Pregnancy Test Kits.. How they works

To support the ongoing pregnancy every Pregnant Women produces a hormone called hCG (Human Chorionic Gonadotrophin). The pregnancy test kits work by detecting this hCG in the urine of the pregnant women. Usually hCG appears in the urine of pregnant women after 20-24 days from the day of her last menstrual period. The level of hCG rapidly increases (almost doubles every 72hrs) till the third month of pregnancy and then maintains a steady state.

Now coming to the question how pregnancy test kits detect hCG.

The pregnancy test kits will have a chromatographic paper enclosed inside a plastic case and you can see the chromatographic paper from the upper side. The Chromatographic paper will be impregnated with hCG and anti hCG (a substance against hCG) at the point marked as ‘C’ and only with anti hCG at the point marked as ‘T’. C stands for “Control” and T stands for “Test”.

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There will a sample well where urine drops has to be poured and the urine will blot through out the chromatographic paper

Now the principle is

hCG + anti hCG + water = color

Let us take an example that a woman is not pregnant and she wants to check whether she is pregnant. She will put her urine in the sample well and the urine will blot through the chromatographic paper and it will cross both the “control” as well as the “Test” bands.

Now apply the principle here

Since the women is not pregnant her urine will not have hCG

At the Control area which is already impregnates with hCG +anti hCG a color band will appear because of the water from the urine but at the test area which is having only anti hCG no color will appear

so What happens at Control Band is?

hCG +anti hCG +water (from urine) = color.

This is basically to ensure that the pregnancy test kit is working.

and What happens at Test Band is?

Anti hCG + water (from urine) = no color

Since the woman is not pregnant her urine will not have hCG. There will be only one pink colored band appearing at the control area and the test band will not be colored.

Let us assume that the woman is pregnant and her urine will have hCG now.

Applying the principle

At Control Band

hCG (already impregnated) + hCG (from urine) + anti hCG + water (from urine) = color

At Test Band

hCG (from urine) + anti hCG + water (from urine) = color

Now two colored bands can be seen at both Control and Test areas.

This means the test is positive. Congrats you are pregnant and it's time for you to consult a Doctor.

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Evolution of Pregnancy Test - II

1900-1970

1903

Research on human reproduction intensified in the early twentieth century. Ludwig Fraenkel described the corpus luteum, the glandular mass that forms in women’s bodies during the normal menstrual cycle that we now know is supported by hCG during pregnancy. He identified some hormones that had a role in female reproduction, naming the hormone that promoted gestation, progesterone. Progesterone was isolated (an important step in the study of hormones) in 1934.

1920s

Independently, scientists in several laboratories across Europe described the presence of a substance that promotes ovary development and growth in rabbits and mice. In Germany, Selmar Aschheim and Bernhard Zondek noted that this substance specifically affected the formation of the corpus luteum.

Scientists recognized that there is a specific hormone (now known as human chorionic gonadotropin (hCG)) that is only found in pregnant women.

1927

Aschheim and Zondek described a test (known as the A-Z test) which identified the presence of hCG in urine. To test for pregnancy, a woman’s urine was injected into an immature rat or mouse. If the subject was not pregnant, there would be no reaction. In the case of pregnancy, the rat would show an estrous reaction (be in heat) despite its immaturity. This test implied that during pregnancy there was an increased production of the hormone. During early studies of the A-Z test, the scientists discovered that testicular tumors could produce hCG as well.

1930s

Hormone research blossomed in this period. Scientists in several different laboratories developed bioassays (special tests using animals or live tissue) to identify hCG by injecting samples to induce ovulation in rabbits, frogs, toads, and rats. These tests were expensive, required the sacrifice of several animals, and slow, often taking days to get results. The tests were also insensitive when measuring hormone levels to diagnose pregnancy because of the similarity between hCG and another substance, luteinizing hormone (LH). Most bioassays were in fact unable to distinguish between the two except at extraordinarily high rates of hCG.

Herbert Evans discovered that when injected with certain fluids from the female glands a female rat would grow an abnormally large corpus luteum. These fluids were hormones now known as gonadotropins.

In the next few decades laboratory scientists increased their level of interest in the study of human reproduction and on the role of ovaries and testes in human development.

1932

The First International Conference of Standardization of Sex Hormones was held in London, marking the culmination of a decade of increased interest in the chemical properties of sex hormones rather than the previously limited focus on biological function.

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1930s-1940s

Popular childbirth books began to encourage women to visit a doctor’s office for confirmation of pregnancy rather than relying on “old wives’ tales” for the diagnosis.

1958

Gonadotropins were first extracted from human pituitary glands.

1960

A “hemagglutination inhibition test” for pregnancy was developed by L. Wide and C.A. Gemzell. Because it used cells in the testing process, this test was an immunoassay rather than a bioassay. The test used purified hCG, mixed with a urine sample and antibodies directed against hCG. In a positive pregnancy test, the red cells clumped, displaying a particular pattern. This test was much faster and cheaper than the old bioassay, but still relatively insensitive, especially for early diagnosis of pregnancy. The test also cross-reacted with various medications.

Mid-1960s

Important disease research in this period led to more knowledge about how hormones, steroids, and antibodies work in the human body. In the next decade, NICHD scientists would transfer these principles to their studies of reproductive hormones such as hCG.

1966

A. R. Midgley described the first radioimmunoassay for hCG, but the test still could not differentiate between hCG and luteinizing hormone. Several other laboratories reported improvements on this test, but did not solve this basic problem.

Evolution of Pregnancy Test - III

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Evolution of Pregnancy Test - I

1350 BCE

One of the earliest written records of a urine-based pregnancy test can be found in an ancient Egyptian document. A papyrus described a test in which a woman who might be pregnant could urinate on wheat and barley seeds over the course of several days: “If the barley grows, it means a male child. If the wheat grows, it means a female child. If both do not grow, she will not bear at all.” Testing of this theory in 1963 found that 70 percent of the time, the urine of pregnant women did promote growth, while the urine of non-pregnant women and men did not. Scholars have identified this as perhaps the first test to detect a unique substance in the urine of pregnant women, and have speculated that elevated levels of estrogens in pregnant women’s urine may have been the key to its success.

Middle Ages through the Seventeenth Century

Using visual aspects of urine to detect pregnancy became a popular method. In Europe, so-called “piss prophets” claimed to be able to diagnose many different conditions and diseases by the color of urine. In a 1552 text, pregnancy urine was described as: “clear pale lemon color leaning toward off-white, having a cloud on its surface.” Other tests included mixing wine with urine and observing the results. Indeed, alcohol reacts with certain proteins in urine, so this may have had a moderate success rate.

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Nineteenth Century

Various theories abounded, such as the possibility that pregnancy urine contained certain identifiable crystals or bacteria. Scientists did not know enough about pregnancy to develop a reliable test. However, for sexually active women, the best method for diagnosing pregnancy remained careful observation of their own physical signs and symptoms (such as morning sickness).

1890s

Many physicians began to describe the workings of chemicals in the body, suggesting that “internal secretions” by certain organs were crucial to an understanding of human biology. Ernest Starling named these chemical messengers “hormones.”

American public health advocates started to encourage women to see their doctors as soon as possible after pregnancy was suspected. Prenatal care was found to improve the health of both infants and mothers, even though most women would not see a doctor or midwife until well into the pregnancy.

Evolution of Pregnancy Test - II

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What is Evaporation line in Pregnancy Test Kits

An evaporation line on a pregnancy test is basically a false positive. While false positives and false negatives are fairly rare, the evaporation line is the closest to having an error on the pregnancy tests.

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The evaporation line is basically caused by reading a pregnancy test after the time limit advised by the manufacturer has passed. This time limit is usually clearly stated in the home pregnancy test kit instructions. The majority of the pregnancy test kits advise you to read the results within a maximum of 10-30 minutes after the urine test results. The evaporation line is only found on urine pregnancy tests. Using a digital pregnancy test will remove the risk of misreading a pregnancy test because of the way the test results announce if you are pregnant or not.

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Late positive line in Pregnancy Test Kit

Most pregnancy tests will tell you to read the results in a certain amount of time, usually from a couple of minutes up until 10 minutes later. So you may take a pregnancy test and read it within the above time period as negative.

If you keep the test around to look at later, you may notice that after that allotted time the test now appears to have a positive result. This is called an evaporation line. It is not indicative of a positive pregnancy test.

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If the negative test results was unexpected or you have tested prior to your missed period, repeat the pregnancy test once again after you have missed your period.

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False Positive Pregnancy Test

A false positive on a pregnancy test means that the test detected the hormone hCG in your urine. The newer, more sensitive early pregnancy tests usually detect lower amounts of hCG in the urine, making them more sensitive.

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For a false positive result, you either have hCG in your body for some other medical reason or the test was not working correctly.

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Can a too early Pregnancy Test be wrong

A urine pregnancy test works by measuring the amount of hCG (pregnancy hormone) in your urine. Typically you need to have missed your period for these to be accurate, particularly a positive result. If you have a positive result a couple of days before this missed period, hCG has been detected and you are pregnant.

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As the tests become more sensitive, more and more women will have positive results earlier. That leaves the problem of taking a pregnancy test prior to your missed period and getting a negative result.

If you get a negative result and you have tested prior to your missed period, you should wait for a couple of days and repeat the test.

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Do any medicines affect the Pregnancy Test Result?

In general there are no medications that will effect the results of your pregnancy test- either positive or negative. The only medications really known to cause false results, false positives in particular, are those medications containing hCG. These drugs are used in conjunction with infertility treatments. In these cases your doctor or fertility center will tell you not to take a pregnancy test until these medications have been excreted from your system.

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Birth control pills and other forms of birth control do not affect the results of a pregnancy test urine or blood.

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What is the time gap for re doing a Pregnancy Test

Most pregnancy tests include instructions that encourage you to wait at least one week between a negative test and taking another pregnancy test. This is to allow time for your body to build up enough hCG to be detected in your urine.

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You may choose to test sooner, knowing that hCG nearly doubles every 48 hours. This is not harmful to anything other than your wallet. You may continue to get a negative result until your body builds up enough hCG to turn the test positive, if you are pregnant.

If you continue to get a negative pregnancy test after a week and you have still not started your period, it is wise to have a physical exam by your doctor to ensure that you are healthy.

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Should the Pregnancy Test be done with first morning urine?

The reason that most pregnancy tests recommend first morning urine is that this urine typically contains the highest concentration of hCG, the pregnancy hormone. However, if you work nights or your sleep is interrupted by bathroom breaks, you may not have the most concentrated urine at this time.

You can recreate first morning urine by not using the bathroom for at least four hours. You must also not drink more than your normal amount of water or else you will dilute your urine. It does not matter if you urinate on the stick or in a cup.

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Typically after the fourth of fifth week of pregnancy your levels of hCG are high enough without having to use first morning urine.

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How soon a Home Pregnancy Test can be done

Home pregnancy tests measure human chorionic gonadotropin (hCG) in your urine. hCG is produced in measureable amounts at about 7 - 10 days after conception. However, it is generally recommended that you wait 14 days (the day your period would normally show) before you test. (This is typically 28 days since your last menstrual period (LMP)).

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There is also the question of how much hCG do each of the test measure. Some tests record as little as 25 mIU of hCG while others measure 250 mIU of hCG. Obviously the more sensitive the pregnancy test, the sooner you will have an accurate answer.

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Accuracy of Home Pregnancy Test

If you follow directions to the letter, home pregnancy tests are 97% accurate. But mistakes do happen, which is why some kits come with two tests. There are several reasons why a home pregnancy test may be negative: you may not be pregnant, or your body may not be making a normal amount of hCG. If you test too early (before the first day of your missed period), you may get a false negative result because not enough of the hormone has built up in your system. If the test comes back negative but you still suspect you're pregnant, wait a few days, read the directions carefully, and try again. False positives, when the test says you're pregnant but you're not, are rare.

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If you have irregular periods, it may be harder to calculate when your period is due. Allow for your longest cycle in recent months before testing. If you have recently stopped taking the contraceptive pill, you may not know how long your natural cycle is so you may end up testing either too soon or too late. If your test is negative in either of these situations, you should test again in three days.

Perform the test first thing in the morning when your urine is most concentrated.

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How to interpret a Home Pregnancy Test

1. Look at the regions marked 'C' and 'T' on the test card. 'C' indicates a control. This band must always appear because this is the comparison band. 'T' indicates the test sample.

2. If only one pink/purple band appear, in the region marked 'C', it means that the test is negative for pregnancy.

3. If two pink/purple bands appear, one in the region marked 'C' and the other in the region marked 'T', it means that the test is positive for pregnancy

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4. In case no bands appear, then the test is invalid. Repeat the test with a new pack of pregcolor cards after 72 hours.

5. If the line formed in region 'T' is faint, this could be due to low levels of hCG hormone. In case of a faint band, repeat the test with a new pack of pregcolor cards after 72 hours.

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Pregnancy Test Kits

• Pregnancy Test Kits.. How they works
• How to do a Home Pregnancy Test
• How to interpret a Home Pregnancy Test

• Accuracy of Home Pregnancy Test
• How soon a Home Pregnancy Test can be done
• Should the Pregnancy Test be done with first morning urine

• What is the time gap for re doing a Pregnancy Test...
• Do any medicines affect the Pregnancy Test Result?...
• Can a too early Pregnancy Test be wrong

• False Positive Pregnancy Test
• Late positive line in Pregnancy Test Kit
• What is Evaporation line in Pregnancy Test Kits

• Evolution of Pregnancy Test - I
• Evolution of Pregnancy Test - II
• Evolution of Pregnancy Test - III

How to do a Home Pregnancy Test

Try to perform the test first thing in the morning for optimal results, though this is not mandatory.

• Pregnancy tests are usually stored in the refrigerator. So, make sure you bring the test kit to room temperature before you use it.
• Collect urine in a clean, dry glass or plastic container. Ensure that there is no detergent residue in the container.
• Take out the pregcolor card (see image below) and place it on a flat surface.
• Draw out a little urine with a dropper (provided with the kit) and put just two drops in the circular test well that is usually marked 'S'. Do not spill urine on the reading strip.
• Wait for three to five minutes (depending on manufacturer's instructions) and then read the test results. Trying to read the results before the stipulated time or waiting too long, can both lead to inaccurate readings.

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Some newly launched pregnancy test kits (for example, Clearview) can be held directly in the urine stream and do not require you to collect urine in a container. These kits are usually more expensive.

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