Pregnancy Test

Pregnancy diagnosis and monitoring
Antenatal Screening (Quad Screen, Aneuploidy Screening)
1. Free bHCG is increased in Trisomy 21
2. Free bHCG is decreased in Trisomy 18
Tumor monitoring
1. Nonseminomatous germ cell tumor monitoring
2. Hydatiform mole diagnosis and monitoring
3. Choriocarcinoma diagnosis and monitoring

Urine Pregnancy Test (UPT) has a False Negative Rate of 1-2% (see below)
1. Despite descriminatory levels as low as urine bHCG of 20 IU, False Negatives occur at higher bHCG levels
2. UPT False Negative Rate is 3.6% in presentations of Pelvic Pain or Vaginal Bleeding (e.g. Ectopic Pregnancy)
3. Confirm a negative Pregnancy Test with a serum HCG in high acuity cases or when clinical suspicion is high
4. Kleinschmidt (2021) J Am Coll Emerg Physicians Open 2(3):e12427 +PMID: 33969349 [PubMed]

HCG Function
1. Prevents corpus luteum from involuting during pregnancy
  1. Allows for continued increase of Estrogen and Progesterone
2. Stimulates Testosterone production in the male fetus Testes
HCG is a Glycoprotein Hormone with subunits a and b
HCG shares the same alpha subunit with other Hormones (while Beta is unique to HCG)
1. Luteinizing Hormone (LH)
2. Thyroid Stimulating Hormone (TSH)
Components
1. Composed of 65% polypeptides by molecular weight
2. Composed of 35% large sugar side chains (8 chains)
  1. Four are N-Linked (2 each on alpha and beta)
  2. Four are O-Linked (all 4 are on beta subunit)
3. Forms of HCG found in blood and urine
  1. Intact HCG with alpha and beta subunits
  2. Nicked HCG and nicked free beta subunit
  3. Free Beta hCG subunit and free alpha HCG subunit
  4. Hyperglycosylated free beta and free alpha subunits
  5. Beta core fragment (present only in urine)
Measurement
1. Urine and Blood HCG tests are specific for beta subunit
2. Serum half life of HCG: 24 to 36 hours
3. Discriminatory values at which bHCG is positive
  1. Urine bHCG: 20-25 IU/ml
  2. Serum bHCG: 10 IU/ml

Estimation in pregnancy for weeks 4 to 8 (plateaus after 10 weeks)
1. Normal pregnancy
  1. bHCG may be detectable as early as 8 days after Ovulation
  2. Typically increases 80% every 48 hours (doubling every 48 to 72 hours)
  3. Minimal expected HCG increases
    1. HCG <1500 mIU/ml: Increases at least 49% in 48 hours
    2. HCG 1500-3000 mIU/ml: Increases at least 40% in 48 hours
    3. HCG >3000 mIU/ml: Increases at least 33% in 48 hours
    4. Barnhart (2016) Obstet Gynecol 128(3): 504-11 [PubMed]
2. Fetal demise
  1. Decreases 20% every 48 Hours
3. Ectopic Pregnancy
  1. Slower than expected rise in bHCG
4. Faster than expected rise in bHCG
  1. Multiple Gestation
  2. Gestational Trophoblastic Disease (extremely high bHCG)
Chart of corresponding Gestational age
1. Day 23 (3.3 weeks): 100 mIU/ml (correlates with blastocyst implantation)
2. Day 28 (4.0 weeks): 250 mIU/ml (approximate time of first missed Menses)
3. Day 35 (5.0 weeks): 1000 mIU/ml
  1. bHCG 1800: Gestational Sac on Transvaginal U/S
  2. bHCG 3500: Gestational Sac on Transabdominal U/S
4. Day 42 (6.0 weeks): 4000 mIU/ml
5. Day 49 (7.0 weeks): 15000 mIU/ml
  1. bHCG 20,000: 5-10 mm Embryo with cardiac activity
6. Day 56 (8.0 weeks): 65000 mIU/ml
7. Decreases gradually after 8 weeks
8. Plateaus after 20 weeks
Ranges of bHCG over each Trimester
1. First Trimester: 30,000 to 100,000 mIU/ml
2. Second Trimester: 10,000 to 30,000 mIU/ml
3. Third Trimester: 5,000 to 15,000 mIU/ml

Commercial Pregnancy Tests (bedside Pregnancy Test)
1. Approved for urine and serum samples
2. Qualitative Urine Pregnancy Test
  1. Test Sensitivity: 95%
  2. Negative Predictive Value: 98%
3. Whole blood may also be used (e.g. Emergency Department)
  1. Wait at least 10 minutes before interpreting test
  2. Bedside whole blood method in 10 minutes
    1. Much faster than the alternative serum result from lab in over 1 hour
  3. Test Sensitivity: 96%
  4. Test Specificity: 100%
  5. Negative Predictive Value: 98%
    1. Misses cases where bHCG is <50
    2. Confirmatory testing is needed for negative tests (or repeat testing in 48-72 hours)
4. References
  1. Fromm (2012) J Emerg Med 43(3): 478-82 [PubMed]
Home Pregnancy Tests
1. Variable efficacy after missed Menses
2. Wide discrepancy between brands
3. Cole (2004) Am J Obstet Gynecol 190:100-5 [PubMed]

HCG-producing tumors in women (extremely high bHCG)
1. Hydatidiform Mole or Choriocarcinoma (Gestational Trophoblastic Disease)
HCG-producing tumors in men
1. HCG is usually undetectable in healthy males
2. Nonseminomatous germ cell tumor (Testicular Cancer)
  1. Used with AFP for monitoring

Causes
1. Heterophilic Antibody (most common)
2. Human anti-mouse Antibody (HAMA)
3. Nonspecific Protein-binding hCG-like substances
4. Red Blood Cell interference
5. Marijuana use
6. Hypogonadism
Confirmation methods in non-pregnant conditions
1. Qualitative urine hCG
2. Serum hCG by different immunoassay method
3. Serial dilutions of serum hCG sample

Dilute urine
High Vitamin C intake
Processing error
Hook Effect
1. Detection is via free and fixed antibodies that bind HCG in a "sandwhich"
2. Excess intact HCG fragments saturate antibodies and prevent detection (False Negative)
Variant Hook Effect
1. HCG variants present later in pregnancy may not be detected by some assays
References
1. Griffey (2013) J Emerg Med 44(1):155-60 +PMID: 21835572 [PubMed]

Interpretation
1. Poor prognosis if bHCG >50,000 mIU/ml at diagnosis
2. Five year survival rate: 50%
Efficacy
1. High Specificity in men
2. Low Test Sensitivity
  1. HCG or AFP increased in 85% of patients
  2. HCG or AFP increased in only 20% of Stage 1 Cancer
Protocol
1. Initial: bHCG with AFP q1-2 months for 1 year
2. Later: bHCG with AFP q3 months for 1 year