Obstetrics and Periodontal Therapy (opt) Study Manual of Operations Version 1 March 3, 2003 Brief Table of Contents



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Lab Collection


– The Study Coordinator or designated study personnel will escort the subject with a

Study Request Card and 10 labels to the OB/GYN clinic.

– The OB/GYN phlebotomist will draw blood into one 10ml red top vacutainer labeled

with one of the study labels. An additional vacutainer tube will be drawn if the first

one is less than three quarters full of blood. When possible the blood draw will

coordinate with the study participants routine monthly blood draws.



Specimen Processing


– The whole blood will be immediately taken to the laboratory of the Center for Oral

health Research and centrifuged at 300 rpm in a 4oC refrigerated slow speed

centrifuge to separate the serum.

– The serum will be separated from the red cell pellet using a sterile pyrogen free

disposable pipette. The sample will be equally divided into four 1ml screw top

sample tubes, each of which is labeled with one of the study labels (contains the

subject’s study number and visit number).

– All serum samples will be recorded in Excel using subject and study visit identifiers

and a hand written map to the box of tubes indicating specimen location.

– The labeled serum tubes will be promptly taken to the -70oC freezer and stored in the

designated place with other serum tubes from this study.

– The Study Coordinator will escort the subject to the research clinic to receive their

appointments for Essential Dental Care and if appropriate, study periodontal

therapy.


III.4.2.3. New York (Harlem Hospital/Columbia University)


Lab Collection


– The Study Coordinator or designated study personnel will escort the subject with a

Study Request Card and 10 labels to the laboratory, (which includes 3 extras, in case

of torn labels).

– The Ob-phlebotomist (Mr. Frazier) will draw blood into two 10-ml SST tubes, each of

which is labeled with one of the study labels.


Specimen Processing


– The whole blood is centrifuged at 300 rpms in a 4oC refrigerated slow speed

centrifuge to separate the serum.

– The serum will be separated using sterile disposable pipettes. The separated serum

will be placed in four 1ml screw top sample tubes, each of which is labeled with one

of the study labels (contains the subject’s ID number and visit number).

– The labeled serum tubes will be promptly taken to the -70oC freezer located in the

OB Clinic, Rm2041, and stored in the designated place with other serum tubes from

this study.

– The Study Coordinator or designated study personnel will escort the subject to the

study office to receive appointments in coordination with their OB appointments if



possible, for Essential Dental Care and periodontal therapy (if appropriate).

– All serum samples will be recorded in Excel, using subject and study visit identifiers,

along with a hand written map to the box of tubes indicating specimen location.

III.4.2.4. Jackson, Mississippi (University of Mississippi)

Lab Collection


- The Study Coordinator or designated study personnel will escort the subject with a

Study Request Card and 10 labels to the laboratory (which includes

3 extras in case of torn labels).

- A phlebotomist will draw blood into two 7-ml SST tubes, each of which will be

labeled with one of the study labels.


Specimen Processing


- The whole blood is centrifuged at 300 rpms in a 4oC refrigerated slow speed

centrifuge to separate the serum.

- The serum will be separated using a sterile pyrogen free disposable pipette. The

sample will be placed in five 2ml screw top sample tubes, each of which is labeled

with one of the study labels (contains the subject’s study number and visit number).

- All serum samples will be recorded in Excel using subject and study visit identifiers

and a hand written map to the box of tubes indicating specimen location.

- The labeled serum tubes will be promptly taken to the -70oC freezer located in the

Medical Mall and stored in the designated place with other serum tubes from this

study.


- The Study Coordinator will escort the subject to the study office to set up their

appointments for Essential Dental Care and if appropriate, study periodontal

therapy.





III.4.3. Shipping Frozen Serum Samples.
At Sites other than Lexington (University of Kentucky), the Study Coordinator is responsible for shipping the frozen serum samples when the storage box in the freezer is full (100 tubes).
When enrollment begins, the Study Coordinator begins a fresh Form 31 (Serum Sample Shipping Form) for two purposes, both related to Item A.7 of the form. The two purposes are: first, to help him/her keep track of the number of serum tubes in the freezer; and second, to serve as a packing list when the box is shipped to the Host Response Lab. (See Section III.4.4. "Form 31 (Serum Sample Shipping Form)".) In particular, Item A.7 of Form 31 provides spaces for 25 labels pre-printed with PIDs, the same labels that are attached to the serum tubes. Each time the Study Coordinator sends a subject to the clinical lab for a blood draw, s/he sticks a label with the subject's PID into one of the 25 boxes on Form 31, Item A.7. For each blood draw, four tubes of serum go into the freezer. Thus 25 blood draws fill the available spaces in Item A.7 of Form 31 and indicate that 4 x 25 = 100 tubes are in the storage box in the freezer. When this happens, the storage box in the freezer is full and should be shipped as soon as practicable. Then the Study Coordinator begins a fresh Form 31 to keep track of tubes in the newly-empty freezer storage box.
Do not store Form 31 in the freezer, because the paper will become brittle and the labels won't stick to it. See Section III.4.4. "Form 31 (Serum Sample Shipping Form)" below for more details.
When the study's allocation of space in the –70°C or –80°C freezer is full, the New York, Minneapolis, and Jackson Sites should ship the samples to the Host Response Lab in Kentucky. Shipping cost is covered by the Host Response Lab. For this purpose, each Site will have a UPS billing number to put on the airbill, ensuring that the Host Response Lab is billed.
To ship the tubes, pack them into a Styrofoam container in crushed dry ice in compliance with IATA packing instruction 650 and ship the box by FedEx to the University of Kentucky for next day delivery. IMPORTANT: Do not ship plaque samples on Thursday or Friday or within two days before a holiday. The extra day is needed in case of a shipping problem. The shipment's packing list is the same Form 31 on which the PID labels have been stuck to monitor the number of tubes in the freezer. Form 31 is described in detail in the section immediately following.

III.4.4. Form 31 (Serum Sample Shipping Form)
This form serves several purposes:
• the Site uses it as the packing slip accompanying the serum samples from the Site to the Host Response Lab;

• the Host Response Lab uses it as a receipt for the box of tubes, and to indicate any damage to the tubes and their frozen contents; and

• the Data Coordinating Center (DCC) uses it to track shipments of tubes from the Sites to the Lab and to collect data monitoring the quality of shipping.

T
hus, part of Form 31 is completed by the Study Coordinator at the Site, who ships the tubes containing samples to the Host Response Lab. The rest of Form 31 is completed by the Host Response Lab upon receipt.


The Study Coordinator completes Part A and the identifying information in the upper right-hand corner of each page. Taken together, these serve as a packing slip.

• Part A and the identifying information on each page are self-explanatory, except for Item A.7. The purpose of Item A.7 is to identify the subjects and visits represented by the serum tubes in the shipping box. To complete Item A.7, each time the Study Coordinator sends a subject to the Site's clinical lab to have a serum sample drawn, s/he sticks a pre-printed label identifying the subject and visit in one of the 25 spaces provided in Item A.7. When the 25 spaces are filled with labels, the box of tubes stored in the Site's –70° C freezer is filled and ready to ship to the Host Response Lab.


After completing Part A and the identifying information on each page, the Study Coordinator faxes the first page of Form 31 to the DCC at (612) 625-0080, makes a photocopy for the Site's files, and puts the original form in the shipping box with the tubes containing the serum samples.
The Host Response Lab completes only Parts B and C (if needed). Part B describes the condition in which the box of tubes arrived, and is used to maintain quality in shipping. In completing Part B, the Lab may make marks on Part C, the box map, indicating missing or thawed tubes.
• Item B.2 asks lab personnel to record the locations of missing tubes. If nine or fewer tubes are missing, this is done using Part C's row labels (letters a through j) and column labels (numbers 1 through 10). If 10 or more tubes are missing, this is done by marking Xs on the box map.
• Item B.3 asks lab personnel if any tubes were thawed. If some but not all were thawed, this is done by marking Os on the box map to record the locations of thawed tubes.
After completing Part B and as much of Part C as is appropriate, lab personnel fax all of Form 31 to the Data Coordinating Center (DCC) at (612) 625-0080 and store the original in the Host Response Lab's files.


III.5. Overview of Serum Assays (Host Response Lab Procedures)
A sequential ELISA methodology is used to optimize the lab's capacity to measure multiple biomarkers in the available serum/plasma samples.
III.5.1. Acute phase proteins
Acute phase proteins will be analyzed using a capture ELISA for quantitating C-reactive protein (CRP) that was developed in our laboratory. This protein was chosen based on previous data suggesting alterations associated with periodontitis and using CRP as a response element for IL-1-type cytokines. This assay will use goat or sheep anti-human CRP (Calbiochem, Cappel) to coat the microtiter plates (Dynatech) at a concentration of approximately 1.0 µg/well. The sera will be diluted approximately 1:100-1:500 and incubated for two hours. After washing, polyclonal monospecific antihuman CRP or Fb will be added and incubated for 2 hours. The plates will be washed and incubated overnight with goat anti-rabbit IgG conjugated with alkaline phosphatase (R&D Systems). The dynamic range of the assay is generally 0.1-500 ng/ml with inter- and intra-plate coefficients of variation of approximately 10%.
III.5.2. Acute phase mediators
Microtiter plates will be coated with 5 µg/ml of mouse monoclonal antibody to IL-1, IL-6, IL-8, PGE2 (Biosource) or TNF (R&D). The sequence for serum/plasma analysis will be IL-1, IL-6, TNF, and IL-8. A pooled recombinant standard of the cytokines will be used on all plates. Each of the cytokines in the pooled standard will be adjusted to 1000 pg/0.2 ml and diluted serially two-fold to 0.5 pg/0.2 ml. This pooled standard will be used as the combination of cytokines that most closely resemble the samples being tested. The ELISA will be developed as described previously. The dynamic range of the assays is 10-5000 pg/0.1 ml with inter- and intra-plate coefficients of variation of approximately 10%.
III.5.3. Matrix Metalloproteinases
MMP-9 (gelatinase) was selected for these studies due to multiple reports describing elevations in this tissue-modifying enzyme in amniotic fluids during preterm birth and associated with amniotic infections. While there have been no studies documenting systemic changes in any MMP associated with preterm birth, numerous studies have identified changes in MMP-9 and other MMPs associated with myocardial infarcts and acute coronary syndromes, degenerative aneurysms, carotid plaque instability, and polycystic kidney disease. Thus, the ability to measure this biomolecule in serum/plasma appears reasonable, and the potential to document systemic changes with local activities contributing to preterm rupture of membranes (PROM) and preterm birth appears biologically plausible. The MMP-9 ELISA will be obtained as a commercial kit (R&D Quantikine) for analysis of this mediator. We have developed a standard procedure for evaluating and validating new reagents for detection of specific biological mediators. Briefly, this entails obtaining multiple sources for capture and developing reagents. This is done since the majority of reagents are developed to the human molecules, although they are not all as efficient in reacting with complementary antigenic epitopes within the capture assays. These reagents are tested with human sera from our library of samples including periodontitis and autoimmune disease patients, through a broad range of dilutions, and compared to a human purified standard. Those reagent combinations deemed optimal for providing signal/noise ratio and a dynamic standard curve are selected.
III.5.4. Serum IgG Antibodies
IgG antibody levels to the 7 bacteria tested in the microbiological studies will be determined by quantitative ELISA using formalin-killed whole bacterial antigens. The microorganisms will be attached to microtiter plates under alkaline conditions at 37°C. The plates will be incubated and developed with either affinity purified goat anti-human IgG (Calbiochem) conjugated with biotin, followed by streptavidin conjugated to alkaline phosphatase (R&D Systems). Each bacterial antigen plate has standard wells coated with an anti-IgG capture antibody. To these wells will be added serially diluted nonhuman primate IgG (R&D Systems) of a known gravimetric measure. The bacterial and standard wells will be developed with the same IgG reagents. Thus, the antibody levels will be estimated to the standard curve and expressed as ng-µg/ml of IgG reactivity. Samples will be assayed in the sequence of least to most prominent antibody level in the sera based upon preliminary studies and the minimal detectable dose for the assays.
The coating of the microtiter plates for the above assays will be performed in carbonate/bicarbonate buffer. After 3-4 hours of incubation at 37°C, the solution will be removed and 1% bovine serum albumin (BSA) in PBS added to block the unbound sites in the wells. Plates then will be stored with the BSA at 4°C overnight. Intra- and inter-plate variability will be accepted with a sample duplicate variation of ≤ 15% and the standard curve between plates requires: (i) maximum OD of at least 1.0 (no more than 20% variation), (ii) significant difference (p > 0.05) in the slopes, and (iii) a background of < 0.15.
III.5.5. Endotoxin Levels
Endotoxin activity will be analyzed using the Endosafe® commercial kit (Charles River), as an indicator of local and systemic challenge with this biological inflammatory stimulant, and as a correlate with increases in Gram-negative bacteria in the host. The serum will be diluted (1:100) with pyrogen-free water and heat inactivated at 70° C for 10 minutes. 100-µl sample and 100 µl of standard endotoxin (E. coli O55:B5) will be transferred to pyrogen-free microtiter plates. 100 µl of the Limulus Amoebocyte Lysate will be added to each well, mixed, and the kinetics of absorbance assessed at 405 nm using a Dynatech microplate reader. The time of onset of color development is inversely related to the amount of endotoxin activity in the experimental sample. Endotoxin levels in the samples will be determined by comparison to a standard curve using E. coli LPS (as per manufacturer’s instructions).
III.5.6. Forms
III.5.6.1. Form 31 (Serum Sample Shipping Form)
This form serves several purposes:
• the Site uses it as the packing slip accompanying the serum samples from the Site to the Host Response Lab;

• the Host Response Lab uses it as a receipt for the box of tubes, and to indicate any damage to the tubes and their frozen contents; and

• the Data Coordinating Center (DCC) uses it to track shipments of tubes from the Sites to the Lab and to collect data monitoring the quality of shipping.

Thus, part of Form 31 is completed by the Study Coordinator at the Site, who ships the tubes containing samples to the Host Response lab. The rest of Form 31 is completed by the Host Response Lab upon receipt.


The Study Coordinator completes Part A and the identifying information in the upper right-hand corner of each page. Taken together, these serve as a packing slip.
• Part A and the identifying information on each page are self-explanatory, except for Item A.7. The purpose of Item A.7 is to identify the subjects and visits represented by the serum tubes in the shipping box. To complete Item A.7, each time the Study Coordinator sends a subject to the Site's clinical lab to have a serum sample drawn, s/he sticks a pre-printed label identifying the subject and visit in one of the 25 spaces provided in Item A.7. When the 25 spaces are filled with labels, the box of tubes stored in the Site's –70° C freezer is filled and ready to ship to the Host Response Lab.
After completing Part A and the identifying information on each page, the Study Coordinator faxes the first page of Form 31 to the DCC at (612) 625-0080, makes a photocopy for the Site's files, and puts the original form in the shipping box with the tubes containing the serum samples.
The Host Response Lab completes only Parts B and C (if needed). Part B describes the condition in which the box of tubes arrived, and is used to maintain quality in shipping. In completing Part B, the Lab may make marks on Part C, the box map, indicating missing or thawed tubes.
• Item B. 2 asks lab personnel to record the locations of missing tubes. If nine or fewer tubes are missing, this is done using Part C's row labels (letters a through j) and column labels (numbers 1 through 10). If 10 or more tubes are missing, this is done by marking Xs on the box map.

• Item B.3 asks lab personnel if any tubes were thawed. If some but not all were thawed, this is done by marking Os on the box map to record the locations of thawed tubes.


After completing Part B and as much of Part C as is appropriate, lab personnel fax all of Form 31 to the Data Coordinating Center (DCC) at (612) 625-0080 and store the original in the Host Response Lab's files.
III.5.6.2. Form 33 (Serum Assay Results)
Lab personnel transcribe each sample's patient ID (PID) and visit number from the Form 31 (Serum Sample Shipping Form) that came with the tubes to the Excel spreadsheet (Form 33, Serum Assay Results) in which the assay results will be transmitted to the Data Coordinating Center. In all the steps of collecting and processing serum samples, this is the only occasion on which a PID and visit number are transcribed, so it is the main opportunity for introducing an error into these data. Thus, transcription of PIDs and visit numbers should be done with great caution, double-checking to ensure that the correct PID and visit numbers have been transcribed.

Form 33, Serum Assay Results
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