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Yale Researchers Develop Score-Based Protein Biomarker Dx for Prenatal Sepsis

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Catalin Buhimschi
Assistant professor
department of obstetrics, gynecology and reproductive sciences, Yale University School
of Medicine
Who: Catalin Buhimschi
 
Position: Assistant professor, department of obstetrics, gynecology and reproductive sciences, Yale University School of Medicine
 
Background: Fellow, maternal-fetal medicine, department of obstetrics, gynecology and reproductive sciences, Yale University School of Medicine, 2003-2004; assistant professor, maternal-fetal medicine, department of obstetrics, gynecology and reproductive sciences, Yale University School of Medicine, 2004 to present.
 

 
Catalin Buhimschi and his colleagues at Yale University School of Medicine have devised a score based on four proteins they had previously identified that may be able to predict preterm delivery and the presence of infection in the placenta and severe infection in newborns.
 
Amniotic fluid was taken from 169 women to create the score, which Buhimschi and his colleagues call the Mass Restricted score. The research and its results, published on the open-access journal, PLoS Medicine, can be found here. The following is an edited version of a conversation between ProteoMonitor and Buhimschi this week.
 
Describe for me the work you did to identify the four proteins that make up the Mass Restricted score.
 
Originally, we analyzed samples of amniotic fluid with the purpose of identifying biomarkers [for] intra-amniotic inflammation and infection. We have been successful in identifying four biomarkers: defensin 1 and 3, and calgranulin A and C. We determined at that particular time that the proteomic profile that we described, which we called the MR score, has very high accuracy in diagnosing intra-amniotic inflammation.
 
Now, one of the biggest issues in proteomic research is: Once you describe the profile, how is the profile going to perform in a real, let’s say, clinical scenario?
 
That is the reason approximately two to three years ago we basically started this prospective study where we analyzed samples of amniotic fluid, immediately following their collection [from] patients who had symptoms of preterm labor and preterm premature rupture of the membrane.
 
We did this in a prospective fashion in consecutive patients. The samples of the amniotic fluid that we used were fresh samples, not stored in any tissue banks. And we then compared the results of our proteomic profile with all the clinical laboratory tests that are proposed for diagnosis of intra-amniotic inflammation and infection.
 
So we tested this profile, and if I’m not wrong, I think this is, in fact, probably the first testing of a proteomic profile in a prospective fashion. And we determined that this profile maintains its high accuracy diagnosing intra-amniotic inflammation, and the addition of the score to the rapid test proposed for diagnosing infection actually improves significantly the accuracy of the clinician in his or her ability to diagnose infection in women with preterm labor and preterm premature rupture of the membrane.
 
How does the MR score work?
 
In the original study, [the researcher] did a manual discrimination. She analyzed SELDI strips from patients who had intra-amniotic inflammation and patients who are normal controls. She did a manual discriminatory analysis and invented, in fact, the six criteria regarding how to identify a peak, how you can say a peak is present or not.
 
In the original phase, we came up with 13 peaks, but we determined [that] by doing a more restrictive analysis, only four of them are necessary to diagnose intra-amniotic inflammation. It was a Boolean type of analysis, and that’s the way that we arrived at these four biomarker peaks.
 
When the peaks are identified, it’s very important. But it’s also very important to know the identity of the peaks. Therefore, there were a lot of proteomic techniques that we employed in order to identify the peaks, including on-chip capture and SELDI-TOF analysis. And we have been able to establish their identity with high accuracy.
 
The Mass Restricted score is actually the combination of all four biomarkers. An MR-0 basically suggests that there are no biomarkers present, while for the presence of each one of the biomarkers, one point is added.
 
So in other words, an MR-0 [means] no biomarkers; an MR-1 suggests that there is one biomarker present; MR-2 [means] two biomarkers present; MR-3 three; MR-4, four of the biomarkers are present.
 
So once you identify them by SELDI, by counting them, you just add a score. But the greatest benefit of the MR score in comparison with all the other clinical tests is that the presence or the absence of a peak is a categorical presence [or absence]. So in other words, it’s a ‘yes’ or ‘no’ [answer], while using all the other clinical tests like glucose LDH you have to rely on certain cut-offs that were previously picked up by other investigators.
 
It’s very difficult to judge four tests at the same time, each one of them having its own cut-off. There’s a big discordance between these clinical tests, while an MR score basically tells you ‘treat,’ ‘don’t treat,’ or ‘send home.’
 
These four peaks, do they have to be present simultaneously to suggest there may be complications in the pregnancy?
 
It varies. What we discovered in the original phase of the study [was] that the presence of one or two of the proteomic biomarkers excludes the inflammation, while the presence of three or four of the biomarkers confirms their presence.
 
This was in the original phase, but without the prospective phase, we wouldn’t have been able to determine that, in fact, the presence of one of two of the biomarkers places the patient at risk of at least preterm labor. And one of the graphs in the PLoS paper shows that patients who have one or two biomarkers present, in almost 93 to 95 percent of the cases, this should be defensin 1 and defensin 3. Their presence in the amniotic fluid determines or identifies a subgroup of patients who are at risk for preterm premature rupture of the membrane.
 
This is one of the most important findings of this prospective study because while proteomic tools like SELDI were able to identify the presence of the biomarkers, none of the clinical tests that are proposed currently for clinical use were able to discriminate and identify this group of patients.
 
Therefore, if you use the clinical tests … the patient will go home and deliver prematurely a few days later, while by using proteomic tools, you will be able to identify them. And we hope in the future to be able to demonstrate in a randomized trial that [by] treating this group of women with one or two of the biomarkers, defensin 1 and 3, we will be to reverse the curve and prolong pregnancy.
 
Are defensin 1 and 3 the most important of the four markers?
 
We think all of them are all important, but the proteomic tools are able to identify this subgroup of patients when one or two of [these] biomarkers are present.
 
What about calgranulin C and A?
 
Once you have calgranulin C and A, basically, I believe the battle is lost. I believe the process is already irreversible.
 
Does a patient with defensin 1 and 3 progress to having calgranulin C and A?
 
In some of the patients where we have serum samples, yes. We publish in this PLoS paper one of the results [that shows] that there is a sequential appearance of the biomarkers. So it’s a progressive disease. And this is another important finding of the study.
 
Did you find any patients that have calgranulin C and A but not defensin 1 and 3?
 
No
 
How accurate is the MR score?
 
In a prospective study, it’s the most accurate test to diagnose intra-amniotic inflammation reported to date. The accuracy of the test is 93 percent.
 
What about for premature births?
 
We didn’t analyze the accuracy of the test in predicting preterm birth. What we know [is] an MR score of zero is associated with longer time between the amniocentesis [and] delivery, 34 days. A score of one or two, four days, while an MR score [of] three or four means it will be only 0.6 or 0.7 days until the delivery of the baby.
 
What is the need for the score? Will it lower the infant mortality rate?
 
Yes.
 
Significantly?
 
[It’s] very difficult to [say].
 
Can it lower health care costs?
 
Absolutely. Because [if] you catch the disease, you will be able to provide the treatment in utero for the fetus while the baby is still not delivered. So you start much earlier, therefore the morbidity for [the] baby can be significantly decreased, and you can avoid cerebral palsy in the future by doing this.

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