Congo Red Dot Test: A Groundbreaking Method to Diagnose Preeclampsia

Many of us may have previously encountered Congo Red in a chemistry lab where it was merely one of many pH indicators used in titration experiments. However, Yale University’s Dr. Irina Buhimschi and her research team have discovered that this dye’s properties can be exploited in the medical setting to detect the body’s pH imbalances. Congo Red can indicate the presence of preeclampsia in a quick and simple method that researchers call the Congo Red Dot Test.

What is Preeclampsia, and Why Does It Matter?

Preeclampsia is a hypertensive pregnancy condition accompanied by high protein levels in the urine that typically occurs after 20 weeks of pregnancy. Although it only affects about 5-8% of pregnant women, preeclampsia has the greatest impact on the mother and infant—especially if left untreated or diagnosed late. Preeclampsia entails blood vessel constriction, which increases blood pressure and reduces blood flow. Thus, in severe cases, the brain, kidneys, and liver can fail. Likewise, the baby is at risk for poor growth, placental abruption (when the placenta separates from the uterus prematurely), and other complications associated with insufficient blood flow to the uterus.

Additionally, preeclampsia is dangerous because it can lead to the development of more serious conditions like HELLP syndrome and eclampsia. Women with HELLP syndrome have Hemolysis (breakdown of red blood cells), Elevated Liver enzymes, and a Low Platelet count, all of which increase the risk for the aforementioned complications of preeclampsia. When left untreated, preeclampsia can evolve into eclampsia, a life-threatening condition involving seizures that can lead to coma. According to the World Health Organization, around 63,000 women die each year from preeclampsia or eclampsia, 99% of whom live in low or middle income countries.

Dr. Buhimschi calls preeclampsia the “disease of theories” because there are no known causes, only hypotheses that emerge and are discarded when stronger theories appear. Therefore, researchers cannot specifically target the disease with drugs, and doctors do not know what exact symptoms to look for in pregnant women. Although doctors know that hypertension and proteinuria (abnormally high levels of protein in the urine) sometimes accompanied by abnormal swelling are common in those with preeclampsia, there are many other diseases that can cause these same symptoms. Also, pregnant women may suffer from the underlying cause of preeclampsia before the symptoms manifest themselves. This uncertainty is problematic because the only cure in that case is labor induction, which can be life-threatening to the child if born premature. Physicians and pregnant women are stuck with the dilemma of choosing an early delivery in fear of developing eclampsia. Doctors can prevent eclamptic seizures by using magnesium sulfate, but this would require diagnosing preeclampsia first. Dr. Buhimschi’s research into a cheap, effective way to diagnose this condition is essential in preventing deaths from preeclampsia by getting appropriate treatment to women more quickly.

Power of Proteomics

Dr. Buhimschi works in a translational lab, meaning that she conducts bench research in a close connection to the patient setting. Her lab is interested in proteomics, the study of the structure and function of proteins. “The interesting part of proteomics,” she says, “is that you’re unbiased, so these proteins unravel to you…you’re not intervening.”

While searching for a diagnostic tool for preeclampsia, Buhimschi’s lab first analyzed urine from a proteomic perspective since preeclampsia produces proteinuria. By looking at samples from women with preeclampsia and those without it, they hoped to find specific proteins unique to preeclampsia. The researchers discovered that women with preeclampsia indeed had specific protein fragments in their urine. They identified these fragments with other proteomic techniques and confirmed their results with more traditional methods like Western blots, a technique used to separate proteins based on size. These Western blots led to the key discovery that would eventually result in the Congo Red Dot Test.

Scientific Serendipity

The Western blots showed that some protein fragments from women with preeclampsia were much heavier and larger than the researchers expected. In fact, they were even larger than the parent sample. Buhimschi postulated that these fragments aggregated and thus could not be broken apart into pieces of the predicted size. The aggregates also indicated the presence of misfolded proteins. This evidence suggested not only that preeclampsia was a disease of misfolded proteins but also, more importantly, that it was congophilic.

More Than Just a pH Indicator

Congophilia is the ability of a substance to be stained by a specific class of dyes of which Congo red is the most prominent example. Due to specific ways of aggregation in misfolded protein complexes, the aggregates have spaces for the dye to enter and stain them. Divry established this method of identification in 1927 for amyloid fibrils in general and in 1934 for the amyloid plaques formed in patients with Alzheimer’s. Other diseases under this category are Creutzfeldt-Jacob disease, prion diseases, and now preeclampsia.

At first glance, the fact that Congo red can be an indicator for several different diseases seems problematic. However, Crezutzfeldt-Jacob and prion diseases tend to be rare, and Alzheimer’s is very unlikely to affect young pregnant women. It may be surprising that the usefulness of this dye in diagnosis has not been realized before, but all the other congophilic diseases, if not rare, are easily identifiable or chronic, in which case a quick diagnosis is not necessary.

What’s Left To Be Done

The next step for these researchers would be to have the Congo Red Dot Test be on the market and available to physicians, but more extensive clinical trials must be done first. Dr. Buhimschi’s translational lab has already done trials involving about four hundred people, but tests of women in different scenarios are still needed. Once the Congo Red Dot Test has been approved, though, this test is likely to become widespread due to the low cost of the Congo red dye.

With this method of reliable diagnosis, researchers can turn their attention to future treatments. Dr. Buhimschi envisions drugs that can break apart the aggregates and possibly prevent them from forming. Together with Dr. Charles Gabe from University of California Irvine, she is working on using antibodies that can be used in conjunction with the test to learn more about preeclampsia. Additionally, she mentions that scientists will now look into drugs that had been created to treat Alzheimer’s but were tossed aside as failures because they were not able to cross the blood-brain barrier to attack the plaques in the brain. Coincidentally, though, that property is exactly what is necessary in treating preeclampsia because reaching the baby could be fatal.

As for her own current research, Dr. Buhimschi will continue to investigate preeclampsia, which she calls one of the “black boxes” of obstetrics and . Specifically, she will study the specific proteins formed in preeclampsia in more detail. Now that she knows these proteins aggregate, she will examine their different compositions and conformations. She hopes to discover if certain aggregates are benign or cause more harm than others and if there are sub-types of preeclampsia. In doing so, better treatments may be designed and the cause of preeclampsia might be finally discovered. “The lesson for us is always believe the data that you have,” she says, “and don’t get too attached to your hypothesis. Because most of the time, the data is true, and you’re not.”

Additional Reading

Buhimschi, Irina, Edmund Funai, Guomao Zhao, Antonette Dulay, Sarah Lee, Christina Han, Erika Werner, Stephen Thung, and Catalin Buhimschi. “20: Assessment of global protein misfolding load by urine “Congo Red Dot” test for diagnosis and prediction of outcome in women with preeclampsia (PE).” American Journal of Obstetrics and Gynecology 201, no. 6, Supplement 1 (December 2009): S12-S13.

Buhimschi, Irina A., Guomao Zhao, Margaret A. Baumbusch, Sabina Janciauskiene, and Catalin S. Buhimschi. “239: Preeclampsia is a disease characterized by specific supramolecular aggregates of misfolded proteins and congophilia.” American Journal of Obstetrics and Gynecology 199, no. 6, Supplement 1 (December 2008): S78.

Buhimschi, Irina A., Guomao Zhao, Edmund F. Funai, Nathan Harris, Isaac E. Sasson, Ira M. Bernstein, George R. Saade, Catalin S. Buhimschi. “Proteomic profiling of urine identifies specific fragments of SERPINA1 and albumin as biomarkers of preeclampsia.” American Journal of Obstetrics and Gynecology 199, no 5 (November 2008): 551.e1-551.e16.

Lachmeijer, Augusta M. A., Guustaaf A. Dekker, Gerard Pals, Jan G. Aarnoudse, Leo P. ten Kate, and Reynir Arngrímsson. “Searching for preeclampsia genes: the current position.” European Journal of Obstetrics & Gynecology and Reproductive Biology 105, no. 2 (November 15, 2002): 94-113.

Saadat, Mandana, Soheila Marzoughian Nejad, Gholamreza Habibi, and Mehrdad Sheikhvatan. “Maternal and Neonatal Outcomes in Women with Preeclampsia.” Taiwanese Journal of Obstetrics and Gynecology 46, no. 3 (September 2007): 255-259.

Sevene, E, S Lewin, A Mariano, G Woelk, A D Oxman, S Matinhure, J Cliff, B Fernandes, and K Daniels. “System and market failures: the unavailability of magnesium sulphate for the treatment of eclampsia and pre-eclampsia in Mozambique and Zimbabwe.” BMJ 331, no. 7519 (October 1, 2005): 765-769.

About the Author: Nancy Huynh is a sophomore prospective Molecular, Cellular, and Developmental Biology major in Silliman College. She is still trying to figure out her way around the medical campus for these YSM interviews.

Acknowledgements: The author would like to thank Dr. Irina Buhimschi for kindly taking the time to come down to Silliman for an interview to explain her research.