J Clin Lab Anal. electronic and paper medical records was performed on 348?samples identified as RhIG and 52 true anti\D samples. The agglutination strength of antibody was recorded for each sample. Results For RhIG, there was an even distribution between the weak to moderate agglutination strength (w+, 1+, and 2+) results (35%, 26%, and 33%, respectively) and just 6% had a 3+ strength. Agglutination strength in patients with high titer (1:16) anti\D showed they often (44.4%) have 1+ or 2+ agglutination reactivity. Conclusions These results show that agglutination strength alone does not provide reliable evidence to distinguish RhIG from high titer anti\D antibodies. We recommend that in cases where there is any uncertainty about whether the anti\D reactivity is due to RhIG, titers should be performed to rule out clinically significant anti\D antibody. Keywords: HDFN, RhIG, transfusion 1.?INTRODUCTION Hemolytic disease of the fetus and newborn (HDFN) is a serious and potentially Preladenant fatal complication due to the formation of maternal alloantibodies following sensitization by a target antigen. Sensitization can occur either through exposure to paternally\derived antigens during pregnancy or secondary to transfusion of Red Blood Cell (RBC)Cbased products containing foreign antigens. HDFN severity can range from mild subclinical hyperbilirubinemia to severe anemia associated with fetal hydrops and dangerously elevated bilirubin in the newborn. While ABO incompatibility is the most common cause of mild symptoms associated with HDFN, severe HDFN is more often caused by the anti\D alloantibody. Sensitization in the latter occurs by maternal exposure to fetal blood during pregnancy or at the time of delivery. Under most circumstances, HDFN secondary to an anti\D alloantibody does not affect the sensitizing (initial pregnancy with antibody detected) pregnancy, but rather subsequent pregnancies. 1 , 2 For women who have developed an anti\D alloantibody, titers are performed during pregnancy, most often monthly or biweekly. Once the maternal titer reaches 1:16, consultation is recommended with a Maternal\Fetal Medicine physician and biweekly ultrasound examinations are indicated to assess for fetal anemia with middle Cd14 cerebral artery (MCA) Dopplers and fetal hydrops. 3 Elevated MCA Dopplers consistent with severe anemia, >1.5?MoM, and/or fetal hydrops are indications for Preladenant percutaneous umbilical blood sampling and intrauterine transfusion. 4 In the US, Rh immunoglobulin (RhIG; 300?mcg/1500?IU) is given prophylactically to RhD negative pregnant women at 28?weeks gestation, following delivery (if the newborn is Rh\positive), and following any vaginal or uterine bleeding to prevent development of the anti\D alloantibody. Gel agglutination or solid phase antibody screens may be positive for anti\D due to passive transmission for up to 3C4?months following administration with strong agglutination (3+) with tube testing using PEG enhancement occurring within the first eight weeks after administration. 5 Some sources found that that large doses may result in a positive anti\D for up to 6?months. 6 , 7 For this reason, it is imperative to Preladenant perform an Preladenant antibody screen prior to RhIG administration to evaluate for a new anti\D alloantibody. In the rare circumstance that a true anti\D alloantibody has developed during pregnancy, it may be incorrectly reported as passive anti\D due to RhIG administration. When high suspicion exists for an anti\D alloantibody, a titer should be drawn. The patient should be treated as having a true anti\D antibody until a subsequent sample demonstrates a negative antibody screen or a decreasing anti\D titer, consistent with RhIG. Currently, there is no routine serological method in a single blood draw to differentiate between a true anti\D alloantibody and RhIG administration. Rather, historical documentation of RhIG administration and prior antibody screens prior to RhIG Preladenant administration are used to make an inference about the cause of anti\D reactivity. Here, we report a case of severe HDFN affecting what we believe is the sensitizing pregnancy (as evidenced by a negative antibody screen.