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AFP - Alpha Fetoprotein

 Alpha-Fetoprotein Analysis


Introduction

Human α-fetoprotein (AFP) is a tumor-associated fetal mammalian glycoprotein involved in ontogenic and oncogenic growth. This tumor marker is encoded by the AFP gene on chromosome 4q25. The fetal protein is a 70-kDa single polypeptide chain containing 3% to 5% carbohydrate. This protein exhibits a triplicate domain structure configured by intramolecular loops dictated by disulfide bridging. AFP occupies an α-1 anodic position in the electrophoretic profile, running slightly slower compared to albumin. AFP is synthesized in the yolk sac, fetal liver, and gastrointestinal tract during pregnancy but is re-expressed in multiple adult tumors of mixed mesodermal or endodermal origin.

In the clinical laboratory, AFP has been employed both as a post-operational tumor marker and as a gestational age-dependent fetal defect marker, demonstrating utility in screening for neural tube defects and aneuploidies. When a fetus has neural tube defects, maternal serum AFP levels are elevated, whereas chromosomal disorders are associated with lower levels. Yolk sac and liver-derived AFP have different carbohydrate content. The half-life of AFP is 4 to 5 days. Similar to albumin, serum AFP binds and transports many ligands such as bilirubin, fatty acids, retinoids, steroids, heavy metals, dyes, flavonoids, phytoestrogens, dioxin, and various drugs. 

AFP can be fractionated by affinity electrophoresis into 3 glycoforms: L1, L2, and L3, based on the reactivity with the lectin lens culinaris agglutinin. AFP-L3 binds strongly to lens culinaris agglutinin through an additional α-1-6 fucose residue attached at the reducing terminus of N-acetylglucosamine, in contrast to the L1 isoform. The L1 isoform is typically associated with non-hepatocellular carcinoma inflammation of the liver disease. The L3 isoform is specific to malignant tumors, and the detected presence can identify patients who need increased monitoring for the development of hepatocellular carcinoma in high-risk populations such as chronic hepatitis B and C and liver cirrhosis.


Alpha-Fetoprotein (AFP) Test 

What is an Alpha-fetoprotein (AFP) Test?

What is an AFP (alpha-fetoprotein) tumor marker test?

An AFP tumor marker test is a blood test that measures the level of AFP (alpha-fetoprotein) in a sample of blood. AFP is a protein that a developing baby makes. Normally, some AFP passes from the baby into the pregnant person's blood. An AFP test is a test that is mainly used to measure the level of alpha-fetoprotein (AFP) in the blood of a pregnant person. The test checks the baby's risk for having certain genetic problems and birth defects. An AFP test is usually done between 15 and 20 weeks of pregnancy. In other patients than pregnant women, the test is usually used to help diagnose certain types of cancer and to check how well treatment is working. Certain conditions can make a baby's body release more or less AFP. 

During pregnancy, if the AFP blood levels are higher or lower than normal, it may be sign that:

- The baby has a high risk of having a genetic disorder, such as:

                - A neural tube defect, which is a serious condition that causes abnormal development of a developing baby's brain and/or spine;

              - Down syndrome, a genetic disorder that causes intellectual disabilities and other health problems.

- The estimated due date is wrong. AFP levels normally rise and fall at set times during pregnancy, so an abnormal AFP may mean that the baby is due earlier or later than estimated. This is the most common reason for abnormal AFP levels. 

- If pregnant with more than one baby. Each baby makes AFP, so the AFP blood levels will be higher with two or more babies.

AFP is a protein that the liver makes when its cells are growing and dividing to make new cells. AFP is normally high in unborn babies. After birth, AFP levels drop very low. Healthy children and adults who aren't pregnant have very little AFP in their blood. 

AFP in non-pregnant people is mainly measured as a tumor marker. Tumor markers are substances that are often made by cancer cells or by normal cells in response to cancer. High levels of AFP can be a sign of cancer of the liver, ovaries or testicles.

An AFP tumor marker test cannot be used by itself to screen for or diagnose cancer. That's because other conditions can increase AFP levels, including liver diseases that aren't cancer. And some people who do have liver, ovarian, or testicular cancer will have normal AFP levels. So, an AFP tumor marker test can't rule out cancer for sure. But when used with other tests and exams, AFP tumor marker testing can help diagnose and monitor cancers that cause high AFP levels. 

Other names: total AFP, AFP Maternal, Maternal Serum AFP, msAFP screen, alpha-fetoprotein-L3 Percent


What is AFP used for?

An AFP tumor marker test may be used during the diagnosis and/or treatment of cancer of the liver, ovaries, or testicles that make high levels of AFP. It is used to:

- Help confirm or rule out a cancer diagnosis when used with other exams and tests;

- Predict how cancer may behave over time;

- Monitor cancer treatment. AFP levels often go up if cancer is growing and go down when treatment is working;

- Check whether cancer has returned after treatment.

In certain cases, results from an AFP tumor marker test may be used to guide treatment choices for specific types of cancer. The test may also be used to monitor your health if you have chronic (long lasting) hepatitis or cirrhosis of the liver. These conditions aren't cancer, but they increase your risk of developing liver cancer.


Why do I need an AFP tumor maker test?

You may need an AFP tumor marker test if:

- A physical exam and/or other tests suggest that you may have cancer of the liver, ovaries, or testicles;

- You are currently being treated for a cancer that causes high AFP levels. Measuring your AFP test can show how well your treatment is working;

- You have completed treatment for a cancer that increased your AFP level. You may need an AFP tumor marker test from time to time to check whether your cancer is coming back.

If you have chronic hepatitis or cirrhosis, you have a higher risk of developing liver cancer. A very high level of AFP or a sudden increase can be an early sign of liver cancer. Most medical experts don't recommend measuring AFP levels to screen for cancer in these diseases. But, some health care providers may still use an AFP tumor marker test with other tests to watch for liver cancer.

If a woman is pregnant, AFP test is routinely offered between the 15th and 20th week of pregnancy. Your provider may especially recommend the test if you:

- Have a family history of birth defects;

- Are 35 years or older;

- Have diabetes;

- Have used medicines or drugs during pregnancy that could harm the baby.


What happens during an AFP tumor marker test?

A health care professional will take a blood sample from a vein in your arm, using a small needle. After the needle is inserted, a small amount of blood will be collected into a test tube or vial. You may feel a little sting when the needle goes in or out. This usually takes less than five minutes.

Will I need to do anything to prepare for the test?

You don't need any special preparations for an AFP tumor marker test. 

Are there any risks to the test?

There is very little risk to having a blood test. You may have slight pain or bruising at the spot where the needle was put in, but most symptoms go away quickly.

What do the results mean?

The most common cause of abnormal AFP test results during pregnancy is an error estimating the due date. But a result that isn't normal may also be a sign of possible problems:

Lower than normal AFP levels may mean that the baby has a genetic disorder that causes intellectual disabilities and health problems;

Higher than normal AFP levels may mean that the baby has an increased risk of having a neural tube defect, such as:

    Spina bifida, a condition in which the bones of the spine don't close around part of the spinal cord;

    - Anencephaly, a condition in which the brain does not develop properly.

High AFP levels may also mean only that there are more than one baby. A false-positive result may also be obtained. That means that the AFP results aren't normal, but the baby is healthy.

If the AFP test results aren't normal, it is likely that more tests are performed in order to help make a proper diagnosis.


Specimen collection

A blood sample is collected from the vein using aseptic techniques, and universal precautions are applied. Serum AFP levels are measured as part  of a maternal triple or quadruple screening test or for other diagnostic purposes in nonpregnant female or male patients. Urine samples collected in plain or universal bottles may also be assayed for AFP levels, although this may be significantly lower compared to serum levels.

Specimens derived from sera are stable at room temperature or 4°C for at least 1 week and can be stored for several months at -20°C. The type of centrifugation and non-stable refrigeration frost-free cycles can affect AFP levels. Thus, monitoring various steps involved in sample collecting, storage, and preparation processes is crucial in maternal serum AFP screening programs.

Amniocentesis is needed to assay AFP levels in the amniotic fluid. A diagnostic amniocentesis involves using an ultrasound-guided, hollow needle through the maternal anterior abdominal wall into the amniotic cavity to draw out amniotic fluid for AFP immunoassay. AFP may also be measured by elution from blood spots collected on filter paper. Serum AFP is somewhat thermostable, and samples may be shipped at ambient temperatures after separation from red blood cells.


Procedure for Amniocentesis (From 15 Weeks Gestation):

The subsequent steps outline the amniocentesis process:

- Confirm the patient's identity and gestational age of the pregnancy;

- Conduct pretest counseling, which typically includes genetic counseling;

- Obtain informed consent from the patient;

- Adhere to hand washing and universal precautions;

- Position the patient appropriately;

- Clean the exposed area of the abdomen;

- Use a local anesthetic if necessary;

- Carefully introduce the ultrasound-guided, hollow needle through the anterior abdominal wall into the amniotic cavity;

- Aspirate 15 to 20 mL of amniotic fluid;

- Send the sample for AFP assay.

Procedure for AFP Analysis:

The procedure for AFP analysis is as follows: 

- Competitive radioimmunoassay has been widely used to quantify AFP;

- Currently, AFP is mainly measured by non-isotopic immunoassays;

- These immunoassay systems employ enzyme, fluorescent, or chemiluminescent labels;

- There are 2 types of immunoassays for measuring AFP: (1) competitive and (2) two-site solid-phase immunometric assays;

- In a typical radioisotopic competitive type, purified iodine-125-labeled AFP is mixed with the sample containing AFP to compete for a limited amount of AFP antibody;

- The 2-site immunometric assays use a solid phase, such as magnetic particles, plastic beads, or microtiter plates;

- The solid phase is coated with anti-AFP antibodies and incubated with patient specimens or standards; 

- AFP in the sample reacts with the anti-AFP antibody and is immobilized in the solid phase;

- Unbound components of the specimen are removed by washing the solid phase;

- A second anti-AFP antibody labeled with an enzyme, radioactive, fluorescent, or chemiluminiscent label is incubated with the solid phase and reacts with AFP bound at a second epitope site;

- Polyclonal and monoclonal antibodies have been used in tandem for each step; 

- Washing the solid phase removes the unreacted label, and any additional reagent needed to generate a signal is added;

- The bound label can be measured directly in the case of a fluorescence or radioactive label;

- The solid phase must be incubated with substrate before the detection step for enzyme labels;

- A reagent must be added for chemiluminiscence labels to generate the chemiluminiscent signal;

- In all cases, the signal generated with the solid phase is directly proportional to the AFP concentration of the standard or unknown;

- AFP-L3% is measured using a microfluidics-based instrument that utilizes immunochemical and electrophoretic techniques.

Indications:

The following lists various situations or medical conditions in which an AFP assay is performed: 

- Advanced maternal age;

- Previous births with chromosomal or congenital disabilities, including neural tube defects;

- Family history of chromosomal or congenital disabilities, such as Down syndrome and spina bifida;

- Screening for certain types of cancers, such as liver cancer, testicular cancer, and ovarian cancer;

- To evaluate the progress of anti-cancer treatment.


Potential Diagnosis

Maternal Serum AFP Levels Elevated:

The following conditions are associated with elevated maternal serum AFP levels:

- Neural tube defects such as spina bifida and anencephaly

- Omphalocele

- Gastroschisis

- Sacrococcygeal teratoma

- Placental abnormalities

- Cystic hygroma

- Renal abnormalities such as polycystic kidney or absent kidney, urinary obstruction, and congenital nephrosis

- Osteogenesis imperfecta

- Threatened abortion

- Decreased maternal weight or intrauterine growth restriction

Maternal Serum AFP Low Levels:

The following factors may contribute to low levels of maternal serum AFP:

- Down syndrome

- Increased maternal weight

- Fetal demise

- Hydatidiform mole

- Trisomy 18, also known as Edwards syndrome

- Incorrect gestational age (older than calculated)

Non-Pregnant Female or Male AFP Levels Elevated:

The following conditions may lead to elevated AFP levels in non-pregnant females or males:

- Hepatocellular cancer

- Metastatic liver cancer

- Liver cirrhosis

- Hepatitis

- Germ cell tumors

- Yolk sac tumor

- Ataxia-telangiectasia


Normal and Critical Findings

Typical findings include:

- AFP levels in men and non-pregnant women vary by age and race but typically range from 0 to 40 ng/mL;

- Between 15 and 20 weeks of pregnancy, AFP levels typically range between 10 and 150 ng/mL;

- Adult blood levels greater than 200 ng/mL in patients with liver cirrhosis strongly indicate hepatocellular carcinoma.


Interfering Factors

The following factors have been implicated in false-positive AFP results:

- A period of 2 weeks after radiodiagnosis involving the use of radioactive tracers;

- Multiple gestations;

- Gestational diabetes;

- Cigarette smoking;

- Race (slightly higher levels in Black women and lower in women of Asian descent compared to Whites);

- Levels adjusted for weight;

- Amniotic fluid specimens contaminated with fetal blood may exhibit abnormally high AFP values, leading to misinterpretation of test results;

- Specimens from patients who have received preparations of mouse monoclonal antibodies may contain human anti-mouse antibodies, which may give falsely high results. Results must always be considered within the clinical context and previous results, especially when serial results monitor a patient's response to treatment.


Complications

Risks associated with phlebotomy include:

- Phlebitis

- Abnormal bruising and bleeding in patients with clotting disorders or those taking blood thinners.

Risks associated with amniocentesis include:

- Miscarriage

- Preterm delivery

Patient Safety and Education

Phlebotomy for blood AFP assay: patients need to be informed about the possible discomforts. This is a screening test. Depending on the outcome, more tests may be ordered to establish a diagnosis. A negative test does not necessarily indicate no risk, as a very low maternal blood AFP is associated with an increased incidence of Down syndrome. Hence, a low maternal blood AFP should be investigated.

Patients undergoing amniocentesis must be counseled about the procedure and the associated risks. The risk of obstetric mishap following amniocentesis exists; a miscarriage can happen in less than 1% of cases. Some rare complications of amniocentesis include preterm labor, infection (amnionitis), iatrogenic trauma, or injury to the developing fetus or mother. Following amniocentesis, patients may experience some cramp-like discomfort in the first few hours. Patients should report back to the hospital in case of vaginal bleeding, vaginal discharge, or increasing abdominal cramps.


Clinical Significance

Maternal blood AFP levels often comprise triple, such as AFP, unconjugated estriol, and human chorionic gonadotropin, or quadruple, such as AFP, estriol, human chorionic gonadotropin, and inhibin A, screening tests for congenital disabilities. Levels are typically interpreted for age, race, weight, and gestational age. As the levels of these markers change with gestational age, the results are expressed as multiples-of-median. The elevated levels imply a significant risk of having congenital disabilities; hence, further evaluation may be required to assess the level of risks. A substantial number of patients with elevated maternal AFP do not develop congenital disabilities, but an increased risk of obstretic complications such as premature membrane rupture, placenta accreta, and increta may exist. 

Low maternal AFP levels may suggest a risk for Down syndrome. In non-pregnant women and men, elevated levels are observed in cancers, especially liver cancer. Levels greater than 200 ng/mL in patients with cirrhosis suggest hepatocellular carcinoma. Elevated AFP levels can also be found in testicular and ovarian carcinoma. AFP is also useful for determining prognosis and monitoring therapy for hepatocellular carcinoma. The concentration of AFP is a prognostic indicator of survival. Elevated AFP concentrations (>10μg/L) and serum bilirubin concentrations greater than 2 mg/dL are associated with a decreased survival time.

The lens culinary agglutinin-reactive fraction of fetoprotein (AFP-L3), a subtype of AFP produced by malignant hepatocytes, is considered specific to hepatocellular carcinoma. AFP-L3 is isolated using immunoassay techniques and quantified using chemiluminescence on an automated platform. The proportion of AFP-L3 to total AFP can be used to diagnose early hepatocellular carcinoma. Early studies have reported that the diagnostic sensitivity of AFP-L3% for hepatocellular carcinoma ranged from 75% to 96.9%, with a specificity of 90% to 92%. The high pre-treatment serum AFP-L3% levels indicated a poor prognosis for patients with hepatocellular carcinoma, and AFP-L3% may have a significant prognostic value for patients with hepatocellular carcinoma, especially those with low AFP concentration.

A cutoff of 10% is used, and patients with chronic liver disease and an elevated AFP-l3% have a 7-fold increased risk of developing hepatocellular carcinoma within 21 months. The test is useful for early detection, particularly in the AFP range of 20 to 200 μg/L, as has been shown in patients with hepatitis C-related cirrhosis. Although AFP-l3% is useful in detecting and prognosis, the value is typically used only when AFP concentrations are elevated.

The AFP concentration is a good indicator for monitoring therapy and assessing changes in the clinical status. Elevated AFP concentration after surgery may indicate incomplete removal of the tumor or the presence of metastasis. Falling or rising AFP concentration after therapy may reveal the success or failure of the treatment regimen. A notable increase in AFP concentration in patients considered free of a metastatic tumor may indicate metastasis development.

Measuring AFP and human chorionic gonadotropin concentration aids in classifying and staging germ cell tumors. Germ cell tumors may be predominantly 1 type of cell or a mixture of various types, including seminoma, yolk sac, choriocarcinomatous elements (embryonal carcinoma), and teratoma. Serum concentrations of AFP are elevated in yolk sac tumors, whereas human chorionic gonadotropin is elevated in choriocarcinoma. Both are elevated in embryonal carcinoma. AFP is not elevated in seminomas, whereas human chorionic gonadotropin is elevated in 10% to 30% of patients with syncytiotrophoblastic tumor cells. Neither marker is elevated in teratoma.

One or both of the markers are elevated in about 90% of patients with non-seminomatous testicular tumors. Elevations are noted in less than 20% of patients with stage I, 50% to 80% with stage II, and 90% to 100% with stage III. These markers correlate with tumor volume and the prognosis of the disease. The combined use of these markers is useful in monitoring patients with germ cell tumors: elevation of either marker indicates the recurrence of the disease or the development of metastasis. The effectiveness of chemotherapy can be assessed by calculating the decrease in the concentration of both markers using the half-lives of AFP (5 days) and human chorionic gonadotropin (12 to 20 hours).          


What do the results mean?

If you haven't been diagnosed with cancer, test results that show:

- High levels of AFP may be a sign of cancer of the liver, ovaries, or testicles. But having a high AFP level doesn't mean you have cancer or that you will get cancer. Liver injury and liver diseases that aren't cancer can also cause high AFP levels. Less often, high levels of AFP may be a sign of other cancers, including lymphoma or lung cancer. Your provider will use your medical history and other test results to make a diagnosis. 

- Normal levels of AFP mean you're less likely to have cancer that causes high AFP levels. But a normal test result doesn't rule out cancer because some people with these cancers have normal AFP levels. 

If you're being treated for a cancer that increased your AFP levels, you may be tested several times during treatment. Your provider will look at all your AFP test results to see how your levels have changed over time. If your results show:

- Your AFP levels are increasing, it may mean that your treatment isn't working;

- Your AFP levels are decreasing, it may mean your treatment is working;

- Your AFP levels have stayed the same, it may mean your disease is stable and hasn't gotten better or worse.

If you've finished treatment for cancer that caused high AFP levels and your test results are:

- Not normal, it may mean that you still have some cancer in your body;

- Higher now than they were shortly after treatment, it may mean your cancer is growing again.

If you've finished treatment for cancer that caused high AFP levels and your test results are:

- Not normal, it may mean that you still have some cancer in your body;

- Higher now than they were shortly after treatment, it may mean your cancer is growing again.

If you have a long-lasting liver disease that's not cancer, you may need other tests to check for liver cancer if your test results show a sudden increase in AFP or your level is very high.


Is there anything else to know about an AFP test?

In more developed countries, the most common AFP tumor marker test measures all forms of AFP in the blood. The AFP form called L3 is a less common AFP test may be used to check the risk of liver cancer in people who have chronic liver disease. An AFP-L3 percent test (AFP-L3%) compares the amount of L3 to the amount of total AFP in the blood. If the portion of L3 increases, it may mean that one might have a high risk of developing a serious form of the most common liver cancer called hepatocellular carcinoma.

As mentioned before, AFP tests are often part of a group of prenatal tests called multiple marker or triple screen tests. These tests can help diagnose Down syndrome, trisomy 18 (Edwards syndrome), and other genetic disorders. A triple screen test includes tests for:

- Alpha-fetoprotein (AFP)

- Human chorionic gonadotropin (HCG), a hormone produced by the placenta

- Estriol, a form of estrogen made by the baby and the placenta

In some cases, a fourth test is included, called an inhibin A test, which helps diagnose Down syndrome.

If you have a high risk for having a baby with certain birth defects, the doctor may also recommend a test called prenatal cell-free DNA (cfDNA) screening. This is a blood test can can be done as early as the 10th week of pregnancy. It can show if the baby has a higher chance of having Down syndrome or certain other genetic disorders.