Lab Diagnosis of PregnancyLab Diagnosis of Pregnancy

Dr. Neelesh BhandariDr. Neelesh BhandariM.B.B.S (AFMC), M.D. (Path)M.B.B.S (AFMC), M.D. (Path)P.G.P in Human Rights.P.G.P in Human Rights.

Several hormones can be measured and monitored

to aid in the diagnosis of pregnancy

The most commonly used assays are for the beta subunit of hCG

Other hormones that have been used include

progesterone

early pregnancy factor (EPF).

HCGThis hormone is only released by trophoblastic tissue,usually is produced by a growing fetus and its associated placenta. (rarely, from a choriocarcinoma or some other germ cell tumours.)

hCG is a glycoprotein similar in structure to follicle-stimulating hormone,

luteinizing hormone (LH),

and thyrotropin.

The free beta subunit of hCG differs from the others in that it has a

30–amino acid tailpiece at the COOH terminus.

Free beta subunits are degraded by macrophage enzymes in the kidney

to make a beta subunit core fragment which is primarily detected in Urine samples.

Detection of HCG in maternal serum and urine is evident only after implantation and vascular communication has been established with the decidua by the syncytiotrophoblast 8-10 days after conception. Time of detection is related to the sensitivity of the assay being used. Most current pregnancy tests have sensitivity to approximately 25 mIU/mL theoretically,But ranges from 25 to 100mIU/mL.

Sample reference list for HCG levels in singleton pregnancies, based on days past ovulation (DPO): * At 14 DPO, the average HCG level is 48 mIU/ml, with a range of 17-119 mIU/ml. * At 15 DPO, the average HCG level is 59 mIU/ml, ( 17-147 mIU/ml.) * At 16 DPO, the average HCG level is 95 mIU/ml, ( 33-223 mIU/ml.) * At 17 DPO, the average HCG level is 132 mIU/ml, (17-429 mIU/ml. )* At 18 DPO, the average HCG level is 292 mIU/ml. (70-758 mIU/ml.) * At 19 DPO, the average HCG level is 303 mIU/ml, ( 111-514 mIU/ml.) * At 20 DPO, the average HCG level is 522 mIU/ml, ( 135-1690 mIU/ml.) * At 21 DPO, the average HCG level is 1061 mIU/ml, ( 324-4130 mIU/ml.) * At 22 DPO, the average HCG level is 1287 mIU/ml, ( 185-3279 mIU/ml.) * At 23 DPO, the average HCG level is 2034 mIU/ml, ( 506-4660 mIU/ml.) * At 24 DPO, the average HCG level is 2637 mIU/ml, ( 540-10,000 mIU/ml.) Accordingly, the key to interpreting the true value of HCG measurements is to look at their progression. In general, the HCG level will double every two to three days in early pregnancy.

Therefore, Testing during early pregnancy ( with Low hCG levels),Or by test kits not sensitive to lower levels of hCG will give false Negative Results

False positive results can result from rare beta-HCG producing tumors like choriocarcinomas.

Currently, 4 main hCG assays are used, (1) radioimmunoassay,

(2) immunoradiometric assay,

(3) enzyme-linked immunosorbent assay (ELISA),

(4) fluoroimmunoassay.

Radioimmunoassay Sensitivity - 5 mIU/mL Time to complete - 4 hours Postconception age when first positive - 10-18 days Gestational age when first positive - 3-4 weeks

Immunoradiometric assay (more sensitive) Sensitivity - 150 mIU/mL Time to complete - 30 minutes Postconception age when first positive - 18-22 days Gestational age when first positive - 4 weeks

Immunoradiometric assay (less sensitive) Sensitivity - 1500 mIU/mL Time to complete - 2 minutes Postconception age when first positive - 25-28 days Gestational age when first positive - 5 weeks

Enzyme-linked immunosorbent assay (more sensitive) Sensitivity - 25 mIU/mL Time to complete - 80 minutes Postconception age when first positive - 14-17 days Gestational age when first positive - 3.5 weeks

Enzyme-linked immunosorbent assay (less sensitive) Sensitivity - Less than 50 mIU/mL Time to complete - 5-15 minutes Postconception age when first positive - 18-22 days Gestational age when first positive - 4 weeks

Fluoroimmunoassay Sensitivity - 1 mIU/mL Time to complete - 2-3 hours Postconception age when first positive - 14-17 days Gestational age when first positive - 3.5 weeks

hCG is detectable in the serum of approximately 5% of patients 8 days after conception and in more than 98% of patients by day 11.

At 4 weeks' gestation (18-22 d postconception), the dimer and beta subunit hCG doubling times are approximately 2.2 days (standard deviatio0.8 d) and fall to 3.5 days (standard deviation1.2 d) by 9 weeks' gestation.

Levels peak at 10-12 weeks' gestation and then plateau before falling.

Failure to achieve the projected rate of rise (slow rise) may suggest an ectopic pregnancy or spontaneous abortion.

On the other hand, an abnormally high level or accelerated rise can prompt investigation into the possibility of

• molar pregnancy, • multiple gestations, • chromosomal abnormalities.

False-positive serum hCG results are usually due to interference by non-hCG substances or the detection of pituitary hCG. Some examples of non-hCG substances that can cause false-positive results include human LH, anti animal immunoglobulin antibodies, rheumatoid factor, heterophile antibodies and binding proteins. Most false-positive results are characterized by serum levels that are generally less than 1000 mIU/mL and usually less than 150 mIU/mL.

Some of the substances that can cause serum false-positive results have much higher molecular weights that are not easily filtered through the renal glomeruli; therefore, they do not produce a positive urine test.

False-negative hCG test results usually involve urine and are due to the qualitative nature of the test. Reasons include• an hCG concentration below the sensitivity threshold of the specific test being used.• a miscalculation in the onset of the missed menses, • delayed menses from early pregnancy loss. • Delayed ovulation or delayed implantation.

Serum progesterone is a reflection of progesterone production by the corpus luteum which is stimulated by a viable pregnancy. •Measurement of serum progesterone is inexpensive •Can reliably predict pregnancy prognosis. Currently, radioimmunoassays and fluoroimmunoassays are available that can be completed in 3-4 hours.

A dipstick ELISA that can determine a serum progesterone level of less than 15 ng/mL is also on the market.

ELISA is helpful as a screening tool for at-risk populations because progesterone levels of greater than 15 ng/mL make ectopic pregnancy unlikely. Serum progesterone levels greater than 25 ng/mL Viable Intrauterine PregnancySerum progesterone levels of less than 5 ng/mL Nonviable pregnancy.

An ectopic pregnancy cannot be distinguished from a spontaneous intrauterine abortion. In the event that the serum progesterone level is 5-25 ng/mL, further testing using US, additional hormonal assays, or serial examinations is warranted to establish the viability of the pregnancy.

Early pregnancy factor The EPF assay may be useful in the future. •EPF is a poorly defined immunosuppressive protein that has been Isolated in maternal serum shortly after conception. •It is the earliest available marker to indicate fertilization.•detectable in the serum 36-48 hours after fertilization. •Peaks early in the first trimester, and is almost undetectable at term.

•EPF also appears within 48 hours of successful in vitro fertilization embryo transfers.

• EPF cannot be detected 24 hours after delivery or at the termination of pregnancy. •EPF is also undetectable in many ectopic pregnancies and spontaneous abortions, indicating that an inability to identify EPF during pregnancy heralds a poor prognosis.

•EPF has limited clinical applications because the molecule is difficult to isolate. Detection of EPF currently relies on a complex and unwieldy assay termed the rosette inhibition test. •EPF may play a more prominent role in the future as the diagnosis of conception prior to implantation elucidates new strategies for contraception, highly accurate dating, and advanced genetic studies.

Home Pregnancy Testskits available for hCG detection in urine via Direct Latex agglutination and/orIndirect Agglutination inhibition tests.

•HPTs are most commonly used in the week after the missed menstrual period (fourth completed gestational week). •Urine hCG values are extremely variable at this time and can range from 12 mIU/mL to greater than 2500 mIU/mL. This variability continues into the fifth week, when values have been shown to range from 13 mIU/mL to greater than 6000 mIU/mL. Both weeks have a percentage of urine hCG values that is below the sensitivities of detection for common HPTs (range 25-100 mIU/mL). 100% accuracy by H.P.T achieved only •when the highest hCG concentration (100 mIU/ml) was used, •an extended reading time was used, ( more then 10 mins.)•faintly discernible results were included as positive.

Early pregnancy detection allows for •the commencement of prenatal care, •potential medication changes, •lifestyle changes to promote a healthy pregnancy (appropriate diet; avoidance of alcohol, tobacco, and certain medications), •early pregnancy termination if so desired.

UltrasoundThe identification of gestational structures by US correlates with specific levels of hCG, termed discriminatory levels. •A discriminatory level is the level of hCG at which the structure in question should always be identified.

•Most experienced TVUS operators should visualize the GS when levels are approximately 1000 mIU/mL.

•The discriminatory level for the GS is approximately 3600 mIU/mL, and if it is not seen at this point, other pathology must be excluded.

• Many use a more conservative discriminatory level for the GS, at 2000 mIU/mL by TVUS and 3600 mIU/ml by TAUS. •The adnexa should be scanned for an ectopic pregnancy.

•One study showed that all viable intrauterine pregnancies had a GS identified by TAUS for hCG levels of greater than 6500 mIU/mL

•Other structures are also anticipated in correlation with specific hCG levels.

•The yolk sac is commonly observed with an hCG level ofapproximately 2500 mIU/mL, although it may not be identified until levels are much higher.

• The embryonic pole usually becomes evident at a level of approximately 5000 mIU/mL,

• Fetal heartbeat can be seen in the vast majority of normal gestations when the hCG level reaches 10,000 mIU/mL.