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



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III.Details on Procedures



PART III.
DETAILS ON PROCEDURES

III.1. Periodontal Measurements
Full-mouth periodontal assessments will be made on all subjects at 13-16 weeks of pregnancy (Baseline/Randomization Visit (Visit 1)) and again at 21-24 weeks (Study Visit 3) and 29-32 weeks (Visit 5).
III.1.1. Who Obtains the Periodontal Measurements?
Only trained and calibrated periodontal examiners will take periodontal measurements on subjects.
III.1.2. Equipment, Standards and Special Situations
Measurements will be made using standardized color-coded periodontal probes (UNC –15; Hu-Friedy Mfg. Co., Chicago IL). These probes have markings at millimeter increments from 1-15 mm. Examiners will use standard clinical examination procedures to obtain the measures, which include the use of a front-reflecting (#5) mouth mirror and fixed overhead dental operatory lighting. Whenever possible, subjects should be examined in a reclined position to facilitate proper lighting and operator view of the areas of interest.
III.1.2.1. Teeth to be Scored or Measured. The Plaque and Calculus Indices will be scored from the six Ramfjord Index teeth. Mean values from these teeth have been shown to correlate well with mean scores from the entire dentition. The Ramfjord Index teeth are the maxillary right first molar (#3 in the Universal system), maxillary left central incisor (9), maxillary left first bicuspid (12), mandibular left first molar (19), mandibular right central incisor (25), and mandibular right first bicuspid (28).
The Gingival Index (GI), Pocket Depth (PD; also called Probing Depth), cementoenamel junction to gingival margin (CEJ·GM), and Bleeding on Probing (BOP) will be assessed on all teeth in the dentition, excluding third molars (i.e., #’s 2-15 and 18-31). NOTE: CAL will be calculated as the difference between the PD and CEJ·GM. (See Section III.1.2.8 for details.)
III.1.2.2. Substitutions for Missing Teeth. If any of the Ramfjord Index teeth is missing, the next most distal tooth in the quadrant will be substituted.
III.1.2.3. Tooth Locations for Scoring or Measuring. Scores or measures will be obtained at the following six locations of each tooth scored or measured: mesiobuccal, mid-buccal (also called "direct buccal"), distobuccal, distolingual, mid-lingual (also called "direct lingual"), and mesiolingual.
Whereas the Plaque, Gingival and Calculus Indices will be assessed along these six surfaces, PD and CEJ·GM measures and BOP will be assessed at specific locations within these surfaces. The tooth surfaces are defined as extending either from the mesial to distal line angles of the tooth (e.g., buccal and lingual surfaces) or from the respective line angle to the mid-point of the proximal surface (e.g., mesiobuccal, distobuccal, distolingual, and mesiolingual surfaces). For example, the Plaque Index for the buccal surface of a tooth will be scored by determining the maximum amount of plaque present between the mesiobuccal and distobuccal line angles of the tooth. The PD at that site, however, will be determined by probing the mid-buccal point of the tooth. This differs from what is normally done in a clinical setting, since the therapist will typically “walk” the probe from the mesiobuccal to distobuccal line angles and record the deepest pocket along that surface.
For measuring PD and CEJ·GM, the following locations will be used:
Radicular (buccal and lingual) sites: PD and CEJ·GM measures and BOP will be scored at the mid-point of the tooth mesiodistally. The probe will be oriented parallel to the long axis of the tooth with the probe tip in contact with the crown or root surface.
Proximal sites: The shaft of the probe will be placed against the proximal contact area. The probe tip should be angled slightly (about 15 degrees) underneath the proximal contact. Care should be taken not to over-angle the probe so that readings are taken obliquely across the tooth or root surface. The tip of the probe should not extend beyond an imaginary line extending apically from the mid-proximal point of the tooth and parallel to the long axis of the tooth. The probe tip should remain in contact with the crown or root surface.
If there are no proximal contacts, the mesiobuccal and distobuccal measures will be obtained by positioning the probe parallel to the long axis of the tooth slightly buccal to the mid-point of the proximal tooth surface. Similarly, the mesiolingual or distolingual measures should be obtained by positioning the probe parallel to the long axis of the tooth slightly lingual to the mid-point of the proximal surface when there are no proximal contacts.
III.1.2.4. Order of Measurement. The clinical measures and indices will be record in the following order:
1. Plaque Index from 6 surfaces on the Ramfjord teeth

2. Gingival Index from 6 surfaces on all teeth

3. PD then CEJ·GM from 6 sites on all teeth

4. BOP from 6 sites on all teeth*

5. Calculus Index from 6 surfaces on the Ramfjord teeth
* BOP will be assessed after probing the buccal or lingual/palatal sites of each quadrant. BOP assessments will not be delayed until after the entire arch or dentition is probed. For example, BOP will be recorded for the facial surfaces of the upper right quadrant immediately after PD and CEJ·GM or CAL measures have been obtained for these same tooth sites.
III.1.2.5. Criteria for the Plaque Index (Löe 1967)
0 = No plaque in the gingival area

1 = A film of plaque adhering to the free gingival margin and adjacent area of the tooth. The plaque may only be recognized by running a probe across the tooth surface.

2 = Moderate accumulation of soft deposits within the gingival pocket, on the gingival margin and/or adjacent tooth surface, which can be seen by the naked eye.

3 = Abundance of soft matter within the gingival pocket and/or on the gingival margin and adjacent tooth surface.


III.1.2.6. Criteria for the Gingival Index (Löe, 1967)

0 = Normal Gingiva.

1 = Mild Inflammation: There is slight change in color and slight edema. There is no bleeding when a blunt instrument (periodontal probe) is run along the soft tissue entrance of the gingival crevice.

2 = Moderate Inflammation: There is redness, edema and glazing. There is bleeding when a blunt instrument (periodontal probe) is run along the soft tissue entrance of the gingival crevice.



3 = Severe Inflammation: There is marked redness and edema. Ulceration may be present and there is marked tendency for spontaneous bleeding (i.e. on gentle palpation of the gingiva with the side of a probe.
III.1.2.7. Pocket depth. Pocket depth is defined as the probeable distance from the free gingival margin (FGM) to the base of the crevice or pocket. Distances will be rounded to the nearest whole millimeter. For example, if the probe penetrates just over 4 mm, for example, 4.1, 4.2, 4.3, 4.4, or 4.5 mm into the pocket, then the PD is recorded as 4 mm. Pocket depths should be recorded as whole numbers, without fractions or decimal places. Similarly, pockets judged to be 4.6, 4.7, 4.8, or 4.9 mm will be recorded as 5mm. Measurements viewed as falling directly between two mm increments (e.g., 4.5 mm) will be rounded down.
III.1.2.8. Clinical Attachment Level. Clinical attachment level (CAL) is defined as the distance from the cementoenamel junction (CEJ) to the base of the pocket. Since both PD and CEJ·GM are recorded in millimeters (mm), the unit of measure for CAL is also mm.
CAL will be calculated from the PD and CEJ·GM measures. The chairside assistant or examiner will calculate CAL at each site immediately after the examination is completed.
Measuring CEJ·GM – The examiner should place the NCP-15 probe in the same general position used to record PD. To detect the CEJ, however, it may be necessary to angle the probe more perpendicular the tooth/root surface (i.e., greater than 15 degrees). Once the CEJ is identified, the examiner will measure (with the probe repositioned to be more parallel to the long axis of the tooth) the distance from this point to the gingival margin. If the gingival margin is coronal to the CEJ, then the CEJ·GM measure is recorded as a POSITIVE number. Conversely, if the gingival margin is apical to the CEJ (i.e., there is visible recession), then the CEJ·GM measure is recorded as a NEGATIVE number. Finally, if the free gingival margin is located at the CEJ, then the CEJ·GM measure is recorded as ZERO. As with PD, the CEJ·GM measure will be rounded to the nearest whole mm. Measures viewed as falling directly between two mm increments (e.g., 3.5mm) will be rounded DOWN. For example, pockets judged to be 3.1, 3.2, 3.3, 3.4 or 3.5 mm in depth will be recorded as 3mm. Similarly, pockets judged to be 3.6, 3.7, 3.8 or 3.9 will be recorded as 4mm.
If the CEJ is obliterated or covered by a restoration, the apical margin of the restoration will be used as the landmark for calculating CAL. In these instances, the CAL measure may not reflect the true attachment level, but this “relative” CAL measure can be used to monitor disease progression as long as the restoration remains intact. If the CEJ is covered by calculus, just enough calculus will be removed with a hand instrument to detect the CEJ.
Calculating CAL – CAL is calculated as the difference between PD and CEJ·GM, or CAL = PD – CEJ·GM. Thus is the CEJ·GM measure is positive (i.e., the gingival margin is located coronal to the CEJ), then the CAL is LESS THAN the PD. Conversely, if the CEJ·GM measure is negative (i.e., there is visible recession at that tooth site), then the CAL is GREATER THAN the PD. This is so because subtracting a negative number is equivalent to adding it. For example, for a tooth site with a PD of 4 mm and 3 mm of visible recession (CEJ·GM = -3), the CAL is 7 mm (4 – (-3) = 7). Finally, if the CEJ·GM measure is zero, then CAL equals PD at that tooth site.
CAL measures should be recorded as whole numbers, without fractions or decimal places.


III.1.2.9. Criteria for Bleeding on Probing
0 = no bleeding following probing

1 = bleeding following probing


BOP will be assessed after probing the buccal or lingual/palatal sites of each quadrant. BOP assessments will not be delayed until after the entire arch or dentition is probed. For example, BOP will be recorded for the facial surfaces of the upper right quadrant immediately after PD and CEJ·GM measures have been obtained for these same tooth sites.
III.1.2.10. Criteria for the Calculus Index (from OHI-S; Greene, 1967)
0 = No calculus present.

1 = Supragingival calculus covering not more than one-third of the exposed tooth surface being examined. No subgingival calculus can be detected.

2 = Supragingival calculus covering more than one-third, but not more than two-thirds of the tooth surface, or the presence of individual flecks of subgingival calculus around the cervical portion of the tooth.

3 = Supragingival calculus covering more than two-thirds of the exposed tooth surface or a continuous heavy band of subgingival calculus around the cervical portion of the tooth.


III.1.3. Training and Calibration Protocols
Periodontal examiners at each enrollment site will be trained and calibrated prior to the start of the trial. Training and calibration will take place at the University of Minnesota's Oral Health Clinical Research Center. Examiners will be calibrated for both for intra and inter-examiner error using Dr. Bryan Michalowicz as the “Gold Standard” examiner. The calibration protocol will involve measuring five representative subjects twice by each examiner and Dr. Michalowicz. Examiners will qualify for the study if they achieve the following criteria:
GI: At least 80% intra- and inter- examiner exact reproducibility plus 95% intra- and inter- examiner reproducibility within ± 1 index unit;
CAL and PD, respectively: At least 95% intra-examiner reproducibility within ± 2 mm for both parameters; at least 75% inter-examiner agreement for PD within ± 1 mm and at least 60% inter-examiner agreement for CAL within ± 1 mm.
The kappa statistic will not be used to assess reproducibility of GI scores because it is sensitive to a skewed distribution, which is common for this index. Determining bleeding on probing is an invasive procedure and sites are more likely to bleed at subsequent passes in a calibration trial. Thus, adequate calibration procedures for BOP do not exist. Instead, examiners will observe each other during training sessions so they may discuss and evaluate the criteria for BOP. In our experience, this helps standardize examiners. The Plaque Index will be handled in a similar manner; it cannot be calibrated because plaque is removed when this index is scored.
Intra-examiner reproducibility will be continually assessed throughout the trial. Examiners will re-measure a randomly-selected dental quadrant in 5% of subjects throughout the duration of the trial. The examiner will have no knowledge beforehand which patients or quadrants will be selected for re-measurement. In the event that examiners do not continue to meet baseline calibration standards, they will be re-trained by the Gold Standard examiner until these standards are re-met.
III.2. Collecting, Storing, and Shipping Dental Plaque Samples (Enrollment Site Procedures)
III.2.1 Equipment and Standards
III.2.1.1. Equipment provided by the Enrollment Site. The Enrollment Site provides the freezer and ice machine for storing the plaque samples. Each Site's location where plaque will be collected should have on site a standard –20º C freezer or refrigerator with a –20º C freezer compartment, for storage of the plaque collection tubes before they are used. (A –70º C or colder freezer may be used if it is convenient). Clinics should also have an ice machine, so that thawed collection tubes can be kept on ice at chair-side during the exams at which samples are collected. After the plaque samples are collected, they must be stored in an ultra-low freezer temperature (–70º C or colder) until they are shipped to the Microbiology Lab at the University of Minnesota.
III.2.1.2. Collection tubes provided by the Microbiology Lab. Plaque collection tubes containing sample buffer will be shipped frozen to each Site by the Microbiology Lab. The tubes will provided in boxes containing 100 tubes each. Two full boxes and one empty box will be sent at the beginning of the study, before enrollment begins. After enrollment begins, one new box full of tubes with sample buffer will be sent from the Microbiology Lab to the Site each time a box of collected samples is received at the Microbiology Lab.
Section III.2.4 "Forms" (below) describes how to complete all relevant forms. Two forms are used to track the plaque collection tubes, one each for
• shipping new plaque collection tubes with buffer from the Microbiology Lab to the Site (Form 29) and

• shipping collected samples from the Site to the Microbiology Lab (Form 30).


III.2.1.3. Standardized curettes and tube labels provided by the DCC. The Administrative Center provides standardized curettes for the plaque sampling. The Administrative Center also trains the examiners in the method used to collect these samples, at the same visit to Minneapolis in which the examiners are trained and calibrated for periodontal measurements.
Before enrollment begins, the Data Coordinating Center (DCC) will send to each site provisional labels intended for use at the Recruitment Visit and the Baseline/Randomization Visit (Visit #1). Specifically, the Study Coordinator should receive a set of provisional labels for each possible Patient ID (PID). When a new subject has her baseline plaque sample taken, a provisional label for her newly-assigned PID is attached to the plaque collection tube. When the subject is randomized, the DCC sends the Site a set of labels preprinted with the subject's PID and Enrollment Code. When the subject has her post-baseline plaque sample taken (usually at Visit #5), one of these latter labels is attached to the plaque collection tube.
The labels for this purpose are cryogenic labels (Teeny Tough Tags™), specially fabricated to adhere to tubes in a –80° C freezer. Use only these labels supplied by the DCC. The DCC will include extra labels for each subject in case some are torn and unusable.
III.2.1.4 Collection of Plaque Samples. Tubes may be thawed to room temperature prior to collection in order to affix the labels. It is recommended that labels be put on the tubes before plaque is collected to insure adhesion. Tubes containing plaque samples must be kept on ice from the moment of collection to prevent the plaque bacteria from degrading. Plaque samples are placed on ice to ensure 1) bacteria will stay inactive, and not produce enzymes like DNAases that might influence the results by degrading sample DNA, and 2) that species do not grow by degrading others. Keeping plaque samples on ice will also reduce variability and increase consistency across sites and participants.

III.2.2. Storing and Shipping Samples
III.2.2.1. Confirming receipt of the new box of collection tubes. When a box of new plaque collection tubes arrives at the Site, examine it for broken or thawed tubes. The box of new tubes will contain a Plaque Collection Tube Shipping Form (Form 29; see Section III.2.4 "Forms" below for details) of which Part A has been filled out by the Microbiology Lab. Fill out Part B of this Form 29 to acknowledge receipt of the boxes and to describe the conditions of their contents. When Part B of the Form 29 is completed, fax it to the Data Coordinating Center (DCC) at (612)625-0080 and keep the original for the Site's records.
At the beginning of the study, before enrollment begins, two boxes of new plaque collection tubes will arrive at the Site; a Plaque Collection Tube Shipping Form (Form 29) should arrive with each one and should be completed and faxed to the DCC.
Save the Styrofoam container in which the freezer box was shipped, so it can be used again when a box of plaque samples is shipped back to the Microbiology Lab.
III.2.2.2. Storage prior to collection. Immediately after examining the tubes, place newly arrived boxes of plaque collection tubes in a freezer (–20º C or –70º C) where you have convenient access to them. The tubes must remain frozen until the day they are used. The empty freezer box (and subsequent empty boxes) should be placed in the

–70º C freezer where collected plaque samples will be stored.


III.2.2.3. Storage on the day of collection. Each day that plaque samples will be taken, remove from the freezer one new tube with buffer for each subject who will be sampled that day. Tubes may be thawed to room temperature prior to collection in order to affix the labels. It is recommended that labels be put on the tubes before plaque is collected to insure adhesion. Tubes containing plaque samples must be kept on ice from the moment of collection. Plaque samples are placed on ice to ensure 1) bacteria will stay inactive, and not produce enzymes like DNAases that might influence the results by degrading sample DNA, and 2) that species do not grow by degrading others. Keeping plaque samples on ice will also reduce variability and increase consistency across sites and participants.
During the actual plaque collection, the tubes should be kept on ice at chair-side. If the labels were not affixed to the tubes prior to plaque collection, carefully dry off the outside of the tube, and then place one of the pre-printed labels around the tube just beneath the upper rim. IMPORTANT: Labels may not stick to the tube properly if the tube is wet. Once the label is firmly attached, the tube may be returned to an ice bucket, refrigerator, or freezer, but the label must not get wet. Do not allow the plaque samples to sit at room temperature.
At the Baseline/Randomization Visit (Visit 1), the plaque sample will usually be taken before it is confirmed that the subject is eligible. (See Section II.2 "The Baseline/Randomization Visit (Visit 1)", and particularly Section II.2.4.3 "Plaque Sampling, Form 11 (Periodontal Measurements), continued".) If the subject's eligibility has not been confirmed, the plaque sample should be kept in a convenient ice bucket, refrigerator, or freezer until:
• eligibility is confirmed, after which the sample should be stored as described immediately below (Section III.2.2.4 "Storage after collection"); or
• the subject is determined to be ineligible, in which case the plaque sample should be discarded following the Enrollment Site's standard procedure for biological waste.
III.2.2.4. Storage after collection. As soon as possible, take the samples to the –70º C freezer for storage. The tubes should be kept cold while in transit, for example, by carrying them in the ice bucket. The –70º C freezer box has its rows and columns labeled for easy reference, the rows being labeled by capital letters, while the columns are labeled by numbers. If possible, tubes should be placed into the –70º C freezer box in numerical order. Start in Row A, column 1, fill the spots in order to row A column 10, then go back to Row B column 1, and so on.
III.2.2.5. The map of the freezer box (Form 30, Plaque Sample Shipping Form). When enrollment begins, the Study Coordinator begins a fresh Form 30 for two purposes, both related to the map of the freezer box that is Part C of Form 30. The two purposes are: first, to help him/her keep track of the number of full plaque-collection tubes in the freezer; and second, to be a packing list and a map of the box when it is shipped to the Microbiology Lab.
In particular, when a subject's full plaque collection tube is stored in the –70° C freezer, the Study Coordinator sticks to the box map a label pre-printed with the subject's PID, the same label that is stuck to the plaque collection tube. The Study Coordinator sticks this label in the row and column of the map corresponding to the tube's row and column in the freezer box.
Do not store the box map in the freezer, because the paper will become brittle and the labels won't stick to it. See Section III.2.4.1 "Form 30 (Plaque Sample Shipping Form)" below for more details.
III.2.2.6. Shipping. The Study Coordinator is responsible for shipping the frozen plaque samples when the storage box in the freezer is full (100 tubes). When the box map (Part C of Form 30) is full, the freezer box is full and needs to be shipped as soon as is practicable. Then the Study Coordinator begins a fresh Form 30 to keep track of tubes in the newly-empty freezer storage box.
To ship the samples, pack the freezer box in crushed or pelleted dry ice. Use the Styrofoam shipping box that was saved from the previous shipment of new collection tubes. Complete Part A of the Form 30 (Plaque Sample Shipping Form) that has been accumulating tube labels, following instructions given in Section III.2.4.1 "Form 30 (Plaque Sample Shipping Form)" below. Part C of that form, the map of the storage box, should have all 100 places filled with labels, unless this is the last shipment to be made from the Site. After completing Part A of the Form 30, fax the form to the Data Coordinating Center at (612) 625-0080, copy the form and keep the copy for the Site's records, and put the original Form 30 in the shipping box with the samples, where it serves as a packing list.
The New York, Lexington, and Jackson Sites should ship the samples to the Microbiology Lab in Minneapolis by FedEx Priority Overnight service or its equivalent (arrival by 10:30 AM next business day). 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 Minneapolis Site (Hennepin County Medical Center) should send samples to the Microbiology Lab by a reliable same day courier.
Finally, the Study Coordinator should call the Microbiology Lab at 612-624-3196 to let them know that a shipment is coming.

III.2.3. Training And Standardization For Plaque Sample Collection
As part of their pre-trial training and calibration, examiners will be trained in these plaque collection methods at the Administrative Center.
III.2.4 Forms
III.2.4.1. Form 29 (Plaque Collection Tube Shipping Form)
This form serves several purposes:

• the Microbiology Lab uses it as the packing slip accompanying the new collection tubes from the Lab to the Site;

• the Site uses it as a receipt for the box of tubes, and to indicate any damage to the tubes and their frozen buffer; and

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




Thus, part of Form 29 is completed by personnel at the Microbiology Lab, which ships the collection tubes to the Sites. The rest of Form 29 is completed by the Study Coordinator at the Site that receives the collection tubes.

The Microbiology Lab completes Part A and the identifying information in the upper right-hand corner of each page, which serves as a packing slip. Upon completing Part A and the identifying information on each page, the responsible Lab person faxes the first page of Form 29 to the Data Coordinating Center (DCC) at (612) 625-0080, makes a photocopy for the Lab's files, and puts the original form in the shipping box with the collection tubes.
The Study Coordinator at the receiving Site completes Parts B and C only. Part B describes the condition in which the box of tubes arrived, and is used to maintain quality in shipping. Part C, the box map, is used by the Site only to describe missing, thawed, or otherwise damaged tubes.
• Item B. 2 asks the Study Coordinator 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 3 asks the Study Coordinator 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.
A
fter completing Part B and as much of Part C as is appropriate, the Study Coordinator faxes all of Form 29 to the Data Coordinating Center (DCC) at (612) 625-0080 and stores the original in the Site's files.

III.2.4.2. Form 30 (Plaque Sample Shipping Form)
This form also serves several purposes:
• the Site uses it as the packing slip accompanying the plaque samples from the Site to the Microbiology Lab;

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

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

Thus, part of Form 30 is completed by the Study Coordinator at the site, which ships the tubes containing samples to the Microbiology lab. The rest of Form 30 is completed by the Microbiology Lab upon receipt.


The Study Coordinator completes Part A, Part C (the box map), 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.




Part C, the box map, is filled in as plaque samples are collected, as described in Section III.2.2.4, "Storage after collection" (above). Specifically, each time a full plaque collection tube is placed into the –70º C freezer box, indicate its location on Part C, the box map, by sticking on the box map a pre-printed label with the subject's Patient ID (PID) and visit number, provided by the Data Coordination Center (DCC). The pre-printed label is stuck on the box map in the row and column of the map corresponding to the row and column in the freezer box where the tube was placed. Rows of the map are indicated by letters (a through j) and columns of the map are indicated by numbers (1 through 10).


After completing Part A, the identifying information on each page, and Part C (the box map), the Study Coordinator faxes the first page of Form 30 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 plaque samples.
The Microbiology Lab completes Parts B and D only. 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. Part D describes the box's location in the Lab's freezer.
• Item B. 2 asks the Study Coordinator 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 3 asks the Study Coordinator 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 Parts B and D and as much of Part C as is appropriate, the Study Coordinator faxes all of Form 30 to the Data Coordinating Center (DCC) at (612) 625-0080 and stores the original in the Site's files.


III.3. Overview of Plaque Assays (Microbiology Lab Procedures)
III.3.1. Shipping Collection Tubes to Enrollment Sites
Plaque is collected at Enrollment Sites in sterile locking-lid DNAase/RNAase-free 1.7 ml microcentrifuge tubes containing 1 ml of collection buffer. Tubes complete with buffer are provided by the Microbiology Lab; see Section III.2.1.2. "Collection tubes provided by the Microbiology Lab".
Plaque collection tubes containing sample buffer will be shipped frozen to each site by the Microbiology Lab. The tubes will be provided in 10 x 10 place freezer boxes containing 100 tubes each. Two full boxes and one empty box will be sent initially. Then, each time a box of collected samples is returned to the Microbiology Lab, one new full box will be sent. The freezer boxes will be packed in dry ice inside reusable Styrofoam shipping containers. The Styrofoam containers are saved by the Site and used to return plaque samples to the Microbiology Lab.
Each box of collection tubes shipped to a Site is documented by a Plaque Collection Tube Shipping Form (Form 29). From the point of view of the Microbiology Lab, this form mainly serves as a packing list that goes with the shipment. The Site also uses it to record information about damage to the shipment, for quality control purposes. After Form 29 is completed, the Lab faxes a copy to the Data Coordinating Center (DCC) at (612) 625-0080, makes a photocopy for its files, and puts the original in the box with the shipment. Detailed instructions on completing Form 29 are given below, in Section III.3.5.1. "Form 29 (Plaque Collection Tube Shipping Form)".
III.3.2. Acknowledging Receipt of Samples
When a Site has filled 100 tubes with plaque samples, the Study Coordinator ships the filled box of collection tubes to the Microbiology Lab, following procedures detailed in Section III.2.2.5. "Shipping", above. When the box arrives at the Microbiology Lab, its receipt and condition are documented on the Form 30 (Plaque Sample Shipping Form) that came in the shipping box with the shipment of plaque samples. Detailed instructions on completing Form 30 are given below, in Section III.3.5.2. "Form 30 (Plaque Sample Shipping Form)". Briefly, Lab personnel indicate on Parts B and C of Form 30 which (if any) tubes were missing, thawed, or otherwise damaged.
III.3.3. Storage of Samples
After opening the shipping container and checking and documenting the condition of its contents, lab personnel complete Part D of the Form 30 (Plaque Sample Shipping Form) that came with the freezer box, indicating the freezer's rack number and shelf number where the box was placed. Detailed instructions on completing Form 30 are given below, in Section III.3.5.2. "Form 30 (Plaque Sample Shipping Form)". Freezer boxes are stored in a –80º C Harris chest freezer, in the location specifically designated for this purpose. The freezer is wired to the building central freezer alarm system. University of Minnesota Facilities Management workers will respond to an alarm, and notify lab personnel as needed.
III.3.4. Speciation and Quantitation Method
The Amplifluor™ System for Quantitative PCR (Gaithersburg, MD) will be used for running qPCR assays to speciate and quantitate the seven target species: B. forsythus, T. denticola, P. gingivalis, C. rectus, F. nucleatum, P. intermedia, A. actinomycetemcomitans. This system uses a manufacturer-designed sequence (Z-tail) that is added to the 5' end of the forward or reverse primers. The Z-tail also constitutes the 3' end of a manufacturer-supplied UniPrimer™ with a 5' hairpin structure. The hairpin brings a fluorescein molecule into close proximity with a quencher molecule (DABSYL). The conserved tailed primer and Z-specific UniPrimer™ is added to the master mix in a ratio of approximately 1 to 10. During the earliest stages of amplification, the tailed primer incorporates the Z-sequence into the PCR product. The complement to the Z-tail then can anneal to the UniPrimer™. As the limited supply of tailed primer is exhausted, the UniPrimer™ and species-specific primers continue to drive amplification. When the specific primers are extended, the hairpin at the 5' end of the UniPrimer™ unfolds. The fluorescein it contains then can be detected. The amount of fluorescence is proportional to the original copy number of template DNA molecules across a range of 102 to 107.
III.3.5. Outline of laboratory procedures
To prepare DNA extracts for standard curves, large quantities of Aa, Pg, Bf, Td, Cr, Fn, Pi are grown from ATCC stocks, using appropriate media and growth conditions for each. After verifying gram staining and morphology, DNA are extracted from washed cells using the Masterpure™ kit protocol. DNA are then quantified using the Picogreen™ kit protocol. Stock DNA dilutions are prepared based on established DNA/cell values for each species. qPCR is then run to verify species identity and standard curve consistency with previous standard curves. Aliquots of DNA stocks will be stored at –80º C.
On Mondays 26 plaque sample tubes are thawed. DNA are extracted in the same tubes using the Masterpure™ kit protocol. DNA are then quantified using the Picogreen™ kit protocol. This is done in a laminar flow template preparation chamber pre-treated with ultraviolet light to minimize DNA contamination. Two black plastic microplates for fluorescence are used. Picogreen DNA standards or 10-fold sample dilutions are loaded in triplicate wells using a pre-established template (unused portions of samples will be stored at 6 º C). Outer wells are excluded to minimize edge effects. Picogreen™ reagent is then added to each well in equal volumes.
As part of the speciation and quantitation method, Lab personnel transcribe each tube's patient ID (PID) and visit number from the box map and/or tube labels to the quantitation software, checking the PID and visit number on the map against the label on the tube. In all the steps of collecting and processing plaque 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.
In the case of the DNA assay, PID numbers are entered into a pre-established template created with Deltasoft plate reader software. Fluorescence is read using a Tecan Spectrafluor microplate reader. The software automatically calculates DNA concentrations based on the standard curve. A hard copy of the concentration report is printed and an electronic copy is saved on disk. Deltasoft is used to export the PID and sample DNA concentration columns to a simple two-column Excel file. Header information required by the DCC are added in Excel, and this DNA data form is sent to the DCC by E-mail.
qPCR master mix is prepared in a laminar flow chamber pre-treated with ultraviolet light to minimize DNA contamination. A dedicated liquid handling system is used with disposable tips. The technician prepares 6.0 ml (Tuesday or Thursday) or 4.5 ml (Wednesday or Friday) master mix excluding primers. The technician then divides the master mix into 4 (Tuesday or Thursday) or 3 (Wednesday or Friday) tubes, and labels the tubes. Then species-specific primers are added to each tube: 1. Aa, 2. Pg, 3. Bf, 4. Td (Tuesday or Thursday); 1. Cr, 2. Fn, 3. Pi (Wednesday or Friday). Then 4 (Tuesday or Thursday) or 3 (Wednesday or Friday) skirted black PCR microplates are labeled. The tech then dispenses 23 µl master mix into cols. 2-11 rows B-G of the microplates. The plates are covered with an autoclaved sealing mat (to be discarded after 10 uses). The plates are transferred to the laminar flow template preparation chamber pre-treated with ultraviolet light to minimize DNA contamination. The technician thaws a DNA standard tube for each species and makes 6 dilutions in opaque tubes: 1. Aa, 2. Pg, 3. Bf, 4. Td (Tuesday or Thursday); 1. Cr, 2. Fn, 3. Pi (Wednesday or Friday). Label 4 (Tuesday or Thursday) or 3 (Wednesday or Friday) of these tubes. Using dedicated pipettors or a repeater with filter or positive displacement tips, the technician loads 2 µl standards/sample in triplicate wells using a pre-established template (include negative controls): Samples 1-13 (Tuesday or Wednesday); Samples 14-26 (Thursday or Friday). The plates are then re-sealed and mixed on plate shaker. The tech then transfers the plates to the thermal cycler room and returns the samples to their previous rack/shelf/box location in–80º C freezer. The tech places each plate in the designated thermal cycler for each species, and runs the pre-set program.
After the pre-set program is run, the plates are returned from thermal cyclers and brought to the Spectrafluor. For each plate, the tech enters PID numbers into the pre-established Spectrafluor template for that species, then reads fluorescence. The software automatically calculates bacteria per µl DNA extract based on the standard curve.
After fluorescence is read, the technician loads 10 µl of the contents of each well into a pre-cast 96-well Invitrogen E-Gel for high throughput electrophoresis. An image of the gel is saved using a Bio-Rad Gel-doc system; a hard copy is also printed at this time. The gel image is used to cross-check Spectrafluor results. For each plate, problem wells are defined as follows: 1. Band, but no fluorescence above baseline; 2. Fluorescence above baseline, but no band; 3. An unusually dense primer-dimer band; and 4. Fluorescence in one replicate is much higher or lower than other two. If one well per triplicate is a problem, Spectrafluor software will be used to mask that well and recalculate bacterial numbers for that sample. If 2-3 wells per triplicate are problem wells, the qPCR for that sample will be re-done. A hard copy of the cross-checked concentration report is printed; the data file is also saved on disk. For each species, Deltasoft is used to export the PID and bacterial count columns to a simple two-column Excel file. Header information required by the DCC is added in Excel, and the DNA data form is sent to the DCC by E-mail.
For a study lasting three years, reproducibility is necessarily a concern. An important potential source of variability is pipetting. Pipettor accuracy will be checked each month. Pipettes will be calibrated annually, or as often as needed. We have observed that negative controls are more prone to primer-dimer problems. Thus contamination is declared only when there are species-specific amplicons in the negative control lanes of the gel. In that case, the PCR areas are thoroughly cleaned with bleach, and reagents checked for contamination. No further samples are run until clean no-template controls are obtained. Samples from the run where contamination was detected then would be re-done. Repeats can also be triggered by a substantial loss of signal strength from the quantitative positive control samples. This is monitored by plotting each standard curve relative to the ten previous ones. If such an event should occur, DNA standards and PCR reagents are checked for loss of activity. After resolution of any problem, affected samples are re-done. We attempt to minimize such problems by preparing qPCR DNA standards from very large batches of pure bacterial cultures. Those extracts are frozen in small aliquots for weekly use. Lot-to-lot variation in reagents is minimized by buying in large quantities (but not in excess of their shelf life).
III.3.6. Forms
III.3.6.1. Form 29 (Plaque Collection Tube Shipping Form)
This form serves several purposes:
• the Microbiology Lab uses it as the packing slip accompanying the new collection tubes from the Lab to the Site;

• the Site uses it as a receipt for the box of tubes, and to indicate any damage to the tubes and their frozen buffer; and

• the Data Coordinating Center (DCC) uses it to track shipments of tubes from the Lab to Sites and to collect data monitoring quality of shipping.
T
hus, part of Form 29 is completed by personnel at the Microbiology Lab, which ships the collection tubes to the Sites. The rest of Form 29 is completed by the Study Coordinator at the Site that receives the collection tubes.
The Microbiology Lab completes Part A and the identifying information in the upper right-hand corner of each page, which serves as a packing slip. Upon completing Part A and the identifying information on each page, the responsible Lab person faxes the first page of Form 29 to the Data Coordinating Center (DCC) at (612) 625-0080, makes a photocopy for the Lab's files, and puts the original form in the shipping box with the collection tubes.
The Study Coordinator at the receiving Site completes Parts B and C only. Part B describes the condition in which the box of tubes arrived, and is used to maintain quality in shipping. Part C, the box map, is used by the Site only to describe missing, thawed, or otherwise damaged tubes.
• Item B. 2 asks the Study Coordinator 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 the Study Coordinator 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, the Study Coordinator faxes all of Form 29 to the Data Coordinating Center (DCC) at (612) 625-0080 and stores the original in the Site's files.
III.3.6.2. Form 30 (Plaque Sample Shipping Form)
This form also serves several purposes:
• the Site uses it as the packing slip accompanying the plaque samples from the Site to the Microbiology Lab;

• the Microbiology Lab uses it as a receipt for the box of tubes, and to indicate any damage to the tubes; 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 30 is completed by the Study Coordinator at the site, which ships the tubes containing samples to the Microbiology lab. The rest of Form 30 is completed by the Microbiology Lab upon receipt.
The Study Coordinator completes Part A, Part C (the box map), 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.
• Part C, the box map, is filled in as plaque samples are collected, as described in Section III.2.2.4, "Storage after collection" (above). Specifically, each time a full plaque collection tube is placed into the –70º C freezer box, indicate its location on Part C, the box map, by sticking on the box map a pre-printed label with the subject's Patient ID (PID) and visit number, provided by the Data Coordination Center (DCC). The pre-printed label is stuck on the box map in the row and column of the map corresponding to the row and column in the freezer box where the tube was placed. Rows of the map are indicated by letters (a through j) and columns of the map are indicated by numbers (1 through 10).
After completing Part A, the identifying information on each page, and Part C (the box map), the Study Coordinator faxes the first page of Form 30 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 plaque samples.

The Microbiology Lab completes Parts B and D only. 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. Part D describes the box's location in the Lab's freezer and is completed when the box is placed in the freezer.
• 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 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 Parts B and D and as much of Part C as is appropriate, lab personnel fax all of Form 30 to the Data Coordinating Center (DCC) at (612) 625-0080 and store the original in the Microbiology Lab's files.

III.3.6.3. Form 32 (Bacterial Species and Quantities/DNA)
Form 32, Bacterial Species and Quantities/DNA:
Obstetrics and Perio Therapy Study

















































































































































































































































































































































































































































OPT Form 32 V1 March 03


























































Two-letter abbreviation of species reported on this specific instance of Form 32
Column 1 Column 2

PID/Visit Species count/µl sample DNA

To be E-mailed to the DCC (e-mail files to helen@ccbr.umn.edu) as attached Excel files.

III.4. Collecting, Processing, Storing, and Shipping Serum Samples (Enrollment Site Procedures)
III.4.1. Equipment and Standards
III.4.1.1. Equipment provided by the Enrollment Site. Each Site or its clinical lab provides the following equipment.
• Any equipment needed for the blood draw, in particular tubes into which blood is drawn. The Host Response Lab recommends 14ml red top vacutainer tubes containing clot activator and separator gel for serum separation.

• Any equipment needed for centrifuging and pouring off serum. In particular, serum must be drawn off the centrifuged samples with transfer pipettes that are single-use sterile plastic (pyrogen-free) pipettes or that are glass fired to burn off pyrogen.



• Space in a –70°C or –80°C freezer, for storing serum samples.
III.4.1.2. Serum-collection tubes provided by the Host Response Lab. Before enrollment begins and periodically thereafter, the Host Response Lab will send to each Site a supply of 2ml screw-top sample tubes that can be stored at –80°C, to hold and store the serum. Unlike the plaque-collection tubes supplied by the Microbiology Lab, the serum-collection tubes need not be kept frozen, so their shipment does not require the formal tracking needed for the plaque-collection tubes. Instead, the Host Response Lab ships the tubes to the Study Coordinator, who confirms their arrival by e-mail, noting any irregularities.
III.4.1.3. Tube labels provided by the DCC. Before enrollment begins, the Data Coordinating Center (DCC) will send to each site provisional labels intended for use at the Recruitment Visit and the Baseline/Randomization Visit (Visit #1). Specifically, the Study Coordinator should receive a set of provisional labels for each possible Patient ID (PID). The first set of labels for the baseline visit will be coded with the letter, “B” followed by the five-digit PID, with “B” representing baseline. When a new subject has her baseline serum sample taken, the Study Coordinator uses these provisional labels, with the subject's newly-assigned PID, to label the serum tubes and sends five more provisional labels with the subject to the clinical lab, for the blood-draw tubes. When the subject is randomized, the DCC sends the site a set of labels preprinted with the letter, “V” followed by the five-digit PID, with “V” representing “visit”. When the subject has her post-baseline blood draw (usually at Visit #5), some of these latter labels are attached to the blood draw and serum tubes.
The labels for this purpose are cryogenic labels (Teeny Tough Tags™), specially fabricated to adhere to tubes in a –80° C freezer. Use only these labels supplied by the DCC. The DCC will include extra labels for each subject in case some are torn and unusable.
III.4.2. Collecting, Processing and Storing Samples
The context for these procedures is given in Section II.2.4, on the sequence of procedures and forms at the Baseline/Randomization Visit (Visit 1), specifically Section II.2.4.5 "Blood Draw for the Serum Sample". The procedure is first described in general terms, then specifics are given for each Enrollment Site.
In general terms, the object of this procedure is to obtain 4 serum-collection tubes containing a minimum 4 to 5 ml of serum total (approximately 1ml of serum per tube). Roughly 10 ml of whole blood must be drawn to obtain this amount of serum, with the exact amount for a subject depending on her hematocrit percentage. If the vacutainer is less than two thirds full of blood, a second vacutainer should be drawn so that enough serum can be obtained. When possible the blood draw will coordinate with the study participants routine monthly blood draws.
The procedure is generally as follows for Sites other than Lexington (University of Kentucky. The subject having blood drawn is escorted to the Site's clinical lab by the Study Coordinator. She carries with her an order for the blood draw, in the format appropriate to the Site (see below). She also carries with her four 2-ml screw-top tubes with PID/visit labels attached and four other pre-printed labels bearing her PID, to be attached to the blood draw tubes. (This includes extras in case of torn labels.) At the lab, her blood is drawn into red top vacutainer tubes containing clot activator and separator gel for serum separation. Following the blood draw, the subject’s blood is centrifuged in a refrigerated slow speed centrifuge for serum separation. The separated serum is drawn off using a sterile pipette, taking care to minimize the inclusion of red blood cells, and aliquoted equally into the four labeled screw top serum tubes. One ml of separated serum is placed in each vial. The serum is immediately taken to the Site's

–70°C or –80°C freezer and stored in the designated place with other serum tubes from the OPT Study.


At the Lexington (University of Kentucky) Site, the subject has her blood drawn in the Ob-Gyn clinic into red top vacutainer tubes containing clot activator and separator gel for serum separation. The whole blood is taken immediately to the Host Response Lab for processing and storage.
Freezing the sample "immediately" should be interpreted as meaning within an hour of the blood draw. The Study Coordinator is responsible for working with the appropriate person in the clinical lab to ensure that this guideline is met consistently.
The rest of the present section gives detailed instructions for each Enrollment Site.

III.4.2.1. Minneapolis (Hennepin County Medical Center).
• The subject arrives at the clinical lab with a Study Request Card and 10 labels (which includes 3 extras in case of torn labels). The Study Request Card is completed as follows:
The study's name for this purpose is: "Pre-term Study". This exact name must be entered here because the lab's technicians use this name to locate this study's protocol in their notebook of protocols.
The "patient name" line should be completed by entering the subject's name only, not her PID. The PID is excluded because putting both the subject's name and PID on the card would make it a sensitive document requiring storage in the Study Coordinator's locked storage. If the Special Request Card contains only the subject's name, it can be stored in the lab's files in the usual manner.
– The instructions on the lower half of the card are:
“• Draw two – 10 ml SST vacutainer tubes of blood

• Spin down and PO 5 serum tubes

• Affix one of the attached labels to each tube

• Freeze at –70º C"


– Except for the subject's name, these Special Request Cards are the same for all subjects. Thus, the Study Coordinator may prefer to prepare them in advance so that only the subject's name needs to be added when she is sent for her blood draw.
– Blood is drawn by a phlebotomist into two 10 ml SST tubes, to each of which a study label is attached.
– The whole blood is centrifuged to separate the serum. One ml of serum is pipetted

into each of four, labeled, 2ml tubes.


– The labeled tubes are immediately taken to the blood bank's –70°C freezer

and stored in the designated place.


III.4.2.2. Lexington, Kentucky (University of Kentucky)
Lab Collection, Processing, and Storing Serum Samples

University of Kentucky Lab Procedures for specimen collection:


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