A New Safe Blood Test to Diagnose Down Syndrome

14 03 2011

ResearchBlogging.org
The established method to prenatally diagnose chromosomal gross abnormalities is to obtain fetal cells from the womb with a fine needle, either by Amniocentesis (a sample of the fluid surrounding the foetus in the womb)  or by Chorionic villus sampling (CVS, a sample of the placenta taken via the vaginal route).
The procedures are not to be sneezed at. I’ve undergone both, so I talk from experience. It is kind of horrifying to see a needle entering the womb near to your baby, also because you realize that there is a (small) chance that the procedure will cause a miscarriage. Furthermore, in my case  (rhesus negative) I also had to get an injection of human anti-D immunoglobulin as a precaution to prevent rhesus disease after birth. Finally it takes ages (ok 2-3 weeks) to hear the results. At that point the fetus is already 14-18 weeks old and there is little time to intervene, if that is what is decided.

en: Karyotype of 21 trisomy (Down syndrome) fr...

Image via Wikipedia

Over the years many non-invasive alternatives have been sought to test for Down Syndrome, the most common chromosomal abnormality,  affecting chromosome 21. Instead of one pair of  chromosomes, there is (usually) a third chromosome 21 (hence trisomy 21) (see Fig).

An older non-invasive test is a simple blood test to check the levels of some proteins and hormones in the mother’s blood, that are somehow related to Down Syndrome. However, this test is not very accurate.

The same is true for another non-invasive method: the ultrasound scan of the neck of the fetus. An increased amount of translucent fluid behind the neck (‘nuchal translucency‘) is associated with Down syndrome and a few other chromosomal defects.

A combination of serum tests and nuchal translucency in the 11th week correctly identifies fetuses with Down syndrome 87 percent of the time, whereas it misidentifies healthy fetuses as having Down Syndrome in 5% of the cases (5% false positive rate).

For this reason these non-invasive tests are usually used to “screen”, not to diagnose trisomy 21.

Ever since circulating fetal cells and free fetal DNA were found in the maternal blood, researchers try to enrich for this fetal sources and try to characterize chromosomal aberrations, using very sensitive molecular diagnostic tools, like polymerase chain reaction PCR (i.e. see this post) . The first attempts were directed at detecting the Y chromosome of male babies in the blood of the mother [1].

This January, Chiu et al published an article in the BMJ showing that Down’s syndrome can be detected with greater than 98% accuracy in maternal blood [2]. The group of Lo tested 753 pregnant women at high risk for carrying a fetus with trisomy 21 with this new blood test and compared it with the results obtained by karyotyping (analyzing number and appearance of chromosomes). The new technique is called multiplexed massively parallel DNA sequencing. This is a high throughput sequencing technique in which many DNA-fragments are sequenced in parallel. Sequencing means that the genetic code is unraveled. It is even possible to analyze 2 to 8 labeled maternal samples in parallel (2- and 8-plex reaction).

This parallel DNA sequencing method is “just” a counting method, in which the overall number of chromosomes is counted and one looks at an overrepresentation of chromosome 21.

With the superior 2-plex approach, 100% of the 86 known trisomy 21 fetuses were detected at a 2.1% false positive rate. In other words  the duplex approach had 100% sensitivity (all known positives were detected) and 97.9% specificity (2 were positive according to the test, whereas they were not in reality).

Thus it is a good and non-invasive technique to exclude Down syndrome in pregnant women known to have a high risk of Down syndrome. The approach might perform less well in a low risk group. Furthermore the study was not fully blinded. A practical disadvantage of this new test is that it is expensive, requiring machines not yet available in most hospitals (A spoonful of Medicine).

Another approach, recently published in Nature Medicine doesn’t have this disadvantage [3]. It involves the application of methylated DNA immunoprecipitation (MeDiP) and real-time quantitative PCR (rt-qPCR), which are accessible in all basic diagnostic laboratories. MedDiP is a technique to enrich for methylated DNA sequences, which are more preponderant in the fetus. Next rt-qPCR (amplification of DNA) is used to assess whether the fetus has an extra copy of the fetal-specific methylated region compared to a normal fetus.

In an initial series of 20 known normal cases and 20 known trisomy 21 cases, the researchers tested several differentially methylated regions (DMRs).  The majority of the ratio values in normal cases were at or below a value of 1, whereas in trisomy 21 cases the ratio values were above a value of 1. A combination of 8 specific DMRs out of 12 enabled the correct diagnosis of all the cases.

Next the authors validated the technique by applying the above method to 40 new samples in a blinded fashion. These samples contained 26 normal cases and 14 trisomy 21 cases (as later defined by karyotyping). Normal and trisomy 21 cases were all correctly identified.

The authors conclude that they achieved 100% sensitivity and 100% specificity in 80 samples. However, the first 40 samples were used to calibrate the test, thus the real validation study was done in a small set of 40, containing only 14 trisomy cases. One can imagine that a greater sample could have a few more false negatives or false positives. Indeed, small initial studies are likely to overestimate the true effect.

Furthermore, there was an overrepresentation of trisomy 21 cases (1/3 of the sample). Thus it is to soon to say that this trisomy 2 method is to be potentially employed in the routine practice of all diagnostic laboratories and be applicable to all pregnancies”, as the authors did. To this end the method should be confirmed in larger studies and in low risk pregnancies.

In conclusion, the relative easy and cheap methylated DNA immunoprecipitation/real-time quantitative PCR combo test, seems a promising approach to screen for Down syndrome in high risk pregnancies. Larger studies are needed to confirm the extreme accuracy of 100% and must demonstrate the applicability to low risk pregnancies. If confirmed, this blood test could eliminate the need for invasive procedures. Another positive aspect is that the test can be performed early, from the 11th week of gestation, and the results can be obtained within 4-5 days. Moreover the researchers can easily adapt the current technique to demonstrate abnormal numbers (aneuploidy) of chromosomes 13, 18, X and Y.

References

  • LO, Y., CORBETTA, N., CHAMBERLAIN, P., RAI, V., SARGENT, I., REDMAN, C., & WAINSCOAT, J. (1997). Presence of fetal DNA in maternal plasma and serum The Lancet, 350 (9076), 485-487 DOI: 10.1016/S0140-6736(97)02174-0
  • Chiu, R., Akolekar, R., Zheng, Y., Leung, T., Sun, H., Chan, K., Lun, F., Go, A., Lau, E., To, W., Leung, W., Tang, R., Au-Yeung, S., Lam, H., Kung, Y., Zhang, X., van Vugt, J., Minekawa, R., Tang, M., Wang, J., Oudejans, C., Lau, T., Nicolaides, K., & Lo, Y. (2011). Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study BMJ, 342 (jan11 1) DOI: 10.1136/bmj.c7401
  • Papageorgiou, E., Karagrigoriou, A., Tsaliki, E., Velissariou, V., Carter, N., & Patsalis, P. (2011). Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21 Nature Medicine DOI: 10.1038/nm.2312

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Screening Can’t Hurt, Can it?

23 11 2009

The next Grand Rounds is hosted by How To Cope With Pain and, not surprisingly, the main theme will therefore be pain. Now, I had a personal story in mind on the downside of testing, but I didn’t have a good title that fit the theme. Till, this Saturday when I a saw a perfect headline in the Los Angeles Times (Nov 21th), reading:

Cancer screening: What could it hurt? A lot, actually

It is a very thoughtful article showing the downside of screening. It was prompted by “the furor over this week’s recommendation from the U.S. Preventive Services Task Force that most women wait until age 50 to start routine mammograms, and then get them only every other year.” (also see kaleidoscope 2009- wk47).

They started their article as follows:

It seemed like a good idea at the time.

In 1984, Japan began screening the urine of 6-month-old infants for neuroblastoma, the most common type of solid tumor in young children. The test was simple and could show signs of cancer long before clinical symptoms arose.

Hundreds of infants went through the ordeal of diagnosis and treatment, but it didn’t reduce the number of tumors, including deadly ones, found later. Almost none of the tumors caught by screening turned out to be dangerous — and more of the screened children died from complications of surgery and chemotherapy than from the cancer itself.

In 2004, health officials ended the program.

The article further describes the potential downsides of current cancer screening protocols, including breast cancer screening.

  • But finding cancers that respond to early treatment is only one of the potential outcomes from a screening test. Many tests produce false positives, prompting additional tests that can be invasive, expensive, time-consuming and anxiety-inducing.(……)
  • Other screening tests produce false negatives, giving patients and their doctors the incorrect impression that they have nothing to worry about.
  • Some detect aggressive cancers whose outcomes aren’t improved by early detection.
  • And some identify small cancers that grow so slowly they’d never compromise a patient’s health. Many would even go away on their own.

All true but the problem is that people see it as their right to be screened (Will Women in Their 40s Be Denied etc). Cancer survivors are furious about the new breast cancer screening guidelines, they think decisions are made on political grounds and/or fear Medicare will no longer cover screening at younger age.

Why people are upset about the softened screening recommendations is because cancer is a frightening and deadly disease and because (as the Los Angeles Times explains so well) it’s easy to identify cancer survivors whose tumors were caught by screening, but it’s nearly impossible to put a face on the woman or man who is hurt by over-screening.

The first time I heard about the downside of screening was in 2004, when I attended a meeting for  Conn patients  to write an article for the patients association NVACP (see page 11-16, Dutch). Prof. Kievit, a surgeon and professor in decision analysis said:

“Imaging techniques (CT-scan or MRI) should only be applied if the stature test is positive and the aldosterone blood levels proven to be abnormally high. This is important because people often have benign nodules. Innocent nodules (incidentalomas) can obscure the diagnosis, worry the patient or even lead to unnecessary interventions. Furthermore it is inefficient to randomly subject people to all kind of tests. And please do not follow the current US trend to ask a CT-scan for your birthday!

That the balance of harm and benefit of diagnostic tests and screening can dip the wrong way can be best understood when you experience it yourself.

from: Wikipedia

During my last pregnancy my daughter was diagnosed with a mild prenatal  hydronephrosis during routine pregnancy ultrasound. Hydronephrosis is distension and dilation of the renal pelvis, usually caused by obstruction of the free flow of urine from the kidney. Since this can lead to progressive atrophy of the kidney, my little girl also had to undergo several tests to check the function of the kidneys and the cause of this anomaly. For one of those tests she had to be injected with radioactive isotopes in the catacombs of another hospital. But everything seemed o.k.: the anatomy (no obstruction) and the kidney functions. It should also be stressed that the dilatation was near-normal and didn’t worsen. Nonetheless, because of complications often seen with children with severe dilatation my daughter had to take daily antibiotics as a preventive measure. We had to regularly visit the polyclinics for an ultrasound and urine testing (to exclude infection and resistance). After a year it was decided to discontinue the antibiotic treatment. Follow-up was not needed.  Later a pediatric urologist told us that the guidelines had been changed: preventive antibiotic treatment was no longer required in case of mild hydronephrosis with no underlying cause.

My daughter developed asthma at the age of 7. Both she and her sister had atopic eczema, a known predicting factor for asthma, when they were toddlers. In line with the hygiene-theory, that states that a lack of early childhood exposure to infectious agents, increases susceptibility to allergic diseases, I often wondered whether 1 year daily antibiotic treatment wasn’t the final trigger for my daughter’s asthma. Indeed @Allergy (Ves Dimov) recently twittered about a study in J Allergy Clin Immunol that showed an association between antibiotic use in the first year of life and current symptoms of asthma in children 6 and 7 years old. A  Systematic Review of observational studies came to the same conclusion: “Exposure to at least one course of antibiotics in the first year of life appears to be a risk factor for the development of childhood asthma.” These studies had some limitations, and don’t  prove there is a causal relationship between antibiotic treatment and asthma, but they do fuel my suspicion.

In any case, although prenatal diagnosis of hydronephrosis may help to prevent later development of serious kidney disease in children with real malformations, it only resulted in “harm” in our case. Unnecessary testing (all results negative), unnecessary polyclinic visits, worries (that stayed until she was 9, when we visited the pediatric urologist to exclude an UTI, because you never know..)), unnecessary antibiotic treatment and -perhaps- the triggering of asthma. Looking back, and knowing what I know now, I wished the somewhat dilated renal pelvis had never been observed.

Last Friday I was at a lottery offered by my Sports Club. The last 2 prices were mystery prices: A total body scan of 1000 Euros each. I heard a lot of “Aaahs” and “Oooohs”. But I whispered “not for me“. The women next to me turned their heads, looked at me perplexed with their eyes blanked. Of course it is difficult to understand why one would refuse such a price, because “if screening doesn’t help, it won’t harm either”.

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Nursing Myths (1): Post-operative Temperature Measurements.

21 09 2008

Patients recovering from surgery at the ward, are frequented by nurses taking their blood pressure, pulse and temperature. What would happen if nurses wouldn’t routinely measure temperature? Would infections be missed? Could this lead to more serious infections and other complications?

Hester Vermeulen and colleagues have performed a study that shows that routine measurements of body temperature in postoperative patients is of limited value. This study was published in Clin Infect Dis. 2005 May 15; 40(10): 1404-10, (see here for HubMed-citation). Previous studies have also pointed in the same direction, but were less robust in design (retrospective, unblinded studies and/or surrogate endpoints). (for study designs see previous post here)

The study of Hester Vermeulen et al. was a prospective, triple-blinded diagnostic study, which means that groups of patients were followed from the beginning and neither the patient, the treating physicians nor the nurses responsible for daily care were informed about the outcome of the measurements. Independent nurses not involved in routine care did the temperature readings. Only patients with non-infectious diseases were included.

Of the 284 enrolled patients, 60 (21%) had a temperature of ≥38ºC, but only 7 out of theses 60 patients really had infections. The sensitivity did not improve for higher febrile temperatures (38,5-39ºC) or when the febrile temperature was measured on more than one occasion.

On the other hand, of the 223 patients (79%) who had a temperature less than 38ºC, 12 patients (>5%) did develop an infection.

Overall, in 19 patients (7%) a postoperative infection was detected (14 on basis of bacterial culture, 5 on clinical/laboratory grounds). Only 7 of these patients had a febrile temperature beforehand.

Eight patients developed a serious infection (pulmonary, intra-abdominal and sepsis), but six of them had no febrile temperature, meaning that infection is often not accompanied by a previous increase in temperature.
Thus routine temperature measuring might even be misleading to nurses and physicians: relying on body temperature might delay diagnosis and subsequently treatment (because a negative result reassures, but does not exclude an infection).

Experts in diagnostic accuracy studies would say that routine temperature measurements in post-operative patients have a very low sensitivity, a low positive predictive value and meaningless likelihood ratios (see Wikipedia)

Freely translated, this test performs lousy.

The study of Vermeulen et al. is part of an continuing program for the development of evidence based local guidelines. On the basis of these results Vermeulen et al adviced to abandon routine postoperative temperature measurements, but to perform these measurements only when indicated.

Still, as I understand, it is not easy to implement the guidelines. Nurses still find that they ought to check temperatures daily -it is in their routine-. And if they do adhere to the protocol many doctors still ask for the temperature data during ward rounds. Last but not least, patients find temperature measurements reassuring and rely heavily on information about the measured values. Furthermore, it is also pleasant that the nurse visits you regularly, if not for the temperature, then for the caring.

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Patienten die op zaal liggen om van een operatie herstellen worden op gezette tijden bezocht door de zuster, die hun pols opneemt, de bloeddruk meet en hen even ‘tempt’. Wat zou er gebeuren als verpleegster niet routinematig de temperatuur zou meten? Zouden infecties over het hoofd gezien worden? Zou dit tot méér ernstige infecties of tot andere complicaties kunnen leiden?

Hester Vermeulen en collega’s hebben in hun studie (Clin Infect Dis. 2005 May 15; 40(10): 1404-10; zie hier voor HubMed-citatie).aangetoond dat routinematige bepaling van de lichaamstemperatuur in geopereerde patienten weinig zinvol is. Eerdere studies wezen hier ook al op, maar hadden een minder goed onderzoeksdesign (retrospectief, niet geblindeerd, surrogaatmarkers) (voor een beschrijving van studie designs, zie eerder bericht hier).

De studie van Vermeulen et al. is een prospectieve, triple-blinded diagnostische studie. Dat houdt in dat patienten vanaf het begin gevolgd worden en dat noch de patient, noch de behandelend arts of de verpleger (die de patient verzorgt) op de hoogte is van de uitslag. Onafhankelijke verplegers doen de temperatuurmetingen. Alleen patienten zonder infectieziekten werden geincludeerd.

Van de 284 geincludeerde patienten, hadden er 60 (21%) een temperatuur gelijk van 38ºC of hoger. Maar slechts 7 van deze 60 patienten hadden ook echt een infectie. De sensitiviteit van de test ging niet omhoog als men alleen mensen van een hogere temperatuur (38,5-39ºC) of met een herhaalde hoge temperatuursmeting in beschouwing nam.

Aan de andee kant: van de 223 patienten (79%) die een normale temperatuur hadden (lager dan 38ºC), kregen er 12 (>5%) toch een infectie.

In totaal, werd in 19 patienten (7%) een postoperatieve infectie geconstateerd. Slechts 7 van de 19 patienten had tevoren koorts.

Acht patienten kregen een ernstige infectie (long- of buikinfectie of sepsis), maar slechts 2 van hen hadden tevoren koorts en 6 dus niet. Hetgeen betekent een infectie niet altijd voorafgegaan wordt door koorts.
Routinematig tempen kan dus zelfs misleidend zijn. Als verplegers en dokters zich hier teveel op verlaten, kan dit een snelle diagnose en therapie in de weg staan (omdat een negatief resultaat ten onrechte geruststelt).

Epidemiologen zouden zeggen dat de routine temperatuurmetingen in post-operatieve patienten een erg lage sensitiviteit, een lage voorspellende waarde en een nietzeggende likelihoodratio hebben. (zie Wikipedia)

Vrij vertaald: knudde met een rietje.

Bovengenoemde studie is er éen uit een reeks, bedoeld om (locale) evidence based richtlijnen te ontwikkelen. Op basis van de resultaten is het advies van Vermeulen et al. om postoperatieve patienten niet langer routinematig te temperaturen, maar alleen als daar aanleiding voor is.

Een duidelijke stelling, maar toch blijkt de praktijk weerbarstiger. Verplegers vinden dat ze toch dagelijks horen te tempen -dat zit in hun routine. En als ze de regels wel opvolgen, vragen sommige dokters tijdens hun visite er toch naar. Last but not least, patienten vinden temperatuurmetingen en andere vaste rituelen geruststellend. Ze hechten ook veel waarde aan de uitkomst van de meting. Verder vinden ze het gewoon prettig als de zuster op vaste tijden bij hen langskomt. Was het niet voor de tempeartuurmeting, dan toch voor wat extra aandacht en zorg.





Thesis Mariska Leeflang: Systematic Reviews of Diagnostic Test Accuracy.

22 08 2008

While I was on vacation Mariska Leeflang got her PhD. The ceremony was July 1st 2008.

Her thesis is entitled: Systematic Reviews of Diagnostic Test Accuracy.

Mariska is a colleague working (part time) at the Dutch Cochrane Centre (DCC). She studied veterinarian science in Utrecht, but gradually noticed that she was more interested in research than in veterinary practice. Four years ago she applied for a job at the dept. of Clinical Epidemiology, Biostatistics and Bioinformatics (KEBB) at the Amsterdam Academic Medical Centre (AMC). Having a cv with all kinds of odd subjects like livestock and courses delivering anesthetic drugs from a distance, she thought she would never make it, but she did.

Those 4 years have been very fruitful. She did research on diagnostic accuracy, is member of the Cochrane Diagnostic Test Accuracy Working Group and first author of one of the Cochrane pilot reviews of diagnostic test accuracy (chapter 7 of thesis). [Note: Cochrane Diagnostic Test Accuracy Reviews are a new initiative; till recently all Cochrane Systematic reviews were about health care interventions].
Mariska also supports authors of Cochrane systematic reviews, gave many presentations and led many workshops. In fact, she also gave in-service training to our group of Clinical Librarians in diagnostic studies and together we have given several courses on Evidence Based Medicine and Systematic Reviews. In leisure time she is Chair of “Stichting DIO” (Vet Science & Development Cooperation)

She will continue to work for the Cochrane Collaboration, including the DCC, but has also accepted a job at the Royal Tropical Institute (http://www.kit.nl).

Because of her backgound Mariska often gives her work a light “vet” touch.

“The cover of her thesis for instance is inspired by Celtic artwork and reflects the process of a systematic review: parts become a whole. The anthropomorphic (human-like) and zoomorphic (animal-like) creatures represent the background of the author. The stethoscopes and the corners refer specifically to diagnostic test accuracy reviews.The snakes eating their own tail stand in Celtic mythology for longevity and the ever-lasting life cycle.”

Also, she often closes her presentations with a slide showing swimming pigs, the pig being symbolic for “luck”.

So I would like to close this post in turn by wishing Mariska: “Good Luck”

Thesis: ISBN: 978-90-9023139-6
Digital Version at : http://dare.uva.nl
Index: (I’ll come back to chapter 1 and 2 another time)
Chapter 1: Systematic Reviews of Diagnostic Test Accuracy – New Developments within The Cochrane Collaboration – Submitted
Chapter 2: The use of methodological search filters to identify diagnostic accuracy studies can lead to the omission of relevant studies – J Clin Epidemiol. 2006;59(3):234-40
Chapter 3: Impact of adjustment for quality on results of meta-analyses of diagnostic accuracy – Clin Chem. 2007;53(2):164-72
Chapter 4: Bias in sensitivity and specificity caused by data driven selection of optimal cut-off values: mechanisms, magnitude and solutions – Clin Chem. 2008; 54(4):729-37
Chapter 5: Diagnostic accuracy may vary with prevalence: Implications for evidence-based diagnosis – Accepted by J Clin Epidemiol
Chapter 6: Accuracy of fibronectin tests for the prediction of pre-eclampsia: a systematic review – Eur J Obstet Gynecol Reprod Biol. 2007;133(1):12-9
Chapter 7: Galactomannan detection for the diagnosis of invasive aspergillosis in immunocompromized patients. A Cochrane Review of Diagnostic Test Accuracy – Conducted as a pilot Cochrane Diagnostic Test Accuracy review

——————-

Mariska Leeflang is op 1 juli 2008 aan de Universiteit van Amsterdam gepromoveerd op het onderwerp:“Systematische Reviews van de Diagnostische Accurratesse”.

Mariska is eigenlijk een collega van mij. We werken samen part time op het Dutch Cochrane Centre (DCC). Zij heeft diergeneeskunde gestudeerd in Utrecht, maar kwam er gaandeweg toch achter dat ze liever onderzoeker dan practiserend dierenarts wilde zijn. Toen ze vier jaar geleden ging solliciteren bij de afdeling Klinische Epidemiologie, Biostatistiek en Bioinfomatica (KEBB) van het AMC gaf ze zichzelf weinig kans met vakken als graslandbeheer en een cursus ‘verdoven op afstand’ op haar cv. Maar ze werd wel aangenomen. En terecht!

Die 4 jaar zijn zeer vruchtbaar geweest. Ze deed diagnostisch onderzoek, is lid van de Cochrane Diagnostic Test Accuracy Working Group en eerste auteur van een pilot diagnostisch accuratesse review (H 7 van proefschrift). Cochrane Systematische Reviews van Diagnostische Accuratessestudies zijn een nieuw type Systematisch Review, naast de bestaande Cochrane Reviews van interventies.
Mariska heeft veel presentaties en workshops gegeven, ook in Cochrane verband. Ze heeft zelfs ons clinical librarians bijgeschoold op het gebied van diagnostische stusies. Samen geef ik met haar EBM-cursussen en de cursus “Systematische Reviews” voor Cochrane auteurs. In haar vrije tijd is ze voorzitter van de Stichting DIO ( Diergeneeskunde in Ontwikkelingssamenwerking).

Ze zal voor de Cochrane Collaboration blijven werken, maar werkt sinds kort ook 2 dagen per week op het Koninklijk Tropeninstituut (KIT).

Vaak zie je dat Mariska vanwege haar achtergrond als diergeneeskundige vaak een link maakt naar dieren.

Op de omslag van haar boekje dat gebaseerd is op Keltisch kunstwerk wordt het proces van een systematisch review als volgt weergegeven: Alle delen worden samen een geheel. The mensachtige en dierlijke wezens vormen Mariska’s achtergrond. De stethoscoop en de hoeken staan voor de diagnostische accuratessereviews. De slangen, die hun eigen staart opeten staan in de Keltische mythologie voor een lang leven en de eeuwigdurende levenscyclus.

Ook sluit ze haar presentatie vaak af met een plaatje met zwemmende biggetjes, die voor “geluk” staan.

Dat lijkt me ook hier een passend slot: Veel geluk Mariska!!