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Necessary and Optional Laboratory Assessments for the Mother, Father, and Fetus

Laboratory Assessments for the Mother

Pre-pregnancy

Antibody Screen With Reflex For ID And Titer (Optional)
This test screens for antibodies, and will identify and titer any antibodies detected. 

Benefits: Have a starting point for antibody levels prior to pregnancy. May detect any new antibodies that have developed due to last delivery. May show critical levels and a need for early MCA ultrasounds. Reference values exist for MCA scans as early as 12-16 weeks gestation.

Disadvantages: May not show all antibodies due to antibody evanescence (ie negative antibody screen despite prior history of alloimmunization). Titers are not an accurate basis for care after a previously affected pregnancy. Low titer levels pre-pregnancy should not rule out the need for MCA ultrasounds. 



First trimester

Antibody Screen With Reflex For ID And Titer (Necessary) 

Beginning upon initial pregnancy labs, repeat antibody titer every 4 weeks until 28 weeks if the titer is below critical. If monitoring via MCA scans, then continued titer draws are unnecessary. 

Benefits: Provides a starting point for antibody levels in early pregnancy. If a four-fold increase is found, or if titers hit critical levels (4 for Kell, 16 for all other antibodies), then MCA scans should be initiated by18 weeks. Note: Anemia requiring IUT is possible at titers below 4 with anti-Kell. Some doctors debate if there is a critical level for anti-Kell, or if scans should be initiated regardless of titer with anti-Kell. 

Disadvantages: Titers are not an accurate indicator for predicting anemia and basing care after a previously affected pregnancy. MCA scans should be started instead.


A Note About Titers:
The AABB, formerly the American Association of Blood Banks, recommended changing how titers were reported to simply reflect the reciprocal value of the titer. Titer results formerly reported as 1:4, 1:8, 1:16, etc., may now be reported as 4, 8, 16, etc.



Second Trimester

cffDNA Test (Optional)

Also called Non-Invasive Prenatal Testing (NIPT), the cffDNA test is a blood draw on the mother which tells the antigen status of the fetus. 

Benefits: Provides confirmation if the fetus is antigen negative (no risk) or antigen positive (at risk). If the fetus is negative, weekly MCA scans and other therapies are unnecessary. If the fetus is antigen positive, MCA scans may be required depending on titer levels. Can also provide information about timing delivery (ie an at risk fetus would be delivered at earlier gestation than an antigen negative fetus). Results can determine if there is a need for RhD Immune Globulin (Rhogam) in an Rh- mother. Antigen results can be determined as early as 12-14 weeks. Unlike amniocentesis which carries a 0.5-1.0% risk of spontaneous abortion, and a 20% risk of transplacental haemorrhage, there is no risk to the fetus from the cffDNA test.

Disadvantages: Cost - in the USA, this test isn’t always covered by insurance unless the provider writes a letter requesting it and providing information about the disease, the cost of the test, and the cost of the weekly ultrasounds which can be spared if the fetus is antigen negative. There is a slight risk of the test being incorrect, but overall accuracy is over 99%. The test may need to be repeated if gestation is early (9-14 weeks).


Third Trimester

Antibody Screen With Reflex For ID And Titer (Necessary) 

Repeat antibody titer every 2 weeks until 36 weeks if the titer is below critical. If monitoring via MCA scans, then continued titer draws are unnecessary. 

Benefits: Allows a rise in titers to be caught quickly. It is common for antibody levels to increase in the third trimester as both maternal and fetal blood volumes increase. 

Disadvantages: None



36-40 weeks

Antibody Screen With Reflex For ID And Titer (Necessary) 

Repeat antibody titer weekly until delivery if the titer is below critical. If monitoring via MCA scans, then continued titer draws are unnecessary. 

Benefits: Will quickly catch an increase in titers during a time when titer levels for many women increase. Some fetal antigens are not fully developed until late third trimester. In some cases, maternal antibodies may not recognize the fetal antigens until late third trimester and then rapidly increase. May help determine delivery timing. A late third trimester increase may indicate a need for induction prior to week 38. 

Disadvantages: None


Laboratory Assessments for the Father

Pre-pregnancy and During Pregnancy

Antigen Phenotype (Required)

This test will determine the antigen status of the father and will show if he is homozygous for the antigen, heterozygous, or negative for the antigen. These results can determine if all children with the same partner are at risk from maternal alloimmunization, if 50% may be antigen positive and at risk, or if there is no risk to any children with this partner. If a mother has an alloantibody that is known about prior to pregnancy, then this test could be run prior to making the decision to become pregnant. If antibodies are detected during a current pregnancy, then this test should be run when antibodies are detected. 

Benefits: Provides information about the potential risk to the fetus. Can help with counseling couples about their options for future pregnancies. Can also lead to more testing (ie heterozygous father means cffDNA testing on the fetus may be an option). Results can determine if there is a need for RhD Immune Globulin (Rhogam).

Disadvantages: Requires absolute certainty of paternity. Laboratory may mistakenly run an antibody screen instead of the antigen phenotype, so results must be checked carefully. Tests may need to be repeated if unexpected results are received (ie father is antigen negative, but mother has antibodies and no prior history of transfusion). 


Laboratory Assessments for the Fetus

Laboratory assessments on the fetus itself is unnecessary unless Intrauterine Transfusions (IUT) are required. If IUTs are performed, it is helpful to have the following lab tests run on fetal blood retrieved prior to transfusion. 


Percutaneous Umbilical Blood Sample (PUBS) - Starting Hematocrit

This test measures the fetal hematocrit or hemoglobin.

Benefits: Will determine if fetus is truly anemic

Disadvantages: Requires an invasive procedure


Antigen phenotype

This test determines the fetal antigen status.

Benefits: If there is any doubt about the fetus having the antigen (or uncertain paternity), it is possible to determine fetal antigen status along with a starting hematocrit to determine if anemia is truly present.

Disadvantages: Requires an invasive procedure and is not worth the risk unless a PUBS was already being performed due to suspected anemia. No additional risk if PUBS is already being performed.


Ending hematocrit

This test measures the fetal hematocrit or hemoglobin.

Benefits: Will help determine the overall success of an IUT. Can help determine the timing of the next IUT, and if fetal hematocrit was raised to a sufficient level.
Disadvantages: None

Additional Reading

Antibody Screens and Titers

Management of pregnancy complicated by RhD alloimmunization Author: Kenneth J Moise Jr, MD https://www.uptodate.com/contents/management-of-pregnancy-complicated-by-rhd-alloimmunization


Management of non-RhD red blood cell alloantibodies during pregnancy. Authors: Kenneth J Moise Jr, MD, Melanie S Kennedy, MD 

https://www.uptodate.com/contents/management-of-non-rhd-red-blood-cell-alloantibodies-during-pregnancy


AABB http://www.aabb.org/


Comparison of total and IgG ABO antibody titers in healthy individuals by using tube and column agglutination techniques. https://www.ncbi.nlm.nih.gov/pubmed/24790910


Comparison of antibody titers using conventional tube technique versus column agglutination technique in ABO blood group incompatible renal transplant https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613419/ 


Antigen phenotyping



Cell Free Fetal DNA

Noninvasive fetal blood group genotyping of rhesus D, c, E and of K in alloimmunized pregnant women: evaluation of a 7-year clinical experience by PG Scheffer, a,b CE van der Schoot, b GCML Page-Christiaens, a M de Haas b,c.  https://www.ncbi.nlm.nih.gov/pubmed/21668766


Diagnostic accuracy of noninvasive polymerase chain reaction testing for the determination of fetal rhesus C, c and E status in early pregnancy. By Gutensohn K et al. https://www.ncbi.nlm.nih.gov/pubmed/20175873


Prenatal non-invasive foetal RHD genotyping: diagnostic accuracy of a test as a guide for appropriate administration of antenatal anti-D immunoprophylaxis. By Manfroi S et al https://www.ncbi.nlm.nih.gov/pubmed/29757138 

USA Sensigene RhD test website:  https://www.sequenom.com/tests/reproductive-health/sensigene-rhd

Australian Red Cross Non-Invasive Prenatal Analysis (NIPA) for RhD website:  https://transfusion.com.au/node/809

Netherlands Sanquin website:  https://www.sanquin.org/products-and-services/diagnostics/non-invasive-fetal-blood-group-genotyping

UK Testing for those in the NHS system: https://ibgrl.blood.co.uk/services/user-guides/ Click on "Fetal RhD Sampling" for anti-D, and click on "Molecular Diagnostics" and "Fetal Genotype Sample Referral Form".

Canadian Blood Service:  https://blood.ca/en/laboratory-services/fetal-genotyping-maternal-plasma-0




Intrauterine Transfusions 

Intrauterine Fetal Transfusion of Red Cells (UpToDate) by Kenneth Moise Jr. https://www.uptodate.com/contents/intrauterine-fetal-transfusion-of-red-cells