SQA-VISION VALIDATION REPORT

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1 Objective SQA-VISION VALIDATION REPORT T test the new cnvenience features f the SQA-Visin vs. the SQA-V and manual semen assessment: Swim-up Density Gradient Lngevity Vitality Mrphlgy Cunter Scanning Debris/Rund Cells SQA-Visin Visualizatin Validatin These parameters were always available t the SQA-V custmers, but were tracked r recrded manually n separate pages r frms. The new SQA-Visin system is based n the SQA-V platfrm (1), but streamlines the assessment and data cllectin prcesses by allwing this infrmatin t be part f the standard patient recrd. The bjective f this trial is t determine that these semen assessment features perfrm as designed, and prvide the crrect results vs. reference methd (SQA-V and manual semen assessment where applicable) and vs. manufacturer claims. Nne f these cnvenience features include new algrithms fr reprting semen values. Additinally, sme f the features are nt run autmatically n the SQA-Visin, but are run manually using the visualizatin system f the SQA-Visin instead f the micrscpe. In these cases, the micrscpe will be cnsidered the GOLD STANDARD fr cmparisn. Study Design Tw clinical trials were cnducted: at MES Ltd., Israel (169 semen samples f the dnrs), at the University Hspital f Nantes, France (57 semen samples frm patients being evaluated at the andrlgy unit). In ttal, 226 human semen samples were tested. Types f semen samples/tests run: Fresh, Washed, Swim-up, Density Gradient, Lngevity, Vitality, Mrphlgy using the SQA-Visin cunter, samples with Debris/Rund Cells, Visualizatin cmpartment validatin. All semen samples were cllected, split int tw aliquts and run accrding t WHO 5 th editin manual (2) standards and per the manufacturer s prtcl fr cmparisn: Split int tw aliquts each hmgeneusly mixed semen sample after cmplete liquefactin If samples d nt liquefy cmpletely r are highly viscus, treat with chymtrypsin (QwikCheck Liquefactin Kit) and nly then split int tw aliquts Perfrm all testing at rm temperature (RT) Mtility and Prgressive Mtility Assessment: Assess ne aliqut first under the micrscpe in duplicate as defined by WHO 5 th editin manual, within 1 hur f cllectin (and directly fllwing sample liquefactin) and in parallel test the secnd aliqut n the SQA-Visin. Manual cncentratin : Assess using an Imprved Neubauer chamber fllwing WHO 5 th table fr cmparing duplicate cunts Cunt a minimum f 200 cells, twice as required by the WHO 5 th editin manual Use a minimum f 100 µl f raw semen fr any dilutins in rder t avid dilutin errrs Use psitive displacement calibrated lab pipettes File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 1 f 17

2 Manual mrphlgy: Prepare Papaniclau stained smears and assess them based n WHO 5 th guidelines fr nrmal/abnrmal mrphlgy SQA-V: Run the samples per SQA-V nscreen instructins fllwing the present prtcl guidelines (fr each sample, run in duplicate using the same capillary) Save results by clicking Imprt Test in the V-Sperm SQA-Visin: Run the samples per SQA-Visin nscreen instructins fllwing the present prtcl guidelines (fr each sample, run in duplicate using the same capillary) SQA-Visin Visualizatin: The SQA-Visin Visualizatin was validated vs. the micrscpe (Neubauer chamber) and vs. SQA-V Visualizatin as fllws: Run the same varying cncentratins f 20 sperm samples and 8 latex beads samples f varying cncentratins n the SQA-Visin Visualizatin screen (manual mde) Run the same samples using the Imprved Neubauer cunting chamber and additinally n the SQA-V Visualizatin and cmpare results. Materials and Methds Equipment and materials SQA-Visin system SQA-V System + V-Sperm Imprved Neubauer chamber Phase cntrast micrscpe Centrifuge with 15 ml tubes fr centrifugatin Freezing vials Egg Ylk Freezing media Pasteur pipettes Standard lab slides and cver slips 22 X 22 mm Fixed cverslip slides SQA-V capillaries CellVu pre-stained mrphlgy slides Papaniclau reagents Esin reagent fr Vitality test (see recipe belw) Optiprep density gradient medium 2.77% and 1.56% NaCl Fresh, Washed, Swim-up, Density Gradient and Lngevity semen samples Lab pipettes + tips, calculatrs, cunters, data cllectin sheets 10 ml cups with caps Glves, Kimwipes QwikCheck Beads, Test Strips, Liquefactin and Dilutin kits SQA-Visin default settings Cncentratin standard: Neubauer Testing criteria: WHO 5 th File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 2 f 17

3 Autmated pening f vide screen fr debris/rund cells scanning: Cncentratin < 15M/ml OR Mtility < 40% Lngevity: 3 tests - initial test is run at 0 pint, then tw mre tests in 2h intervals; all three recrds are shwn in ne reprt Manual Mrphlgy set t Nrmal/Abnrmal Vitality: Yes QC: QwikCheck beads accrding t the kit labeling (Run 3 levels f QwikCheck beads cntrl daily befre testing samples). Sample inclusin/exclusin criteria Highly viscus r incmpletely liquefied semen samples treated with the QwikCheck Liquefactin kit are included in the trial Semen samples with vlume < 1 ml are excluded frm the trial Samples with methdlgical issues are excluded frm the trial (6 samples) Sample cllectin and preparatin Fresh Samples: Cllect Fresh samples accrding t WHO 5 th manual prcedure Liquefy fr up t 1 hur. If nt liquefied, treat with 1 vial f QwikCheck Liquefactin Kit (α-chymtrypsin) and test after cmplete liquefactin (within 10 minutes) Mix each sample thrughly befre prceeding Mark each sample with a unique sample #, and then split int tw aliquts: SQA-Visin and SQA-V: ml Micrscpe: remaining sample with minimum f 100µl D nt dilute the sample befre splitting it int aliquts. If diluting, fllw the manufacturer s instructins based n the methd used Run the sample in the Fresh mde f the SQA-Visin and SQA-V Washed Samples: Add 2 ml semen and up t 10 ml Earle's buffer t the centrifuge tube, mix thrughly Centrifuge at 220g fr 10 min Remve the supernatant Re-suspend the pellet with 1 ml Earle's buffer Run the sample in the Washed mde f the SQA-Visin and SQA-V Swim-up Samples: Transfer 2 ml f a Fresh semen sample int the test tube. Layer 2ml Earle's buffer n the tp (d nt mix with sample) Incline the tube t a 45 0 angle Incubate the tube fr 1 hur at 37 0 C Cllect the upper layer f Earle's buffer (~ 2 ml) Run the cllected sample in the Swim-up mde f the SQA-Visin and the lw vlume washed mde f the SQA-V File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 3 f 17

4 Density Gradient Samples: Prepare the Density Media: 40% OptiPrep: 1.35 ml f stck OptiPrep ml 2.77% NaCl 25% OptiPrep: 0.85 ml f stck OptiPrep ml 1.56% NaCl Place up t 1 ml (abut half f the semen vlume) f 40% Gradient slutin int a tube. Carefully pur up t 1 ml (abut half f semen vlume) f 25% Gradient slutin, alng the wall f the tube in rder t create tw layers. Mix the semen sample and add the semen gently alng the tube wall t create a third layer. If the semen vlume is mre than 3ml distribute the samples int tw tubes. Centrifuge at 300g (1350 rpm) fr 20 minutes. Cllect the pellet int a new tube. Re-suspend the pellet with 1 ml Earle's buffer. Run the cllected sample in the Density Gradient mde f SQA-Visin and lw vlume washed mde f the SQA-V. Lngevity Test: Fill the capillary with fresh sample, run the initial test in the SQA-Visin Lngevity mde and in the SQA-V Expel the sample frm the capillary int a 10-ml vial and incubate fr 2 hurs After incubatin, re-run the test in the SQA-Visin Lngevity mde and manually fr mtility and prgressive mtility Save results Repeat the same prcedure after 4 hurs Running Samples Parameters fr cmparisn: The SQA-Visin semi-autmated results btained by running fresh, washed and lngevity samples with mderate/high level f debris/rund cells r Cncentratin r MSC beynd autmated dynamic range (<2M/ml) vs. the micrscpe (manual): cncentratin, mtility, prgressive mtility and mrphlgy The SQA-Visin fully autmated mde (fresh, washed and lngevity samples with a lw level f debris/rund cells and Cncentratin and MSC >= 2M/ml) vs. the SQA-V: Cncentratin, mtility, prgressive mtility and mrphlgy The SQA-Visin fully autmated mde fr swim-up and density gradient samples vs. the SQA-V: Mtile Sperm Cncentratin (MSC) and Prgressively Mtile Sperm Cncentratin (PMSC) reprted by bth systems The SQA-Visin CellVu Mrphlgy t micrscpe (manual) Papaniclau (PAP) Mrphlgy The SQA-Visin Vitality t manual (micrscpe) Vitality SQA-Visin and SQA-V sample testing: Turn n the SQA-Visin and the SQA-V. Activate the Visin HOME screen and V-Sperm. Set the SQA-V and SQA-Visin default settings accrding t the setting instructins listed abve. File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 4 f 17

5 Run 3 levels f QwikCheck Beads cntrl material befre testing samples. Prepare the Mrphlgy slide in advance: CellVu pre-stained slide, 5 µl semen and 22mm x 22mm cverslip pressed by finger t make a cell mnlayer. Prepare in advance Vitality slide: Reagent Preparatin 1. NaCl, 0.9% (w/v): disslve 0.9 g f NaCl in 100 ml purified water 2. Esin Y, 0.5% (w/v): disslve 0.5 g f esin Y (clr index 45380) in 100 ml f 0.9% NaCl Prcedure 1. Mix the semen sample well 2. Remve an aliqut f 5 µl f semen and cmbine with 5 µl f esin slutin n a micrscpe slide 3. Mix with a pipette tip, swirling the sample n the slide 4. Cver with a 22 mm x 22 mm cverslip and leave at least fr 30 secnds Enter the patient/sample data. Assess the ph and WBC using QwikCheck Test Strips and enter their values where apprpriate. Fllw the nscreen instructins f each system and run the test. If the results fall belw the cut-ffs fr lw quality semen (Cncentratin r MSC <2M/ml), the SQA- Visin will pen the manual sample assessment screen autmatically. The SQA-V autmatically displays a limited reprt in this case (Cncentratin <2M/ml, etc.); Then the SQA-Visin manual results will be cmpared t the micrscpe manual data. T use the Visin Manual Assessment Screen: Fill a fixed cverslip slide with semen sample per prduct and SQA-Visin nscreen instructins. View the slide in the visualizatin cmpartment f the SQA-Visin and cunt the mtile and immtile sperm cells manually (at least 200 spermatza in ~ fields) Enter the manual data in the fields prvided and btain a reprt. Run a secnd testing cycle using the same testing capillary and sample. Run the Mrphlgy (CellVu) and Vitality (esin) tests. Insert the Mrphlgy slide int the SQA-Visin visualizatin cmpartment and assess nrmal and abnrmal spermatza using the cunter (at least 200 spermatza in apprx fields f view). Insert the Vitality slide int the SQA-Visin visualizatin cmpartment and assess using the cunter. Cunt live (nn-stained) and dead (stained) spermatza (at least 200 spermatza twice in apprx fields f view) at zm ut. In parallel, examine the slides under the micrscpe. Save the data. Manual Mtility: Assess mtility within ne hur f cllectin, immediately after full liquefactin f the sample. Run all tests in the SQA-Visin, SQA-V and the micrscpe in parallel r within 2 minutes f each ther t eliminate the effect f time difference. Assessing mtility under the micrscpe 1. Cunt each sample in duplicate 2. Mix the semen sample well File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 5 f 17

6 3. Remve an aliqut f semen immediately after mixing, allwing n time fr the spermatza t settle 4. Fr each replicate, prepare a wet preparatin apprximately 20 µm deep 5. Prepare standard slides accrding t WHO 5 th manual (p. 21): Lad 10 µl f semen nt a standard micrscpe slide and cver with 22mm X 22mm cverslip prviding 20-micrn depth 6. Wait fr the sample t stp drifting (within 60 secnds) 7. Examine the slide under phase-cntrast ptics at x200 r x400 magnificatin 8. Assess 200 spermatza per replicate t determine the percentage f different mtile categries 9. Per WHO 5 th ed. manual (p. 22), the mtility f each spermatzn is graded as fllws: Prgressive mtility (PR): spermatza mving actively, either linearly r in a large circle, regardless f speed Nn-prgressive mtility (NP): all ther patterns f mtility with an absence f prgressin, e.g., swimming in small circles, the flagella frce hardly displacing the head, r when nly a flagellar beat can be bserved Immtility (IM): n mvement 10. Cmpare the replicate values t check if they are acceptably clse per WHO 5th table 2.1. If s, average results; if nt, prepare and assess new samples 11. Recrd Mtility values f each sample in the Assessment Frm Manual Sperm Cncentratin: 1. Use the Neubauer cunting chamber fr the assessment f cncentratin fllwing the WHO 5 th table (table 2.3 belw) 2. Cunt a minimum f 200 cells twice 3. T cmpare duplicate cunt accuracy use WHO 5th table 2.4 (belw) 4. Recrd the Sperm Cncentratin values fr each sample in the Assessment Frm prvided Manual Mrphlgy: Fllw the steps utlined in the WHO 5 th manual (Sectin 2.13 Sperm mrphlgy, p. 56) recmmended fr testing mrphlgy: 1. Prepare a minimum f tw smears f fresh semen (10µl) fr duplicate measurements 2. Stain using Papaniclau methd 3. Munt the slide with a cverslip if the slide is t be kept fr a lng time Mrphlgy Cunting 1. Examine the slide under il immersin with bright field ptics at x1000 magnificatin 2. Mrphlgical evaluatin shuld be perfrmed in several systematically selected areas f the slide 3. Cunt all spermatza per WHO 5 th criteria, nting nly the # Nrmal and # Abnrmal frms (nt the differential f defects) in rder t establish the % nrmal frms. If apprpriate, cunt differential mrphlgy as well 4. Cmpare replicate values t WHO 5th table 2.1 t determine acceptability; If nt, re-read the slides 5. Recrd the Mrphlgy values f each sample in the Assessment Frm prvided File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 6 f 17

7 File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 7 f 17

8 Statistics and Acceptance Criteria Statistical analysis was perfrmed using the MedCalc and Excel prgrams: Crrelatin Sensitivity (an ability t detect abnrmal cases vs. reference methd) Specificity (an ability t detect nrmal cases vs. reference methd) Precisin: CV (cefficient f variatin) Acceptance Criteria is based n the SQA-Visin prduct perfrmance claims (applicable t any sample type/test results including Lngevity presented in the study): Crrelatin: Sperm Cncentratin: >= 0.90 Ttal Mtile (PR + NP): >= 0.80 (in Lngevity, each step is cmpared vs. reference methd as a separate test) Prgressive Mtility (PR): >= 0.80 (same as abve) MSC: >= 0.85 PMSC: >= 0.80 Vitality: >= 0.90 Sensitivity: Sperm Cncentratin: >= 90% Ttal Mtile (PR + NP): >= 85% Prgressive Mtility (PR): >= 85% Nrmal Frms: >= 80% MSC: >= 85% PMSC: >= 85% Vitality: >= 90% Specificity: Sperm Cncentratin: >= 85% Ttal Mtile (PR+NP): >= 80% Prgressive Mtility (PR): >= 80% Nrmal Frms: >= 90% MSC: >= 80% PMSC: >= 80% Vitality: >= 90% SQA-Visin cefficients f variatin (CV) fr Cncentratin, Ttal Mtile and Prgressive Mtility parameters <10% SQA-Visin Visualizatin: Crrelatin t Neubauer >=0.9 File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 8 f 17

9 Results Sperm Cncentratin The Sperm Cncentratin results frm a variety f semen sample types run n the SQA-Visin were statistically cmpared t the SQA-V and t manual micrscpe results (fr semi-autmated r the manual mde f the SQA- Visin). The results are summarized in the Table 1 belw: TP TN FP FN Table 1: SQA-Visin Sperm Cncentratin Sensitivity 1,2 Specificity 1,2 Crrelatin (r) SQA-Visin Intra-device CV MES Ltd., Israel (n = 169) University Hspital f Nantes, France (n = 57) Sperm Cncentratin reference value used fr calculatin sensitivity and specificity per WHO 5th ed. manual is 15 M/ml 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The crrelatin cefficients f the SQA-Visin Sperm Cncentratin t the SQA-V + manual results are high (0.98; 0.95). They exceed the acceptance criteria. This high crrelatin shws strng agreement between the methds. The tight relatinship between Sperm Cncentratin reprted by the SQA-Visin and SQA-V + manual methds is demnstrated graphically in Figure 1: Figure 1. SQA-Visin vs. SQA-V and manual Sperm Cncentratin The SQA-Visin Sperm Cncentratin Sensitivity and Specificity btained in the different trials greatly exceeds the acceptance cutff f 90% and 85% crrespndingly. The SQA-Visin als demnstrated excellent precisin fr assessing Sperm Cncentratin with lw cefficients f variatin f 4.5% and 4.1%, which meet the acceptance requirements. File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 9 f 17

10 Ttal Mtility (PR + NP) The Ttal Mtility sperm results frm a variety f semen sample types run n the SQA-Visin were statistically cmpared t the SQA-V and t manual micrscpe results (fr semi-autmated r the manual mde f the SQA- Visin). The results are summarized in the Table 2 belw: TP TN FP FN Table 2: SQA-Visin Ttal Mtility Sensitivity 1,2 Specificity 1,2 Crrelatin (r) SQA-Visin Intra-device CV MES Ltd., Israel (n = 178) University Hspital f Nantes, France (n = 54) Ttal Mtility reference value used fr calculatin sensitivity and specificity per WHO 5th ed. manual is 40% 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The crrelatin cefficients f the SQA-Visin Ttal Mtility t the SQA-V + manual results are high (0.92; 0.85) and these results equal r exceed the acceptance criteria. This high crrelatin shws strng agreement between the methds. This tight relatinship between Ttal Mtility reprted by the SQA-Visin and the SQA-V + manual methds is demnstrated graphically in Figure 2: Figure 2. SQA-Visin vs. SQA-V and manual Ttal Mtility The SQA-Visin Ttal Mtility Sensitivity and Specificity are high and exceed the acceptance cutff. The SQA- Visin als demnstrated excellent precisin when measuring Ttal Mtile sperm demnstrated by lw cefficients f variatin f (6.2%; 5.0%) which meet the acceptance requirements. File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 10 f 17

11 Prgressive Mtility (PR) The Prgressive Mtility results frm a variety f semen sample types run n the SQA-Visin were statistically cmpared t the SQA-V and t manual micrscpe results (fr semi-autmated r manual mde f the SQA- Visin). The results are summarized in the Table 3: TP TN FP FN Table 3: SQA-Visin Prgressive Mtility Sensitivity 1,2 Specificity 1,2 Crrelatin (r) SQA-Visin Intra-device CV MES Ltd., Israel (n = 161) University Hspital f Nantes, France (n = 36) Prgressive Mtility reference value used fr calculatin sensitivity and specificity per WHO 5th ed. manual is 32% 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The crrelatin cefficients f the SQA-Visin Prgressive Mtility results vs. the SQA-V + manual results are high (0.91; 0.85) and are equal t r exceed the acceptance criteria. This high crrelatin shws strng agreement between the methds. This tight relatinship between Prgressive Mtility reprted by the SQA-Visin and the SQA-V + manual methd is demnstrated graphically in Figure 3: Figure 3. SQA-Visin vs. SQA-V and manual Prgressive Mtility The SQA-Visin Prgressive Mtility Sensitivity and Specificity are high and exceed the acceptance cutff. The data shws that nly a few samples were categrized differently between the tw systems. This can be due t the subjectivity f the manual Prgressive Mtility assessment. The SQA-Visin als demnstrated excellent precisin fr assessing Prgressively Mtile sperm as demnstrated by lw cefficients f variatin which meet the acceptance requirements. File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 11 f 17

12 Samples with Debris and/r Rund Cells An ejaculate cntains cells ther than spermatza, sme f which may be clinically relevant. These include epithelial cells frm the geniturinary tract, as well as leukcytes and immature germ cells, the latter tw cllectively referred t as rund cells (3). Besides these elements, the ejaculate may cntain debris (particles f brken cells). These inclusins are interfering factrs fr bth manual and autmated semen assessment (2). The results f running semen samples cntaining debris and/r rund cells n the SQA-Visin as cmpared t running these same samples manually under the micrscpe are summarized in Table 4 belw. Table 4: SQA-Visin Samples with Debris and /r Rund Cells Semen Parameters TP TN FP FN Sensitivity 1,2 Specificity 1,2 Sperm Cncentratin (x10 6 /ml) n = 38 Ttal Mtile PR + NP n = 38 Prgressive PR n = 36 MES Ltd., Israel SQA-Visin Intradevice CV University Hspital f Nantes, France Sperm Cncentratin (x10 6 /ml) n = 17 Ttal Mtile PR + NP n = Prgressive PR n = The reference values fr semen parameters used fr calculatin sensitivity and specificity were per WHO 5th ed. manual 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The results demnstrate that the SQA-Visin Sensitivity and Specificity fr Sperm Cncentratin, Ttal Mtility and Prgressive Mtility are very high which equals r exceeds the acceptance criteria. The SQA-Visin als demnstrated very gd precisin as shwn by cefficients f variatin varying frm 3.1% t 8.5%, which meet the pass criteria. Swim-up and Density Gradient Samples Swim-up and Density Gradient prcedures are used when spermatza need t be separated frm the seminal plasma. The separatin f human spermatza frm the seminal plasma t yield a final preparatin cntaining a high percentage f mrphlgically nrmal and mtile cells, free frm debris, nn-germ cells and dead spermatza, is imprtant fr clinical practice (2). The chice f sperm preparatin technique is dictated by the nature f the semen sample (4). Fr example, a direct swim-up technique is ften used when the semen samples are cnsidered t be largely nrmal, whereas in cases f severe ligzspermia, teratzspermia r asthenzspermia, density gradients are usually preferred File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 12 f 17

13 because f the greater ttal number f mtile spermatza recvered. Density-gradients can als be altered t ptimize the handling f specific prperties f individual samples (2). The Swim-up and Density Gradient mdes f the SQA-Visin and the Washed lw vlume mde f the SQA-V require a minimum f 20 µl f semen vlume (nly the frnt sectin f the testing capillary is filled). In these cases, mtility-related parameters such as Mtile and Prgressively Mtile Sperm Cncentratin (MSC and PMSC crrespndingly) are reprted. The results f running Swim-up and Density Gradient samples n the SQA-Visin cmpared t the SQA-V are summarized in Table 5: Table 5: SQA-Visin Swim-up and Density Gradient Samples Semen Parameters TP TN FP FN Sensitivity 1,2 Specificity 1,2 Crrelatin (r) SQA-Visin Intra-device CV MES Ltd., Israel MSC (x10 6 /ml) n = PMSC (x10 6 /ml) n = The MSC and PMSC reference values used fr calculatin sensitivity and specificity are 6.0 and 5.0 (x10 6 /ml) crrespndingly 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The MSC and PMSC Sensitivity and Specificity fr bth systems were 100%. The crrelatin cefficients f these parameters btained using the SQA-Visin system vs. SQA-V are 0.99 (bth) which exceeds the acceptance criteria. The high sensitivity, specificity and crrelatins demnstrate a very high level f agreement between the methds. This tight relatinship between the semen parameters reprted by the SQA-Visin and the SQA-V is demnstrated graphically in Figures 4 and 5 belw. Figure 4. SQA-Visin vs. SQA-V MSC Figure 5. SQA-Visin vs. SQA-V PMSC The SQA-Visin als demnstrated gd precisin when assessing MSC and PMSC as evidenced by cefficients f variatin belw 10% which meet the acceptance requirements. File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 13 f 17

14 Lngevity Lngevity testing is a requirement in many labratries. The length f time sperm are able t remain viable after ejaculatin may be f cncern in sme cases. The sperm lngevity test assesses percent Ttal Mtile and Prgressive sperm at given time intervals. The results f running lngevity tests n the SQA-Visin cmpared t the SQA-V and the manual methd (fr cmparing the semi-autmated r manual mde f the SQA-Visin) are summarized in Table 6 belw: Table 6: Lngevity Semen Parameters TP TN FP FN Sensitivity 1,2 Specificity 1,2 Crrelatin (r) MES Ltd., Israel Ttal Mtile PR + NP n = Prgressive PR n = University Hspital f Nantes, France Ttal Mtile PR + NP n = Prgressive PR n = T Reference values fr semen parameters used fr calculating sensitivity/specificity are per WHO 5th ed. manual 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The results demnstrate high crrelatin cefficients fr the SQA-Visin percent Ttal Mtile and Prgressive Mtility parameters t the SQA-V + manual results; varying frm 0.84 t 0.93 which exceeds the acceptance criteria. This high crrelatin shws gd agreement between the methds. The relatinship between Ttal Mtile and Prgressive Mtility parameters reprted by the SQA-Visin and SQA-V + manual methd in the Lngevity samples is demnstrated graphically in Figures 6 and 7: Fig. 6. SQA-Visin vs. SQA-V & manual Ttal Mtile Fig. 7. SQA-Visin vs. SQA-V & manual Prgressive The sensitivity f Ttal Mtile and Prgressive Mtility parameters assessed by the SQA-Visin fr Lngevity testing is high varying frm 87.5% t 100% which exceed the acceptance cutff. The specificity f these parameters fr the Lngevity tests is als high varying frm 83.3% t 100% which is greater than the acceptance criteria. File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 14 f 17

15 Vitality Sperm Vitality, as estimated by assessing the membrane integrity f the cells, may be determined rutinely n all samples, but is especially imprtant fr samples with less than abut 40% prgressively mtile spermatza. This test can prvide a check n the mtility evaluatin, since the percentage f dead cells shuld nt exceed (within sampling errr) the percentage f immtile spermatza. The percentage f viable cells nrmally exceeds that f mtile cells. The percentage f live spermatza is assessed by identifying thse with an intact cell membrane, using dye exclusin r hyptnic swelling. The principle f the dye exclusin methd is that damaged plasma membranes, such as thse fund in nn-vital (dead) cells, allw entry f membrane-impermeant stains (2). The results f assessing esin-stained semen fr Vitality using the SQA-Visin visualizatin cmpartment cmpared t the micrscpe manual methd are summarized in Table 7 belw: Table 7: Vitality (% Live) TP TN FP FN Sensitivity 1,2 Specificity 1,2 Crrelatin (r) MES Ltd., Israel (n = 49) Vitality reference value per WHO 5th ed. manual is 58%. 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The crrelatin cefficient f the SQA-Visin Vitality t the manual results is 0.91 which exceeds the acceptance criteria. This high crrelatin shws gd agreement between the methds. The tight relatinship between Vitality reprted by the SQA-Visin and the manual methd is demnstrated graphically in Figure 8: Figure 8. SQA-Visin vs. manual Vitality The sensitivity and specificity f the SQA-Visin t assess Vitality vs. the manual results is very high: 100% and 94.9% respectively which exceeds the acceptance cutff. File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 15 f 17

16 Mrphlgy Summarized in Table 8 belw are the results f Mrphlgically Nrmal Frms btained by: SQA-Visin autmatically The use f CellVu pre-stained slides cunted in the SQA-Visin visualizatin cmpartment Manual assessment f Papaniclau (PAP) smears Table 8: Nrmal Frms (Mrphlgy) Semen Parameters TP TN FP FN Sensitivity 1,2 Specificity 1,2 MES Ltd., Israel SQA-Visin aut vs. CellVu (n = 45) SQA-Visin CellVu vs. Manual PAP (n = 50) University Hspital f Nantes, France SQA-Visin aut vs. Manual PAP (n = 43) Nrmal Frms (Mrphlgy) reference value per WHO 5th ed. manual is 4%. 2 Sensitivity = TP / (TP + FN) * 100; Specificity = TN / (TN + FP) * 100 Ntes: TP - True Psitive (crrectly classified as psitive - presence f disease) TN - True Negative (crrectly classified as negative - absence f disease) FP - False Psitive (nt crrectly classified as psitive - absence f disease) FN - False Negative (nt crrectly classified as negative - presence f disease) The sensitivity and specificity f the SQA-Visin nrmal mrphlgy assessed autmatically vs. the SQA-Visin CellVu data is: 80.0% and 91.4% respectively, which exceed the acceptance cutff. A cmparisn f the SQA- Visin CellVu mrphlgy with the manual PAP data resulted in sensitivity and specificity f 80.0% and 92.5% crrespndingly which als exceeds the acceptance criteria. The SQA-Visin autmated percent nrmal frms cmpared t manual PAP data shwed sensitivity f 83.3% and specificity f 96.8% (bth values are higher than the acceptance criteria). Based n this data it is pssible t cnclude that all three methds are in agreement. SQA-Visin Visualizatin Screen Validatin The SQA-Visin Visualizatin Screen was validated by cunting varying cncentratins f spermatza (20 samples) and latex beads (8 samples) using: SQA-Visin Visualizatin Imprved Neubauer cunting chamber Additinally, the SQA-Visin Visualizatin was validated vs. the SQA-V vide screen by cunting 8 sperm samples and 8 samples f latex beads. The results are summarized in Table 9 belw: Parameter Table 9: SQA-Visin Visualizatin Validatin SQA-Visin Visualizatin Cunt Manual Cunt Average SD Average SD Crrelatin (r) MES Ltd., Israel Cunt vs. Neubauer (x10 6 /ml) n = Cunt vs. SQA-V visual screen (x10 6 /ml) n = It is seen that the average values btained by manually cunting sperm cells and latex beads using the SQA-Visin Visualizatin System and the Imprved Neubauer cunting chamber are similar and the standard deviatins (SD) are very clse. A similar relatinship is demnstrated when cmparing the results f cunting sperm cells and File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 16 f 17

17 latex beads using the SQA-Visin vs. SQA-V visualizatin screen. This indicates that there are n systematic discrepancies between the methds which were cmpared. The data set als demnstrates a high level f crrelatin which is shwn graphically belw: Cnclusins Based n results f the multicenter trials, the SQA-Visin demnstrated that all assessed parameters passed the acceptance criteria. A high level f crrelatin between the SQA-Visin and SQA-V autmated + manual results (used as the standard when apprpriate) demnstrated agreement between these methds. The Sensitivity and Specificity levels fr all assessed parameters were higher than the acceptance criteria. The SQA-Visin demnstrated a high level f precisin (all CVs are belw 10%). The SQA-Visin Visualizatin can be used fr manual cunting as an accessry tl. The verall cnclusin is that the SQA-Visin system successfully passed clinical validatin shwing substantial equivalence with the SQA-V system. References 1. Agarwal A., Sharma R.K. (2007). Autmatin is the key t standardized semen analysis using the autmated SQA-V sperm quality analyzer. Fertility and Sterility, 87(1): WHO labratry manual fr the examinatin and prcessing f human semen - 5th ed., Wrld Health Organizatin Jhanissn E et al. (2000). Evaluatin f rund cells in semen analysis: a cmparative study. Human Reprductin Update, 6: Canale D et al. (1994). Inter- and intra-individual variability f sperm mrphlgy after selectin with three different techniques: layering, swimup frm pellet and Percll. Jurnal f Endcrinlgical Investigatin, 17: File: SQA-VISION VALIDATION REPORT_12_NOV_14 Page 17 f 17

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