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Alpha Fetoprotein

Editor: Shailesh Khetarpal Updated: 1/18/2023 5:56:51 PM


Human alpha-fetoprotein (AFP) is a tumor-associated fetal mammalian glycoprotein involved with both ontogenic and oncogenic growth.[1] This tumor marker is encoded by the AFP gene on chromosome 4q25.[2] The fetal protein is a 70-kDa single polypeptide chain containing 3% to 5% carbohydrate; it exhibits a triplicate domain structure configured by intramolecular loops dictated by disulfide bridging.[3] In the electrophoretic profile, alpha-fetoprotein (AFP) occupies an alpha-1 anodic position running slightly slower than albumin.[4] 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/endodermal origin.[5]

In the clinical laboratory, alpha-fetoprotein (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.[6] While maternal serum alpha-fetoprotein (MS-AFP) concentrations associated with neural tube fetal defects are elevated, the chromosomal disorders are associated with much lower levels.[7] Yolk sac and liver-derived AFP have different carbohydrate content. The half-life of AFP is 4-5 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.[8] 

Alpha-fetoprotein (AFP) can be fractionated by affinity electrophoresis into three glycoforms: L1, L2, and L3, based on the reactivity with the lectin lens culinaris agglutinin (LCA).[9] AFP-L3 binds strongly to lens culinaris agglutinin (LCA) via an additional α 1-6 fucose residue attached at the reducing terminus of N-acetylglucosamine; this is in contrast to the L1 isoform. It is the L1 isoform that is typically associated with non-hepatocellular carcinoma (HCC) inflammation of the liver disease.[10] The L3 isoform is specific to malignant tumors, and its detected presence can serve to identify patients who need increased monitoring for the development of hepatocellular carcinoma (HCC) in high-risk populations (i.e., chronic hepatitis B and C and/or liver cirrhosis).[11]

Specimen Collection

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Specimen Collection

A blood sample is collected from the vein using aseptic techniques and observing universal precautions. Serum alpha-fetoprotein levels are measured either as part of a maternal triple or quadruple screening test or for other diagnostic purposes in non-pregnant female or male patients.[12] Urine samples collected in plain or universal bottles may also be assayed for alpha-fetoprotein levels, although this may be significantly lower than serum levels.[13]

Specimens derived from sera are stable at room temperature or at 4 C for at least one week and for several months at −20 C. The type of centrifugation and non-stable refrigeration frost-free cycles can affect AFP levels.[14] Thus, in maternal serum AFP screening programs, it is crucial to monitor various sample collecting, storage, and preparation processes.[15]

Amniocentesis is needed to assay alpha-fetoprotein 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.[16] 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.[17] 


Blood Sample Collection (phlebotomy)

  1. Introduce yourself to the patient and confirm the patient's name and date of birth.
  2. Explain the procedure to the patient, warning the patient of possible discomfort from the needle prick.
  3. Position the patient appropriately, exposing the upper arm.
  4. Follow hand washing and basic universal precautions.
  5. Tie the tourniquet around the upper arm.
  6. Identify a prominent vein and clean it with an alcohol swab.
  7. Introduce the needle attached to a vacutainer.
  8. Remove the tourniquet and remove the needle after obtaining a sample.
  9. Apply the cotton ball to the needle site with pressure to stop bleeding. 
  10. Label sample bottles appropriately.
  11. Send a sample for alpha-fetoprotein (AFP) assay.

Procedure for Amniocentesis (done from 15 weeks gestation)

  1. Introduce yourself and confirm the patient's identity and gestational age of the pregnancy.
  2. Conduct pretest counseling. This usually includes genetic counseling as well.
  3. Obtain informed consent.
  4. Follow hand washing and universal precautions.
  5. Position the patient appropriately.
  6. The clean exposed area of the abdomen.
  7. Use a local anesthetic if necessary.
  8. Carefully introduce the ultrasound-guided, hollow needle through the anterior abdominal wall into the amniotic cavity.
  9. Aspirate 15 ml to 20 ml of amniotic fluid.
  10. Send for alpha-fetoprotein (AFP) assay.

Procedure for Alpha-fetoprotein Analysis

  1. Competitive radioimmunoassay has been widely used to quantitate AFP.
  2. Currently, AFP is mostly measured by non-isotopic immunoassays.
  3. These immunoassay systems employ enzyme, fluorescent, or chemiluminescent labels.
  4. Immunoassays that are employed to measure AFP are of two major types: (1) competitive and (2) two-site solid-phase immunometric.
  5. In a typical radioisotopic competitive type, purified iodine-125 labeled AFP is mixed with the sample containing AFP and allowed to compete for a limited amount of AFP antibody.
  6. In the two-site immunometric assays, a solid phase such as magnetic particles, plastic beads, or microtiter plates is used.
  7. The solid phase is coated with anti-AFP antibodies and incubated with patient specimens or standards.
  8. AFP present in the sample reacts with the anti-AFP antibody and is immobilized in the solid phase.
  9. Unbound components of the specimen are removed by washing the solid phase.
  10. A second anti-AFP antibody labeled with an enzyme, radioactive, fluorescent, or chemiluminescent label is incubated with the solid phase and reacts with AFP bound at a second epitope site.
  11. Polyclonal and monoclonal antibodies have been used in tandem for each step.
  12. The unreacted label is removed by washing the solid phase, and any additional reagent needed to generate a signal is added.
  13. In the case of a fluorescence or radioactive label, the bound label can be measured directly.
  14. For enzyme labels, the solid phase must be incubated with substrate before the detection step.
  15. For chemiluminescence labels, a reagent must be added to generate the chemiluminescent signal.
  16. In all cases, the amount of signal generated with the solid phase is directly proportional to the AFP concentration of the standard or unknown.
  17. AFP-L3% is measured using a microfluidics-based instrument that utilizes immunochemical and electrophoretic techniques.

 Quality Control Procedure

  1. For non-waived tests, laboratory regulations require, at the minimum, analysis of at least two levels of control materials once every 24 hours.
  2. If necessary, laboratories can assay QC samples more frequently to ensure accurate results.
  3. Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance. 
  4.  The laboratory can develop and implement an individualized quality control plan (IQCP) for certain analytes in which QC requirements are different.
  5. The individualized quality control plan (IQCP) involves performing a risk assessment of potential sources of error in all phases of testing.
  6. After risk assessment, a QC plan is formulated to reduce the likelihood of errors.
  7. Westgard multi-rules are used to evaluate the quality control runs.
  8. If a run is declared out of control, investigate the system (instrument, standards, controls, etc.) to determine the cause of the problem.
  9. Do not perform any analysis until the problem has been resolved. 

Lab Safety

  1. Consider all specimens, control materials, and calibrator materials as potentially infectious.
  2. Exercise the normal precautions required for handling all laboratory reagents.
  3. Disposal of all waste material should be in accordance with local guidelines.
  4. Wear gloves, a lab coat, and safety glasses when handling human blood specimens.
  5. Place all plastic tips, sample cups, and gloves that come into contact with blood in a biohazard waste container.
  6. Discard all disposable glassware into sharps waste containers.
  7. Protect all work surfaces with disposable absorbent bench top paper, which is discarded into biohazard waste containers weekly, or whenever blood contamination occurs.
  8. Wipe all work surfaces weekly.


The following are reasons for AFP assay:

  • Advanced maternal age.
  • Previous births with chromosomal or congenital disabilities (e.g., neural tube defects).
  • Family history of chromosomal or congenital disabilities (e.g., down syndrome, spina bifida).
  • Screening for cancers (e.g., liver, testicular, ovarian).
  • To evaluate the progress of anti-cancer treatment.

Potential Diagnosis

 Pregnant Maternal Serum AFP Levels Elevated

  • Neural tube defects (e.g., spina bifida, anencephaly)
  • Omphalocele
  • Gastroschisis
  • Sacrococcygeal teratoma
  • Placental abnormalities
  • Cystic Hygroma
  • Renal abnormalities (polycystic kidney or absent kidney, urinary obstruction, congenital nephrosis)
  • Osteogenesis imperfecta
  • Threatened abortion
  • Decreased maternal weight or Intrauterine growth restriction

Pregnant Maternal Serum AFP Low Levels

  • Down syndrome
  • Increased maternal weight
  • Fetal demise
  • Hydatidiform mole
  • Trisomy 18 (Edward Syndrome)
  • Incorrect gestational age (older than calculated)

Non-pregnant Female or Male AFP Levels Elevated

  • Hepatocellular cancer
  • Metastatic liver cancer
  • Liver cirrhosis
  • Hepatitis
  • Germ cell tumors
  • Yolk sac tumor
  • Ataxia-telangiectasia

Normal and Critical Findings

Typical findings include:

  • Alpha-fetoprotein levels in men and non-pregnant women vary for age and race but mostly range from 0 ng/ml to 40 ng/ml.
  • Maternal AFP levels in pregnancy start to rise from about the 14th week of gestation up until about 32 weeks of gestation.
  • Between 15 and 20 weeks, AFP levels usually range between 10 ng/ml to 150 ng/ml.
  • Adult blood levels greater than 200 ng/ml in patients with liver cirrhosis strongly indicate hepatocellular carcinoma.

Interfering Factors

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

  • Two 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 as compared to whites)
  • AFP levels are also adjusted for weight
  • Amniotic fluid specimens contaminated with fetal blood may exhibit abnormally high AFP values, which may lead to misinterpretation of test results.
  • Human anti-mouse antibodies (HAMA) 

Human Anti-mouse Antibodies (HAMA)

Specimens from patients who have received preparations of mouse monoclonal antibodies may contain human anti-mouse antibodies (HAMA), which may give falsely high results. Results must always be considered concerning the clinical situation and previous results. This is particularly important when serial results are used to monitor a patient’s response to treatment.


Risks associated with phlebotomy include:

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

The risk associated with amniocentesis:

  • Miscarriage
  • Preterm delivery

Patient Safety and Education

Phlebotomy for blood alpha-fetoprotein assay requires little or no preparations as this is mainly safe when performed by qualified health workers. However, patients need to be counseled on the possible discomfort from the needle prick, although mostly bearable.[18] Patients must also be asked about their use of blood thinners (e.g., aspirin, warfarin). Patients must also be given appropriate information regarding possible outcomes and implications of the test.[19]

It is pertinent to explain that this is a screening test. Depending on the outcome, more tests may be ordered to establish a diagnosis.[18] A negative test does not necessarily indicate no risk, as very low maternal blood alpha-fetoprotein is associated with an increased incidence of down syndrome. Hence, a low maternal blood alpha-fetoprotein should also be investigated.[20]

Patients with amniocentesis must be duly counseled about the procedure and the associated risks. There is a risk of obstetric mishap following amniocentesis; a miscarriage can happen in less than 1% of cases.[21] Some rare complications of amniocentesis are preterm labor, infection (amnionitis), iatrogenic trauma, or injury to the developing fetus or mother.[22]

Following amniocentesis, patients may experience some cramp-like discomfort in the first few hours but are usually allowed to go home after a short rest.[23] Oral paracetamol (acetaminophen) may be prescribed. Patients should report back to the hospital in case of vaginal bleeding, vaginal discharge, or increasing abdominal cramps.[21]

Clinical Significance

Maternal blood AFP levels are often part of triple (AFP, unconjugated estriol, and HCG) or quadruple (AFP, estriol, HCG, and inhibin A) screening tests for congenital disabilities.[24] Levels are usually interpreted for age, race, weight, and gestational age. As the levels of these markers change with gestational age, the results of these markers are expressed as multiples-of-median (MoM).[25] The elevated levels imply a significant risk of having congenital disabilities; hence, further evaluation may be required to assess the level of risk.[26] A substantial number of patients with elevated maternal AFP do not develop congenital disabilities, but there may be an increased risk of obstetric complications like premature membrane rupture, placenta accreta, and increta.[24]

Low maternal AFP levels may be suggestive of risk for down syndrome.[27] In nonpregnant women and men, elevated levels are seen in cancers, especially liver cancer. Levels greater than 200 ng/ml in cirrhotic patients suggest hepatocellular carcinoma. Elevated alpha-fetoprotein levels can also be found in testicular and ovarian carcinoma.[28]

AFP is also useful for determining prognosis and monitoring therapy for hepatocellular carcinoma.[29] The concentration of AFP is a prognostic indicator of survival. Elevated AFP concentrations (>10 μg/L) and serum bilirubin concentrations greater than 2 mg/dL are associated with a decreased survival time.[30]

Lens culinary agglutinin-reactive fraction of fetoprotein (AFP-L3), a subtype of AFP generated from malignant hepatocytes, is considered specific to hepatocellular carcinoma (HCC).AFP-L3 is isolated via an immunoassay and quantified using chemiluminescence on an automated platform.[31] The proportion of AFP-L3 to total AFP can be used to diagnose early hepatocellular carcinoma (HCC). Early studies reported that the diagnostic sensitivity of AFP-L3% for HCC ranged from 75.0% to 96.9%, with a specificity of 90.0–92.0%.[32] The high pre-treatment serum AFP-L3% levels indicated a poor prognosis for patients with HCC, and AFP-L3% may have significant prognostic value in HCC patients with low AFP concentration.[33]

A cutoff of 10% is used, and those patients with chronic liver disease and an elevated AFP-L3% have a seven-fold increased risk of developing hepatocellular carcinoma within 21 months.[34] 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. In practice, although AFP-L3% is useful in the detection and prognosis, it is typically used only when AFP concentrations are elevated.[28]

The AFP concentration is a good indicator for use in monitoring therapy and the change in clinical status. Elevated AFP concentration after surgery may indicate incomplete removal of the tumor or the presence of metastasis.[35] 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 the development of metastasis.[5]

The combination of measuring the concentration of both AFP and human chorionic gonadotropin (HCG) helps classify and stage germ cell tumors.[36] Germ cell tumors may be predominantly one type of cell or maybe a mixture of (1) seminoma, (2) yolk sac, (3) choriocarcinomatous elements (embryonal carcinoma), and (4) teratoma. Serum concentrations oF AFP are elevated in yolk sac tumors, whereas human chorionic gonadotropin (HCG) is elevated in choriocarcinoma. Both are elevated in embryonal carcinoma.[37] In seminomas, AFP is not elevated, whereas human chorionic gonadotropin (HCG) is elevated in 10% to 30% of patients who have syncytiotrophoblastic cells in the tumor. Neither marker is elevated in teratoma.[38]

One or both of the markers are elevated in about 90% of patients with non-seminomatous testicular tumors.[36] Elevations are noted in less than 20% of patients with stage I disease, 50% to 80% with stage II disease, and 90% to 100% with stage III disease. These markers correlate with tumor volume and the prognosis of the disease.[37] 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 success 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 (HCG) (12 to 20 hours).[39]



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