this device is the FDA document entitled “Class II Special Controls Guidance Document: Premarket Notifications for Automated Differential Cell Counters for Immature or Abnormal Blood Cells; Final Guidance for Industry and FDA.”