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BIOMEDICAL TECHNOLOGY WINGSREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL SCIENCES AND TECHNOLOGY

Thiruvananthapuram, INDIA - 695 012

STUDY REPORT

Study Title: Evaluation of Coronary Stent for in vitro hemo compatibility and cyto

compatibility

Study number: TPTRU017

Report number: SRTRU003.07

Revision No: 000

of 15

Date effective0 Issued to

Name & Address of theSponsor

Sahajanand Medical Technologies Pvt LtdSaidpura, Surat-395009

Gujarat

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1 Summary of the study

The objective of this study was to evaluate and compare the

hemocompatibility of modified stents (devices) against bare metal stents

submitted by the sponsor. Tests recommended in IS010993 part4 were

selected for evaluation of hemocompatibility. Devices were exposed to

anticoagulated human blood or platelet rich plasma and analysis of

blood/PRP before and after exposure was done for various parameters, to

determine the effect of the device on blood components/systems. Devices

were analyzed for qualitative/quantitative leukocyte and platelet adhesion.

Hemocompatibility studies using whole blood showed significant hemolysis

with all types of devices including the modified stent (Supracore). Though

there is reduction in platelet count after exposure to whole blood, PRP

exposure and 1-125 PRP exposure results indicate that platelet adhesion is

significant only on bare devices. Fibrinogen consumption and related

prolongation in plasma clotting is also observed in the case of all devices as

compared to reference. In studies with platelets, some effect is seen on

platelet aggregability and secretion which is the least with polymer alone

coated samples. Overall, there is no severe effect on any of the parameters

that were studied to assess hemocompatibility, except that hemolysis is

significant.

2 Identification of study

2.1 Purpose: To evaluate the in vitro hemocompatibility of thedevices using anticoagulated human blood.

2.2 Test Item & reference Item

2.2.1 Name of the Test Item:

Stents: Co-Cr, Polymer alone coated and Supracore(6 Replicates for each device)

Signature ofStudy Director ch~ BMT WING

SCTIMST

ReportNo: SRTRU003.07 Volume No. 02STUDY Page No: 2 of 15 Date effective: 17 Feb 2007

REPORT ControlStatus:

Evaluation of Coronary Stent for in vitro hemo compatibility

2.2.2

2.2.3

2.2.4

2.2.5

2.2.6

2.2.7

Preparation of the Test Item, if applicable

Source of the Test Item: Sahajanand Medical TechnologiesPrivate Ltd

Characterization of Test Item, if applicable: NA

Name of the Reference Item: None

Preparation of the Reference Item: NA

Source of the Reference Item: NA

3 Study Sponsor & Test Facility

3.1 Name and address of the sponsor: Ms. Prathibha, Sahajanand MedicalTechnologies Pvt Ltd

3.2 Name of the study monitor, if any.

3.3 Name and address of the test facility and test sites involved:Thrombosis Research Unit

Biomedical Technology Wing,

Poojapura

Thiruvananthapuram- 695012

Kerala, INDIA.

3.4 Name and address of the study director:

Lissy K. Krishnan, Scientist F, TRU, BMT Wing, SCTIMST, Trivndrum-695012.

3.5 Name and address of the principal investigator, if any.

Krishna Prasad C, Project Scientist, TRU, BMT Wing

4 Study dates

4.1Dateof Samplesubmission:22-4-06to 1-7-06

4.2 Dates of Testing: 23-08-06 to 15-01-07

Signature ofStudy Director ~ BMT WING

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5 Description of Methods

5.1 Interaction of devices with Whole Blood (WB):

Interactionof materialswith WB was done underagitation to analyzethe

effect of device on haematologyparameters.

5.1.a Exposure of devices to WB

Blood from human volunteer was collected into the anticoagulant, CPO.

Replicates of test devices were placed in separate polystyrene vials and were

immersed in phosphate buffered saline for 5 min before they were exposed to

blood. To each vial that contains the test devices, blood was added and an initial

sample was collected immediately for analysis. The remaining blood was

exposed to the respective device for 30 min under agitation at 75 :t 5 rpm using

an Environ shaker thermo stated at 35 :t 2°C (WPTRU012). The blood sample

and the device were then analyzed. Six numbers of empty polystyrene vials

were exposed with blood as reference.

5.1.b. Consumption of platelets and leukocytes (WBC) by cell counts

The count reduction in blood was estimated by detecting the counts in

initial and 30 min samples collected from step 5.1.a., using Haematology

Analyzer Sysmex K 4500 as per WPTRU015. The equipment calibration was

verified using traceable control.

5.1.c. Plasma coagulation (Fibrinogen and Partial Thromboplastin timeassay)

The initial and 30 min samples collected from step 5.l.a, were centrifuged

at 4000 rpm for 15 min as per WPTRU006 and platelet poor plasma (PPP) was

aspirated. Fibrinogen was measured in each PPP sample by using a reagent kit

obtained from Oiagnostica Stago (France) on Start 4, coagulation analyzer by

method WPTRU026. Partial thromboplastin time in each PPP sample was

detected using a reagent kit obtained from Oiagnostica Stago (France) oh Start

4, coagulation analyzer as per WPTRU023.

Signature ofStudy Director d~A~~

BMT WINGSCTIMST

ReportNo: SRTRU003.07 Volume No. 02STUDY


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5.1.d. Percentage Hemolysis

The total hemoglobin in the initial samples was measured using automatic

haematology analyzer (Sysmex K 4500) as per WPTRU015. The free

hemoglobin liberated in to plasma (separated as in step 5.l.c) after 30 min

exposure was measured in each sample using Diode Array Spectrophotometer

as per WPTRU022 and the percentage hemolysis was calculated using the

formula (Free Hb/Total Hb) x 100.

5.1.e. Qualitative Analysis of Leukocyte Adhesion

The devices exposed to blood were rinsed immediately with PBS to

remove blood completely, fixed with 1% glutaraldehyde for 1h and the cells

attached to the devices were identified using Geimsa stain and were analyzed as

per work procedure WPTRU028. The devices were cut open and the luminal side

was analyzed under light microscope (Leica IMR) for presence of leukocytes.

Representative photomigraphs are taken and documented.

5.2 Interaction of Device with Platelet Rich Plasma (PRP)

Interaction of device with platelet rich plasma was done under dynamic

condition to understand the effect of device on platelets and deposition of

platelets to the device.

5.2.a. Exposure of Devices to PRP

Blood from human volunteer was collected into the anti coagulant; CPO.

Blood was centrifuged at 2500 rpm for 5 min as per the method described in

WPTRU005. The platelet rich plasma (PRP) was collected. The test devices that

were expanded into the lumen of silicone tube (done by sponsor) was connected

and perfused with phosphate buffered saline for 5 min. After draining the PBS,

PRP was perfused as per WPTRU010 at a flow rate of 50 ml min-1 and within 1

min an initial sample was collected for analysis. After perfusion for 30 min, the

PRP samples were collected for analysis. Empty silicone tubes of the same

dimension without devices were perfused with PRP as reference. Devices were


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rinsed thoroughly and were processed for analysis using scanning electron

microscope.

5.2.b. Platelet Count ReductionThe count reduction was estimated by detecting the counts in PRP

samples collected from step 5.2.a, using Haematology Analyzer Sysmex K 4500

as per WPTRU015. The equipment calibration was verified using traceable

control.

5.2. c. Platelet Function

The amplitude of platelet aggregation in response to agonists, ADP and collagen

were determined as per the work procedure WPTRU017, in samples from step 5.

2.a.

5.2. d. Platelet SecretionThe PRP samples collected in step 5.2.a were centrifuged and the PPP

was obtained using the procedure WPTRU006. The platelet factor 4 (PF4) was

analyzed with commercially available ELISA kit (Hyphen Biomed, France) as per

the method WPTRU019. The difference in PF4 between the samples obtained at

1 min and 30 min was estimated to detect platelet activation induced by the

device.

5.2.e. Analysis of Platelet Adhesion

The devices exposed to PRP were rinsed with PBS to remove PRP

completely, fixed with 2% glutaraldehyde and dehydrated as per WPTRU030.

The devices were cut open and the luminal side was analyzed for platelet

deposition. Before analysis test devices were critical point dried, gold sputter

coated and were analyzed under ESEM as per WPSEM001.

Deviation: In the original study protocol, use of high voltage-high vacuum SEM

analysis was suggested. However, there was charging and cracking in the case

of the entire polymer coated devices. Therefore, samples were analyzed using

environmental SEM at low voltage.


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5.3. Interaction of Devices with Radio labeled Platelet Rich Plasma (1251-PRP)

Interaction of devices with radio labeled platelets was done to quantify the

number of platelets adhered to the device during the exposure period (Resmi

K.R., Nissey Varghese and Lissy K. Krishnan. (2004) Procedure for quantification

of platelet adhesion to biomaterials by Radioscintigraphy. Thrombosis Research

114; 121-128).

5.3.a. Exposure of devices with radio labeled PRP

Blood from human volunteer was collected into the anticoagulant; CPO.

Blood was centrifuged to obtain platelet rich plasma (PRP) at 2500 rpm for 5 min

as per the method described in WPTRU005. Platelets from PRP was collected,

washed and labeled with 1-125,as described in work procedure WPTRU008. The

labeled platelets were re-suspended in the same donor's platelet poor plasma

and the count was adjusted to get 2x108 to 2.5x1 08 per ml PRP. The test devices

were exposed to 1-125labeled platelets for 30 min under agitation at 75 :t 5 rpm

usingan Environshaker thermo stated at 35 :t 2°C (WPTRU012).

Deviation: Most of the stent had come out of the silicone tube by the time we

received the samples, probably because they were not tight fit into the lumen,

when the sponsor prepared the samples. Therefore, the exposure of 1-125

platelets by perfusion that was proposed in the Study Plan was not done;

instead, devices were exposed under aqitation.

5.3.b. Radioscintigraphy

The exposed devices were rinsed with PBS to remove the PRP completely

and fixed with glutaraldehyde. The devices were then dried and phosphor

imaged to detect the number of platelets deposited on the device as per the

WPTRU029. Aliquots of radio labeled platelets with known number of platelets

were imaged to make the calibration curve. The intensity of radio images of each

device was used for estimation of platelet adhesion on to it based on the

calibration curve.


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6 Study Result

For all quantitative data percentage difference between the 1 min (initial)

value and 30 min (final) value is determined and average deviation (n=6) of each

type sample is calculated. The results are presented for all types of materials in

table format for each parameter. For percentage hemolyis and quantification of

platelet adhesion by radioscintigraphy the values shown are that was detected

after 30 min exposure.

6.1. Whole Blood Exposure

6.1. a. Table 1 Consumption of Platelets and WBC during whole blood

exposure

Observation: No significant consumption in WBC is observed. Platelet

consumption is significant in the case of all samples, as compared to reference.

However, non specific retention of platelets on the device and tubing may have

contributed to the difference seen.

6.1.b Table 2. Plasma Coagulation


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Sample ID Platelet Consumption WBC Consumption

(% Count Reduction) (% Count Reduction)

Co-Cr bare stents 39.54::1:9.33 2.92::1:1.78

Polymer alone 21.28::1:4.60 2.07::1:1.11

Coated

Supracore 44.19::1:14.75 4.66::1:2.62

Reference 1.84::1:1.25 1.43::1:0.60

Sample ID Plasma PTT

Fibrinogen (% Delay in Clotting time)

(% Reduction)

Co-Cr bare stents 12.28::1:9.0 14.61::1:5.89

Polymer alone 14.27::1:3.05 9.03::1:3.93

Coated

Observation: There seems to be a reduction in clottable fibrinogen after exposure

of blood for 30 min, more significantly with polymer alone coated devices.

Intrinsic coagulation seems to be affected by all types of devices because PTT is

prolonged significantly as compared to reference.

6.1.c Table 3 Percentage Hemolysis

Observation: All samples showed significant hemolysis as compared to

reference.

6.1.d Fig1. Qualitative Analysis of Leukocyte Adhesion

The representative photomicrographs are given in the figure 1. The

leukocyte adhesion is highest on bare stents. On polymer coated devices, very

few leukocytes were found. On drug loaded stents, a staining pattern was

observed which is not specific for leukocytes. A uniform patterning is seen which

may be due to retention of stain to the porous structure generated during

polymer coating for drug loading, which needs to be confirmed.


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Report No: SRTRU003.07 Volume No. 02STUDY Page No: 9 of 15 Date effective: 17 Feb 2007



Supracore 8.99:t14.75 10.44:t2. 98

Reference 2.43:t3.14 - 0.04:t0.04

Sample ID Hemolysis (% after

exposure)

Co-Cr bare stents 0.30:t0.04

Polymer alone Coated 0.26:t0.05

Supracore 0.19:t0.05

Reference 0.07:t0

Bare

Supra core

Polymer

Fig.1 Light microscopic images of leukocyte adhesion: Representative Fields

shown are of different types of stents after exposure to whole blood for 30min.

The stained cells are leukocytes adhered to the stent. The uniform pattern

observed on drug loaded stents are not specific for leukocytes, and may be due

to retention of stain in the porous structure generated during polymer coating for

drug loading.


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6.2 PRP Exposure

6.2.a Table 4 Platelet Consumption during PRP exposure

Observation: Platelet consumption during exposure of devices to PRP seems to

be low and is comparable, but slightly elevated in the case of Supracore as

compared to reference.

6.2.b Table 5 Platelet Function

Observation: In the case of ADP induced aggregation, devices seem to have

affected the aggregatory response; however, the S.D. is high. Exposure of PRP

to both bare and Supracore devices have affected the collagen-induced

aggregation of exposed PRP.


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Sample 10 Platelet Consumption

(% Count Reduction)

Co-Cr bare stents 5.68:t2.76

Polymer alone Coated 5.95:t1.45

Supracore 7.52:t4.85

Reference 3.32:t2.05

Sample 10 % Change in Aggregation % Change in

in response to ADP Aggregation in

response to

Collagen

Co-Cr bare stents 15.48:t7.29 11 .51 :t7.08

Polymer alone

Coated 10.11:t12.71 5.26:t4.34

Supracore 10.62:t8.66 11.23:t7.94

Reference 5.52:t3.86 6.72:t2.85

6.2.c Table 6 Platelet secretion

Observation: Except after exposure to polymer alone coated stent, in other

samples platelet granule secretion is high as compared to reference.

6.2.d Fig. 2 Analysis of Platelet Adhesion

The SEM images of the devices are given in the figure 2. Platelets

adhesion is highest on bare stents. Aggregates of platelets were found especially

near the bends. On polymer coated devices, the platelet adhesion was found be

very minimal. A representative lower magnification picture (500x) and High

magnification picture (3000x) are given side by side for each type of devices.

Observation: On Bare Devices, the black colored spots on Figure A are platelets.

In the higher magnification image (B) a large aggregate of platelet and lots of

small aggregates are seen (black structures). On Polymer alone coated devices,

platelets are rarely seen. Some single platelets are seen in white color. The

difference in color is because, the image was captured in backward scatter mode

to improve the contrast between the device surface and adhered surface. Since

polymer coating was grayish, the platelets appear as white. In the higher

magnification image (D), the polymer was seen to be peeling off.

Signature ofStudy Director cl~ BMT WING

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Sample ID % Increase in PF4

Release on Exposure

(IUlml)

Co-Cr bare stents 26.72::1:4.53

Polymer alone Coated 7.72::1:2.49

Supracore 39.51::1:22.70

Reference 8.86::1:2.53

Bare

Polymer alone

Supracore

Fig.2 Electron micrograph of Bare, Polymer coated and drug loaded devicesexposed to platelet rich plasma. A, C and E 500x ; B, D and F 3K. Scale baris shown in each figure.


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On Drug loaded devices also, the platelets appear as white spots. Platelet

adhesion is less on these devices, and in high magnification, a uniform pattern

due to coating with polymer was seen (E; F). In Figure F, platelet adhesion was

negligible, but the polymer was seen peeling off. These breaks might have

occurred during the High voltage SEM analysis.

6.2.e Quantification of platelet adhesion

Standard curve generated using log platelet count against respective

radioactivity on each aliquot is shown in Fig 3. The number of platelets deposited

on each material is given in the Table. Out of the total number of platelets in 1251-

PRP to which the materials were exposed, the percentage of cells adhered to the

material is calculatedand given in the Table 7.

.//

#

A/

Signature ofStudy Director 0~'/'~ ~

BMT WINGSCTIMST

ReportNo: SRTRU003.07 Volume No. 02STUDY Page No: 14of 15 Date effective: 17 Feb 2007



Fig.3. Standard Curve used for estimating adhered platelet number. Curve

showed a correlation coefficient 0.9992. Red marks are of known standard

aliquots and green are of tests (Platelets adhered to materials)

Table 7 . Number of platelets adhered on devices

Observation: Platelet deposition is highest on bare stents and minimum on

polymer alone coated stents.

7 Archive

No Archive

8 Documentation

8.1 All recordsgeneratedduringthe study

9 Authorized signatures


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Sample ID Number of Percentage

Platelets on each adhered w.r.t total

devices platelets in PRP

Co-Cr bare stents 5415:f:1133 0.0012:f:0.0003

Polymer alone

Coated 1315:f:538 0.0003:f:0.0001

Supracore 2075:f:578 0.0005:f:0.0001

Authority I Name I Designation I Signature I Date

Study \l FdJ.Director Lissy K.Krishnan ScientistF '"

Technical/oV/oManager G.S. Bhuvaneshwar Head, BMT WIng

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