Tear Based Sample Collection for Breast Cancer Detection
Conditions: Breast Cancer, Tear, Breast Cancer Female
Sponsor: Namida Lab

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Title: Tear Based Breast Cancer Detection 
 
Principal Investigator:  V. Suzanne Klimberg MD. PH.D. 
 
Funding Sponsor: Namida Lab, Inc. 
 
Version: 2 
 
Date: 4/13/2021 
 
 
 
 
 
 
 
 
 
 
 
 

1. Introduction and Purpose: 
 
Namida Lab, Inc. is a biotechnology company located in Fayetteville, AR, with a certified high-complexity 
CLIA lab. In January of 2021, Namida Lab, Inc. completed Melody® analytical and clinical validation, a 
screening test for breast cancer based on proteins from tears. The validation process was carried out 
manually and now needs to be conducted on the Hamilton StarPlus automated ELISA system. To do this, 
Namida Lab, Inc. needs fifty tear samples collected from women recently diagnosed with breast cancer 
but who have not undergone any treatment.  
 
2. Background: 
 
What is the Problem? 
Most women recognize screening mammography as the current gold standard in breast cancer screening. 
However, disparities are present, leaving groups of women without adequate access to screening. Gaps in 
access to screening exist due to geographic location, socio-economic status, race, and ethnicity 
(references). In addition, studies show women who have received a false-positive result are more likely to 
lose trust in the current breast health continuum of care. Therefore less likely to return for follow-up 
screening.  
 
How can Melody® help? 
Currently, Melody® is not intended to be a replacement for screening mammograms but an additional 
tool to facilitate discussion between providers and patients. Tear sample collection can be done during 
any routine office visit and does not require specially trained personnel. The simple collection process 
allows patients to be tested in a clinic they are already familiar with by personnel they already know and 
trust. Melody® early iteration goals are one additional piece of information gathered. One more 
conversation about breast health may be the key to increasing participation in annual or biennial 
screening mammograms.  
 
Key terms 
▪ 
The Clinical Laboratory Improvement Amendment (CLIA) passed in 1988 was established to 
provide quality standards, strengthen Federal oversight of clinical laboratories, and ensure the 
reliability and accuracy of patient test results. This program allows clinical labs to develop and 
validate the testing protocols, which can only be run in the lab where the protocol was validated.  
▪ 
Namida Lab, Inc. is a CLIA certified high complexity lab. 
▪ 
The FDA is responsible for developing and assigning CLIA test complexity categorization rules and 
guidance.  
▪ 
Melody® is a high complexity lab-developed test that has been developed and validated by 
Namida Lab, Inc. and will be run only by Namida Lab, Inc.  
▪ 
A clinical utility study evaluates the usefulness, benefits, and drawbacks of an intervention 
(Smart, 2006).  
 
What is the Validation process for an LDT? 
CLIA clearly outlines requirements for validation of a lab-developed test. In short, each assay must meet 
guidelines for precision, accuracy, sensitivity and specificity (both analytical and clinical), linearity and 
assay range. Each laboratory drafts an umbrella standard operating procedure (SOP) to conduct validation 

of new assays. This SOP is then customized to each specific test the lab intends to offer. The medical 
director must approve all SOPs and subsequent changes to SOPs in the CLIA lab.  
 
After completion of validation experimentation, all results are compiled in a validation report and then 
reviewed and approved by the medical director. All CLIA labs are under the jurisdiction of their respective 
state CLIA licensing boards and undergo an inspection every two years. At the time of inspection, the lab 
must provide validation reports for all assays available on the testing menu for review.  
  
What was the outcome for the validation of Melody®? 
Designing the validation of Melody® was unique because there are no lab developed tests using protein 
biomarkers in tears to Namida Lab, Inc.'s knowledge. The nature of the sample alone provided a new level 
of challenge that made the validation process more stringent. Both analytical and clinical validation of 
Melody® was completed in January 2021. A summary of parameters analyzed and the results for the 
validation can be found in tables 1-2. All performance criteria were successfully achieved, and clinical 
validation resulted in a sensitivity of 92% and a specificity of 54%. These values were in line with 
previously observed sensitivity and specificity values from development work.  
 
Table 1: Summary of Analytical Validation 
Study Parameter 
Sample Description (Name, 
Number, Replicates) 
Results 
Comparison to 
Acceptance Criteria 
(Pass/Fail) 
Accuracy 
20 spiked LGF samples 
Percent recovery between 80-
120% for all samples 
Pass 
Intra-Assay Precision 
24 replicates of 3 concentrations 
per analyte 
All %CV less than or equal to 15% 
Pass 
Inter-Assay Precision 
Duplicates of 3 concentrations 
1xday for 5 days. 
All %CV less than or equal to 15% 
Pass 
Sensitivity 
20 replicates of blank per protein 
S100A8 11% 21.10 pg/ml S100A9 
14% CV 24.32 pg/ml 
Pass 
Linearity 
7 unknown concentrations 
Percent difference less than 15% 
for all samples 
Pass 
 
Table 2: Summary of Clinical Validation 
Study Parameter 
Sample Description (Name, Number, 
Replicates) 
Results 
Comparison to Acceptance 
Criteria (Pass/Fail) 
Accuracy 
26 samples 3 replicates each sample 
92% 
Pass 
Sensitivity 
52 LGF samples in duplicate 
92% 
Pass 
Specificity 
50 LGF samples in duplicate 
54% 
Pass 
Stability of LGF samples 
1 pooled disease LGS 
1 pooled non-disease LGS 
N/A 
Refer to Deviations and 
Section B, Number 4 
 
 
 
3. Concise Summary of Project: 
 
What exactly is Melody®? 
Part 1: Tear sample collection 

Tear samples are collected using a Schirmer strip, a class I medical device, typically used to test for dry 
eye. The filter paper strips have been repurposed to collect proteins from the ocular cavity following 
protocols outlined in the literature (references).  
 
 Tear collection is conducted through the following steps (figure 1):  
1.  The strip is placed in the participants' lower eyelid, and having a foreign object in the eye will 
cause the eye to water. The fluid released washes the inner surface of the eyelid and the ocular 
lens itself, allowing proteins to adhere to the strip.  
2. The strip is left until the liquid reaches the 25 mm mark or 5 minutes has passed.  
3. The strip is then placed in a tube containing a buffered solution and is shipped overnight to 
Namida Lab, Inc.  
 
 
Figure 1: Collection of tear samples.  
 
Part 2: Clinical Lab assay 
During shipment, the proteins leave the strip and go into the solution in the tube. It is this solution that is 
evaluated using standard ELISA techniques in the high-complexity CLIA lab. Namida Lab, Inc. is outfitted 
with a Hamilton Star Plus automated ELISA system. The data generated from the ELISA assay is used 
together with demographic information to generate a Melody® score. The score is then compared to the 
diagnostic threshold, and classification of High and Low will be reported.  
 
A reference range has been developed using the distribution of Melody® scores from the clinical studies 
previously conducted (figure 2). In the future, this scale will be further optimized and provided to 
clinicians to guide their breast health discussions with patients.   
 
Figure 2: The Melody® scale was generated using 705 samples evaluated during development and validation. 
Percentages in each range show the distribution of normal and breast cancer patients that fall within each range.  
 
With validation manually completed, Namida Lab, Inc. needs to do an equivalency study on the 
automated robot to demonstrate consistency between manual and automated results. This analysis will 

be carried out using an equal number of samples. The samples are collected from normal (i.e., recent 
mammogram came back with no new findings) and recently diagnosed breast cancer patients. 
 
Namida Lab, Inc.'s Clinical Laboratory Supervisor has defined qualification criteria of 100% agreement 
between manual and automated validation for the validation to be considered successful.   
 
4. Study Procedures: 
 
Fifty participants will be enrolled following the timeline in Table 3. The participation is limited to a one-
time sample collection.  
 
 
i. Data acquisition: Each participant will be provided with a questionnaire to be filled out at the time of 
sample collection. The study team will assist participants in answering questions such as type of breast 
cancer, staging information, and tumor size. If known at the time of sample collection or after the biopsy 
results are received. In addition, a clinical questionnaire will be filled out for each participant with specific 
information relating to their breast health and current diagnosis.  
 
ii. Clinic: Visit #1 
• Participant is seen 
• Study eligibility is assessed 
• Informed consent process 
• Registration 
• Questionnaires completion 
• Tear sample collection 
 
The tear sample, study participant questionnaire, and clinical info questionnaire will be shipped overnight 
to Namida Lab, Inc. 
 
5. Sub-Study Procedures: 
 
N/A 
 
6. Criteria for Inclusion of Subjects: 
 
Subjects may be invited to participate in this study if the individual is an adult woman and has recently 
been diagnosed with breast cancer. 
 
7. Criteria for Exclusion of Subjects: 
Table 3: Study Timeline
Objective
Wk 1 Wk 2 Wk 3 Wk 4 Wk 5 Wk 6 Wk 7 Wk 8 Wk 9 Wk 10 Wk 11 Wk 12
Namida Lab clinical team to train UTMB clinic 
staff on tear collection procedures.
Begin Study enrollment and sample acquisition
Conduct study enrollment and sample acquisition
Enroll last patient
Finalize any outstanding clinical forms
Close study

Subjects must be excluded from this study if they have already started treatment for the breast cancer 
diagnosis.  
Age less than 18 or more than 100 years. 
 
8. Sources of Research Material: 
 
Enrolled participants will be asked to complete a brief questionnaire with questions around reproductive 
health, personal history of cancer, and family history of cancer. In addition, a clinical questionnaire will be 
filled out for each participant with specific information relating to their breast health and current 
diagnosis. Both questionnaires can be found in the appendix section of this document.  
 
Tear specimens will be collected from each participant.  
 
9. Recruitment Methods and Consenting Process: 
 
Potential participants will be recruited from the patient population of the study's principal investigator 
(PI). Suppose there are potential study participants who are not patients of the principal investigator. In 
that case, the potential participant will not be contacted by the study staff. Unless the potential 
participant has been informed of the study by their medical provider, has expressed interest in receiving 
more information, and/or enrolling in the study. 
 
Individuals will be informed of the study and the opportunity to participate after diagnosis and before any 
treatment(s). Potential participants will be provided the information about the study, given time to 
review the informed consent form (IFC) and ask any questions before signing.    
 
 
10. Potential Risks: 
 
Possible risks from the collection of tears are rare but include conjunctivitis or scleral or corneal abrasion. 
There are no known occurrences of any of these risks from the previous IRB-approved study. 
Any time information is collected, there is a potential risk for loss of confidentiality. Every effort will be 
made to keep the information confidential; however, this cannot be guaranteed. 
 
11. Subject Safety and Data Monitoring: 
 
The Principal Investigator will monitor this study. 
 
Due to the minimal risk to the participants and short duration of participation, this study will be reviewed 
on an as necessary basis from the time the IRB approves it.  Suppose abnormal values are found or the 
testing fails in some manner. In that case, all investigators and collaborators will be informed 
immediately, and enrollment halted until the matter can be resolved.  The patients will not be informed 
of their results, and the tests will not be used to decide their treatment in any way. Any unexpected or 
adverse events that occur, the information on events will be sent to the IRB within 30 working days. 
 
12. Procedures to Maintain confidentiality: 
 
After sample collection, the sample will be placed in a tube with a three-to-four-digit code generated by 
Namida Lab, Inc. (For example, ADX-001). Corresponding labels will be provided for the study participant 
questionnaire, informed consent, and HIPPA form to ensure the sample and paperwork can be matched. 

Completed paperwork will remain locked in the Namida Lab, Inc. facility. It will only be accessible by the 
clinical study staff at Namida Lab, Inc. 
 
Any information obtained in this study will be kept confidential. Representatives from the UTMB 
Institutional Review Board, the United States Food and Drug Administration (FDA), or other institutional 
oversight offices may review records at any time.  If the results of this study are published in medical 
literature, participant's names will not be identified. 
 
13. Potential Benefits: 
 
Each study participant will receive a $25.00 gift card provided by Namida Lab, Inc. for participation. 
 
The results of the tear evaluation will not be disclosed to the patient or be used for their medical 
assessment or treatment.   
 
14. Biostatistics:  
 
Clinical acceptance criteria are defined as 100% agreement between the manual and automated 
classification of each sample.  
 
Namida Lab, Inc.'s CLIA Lab Medical Director established the sample size.  
 
 
15. Appendix 
 
 
 
 
 
 
Tear Based Breast Cancer Detection Participant Questionnaire 
 
Sample ID: 
 
Thank you for taking the time to participate in our study. Please answer the following questions to the 
best of your knowledge. 
 
Initials: ____________ 
 
Age: ____________                                          Sex: 
Male     
Female 
 
Height  
 
 
 
Weight 
 
Race: 
African/African American 
Arab/Persian 
Asian 
East Indian 
Hispanic/Latino/Latina 
Native American 
White/Caucasian 
Multiracial 
Prefer not to answer

Section 1:  In section 1 we need you to answer a few questions about your eye health: 
 
1. Have you been diagnosed by a physician with any of the following ocular disorders? 
Glaucoma                             
Macular Degeneration          
Cataracts                              
Dry Eye    
 None                          
 
2. Have you had Lasik surgery?   
Yes    
No        
 
3. Are you currently wearing contact lenses?   
No     
 Yes  
right eye only 
left eye only                    
 
4. Have you used of the following types of eye drops today? 
Red eye reducing    
OTC for allergies 
Prescription steroid for allergies     
General hydration 
Saline 
Drops for Glaucoma  
Other (Please describe):  
None 
 
Section 2: In section 2 we will cover a few questions about your reproductive health: 
We are asking the next few questions to gather information about additional hormones that you may be 
taking.  
 
5. Are you currently using any of the following types of Birth Control? 
Pill  Name if known: 
Implant 
Contraception Injection 
Contraceptive Patch                  
Intrauterine device (IUD) 
Vaginal Ring 
None 
 
6. Are you currently using any type of Hormone replacement therapy?  
Yes 
No 
 
7. Please select what hormones you are taking and/or the name of your medication: 
Testosterone 
Estrogen 
Progesterone 
Thyroid 
Other: 
 
8. Medication Name if known:  
 
9. At what age did your periods begin?  
 
10. Do you still have regular periods?     
Yes 
No 
 
11. If you answered No on question 10, please select one of the following reasons: 

 Perimenopause 
 Menopause 
 Hysterectomy 
 Response to Birth Control 
 Uterine Ablation 
 Unknown 
 Other (please describe):  
 
12. Do you still have one or both ovaries?    
 
Both       
One 
None 
 
 
Section 3: In section 3 you will find questions about medications you may have taken today. 
 
13. Have you taken any over the counter anti-inflammatory medication in the past 8 hours?   
No   
Yes  Describe: 
 
 14. Have you taken any prescription anti-inflammatory medication in the past 24 hours? 
No   
Yes  Describe: 
 
15. Have you taken any immune suppressant medication today?   
No  
Yes  Describe: 
  
Section 4: In section we need information on your history and your family history of cancer.  
16. In the chart below please list any cancers you have had.   
 
Cancer Type 
Year of diagnosis 
Treatment 
Treatment ongoing 
 
 
Chemo 
Surgery 
Radiation 
 
Yes 
No 
Further details: 
 
 
 
17. Please list below, any members of your family that have or have had: Breast, Ovarian, Prostate, 
Melanoma, Pancreatic, Stomach, Uterine, Thyroid, Colon, and/or Sarcoma.  
 
No Family history of cancer    
Family history unknown  
 
Immediate Family 
Mother  
 
Father     
 
Sibling   
 
Maternal Family 
Grandmother  
 
Grandfather    
 
Aunt   
 
Uncle 
 
Cousin 
 
Paternal Family 

Grandmother 
 
Grandfather 
 
Aunt 
 
Uncle 
 
Cousin  
 
 
18. What Procedure/s are you here for today? 
☐Screening Mammogram 
☐Diagnostic Mammogram 
☐Screening Ultrasound 
☐Diagnostic Ultrasound 
☐Biopsy 
☐Screening MRI 
☐Diagnostic MRI 
☐Other:  
 
 
 
 
 
Study Participant Stop Here 
 
 
Collection Date 
Collection Time 
Collectors Initials 
Distance for 
sample A 
Distance for 
Sample B 
 
 
 
 
 
Sample Collection 
Notes: 
 
 
 
 
 
For lab use only: 
Date Received 
 
Date Stored 
 
Time Received 
 
Time Stored 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Tear Based Breast Cancer Detection Clinical Information 
 
 
Sample ID: 
 
 
       Collection date:  
 
                Collection time:  
 
Initials: ____________ 
 
Age: ____________                         Patient Height: ________                Patient Weight: ___________                
 
1. Please select Patient's Breast Density Category: 
Fatty 
Scattered Fibroglandular Densities 
Heterogeneously Dense 
Extremely Dense 
 
2. Has patient had previous biopsies? 
Yes 
No 
 
3. Has the patient had at least one biopsy with atypical hyperplasia? 
Yes 
No 
 
4. For the procedures below, if conducted at your clinic, indicate procedure outcome: 
Screening Mammogram 
 
Normal  
Called back Date:   
Screening Ultrasound 
 
Normal  
Called back Date: 
Screening MRI 
 
 
Normal 
Called Back Date: 
 
Diagnostic Mammogram 
 
Normal 
Called Back Date: 
Diagnostic Ultrasound 
 
Normal 
Called Back Date: 
Diagnostic MRI 
 
 
Normal 
Called Back Date: 
 
Breast Biopsy    
Benign 
Positive 
Atypical 
 
Date:  
 
5. BiRads Score 
 1 
 2 
 3 
 4 
 5  
 6 
 
6. Which of the following was recommended for patient? 
6 month follow up (not high risk) 
6 month follow up (high risk screening protocol)  
Returned to normal screening 
Clinical Exam follow-up (patient to young for yearly mammogram screening) 
 
7. Were calcifications identified? 
Yes 
No  
 
8. If patient received a biopsy with Atypical results please indicate type of atypia:  
LCIS 
Atypical Ductal Hyperplasia  
Atypical Lobular Hyperplasia 
Other (describe): 
 

9. If Benign please select or describe: 
Hyperplasia of usual type 
Benign Calcifications 
Papilloma 
Sclerosing Adenosis 
Fat Epithelial Atypia 
Fibroadenoma 
Radial Scar 
Stromal Fibrosis 
Calcifications 
 Fibrocystic Change 
 
10. The following information is requested for positive diagnosis: 
Breast Cancer type and location 
    
 
 
Receptors:  
DCIS                 
 left 
right 
 bilateral 
 
ER+ 
IDC  
              
 left 
right 
 bilateral 
 
ER- 
ILC  
              
 left 
right 
 bilateral 
 
HER2 + 
Inflammatory             
 left 
right 
 bilateral 
 
HER2 – 
IMC  
              
 left 
right 
 bilateral 
 
PR + 
Other:  
 
 
 
 
 
               
PR- 
 
11. Metastasis: 
Yes: 
No 
 
12. Tumor Grade 
 
                                       13. Stage if known: 
GX  
 
 
 
 
 0 
G1-low grade 
 
 
 
 Ia 
 Ib 
G2-intermediate grade 
 
 
 IIa 
 IIb 
G3-high grade 
 
 
 
 IIIa 
 IIIb   
 IIIc 
G4-high grade 
 
 
 
 Iva   
 IVb    
 
14. Ki67 
 
 
 
 
 
15. Tumor/s Size 
High 
Borderline 
 
 
 
 
 
Low