Link to : Motion To Dismiss Amended Complaint

            - FDA denial pg. 16 of Amended Complaint








HIFI DNA TECH, LLC                      :


PLAINTIFF,                         :


V.                                      :




MICHAEL O. LEAVITT,                     :

ANDREW VON ESCHENBACH                   :    APRIL 10, 2008






HiFi understands that FDA has discretion granted it by Congress, and that the Court should be deferential, and that DNA PCR is a highly technical area.  But the accepted use of PCR-based DNA fingerprinting in courts and the rest of the world is beyond question.  The science is clear and convincing – indeed, it is accurate beyond any reasonable doubt, as determined by courts in the United States.  HiFi’s device uses publicly available information and readily available materials to identify the presence of HPV DNA.  It relies on globally accepted technology.  It proved more accurate and effective than the only similar device already approved by FDA – a device created before the technology used by HiFi’s device even existed.  It should have been a Class II virology test device.

FDA counters these facts by raising the specter of cancer, misconstruing HiFi’s petition and claiming that the device is important in preventing the impairment of human health and presents an unreasonable risk of illness.  FDA claims that no protections are sufficient to mitigate this risk to human health.  In this FDA hyperbolizes.  In fact, the device does not test for cancer, but rather for a common virus.  Even FDA’s own statements regarding HPV have noted that the current HPV test kit approved to be marketed detects only a virus and does not indicate cancer. Whatever discretion FDA may have, Congress has not given FDA sweeping discretion to make arbitrary medical device decisions without scientific basis and with unreasonable demands.  All arguments concerning cancer, impairment of health and risk of injury are red herrings and should be denied.

In sum, FDA’s denial of HiFi’s petition is arbitrary, capricious and an abuse of the discretion delegated to it by Congress because (1) it ignores the current state of the science of DNA testing, (2) it ignores court recognition of this science as legally acceptable, (3) it violates FDA’s own statements regarding the type of test being done, (4) it ignores the evidence presented as to safety and efficacy of the device, and (5) it misapplies the standards regarding classification of devices.  For these reasons, the denial of the petition should be reversed and the device reclassified as Class II.



     HiFi acknowledges the standard of review as stated by FDA’s attorneys.  HiFi understands it has a high burden in asking a Court to overrule an agency decision, especially one so scientifically based.  HiFi accepts this burden and shows the Court that FDA has abused its discretion and acted arbitrarily and capriciously in contravention of proven scientific fact.



     Attached as Exhibit A to this Objection is a scientific discussion of the process of DNA PCR.  The following paragraphs are a simplification of the process.

     The genetic material of most living organisms is deoxyribonucleic acid (DNA), or deoxynucleotide.  DNA consists of a chain of individual deoxynucleotides chemically linked in specific sequences.  Each deoxynucleotide contains one of four nitrogenous bases, designated as A, C, G or T.  All DNA-based life has strands of DNA with multiple deoxynucleotides arranged in different sequences.  For example, in a hypervariable region of DNA, ACGTACGT represents the genetic information for one organism while AGGATTCC represents that for another organism, although in actual organisms these strands are many times longer.  Natural DNA is double-stranded, consisting of two parallel single-stranded DNA molecules bonding together through a loose association, called a hydrogen bond, between the bases of their individual deoxynucleotides in a strict pairing relationship of "A-T" and "C-G" matches so that if one strand is ATCG, its match will be TAGC.

     Polymerase chain reaction (PCR) is the process of copying a targeted segment of a long DNA repeatedly and exponentially, called DNA amplification or replication, in the test tube.  The first step to perform PCR is to determine the target DNA to be amplified.  This can be done by reviewing the database of DNA sequences stored in the GenBank maintained by the National Institutes of Health (NIH).  NIH has mapped the DNA sequences of the genomes of various organisms and this information is publicly available.

After finding the DNA sequence of the target DNA, two short single-stranded DNA molecules are synthesized by chemical means. These short DNA molecules must match the two end pieces flanking the target DNA in correctly matched base sequence, one matching the sequence of an end piece in one of the two single strands, and the other matching the sequence of the pairing single strand in the opposite end. For example, if the double-stranded target DNA segment to be amplified has a segment of 20 “A” bases, namely AAAAAAAAAAAAAAAAAAAA, at one flanking end in one of the two single strands and a segment of 20 “C” bases, namely CCCCCCCCCCCCCCCCCCCC, at the other flanking end of the pairing single strand, the two 20-base short synthetic single-stranded DNA molecules must have a sequence of TTTTTTTTTTTTTTTTTTTT, and a sequence of GGGGGGGGGGGGGGGGGGGG, respectively. These synthetic short single stranded DNA molecules are referred to as “primers”. During PCR, these primers seek and bind to their respective single stranded DNA pairing partners when the double-stranded target DNA open up or split into two single-stranded DNA at high temperature.

The function of the DNA polymerase is to add the individual deoxynucleotides one-at-a-time to the end of a primer advancing in the direction toward the other primer in the opposite end of the target DNA. The newly formed DNA as an extension of the primer will become the new pairing partner of the single-stranded DNA molecule which the primer binds to, with a perfect “A-T”, “C-G” base pairing relationship between the new single-stranded DNA and the old single-stranded DNA, often referred to as the “template” in enzymatic DNA polymerization. Thus a DNA polymerase catalyzes the extension of a DNA primer in generating a longer DNA polymer if a template (target DNA) for the primer is available. DNA polymerase does not create new DNA. Although the target DNA strand may be hundreds, or thousands, of deoxynucleotides long in PCR, the primers match only the first 20-50 deoxynucleotides and the last 20-50 deoxynucleotides of the pairing single strands of the target DNA in the two flanking ends of the long molecule. Primers can be purchased readily on the open market from any number of vendors, or be created by a machine called DNA synthesizer.

Next, a sample of genetic material (e.g., blood, saliva) that might contain the target DNA (that is, the DNA of the suspected organism) must be obtained.  The sample is placed in a test tube together with the primers for target DNA amplification and a heat-resistant or thermostable DNA polymerase.

     The test tube is then heated and cooled such that the target DNA splits apart into two single-stranded DNA molecules under high temperature and the single-strands bond with the primers.  Because the primers are designed to match only the target DNA, no other DNA will be amplified: that is, if the target DNA is present in the sample, two copies are created after each heating/cooling cycle where only one existed, while if the target DNA is not present, nothing will happen.  Repeating this process 20 times will potentially create one million copies of one single target DNA molecule in the original sample.

     If target DNA exists in the sample, this selective amplification of the target DNA molecules results in an enormous number of copies of target DNA at the completion of PCR compared to all other DNA in the sample.  The final step of PCR is to subject the now amplified sample to a process of gel electrophoresis, in which different sized DNA molecules run at different speeds under the pull of a positive electric charge. Since the target DNA, if present, would have been “amplified” several million times, these overwhelming copies of the target DNA, all of the same size would form a distinct band visualized to the naked eyes under ultraviolet light after dye staining. No such band would be seen if there is no target DNA in the original sample.   

If a positive band of target DNA is visualized, final identification of the DNA in the PCR product, or amplicon, depends on deciphering the DNA sequence of the target DNA generated between the two synthetic primers. There are several techniques in deciphering DNA sequence, including probe hybridization, restriction fragment length polymorphism (RFLP), and DNA sequencing. DNA sequencing is the most accurate. Short tandem repeat analysis is a form of DNA sequencing applied in forensic science.





     The efficacy and acceptance of PCR is beyond argument, as noted in U. S. v. Morrow, 374 F. Supp. 2d 51, 61, (2005):

Over the past decade, numerous federal courts in a variety of jurisdictions have analyzed whether the introduction of DNA evidence garnered from the FBI Laboratory's use of PCR/STR analysis comports with the requirements laid down in Daubert. These courts have been virtually unanimous in finding that the use of PCR DNA testing is admissible, and many of these courts have taken judicial notice of the general reliability of such tests. See, e.g., United States v. Wright, 215 F.3d 1020, 1027 (9th Cir. 2000), cert. denied, 531 U.S. 969, 121 S. Ct. 406, 148 L. Ed. 2d 313 (2000); Hicks, 103 F.3d at 846-47; Beasley, 102 F.3d at 1448 (taking judicial notice of general reliability of PCR testing); Shea, 957 F. Supp. at 338-39; Ewell, 252 F. Supp. 2d at 106 (looking specifically at PCR/STR testing and listing twelve state appellate court cases finding PCR/STR DNA testing to be scientifically reliable); United States v. Cuff, 37 F. Supp. 2d 279, 282 (S.D.N.Y. 1999); Gaines, 979 F. Supp. at 1433-36 n.4 (collecting at least twenty state appellate court cases finding PCR DNA testing to be scientifically reliable); Trala, 162 F. Supp. 2d at 351 (looking specifically at PCR/DNA testing); United States v. Lowe, 954 F. Supp. 401, 416-17, 420-21 (D. Mass. 1997) (collecting approximately twenty state appellate court cases finding that PCR testing methodology comports with Daubert).  As the district court in Shea explained in 1997,


although PCR is a relatively new technology, it is based on sound scientific methods and it has quickly become a generally accepted technique in both forensic and non-forensic settings. Perhaps the strongest evidence on this point is the conclusion reached by the National Research Council's Committee on Forensic DNA Science that "the molecular technology [on which PCR is based] is thoroughly sound and … the results are highly reproducible when appropriate quality-control methods are followed." NRC II, supra, at 23; see also Mange, supra, at 287 (noting PCR's "widespread and growing applications [in the field of molecular biology]").




The crux of this case is whether sufficient information exists to establish special controls to reasonably assure the safety and effectiveness of the device so as to classify it as Class II.  See 21 U.S.C.A. § 360c(a)(1)(B).  As opposed to cases “where the benefits are essentially impossible to determine” because of the complete lack of scientific proof for the device (General Medical Co. v. FDA, 770 F.2d 214, 221 (1985)), the science underlying HiFi’s device is universally accepted.  HiFi proved, and submitted the proof to the FDA in its petition, that the device is safe and effective through testing and comparison with an FDA approved test for HPV.  The petition should have been granted.

1.  HiFi Submitted Valid Scientific Evidence Regarding The Safety And Effectiveness Of The Device In Accordance With FDA Regulations.


The safety and effectiveness of the device when used with special controls was proven in the petition as required by 21 CFR §860.7.  HiFi addressed all of the factors listed for consideration in §860.7(b):

1) The persons for whose use the device is represented or intended:  The device is a test for sexually active women at risk for infection with HPV.  AR 112.  There are no risks of overdose, infection, or other dangers related to implanted devices or drugs.  Indeed, a patient will likely never come in contact with the device at all.

2) The conditions of use for the device, including conditions of use prescribed, recommended, or suggested in the labeling or advertising of the device, and other intended conditions of use:  Because of the technical nature of the device, it is to be used only by qualified professionals within state- and federal-certified laboratories under supervision of a medical director.  This is not a device to be placed on a pharmacy shelf and used by an untrained consumer.  AR 125.

3) The probable benefit to health from the use of the device weighed against any probable injury or illness from such use:  The benefit is clear – the identification of HPV DNA with the ability to obtain type-specific HPV DNA to determine whether specific persistent infection exists.  AR 125.  The potential injury is minimal, if any – the device itself cannot harm the patient, and it is used in conjunction with (and not in lieu of) Pap smear cytology examinations, and never generates a stand-alone diagnosis of disease (AR 111, 114, 117), to ensure that a physician’s judgment based on all relevant factors is paramount in any patient decision (AR 125).  See, infra, discussion of unreasonable risk of injury.

4) The reliability of the device:  Again, the science is unassailable.  More convincing, this device outperformed the Digene HC2 device, currently FDA-approved for HPV testing.  AR 142-148.  Specifically, out of 513 patient samples, the device detected 107 cases of HPV, including 74 high risk cases, while the Digene test detected only 67 cases, including 50 high risk cases.  AR 147-148.  Also significantly, the HiFi device detected all of the cases Digene’s device detected – in other words, HiFi’s device did not miss any Digene hits, but Digene missed many of HiFi’s hits.  Digene’s device uses technology from 1988, before DNA PCR was developed.  Further, HiFi’s device could detect as few as 10 copies of target DNA (AR 140), while Digene’s could only operate with approximately 100,000 copies of DNA present (AR 119).  The results of these tests were reproduced in parallel testing to confirm accuracy.  AR 149.  Further, the all PCR results were confirmed by DNA sequencing, proving that the DNA identified was that of HPV, each validated by the GenBank sequence database for specific HPV genotyping, beyond a reasonable doubt.  AR 140.  FDA has not addressed this in either its denial of the petition or its motion to dismiss.

All of the above evidence resulted from studies controlled and performed as detailed in the petition (AR 142-151) as required by §860.7(c)(2) and (f). 

Because the benefits of the device outweigh the risks when used according to the proper conditions and under a physician’s care, the device is safe in accordance with §860.7(d).

Because the device provides clinically significant results (indeed, more accurate results than another approved device) in the target population, the device is effective in accordance with §860.7(e).

Simply put, all regulations have been satisfied.  The proven scientific basis and the specific relation to an approved test, in addition to the specific, independent reproducibility of the test, prove that the device is safe and effective.


2.   FDA’s Reasons For Denial Fail.

     All of the reasons cited by FDA for denial of the petition fail when viewed against the scientific basis of the device, the proven safety and effectiveness of the device, and the record.  FDA’s denial is based solely on unsupportable, arbitrary judgments, perhaps due to FDA’s failure to understand the science, refer the matter to a panel for review, and its rush to enter a decision after its failure to comply with the statutory time limitations.


     A.   The Device Is Not A Cancer Test.
    FDA’s argument that a “false negative” will inhibit women from further testing, resulting in untreated cancer (AR 496-498) proceeds from the fundamental error that the device screens for cancer.  AR 495, see also AR 500 (“Without knowing whether the [test] subjects are positive or negative for cervical precancer or cancer, FDA cannot assess clinical sensitivity, meaning the proportion of individuals who have precancer/cancer who test positive on the HPV DNA test”).  It does not.  The device merely amplifies HPV DNA, regardless of whether the HPV detected is one of the high risk types.  See AR 147 (showing all genotypes detected).  It does not indicate whether a persistent infection exists, which is the main indicator of risk.  Def.s’ Br. at 10.  It does not even indicate whether a “high-risk” type of HPV exists in the sample.  R 124.  It merely amplifies HPV DNA, which can (1) if deemed appropriate by the qualified professionals of a certified clinical microbiology laboratory and (2) in a completely separate procedure unrelated to the device, be tested for the presence of specific types of HPV.  AR 147.  Both of the intended uses for the device (AR 496) state that its purpose is to screen the presence of HPV DNA and provide suitable materials for accurate HPV genotyping by direct DNA sequencing, not to indicate potential cancer.  Even FDA’s own statements regarding HPV tests confirm that they do not test for cancer: on March 31, 2003, an official FDA News Release (P03-26) from the Commissioner’s office stated:

The FDA today approved expanded use of a laboratory test to detect the presence in women of human papillomavirus (HPV), one of the most common sexually transmitted infections.


The HPV DNA test does not test for cancer, but for the HPV viruses that can cause cell changes in the cervix. If left untreated, these changes can eventually lead to cancer in some women.


     This is a test for a virus, used in conjunction with existing technologies, and more effective than other tests currently available.  It is not a cancer test and should not be viewed as one.


B.   HiFi’s Data Supporting Reclassification Is Sufficient To Prove That The Device Is Safe And Effective.


As shown above, HiFi tested the device itself, compared the results to the FDA-accepted device, and confirmed the results by DNA genotyping.  All of the bases for the denial represent either a misunderstanding of the science involved in DNA PCR or a refusal by FDA to acknowledge the basic efficacy of the device.


1.   Cross-Reactivity Does Not Exist In Genotyping

FDA's arguments that no studies were performed for cross-reactivity and interfering substances misunderstands PCR technology.  In the HPV DNA PCR procedures, "promiscuous" primers are used to amplify the target HPV DNA of numerous genotypes; that is, the primers will bond to any DNA that matches the target ends, even if it is not HPV.  Because the primers replicate any DNA with two segments that match the primers, there is a chance that some other types of DNA may be amplified.  However, the sizes of these non-specific PCR products are different from those of HPV DNA, and can be readily recognized and excluded by the scientists performing the test.  The purpose of PCR amplification is to screen the presence of HPV DNA and to provide a presumptive evidence of possible presence of HPV DNA in the sample.  This is the sole use of the device: to amplify DNA that is presumptively HPV.  Final confirmation of the HPV DNA and its genotype, if detected, depends on additional analytical techniques in the section of cross-reactivity, the most accurate of which is DNA sequencing, similar to that used in criminal court cases and paternity testing.  Since HiFi recommends DNA sequencing to be used as the standard technique for validation of HPV DNA PCR products and for accurate HPV genotyping, cross-reactivity is not an issue in using HiFi’s device.

Similarly, because the device can perform with miniscule amounts of DNA present, interfering substances do not affect the results as significantly as with current probe technology used by other HPV tests currently marketed.  In any case, the results of the HiFi device are more accurate than those of the the currently approved Digene HC2 device (AR 147-148) and should therefore be acceptable to FDA.


2.   Sensitivity Of The Device Was Established.

As stated before, the sensitivity of the device was established though comparison with the Digene device, the only FDA-approved standard.  Further, sensitivity was confirmed through testing diluted specimens of known quantity.  AR 140.  FDA claims that these tests did not mimic “real clinical specimens,” although FDA gives no indication of the ability to measure the amount of DNA present in such specimen before amplification. There are more than 100 genotypes of HPV and at least 40 genotypes of them are clinically relevant in the female anogenital area. Each HPV genotype is amplified by different primer pair with different efficiencies. There is no generally accepted standard to measure sensitivity of HPV detection in clinical specimens because the standard genotypes cannot be decided with general agreement. It is impractical to set 40-100 standards for HPV detection sensitivities. The FDA has not set such a standard for evaluation of HPV DNA testing devices. FDA’s other statements in this paragraph have no support whatsoever.  What FDA considers “statistically significant” is never stated and simply arbitrary, and testing for all high-risk types of HPV is redundant as the issue here is sensitivity of the device and procedure itself, not the specific results of the device. The specificity of the device is dealt in preceding section.


3.   The Device’s Sensitivity Related To Women Was Established.


FDA should recognize that, as the device is intended to detect HPV in vaginal samples, the target population is women.  Samples from women were submitted from doctors in and around New Haven, and those were tested.  AR 142.  FDA has cited no authority, and HiFi is aware of none, that shows that HPV is a strictly age-based infection. All sexually active women exposed to HPV-infected sexual partners are susceptible to HPV infection. All of the language regarding precancer and cancer further shows that FDA misapplies the current understanding of HPV and its relation to cancer.


4.   FDA’s Specificity Argument Simply Restates Other Arguments And Ignores The Petition.


To the extent that the specificity argument (AR 500) merely restates the sensitivity argument (AR 500) and its focus on cancer, the argument fails for the reasons stated above.


FDA also argues that it cannot identify “the precise degree” of low-risk HPV types detected.  AR 500.  The petition, however, clearly states the types detected, including low-risk types. AR 147.  Further, this alleged insufficiency could certainly be cured by limiting the authorized use of the device to high-risk HPV types.  The definition of “high-risk” types may be modified as medical science advances. HiFi’s device is designed to detect all potentially clinical relevant HPV genotypes. The physicians and the epidemiologists will decide which HPV genotypes are of high risk, which may be related to genetic make-up of the host as some cited references in the Petition have indicated.  Therefore, this “precise” language seems designed to inhibit any serious consideration of the issue by requiring HiFi to adhere to an unknown, undefined standard.


5.   Genotyping Is Not Related To Cancer.  

Again, FDA erroneously conflates HPV with cancer.  AR 500-501.  Accurate HPV genotyping can be accomplished though the use of DNA sequencing using the GenBank database maintained by the National Institutes of Health.  This identifies the virus at issue, not the presence of or risk for cancer.


6.   Reproducibility Has Been Proven.

FDA engages in pure speculation when it states that the study failed to account for variables in the testing.  AR 501.  No variables were shown or alleged at any point.  Indeed, the entire issue of testing procedure is the subject of the special controls recommended by HiFi for the use of the device, discussed below.  The test HiFi performed was reproduced, and the use of the device by laboratory professional will ensure such procedures are followed.


7.   Stability Of The Device Was Established.

Stability of the device over time and at different temperatures was established through testing.  The results of the tests were reported.  AR 150.  Again, FDA has simply chosen to state that HiFi failed without providing any reason why.


C.  HiFi’s Controls Are Adequate To Provide Assurances Of Safety And Effectiveness Of The Device.


FDA’s assertions that special controls will not suffice to ensure the accuracy of the device are similarly faulty.  AR 501-502.  The controls reviewed by FDA show that the device will only be used by professionals, thus allaying fears of user error.  Warnings that the device should be used only in conjunction with Pap smears and physician oversight further protect against relying too heavily on the device as the final arbiter of patient treatment.  Genotyping will confirm positive results, and this process is clearly understood by the scientific community.  False negatives are morerare than with Digene’s approved device, so the danger of delayed treatment is greater now than if the device were reclassified.  In sum, all of the issues discussed in the prior sections can be addressed with the use of these controls.  There is little question as to the safety and effectiveness of the device, and the special controls further ensure it.


D.   Sufficient Information Exists To Establish Special Controls Such That The Device Can Be Reclassified As Class II.


In its final argument, FDA claims that insufficient information exists to establish controls to ensure the safety and effectiveness of the device.  AR 503.  In this FDA makes the same errors as throughout its denial letter.

FDA states that the device may not be effective with actual patients.  Only actual patients were used in the study supporting the petition; actual patients and samples therefrom gathered by area doctors.  FDA states that the device has not been demonstrated to determine what portion of the patients have cancer – but the device is not a cancer test.  FDA continues to ignore the performance of the Digene test with the HiFi device.

Even while stating that “many reasons” exist as to why the device may not be effective, FDA cites only three:

1.  FDA claims that because the device uses a small sample, the device may not work if few cells containing target DNA exist in the sample.  This ignores the data submitted with the petition showing as few as 10 copies of DNA are required for a successful test.  This alleged defect is also simply curable – require a larger sample.  FDA has no basis for believing a simple control as to the sample size will not cure this defect.  (Indeed, it seems that the great ability of the device to detect small amounts of DNA is being used against it.)

2.   FDA claims that the risk level of each type of HPV is the subject of debate.  Why debate about the risks of one type of HPV over another should prevent a device that can detect both types from being used is puzzling.  If a physician diagnoses a type of HPV, the physician (not the device) will determine the risk related to that type.  It makes no sense that the medical debate about the effect of certain viruses should impede attempts to detect the virus.    

3.   FDA claims that probe design has not been sufficiently shown.  Probes are not used.  Primers, as discussed above, are used.  The dictionary defines a “probe” as “a piece of dNA or rNA corresponding to a gene or sequence of interest, that has been labeled either radioactively or with some other detectable molecule, such as biotin, digoxygenin or fluorescein. As stretches of DNA or RNA with complementary sequences will(hybridise), a probe will label viral plaques, bacterial colonies or bands on a gel that contain the gene of interest.”  The glossary of defines a probe as a “defined nucleic acid (DNA or RNA) that can be used to identify, usually through autoradiography, specific DNA or RNA molecules bearing the complementary sequence.”  In essence, a probe identifies DNA molecules.

     In contrast, defines a primer as “a short pre-existing polynucleotide chain to which new deoxyribonucleotides can be added by dNA polymerase,” while “a short sequence (of RNA or DNA) from which DNA replication can initiate.”  In other words, a primer is used to replicate DNA, not identify it.

That the Defendants’ Brief attempts to claim FDA’s definition of “probe” includes primer demonstrates the carelessness with which FDA reviewed the petition in relation to current science.  Def.s’ Br. at 26.  Indeed, Defendants’ Brief attempts to overwrite the error by claiming that the “FDA’s general definition” of a probe includes those processes used to identify DNA for replication.  Id.  In contrast, the actual language of FDA in the record clearly shows that probes are meant to “recognize” DNA, not replicate it.  AR 306, n. 40.  For FDA not to know the basic difference between DNA amplification with primers and DNA detection with probes certainly calls into question FDA’s denial of the petition.



     When FDA wrote “We have determined that your type of device is classified as a class III device by the approval order for the VRAPAP [ViraPap] Human Papillomavirus DNA Detection Kit dated December 23, 1988” (AR 20), it violated the law regarding de novo classification and the science of DNA PCR developed since 1988.  HiFi’s device is nothing like the 1988 device.  As noted by Defendants’ Brief at 7-8, the devices can only be considered the same “type of device” under 21 C.F.R. §860.3(i) if they do not “differ significantly in purpose, design, materials, energy source, function, or any other feature related to safety and effectiveness, and for which similar regulatory controls are sufficient to provide reasonable assurance of safety and effectiveness” for all devices of that generic type.  As shown, in the petition (AR 119), and as FDA should know, HiFi’s device uses primers to amplify DNA, while Digene’s uses enzyme-labeled probe to detect target DNA.  Therefore, the devices differ significantly in design, materials, and underlying scientific theory.  Like wearing glasses versus contact lenses versus corrective surgery, the end goal is not determinative of the procedure and does not make the device the same.  Because the device is a “type of device” that has not been classified, it should have been subject to de novo review.  See Def.s’ Br. at 37.



     The science underlying HiFi’s device is universally accepted – there is no doubt about the benefit of detecting viruses.  The device was successfully tested for safety and favorably compared to an accepted device.  FDA’s errors and arbitrary decisions related to the device do not provide a reasoned basis for the denial as required by law.  The decision of FDA should be reversed, the petition should be granted, and the device classified as Class II.

                   THE PLAINTIFF

                   HIFI DNA TECH, LLC








                        Anthony J. Musto

Attorney for Plaintiff

                             2 Sherman Court

                             Fairfield, CT 06824

                             203)259-4488 / Fx:(203)268-9661


                             Fed. Bar No. CT25373







Certificate of Service


I hereby certify that on the above date a copy of the foregoing Memorandum In Opposition To Defendants’ Motion To Dismiss Amended Complaint was filed electronically and served by mail on anyone unable to accept electronic filing. Notice of this filing will be sent by e-mail to all parties by operation of the court’s electronic filing system or by mail to anyone unable to accept electronic filing as indicated on the Notice of Electronic Filing. Parties may access this filing through the court’s CM/ECF System.


Attorney Drake Cutini

Office of Consumer Litigation

U. S. Dept. of Justice

P.O. Box 386

Washington, DC  20044




                        Anthony J. Musto

Attorney for Plaintiff

                             2 Sherman Court

                             Fairfield, CT 06824

                            (203)259-4488 / Fx:(203)268-9661


                             Fed. Bar No. CT25373



Exhibit A


                                        List of Contents    


I.      DNA sequencing                                                                                              

II.    Polymerase chain reaction (PCR)                                                                    

III.  The HPV DNA PCR Device proposed                                                            

IV.  Digene HC2 HPV DNA assay v. HPV DNA PCR                                                        


I. DNA sequencing


The genetic material of all known living organisms is deoxyribonucleic acid (DNA), except in certain viruses whose genetic material may be ribonucleic acid (RNA). DNA consists of a chain of individual deoxynucleotides chemically linked in specific sequences. Each deoxynucleotide contains one of the four nitrogenous bases which may be adenine (A), cytosine (C), guanine (G) or thymine (T), and a deoxyribose, which is a pentose (a sugar molecule containing 5 carbon atoms), with a hydroxyl group attached to its 3' position and a phosphate group attached to its 5' position.


RNA is very similar to DNA, but is usually single stranded, while DNA is usually double stranded. RNA nucleotides contain ribose while DNA contains deoxyribose (a type of ribose that lacks one oxygen atom), and in RNA the base “uracil” (U) of the nucleotide substitutes for the base “thymine” (T) which is present in DNA.


The contiguous deoxynucleotides that form the DNA chain are connected to each other by a phosphodiester bond linking the 5' position of one pentose ring to the 3' position of the next pentose ring in such a manner that the beginning of the DNA molecule always has a phosphate group attached to the 5' carbon of a deoxyribose. The end of the DNA molecule always has an OH (hydroxyl) group on the 3' carbon of a deoxyribose. Replication of DNA molecules catalyzed by an enzyme called DNA polymerase, the fundamental process of life, always proceeds by attaching one single nucleotide at a time to the 3’ end in a 5’ to 3’ direction of an existing segment of DNA, referred to as the primer which is annealed to a template. The selection of the particular one of the four single nucleotides to be added to the 3’ end of a DNA primer during enzymatic DNA extension depends on the base on the template, which is opposite to the immediate vacant slot next to the 3’ end of an extending primer.

DNA usually exists as a double-stranded molecule in which two antiparallel DNA strands are held together by hydrogen bonds between the bases of the individual nucleotides of the two DNA strands in a strictly matched "A-T" and "C-G" pairing manner. It is the order or sequence of the bases in a strand of DNA that determines a gene which in turn determines the type of protein, the basic substance of a living matter, to be synthesized. Therefore, determination of the sequence of the bases in a DNA strand which also constitutes the genetic code for a protein – the basic substance of life, for an individual person, for a species, and for a genotype within a species, including the microorganisms and viruses, is the scientific basis of modern biology at the molecular level.

The base sequence of a DNA molecule is like a “cryptic” passage of letters arranged in a specific order and is not legible unless the sequence of the letters written in the passage is deciphered. The process used to determine the sequence of the bases in a DNA molecule is referred to as DNA sequencing. Among the techniques of determining the base sequences of a DNA molecule, also called the template, the most popular method was developed by Fred Sanger, a
1980 Nobel Prize winner in Chemistry. It is based on the ability of a DNA polymerase to extend a primer annealed to the DNA template to be sequenced in the presence of four normal deoxynucleotide triphosphates (dNTPs), namely, dATP, dCTP, dGTP and dTTP, and on the ability of the nucleotide analogs, the dideoxynucleotide triphosphates (ddNTPs), namely, ddATP, ddCTP, ddGTP and ddTTP, to terminate the extension of an elongating primer, namely a deoxynucleotide polymer, at various lengths.


In the dye terminator technology for DNA sequencing which is a modified automated Sanger method, the fluorescent dye-labeled ddATP, dye-labeled ddCTP, dye-labeled ddGTP and dye-labeled ddTTP are coupled with different fluorescent dyes, each emitting a specific light spectrum, thus directly reporting the type of ddNTP at the 3' terminus of the DNA fragment. These dye-labeled ddNTPs serve the dual function of a specific base terminator and a "color marker". The polymerase DNA extension reaction is performed in a single test tube to generate the required specifically terminated and specifically dye-labeled DNA fragments of various sizes, each having the same starting sequencing primer on one end and differing from the fragment of the immediate next size by only one base in length. Since all the newly formed negatively charged DNA fragments which are composed of the same number of bases (size) and labeled by the same colored terminator identifying their end base (A, C, G or T), migrate at the same speed in the capillary gel under the positive attraction of the anode, a ladder of bands of DNA fragments, each band of fragments carrying the same specific colored terminal base and each band composed of fragments of the same one-size DNA molecules differing from those in the adjacent band by one base only, will be formed during electrophoresis, representing the DNA sequence of the template.


The "DNA sequence of a gene" or "of a gene region" is often regarded as the ultimate result or the gold standard of a molecular genetic test for humans, animals, microbes and viruses alike.



The uniqueness of a hypervariable sequence of the nucleotides in a chain of DNA and the power of DNA sequencing in discriminating different species or genotypes can be illustrated by a simple exercise with a calculator as follows.


Theoretically, if the target hypervariable DNA sequence of a species is composed of randomly distributed DNA bases of four letters, namely A, C, G and T, and the number of bases of a hypothetical DNA sequence is one, say ”A”, the probability of achieving a 100% sequence match between two DNA sequences, each having one base, by meaningless pure random chance association rather than by mathematical calculation supported by science based on the laws of physics (e.g. interaction between molecules, electrons, electrical charges, etc) is one-to-four, i.e. one out of four alignment attempts (1/4). If the number of bases in the target DNA sequence is two, say “AA”, the probability of achieving a 100% sequence match between the two DNA sequences by random chance association is reduced to 1/(4x4) = 1/16. If the number of bases is 3, say “AAA”, the probability of 100% match by random chance association is further reduced to 1/(4x4x4) = 1/64. If the number of bases is 16,say “AAAAAAAAAAAAAAAA”, the probability of 100% match of two DNA sequences by random chance association is 1/( 416 ) = 1: 4,294,967,296. The latter denominator exceeds the entire length of the human genome which consists of 3 billion nucleotides (bases). Therefore, the probability of a 100% match between two hypervariable DNA sequences of 20-30 bases by random chance association and not due to specific unique genotyping match governed by the laws of physics with mathematically definable reproducible results is infinitesimal.


II. Polymerase chain reaction (PCR)


In order to perform DNA sequencing determination on DNA samples collected from the crime scene, the criminal suspect, the suspected microbe or virus, a DNA amplification is needed to replicate the target DNA of interest to numerous copies. Otherwise, the quantity of target DNA in the sample collected is not adequate for direct DNA sequencing. 


Polymerase chain reaction (PCR) is a technique used to amplify the number of copies of a specific region of DNA in the test tube. Kary Mullis was awarded the Nobel Prize in Chemistry in 1993 for his development of this technology. With PCR procedures, tiny amounts of a DNA with a specific base (A, C, G, T) sequence can be copied exponentially within hours. Thus, getting sufficient DNA for analysis has become much easier. Before the advent of the PCR technology, a target DNA molecule must be inserted into a vector which would have to be introduced into a host cell, like an E. coli bacterium, to be cloned for replication to obtain sufficient DNA for sequencing.  


PCR is a process of primer-defined amplification of DNA. The first step for PCR is to synthesize a pair of "primers" of about 20 bases-long each, using a machine which can link the bases (A, C, G, T) together in the order desired, by adding one base-at-a-time to the machine (DNA synthesizer). After adding the unknown sample, the primers, a heat-stable DNA polymerase and the building blocks of dATP, dCTP, dGTP and dTTP into a test tube, the mixture of the tube is subjected to cyclic heating and cooling for 20-40 rounds at three steps for each round in the following schedule:

1) Denaturing of the double-stranded target DNA by heating the sample to a high temperature, e.g. 85-95°C, which would cause the paired double strands to separate (single strands now accessible to primers which are always added in excess).

2) Lowering the temperature to 40-55°C, which would allow the primers to anneal (bind) to the two opposing ends of the paired single strands of the target DNA to be amplified, one primer to one end of a single strand and the other to the opposite end of the opposite single strand.

3) Raising the temperature to 65-75°C, which would allow the DNA polymerase to function, adding the correct nucleotides one at-a-time to the 3’ end of the extending primer in order of a mirror image sequence of the template (target DNA).

4)Repeat the above cycle 20 to 40 times in a thermocycler.


In about 20 cycles, the PCR process will theoretically produce approximately one-million copies (2 raised to the 20th power) of one original target DNA molecule flanked by the pair of primers on both ends, if the target DNA molecule is present, whereas other concomitant non-target DNA molecules in the sample that have no matched sequences with the primers are not amplified. The amplified target DNA now in excess quantity over the others can be detected after gel electrophoresis as a fluorescent band in the expected molecular weight range. This presumptive target DNA PCR product, or amplicon, can be confirmed by determining the base sequence of the region between the primers by various methods, including probe hybridization and DNA sequencing, as final validation of the target DNA observed. The direct DNA sequencing technology after PCR amplification does not use probes to determine DNA sequence of the target DNA. It is based on the DNA polymerase-catalyzed enzymatic nucleotide extension and termination reaction, a research tool in molecular biology first developed by Fred Sanger around 1980 and perfected in the past 20 years, in part to expedite the completion of the National Human Genome Project.     


III. The HPV DNA PCR Device proposed


The HPV DNA PCR device proposed for the FDA to evaluate can be summarized as follows:


(1)     The HPV DNA Nest PCR Test (the device) provides reagents to replicate target HPV DNA molecules. Since the PCR product contains a large number of newly formed identical copies of DNA (the now detectable mass of identical DNA molecules), it is suitable for direct automated DNA sequencing if visualized by the nested PCR system, performing two PCR amplification with two sets of primers in tandem.


(2)     The proposed nested PCR procedure and the subsequent automated DNA sequencing are performed by qualified highly trained health care professionals in a CLIA-certified high-complexity testing laboratory, for example a hospital clinical microbiology laboratory, under a qualified medical director. (CLIA = Clinical Laboratory Improvement Amendments of 1988)


(3)     The health care professionals in a CLIA-certified laboratory use the materials prepared with the HPV DNA Nest PCR Detection reagents (the device) to perform HPV DNA sequencing with equipment and reagents supplied by other vendors specialized in DNA sequencing technologies.


(4)     The health care professionals interpret the DNA sequencing results using the GenBank database maintained by the National Institutes of Health (NIH) to reach an accurate HPV genotyping.


(5)     The certified laboratory health care professionals report the interpretation of the HPV genotyping determination to the clinician who ordered the HPV test.


(6)     The clinician interprets the report of HPV genotyping against the history of the patient, the clinical observation, the clinical history of the patient, the Pap smear cytology results, and the past HPV genotyping information on the patient to make a patient management decision.


(7)     In conclusion, the device only makes identical copies of an HPV DNA for DNA sequencing. Its role in determining a clinical decision is that of a complementary factor to be considered along with the available patient information in toto.


IV. Digene HC2 HPV DNA assay v. HPV DNA PCR 


The present FDA-approved HPV DNA detection kit (the Digene HC2 Hybrid Capture HPV DNA detection test) is a DNA test developed before 1988, prior to introduction of the PCR technology into medical science. It depends on RNA probes designed to bind the target DNA to determine the DNA sequences of the HPV in the clinical samples. The RNA probes composed of the four bases (A, C, G, U) of nucleic acid similar to DNA except that the in the RNA molecules the base “T” has been replaced by a base “U” and that the sugar component, pentose, is a ribose instead of a deoxyribose. The probes are probably of about 25 bases long, and are proprietary. If an HPV DNA molecule has a DNA sequence complementary to the designed RNA probe, an RNA/DNA hybridization (one single stranded RNA molecule and one single stranded DNA molecule binding together) will take place, forming an RNA/DNA hybrid. The resulting RNA/DNA hybrid molecules will be “captured” on the test tube wall coated with an RNA/DNA-capturing antibody raised against any RNA/DNA hybrids. A second enzyme-labeled RNA/DNA detecting antibody raised against any RNA/DNA hybrids will be used to bind to the captured RNA/DNA hybrids already attached to the test tube wall. A substrate of the enzyme attached to the second antibody is added to the test tube to measure how much enzyme, if any, is attached to the test tube wall since the enzyme can make the substrate to emit light. The intensity of the light emitted is in proportion to the enzyme which is a reporter molecule for the second antibody against the RNA/DNA hybrids. A threshold level of light emitted above a base line is taken to be the evidence to indicate the presence of bound enzyme, in turn the presence of an RNA/DNA hybrid-detecting antibody, and in turn an RNA/DNA hybrid bound to the antibody fixed to test tube wall, and in turn the presence of HPV DNA with a signature sequence complementary to that of the RNA probe. Since one original RNA probe has been designed to bind a certain HPV DNA sequence only, it is assumed that a threshold intensity of the emitted light indicates the presence of the target HPV DNA sequence provided one single RNA probe is used in the RNA/DNA hybrid capture test. 


Since the Digene HC2 kit needs to detect 13 high-risk HPV types, at least 13 different RNA probes must be designed and incorporated in the HC2 reagents. A positive HC2 test indicates the presence of anyone or any combination of the 13 genotypes of HPV DNA in the sample. Because all HPV genotypes share closely related DNA sequences in this “hypervariable” region of the L1 gene encoding the major HPV capsid protien, the FDA Order of December 14, 2007 correctly expressed its concerns about “cross-reactivity” in all probe hybridization methods for HPV genotyping. Due to cross reactions between its RNA probes and non-target DNA molecules in the complex cervicovaginal specimens, and due to the lack of PCR target DNA amplification in the procedure, the analytical specificity and sensitivity of the HC2 HPV DNA test cannot meet the standard of the 21st century medical science.


All DNA tests involve determination of the order of the nucleotide bases, namely the DNA sequence, of the DNA molecule of interest. The “hybrid capture” HC2 test depends on signal amplification to express its final result in units of emitted light. However, the number of light units emitted in the HC2 test is determined by many factors unrelated to the target DNA sequence being tested. For example, the final number of photo units emitted is the function of a reaction between an enzyme linked to an antibody and its substrate. The activity of the enzyme is a function of the enzyme protein molecules linked to the second RNA/DNA hybrid-detecting antibody. The titer of the first RNA/DNA capturing antibody and the titer of the second enzyme-linked RNA/DNA detecting antibody all have important impacts on the number of light units emitted as the final result of the test. Using a quantitative light signal subject to the influence of so many independent or only remotely related factors, to determine the presence or absence of a highly specific signature sequence of HPV DNA is bound to generate a great number of false positive and false negative results, as reported in the recent medical literature.


In contrast to the HC2 HPV test kit, the proposed HPV DNA PCR device does not perform or claim to perform any HPV type determination. The device is merely used to prepare nested PCR product suitable for HPV genotyping by direct DNA sequencing to be performed in CLIA-certified high-complexity-testing clinical microbiology laboratories.


Neither the HPV DNA PCR device, nor the DNA sequencing molecular technology for HPV genotyping uses any probes, or depends on probe designs to determine DNA sequences. To perform DNA sequencing, the microbiologist first uses one or two pairs of general consensus PCR primers to amplify a highly conserved segment of the L1 gene DNA known to exist in all clinically relevant HPV genotypes. It relies on a pair of nucleotide primers flanking the region of target DNA for replication of a minute quantity of DNA to numerous copies. When a presumptive positive target DNA PCR product is observed, the PCR product composed of numerous copies of identical, newly formed DNA fragments is subjected to direct DNA sequencing for confirmation of the nature of PCR product and for accurate HPV genotyping. Accurate HPV genotyping depends on an automated computer-assisted DNA sequencing to decipher the individual bases of a “hypervariable” region flanked by the general consensus primers, similar to the “PCR fingerprinting” technology used to link the DNA of a suspect to the crime scene in forensic science. No probes are used in either case.


Automated modified Sanger DNA sequencing is a direct method for accurate DNA sequence determination, based on cyclic specific DNA polymerase-catalyzed enzymatic nucleotide extension and termination reactions. Standard step-by-step DNA sequencing procedures are well characterized in various published technical texts and generally supplied by the manufacturers of DNA sequencing equipment and reagents. In practice, after the sequencing procedure is completed, a segment of the DNA sequence is excised from the computer-generated electropherogram to confirm that the obtained DNA sequence is that of an HPV DNA and to determine the correct HPV genotype by BLAST sequence alignment algorithms against the GenBank database maintained by the National Institutes of Health (NIH). Each HPV DNA genotyping is established beyond a reasonable doubt when a signature sequence is validated by the GenBank database with 100% base matches between the Query and Subject sequences. There is no false positive result or false genotyping if the CLIA-certified clinical laboratory follows the standard procedures recommended by the NIH in using the GenBank database as an on-line genotyping tool.


The similarities and dissimilarities between the HPV DNA Nest PCR and the Digene HC2 test are tabulated for comparison on the next page, as presented in the Petition (FDA Docket No. 2007P-0210).








Digene hc2

Intended Use              

Qualitative detection of all HPV types, with end-product suitable for direct DNA sequencing        

Qualitative detection of 13 high-risk HPV types, not to be genotyped

Indications for its use


Screen patients with ASCUS Pap smear results

Genotyping results used adjunctively with Pap smear in women 30 years and older

Screen patients with ASCUS Pap smear results

Used adjunctively with Pap smear in women 30 years and older

Target molecules for detection

HPV genomic DNA

HPV genomic DNA





Digene hc2

Method of amplification

Nested PCR amplification of target DNA

Signal amplification

Mechanism of amplification

By consensus primer PCR

By reporter molecules

Results detected by

Agarose gel electrophoresis of target DNA PCR products

Chemiluminescence generated by enzyme substrate

Validation of end-product by direct DNA sequencing

Possible and recommended

Not possible

Sensitivity of detection
(purified HPV DNA)

1-10 copies of HPV genomic DNA

105-106 copies of HPV genomic DNA

Sensitivity of detection

(clinical specimens)


37.9-100% depending on genotypes

(clinical specimens)

100% with validation by DNA sequencing for genotyping     

74.6% (high-risk HPV) confirmed by DNA sequencing in split parallel clinical specimens

Specimen types

Liquid-based alcohol-preserved cell suspensions

Liquid-based alcohol-preserved cell suspensions and tissue biopsies

Multiple HPV infections

DNA sequencing not possible for specific genotyping

Capable of detecting 13 high-risk HPV types as a group




DNA is a molecule that encodes the genetic information in all living organisms, as well as viruses because of its unique DNA base (A, C, G, T) sequence. These bases are also referred to as nucleotides. The DNA in a person’s blood is the same as the DNA found in his saliva, sweat, bone, the root and shaft of hair, earwax, mucus, urine, skin tissue, and vaginal and rectal cells. Most importantly, because of polymorphisms in human genetic structure, no two individuals have the same DNA, with the notable exception of identical twins.

With PCR procedures, tiny amounts of a DNA with a specific base (A,C,G,T) sequence can be copied exponentially within hours. Thus, getting sufficient DNA for analysis has become much easier since it became possible to reliably amplify small samples using the PCR method.

With the PCR-amplified DNA, the forensic laboratory and forensic scientists can conduct DNA typing using short tandem repeat (STR) analysis, restriction fragment length polymorphisms or the direct DNA sequencing technology to determine the DNA sequences to identify the possible connection of the suspect and the human materials found in the crime scene. The so-called “PCR-fingerprinting” technology has been widely accepted by the U.S. Courts as evidence linking a suspect and the human materials found in the crime scene.

As a judge deliberating PCR/fingerprinting evidence, a physician would use HPV genotyping data in evaluating the conditions of a patient.  The events involved in the use of HPV DNA PCR in preparing clinical specimens can be summarized for a schematic analysis as follows:


      (1) The HPV DNA Nest PCR Test (the device) provides reagents to replicate target HPV DNA molecules. Since the PCR product contains a large number of newly formed identical copies of DNA (a detectable mass of identical DNA molecules), it is suitable for direct automated DNA sequencing.


      (2) Automated DNA sequencing is performed by qualified highly trained health care professionals in a CLIA-certified high-complexity testing laboratory under a qualified medical director.


      (3) The health care professionals in a CLIA-certified laboratory use the materials prepared with the HPV DNA Nest PCR Detection reagents (the device) to perform HPV DNA sequencing with equipment and reagents supplied by other vendors specialized in DNA sequencing technologies.


      (4) The health care professionals interpret the DNA sequencing results using the GenBank database maintained by the National Institutes of Health (NIH) to reach an accurate HPV genotyping.


      (5) The certified laboratory health care professionals report the interpretation of the HPV genotyping determination to the clinician who ordered the HPV test.


      (6) The clinician interprets the report of HPV genotyping in conjunction with the clinical observation, the clinical history of the patient, the Pap smear cytology results and the past HPV genotyping information on the patient to make a patient management decision.


In conclusion, the HPV DNA PCR device only makes identical copies of an HPV DNA for DNA sequencing. Its role in determining a clinical decision is complementary to the above patient information in toto