12.2 HDFN Detection

12.2 HDFN Detection

Quick Answer: Every prenatal patient gets a type and screen. A clinically significant IgG goes to identification and an AHG titer. The critical titer is method-dependent — many U.S. tube-AHG labs use 16 (some 32); ≥16 commonly triggers MCA-PSV Doppler. Anti-K does not wait for a critical titer. Type or genotype the father and use cell-free fetal DNA for RHD when it will change care. On the cord or newborn: ABO/Rh, weak D if the mother is D-neg, DAT, eluate, Hb, and bilirubin. Rosette is a qualitative FMH screen (D-neg mother, D-pos infant). Kleihauer–Betke or flow cytometry quantifies the bleed so you can dose RhIG.

III.B.3 on the June 9, 2026 outline is half mechanism and half what you actually order. Detection has a prenatal arm and a delivery arm. Mix them up and you will either miss a hydropic fetus or withhold RhIG from a woman who just had a 40 mL FMH.

Prenatal type, screen, identify, titer

First prenatal visit: ABO, D type, and antibody screen. D-neg women who type D-neg at immediate spin are managed as D-neg for transfusion and RhIG; do not “upgrade” them to D-pos on a weak-D afterthought unless RHD genotyping says they are a type 1, 2, or 3 weak D (that decision lives in Chapter 7). The screen is looking for immune IgG.

If the screen is positive, identify the antibody. Then ask the only question that matters: is it clinically significant IgG that can cause HDFN? If yes — anti-D, -c, -E, -C, -K, -k, -Fya, -Jka, -S, -s, -U, and the other AHG villains — titer it. If no — anti-Le(a), anti-P1, cold-only anti-M — do not titer, do not start serial MCA-PSV, and do not pretend this is anti-D.

Titer method: master dilution of maternal plasma against target antigen-positive red cells, read at AHG. Report the reciprocal of the last 1+ tube (lab-defined endpoint). Use the same method, same cell phenotype, same technologist discipline every time so a rise is real. A two-tube (fourfold) rise is the usual significant change.

Critical titer is not a universal number. It is method-dependent. Classic saline-AHG tube methods often use 16; some laboratories use 32. Gel/column titers run higher, so a gel “16” is not the same as a tube “16.” On the exam, say method-dependent, and remember the common teaching line: an AHG titer ≥16 usually prompts referral for MCA-PSV Doppler. Once the critical titer is reached, more titers do not manage the fetus — ultrasound Doppler does. Exception: anti-K (and sometimes other Kell-system antibodies) — do not wait for 16. Refer for Doppler when anti-K is identified and the father is K+ or unknown.

Passive anti-D after antenatal RhIG is a special case. The titer is usually low (≤4, sometimes 8) and does not rise. Do not promote that result into an HDFN workup, and do not skip the postpartum RhIG dose because the screen is positive. Section 12.3 finishes that trap.

MCA-PSV, the father, and cell-free fetal DNA

Middle-cerebral-artery peak systolic velocity (MCA-PSV) is the noninvasive test for fetal anemia. An anemic fetus has thinner blood and a higher cardiac output, so peak velocity rises. The threshold you will be handed is ≥1.5 multiples of the median (MoM) for gestational age. That number, not a Liley curve, is what sends the patient to cordocentesis / intrauterine transfusion in current practice. ΔOD450 still exists in older stems; know what it is, and know that MCA-PSV replaced it, especially for anti-K.

Type the father for the implicated antigen. Better: genotype him when serology cannot settle zygosity (D zygosity is a molecular question; serologic D+ does not tell you RHD copy number).

• Father antigen-negative, paternity reliable → fetus is negative → stand down.
• Father homozygous antigen-positive → fetus is positive → monitor.
• Father heterozygous → 50% chance → fetal typing.

Cell-free fetal DNA (cffDNA) in maternal plasma can determine fetal RHD noninvasively. Many services also offer KEL01 (K) and selected other targets. A D− fetus in a mother with anti-D does not need weekly Doppler. An RHD-negative fetal result also lets some systems skip antenatal RhIG — that is a policy decision; the science is that a D− fetus cannot immunize her to D. Invasive amniocyte genotyping remains a backup when cffDNA is unavailable or inconclusive.

Never phenotype the father from a chair. A verbal “I’m A-positive” is not a Kell type.

Cord and newborn workup

At delivery of an at-risk infant — or any infant of a D-neg mother, or a jaundiced neonate — the blood bank wants:

• ABO and D type on cord or heel-stick. Forward type only on newborns; reverse type is not reliable because isoagglutinins have not been made.
• Weak D testing on the newborn if the mother is D-neg. This is a required serologic weak-D setting (with allogeneic donors). A weak-D infant counts as D-positive. The mother gets RhIG. Miss this and you send a D-neg mother home unprotected.
• DAT (anti-IgG; C3 optional). A positive DAT means IgG is on the cells. It does not name the antibody.
• Elution when the DAT is positive and you need the specificity — especially if the maternal antibody is unknown or you must distinguish ABO from a second alloantibody.
• Hemoglobin / hematocrit and bilirubin (total and direct). These, not the DAT strength, decide phototherapy and exchange.

A group O mother, group A infant, weak or negative DAT, spherocytes, and a climbing bilirubin is ABO HDFN. Do the elution if the DAT is positive; anti-A in the eluate closes it. Do not refuse the diagnosis because the DAT is 0 or w+.

If the mother has anti-D and the infant is D-pos, the DAT should be positive and the eluate anti-D. If the DAT is positive and the maternal screen is negative, think ABO, a low-prevalence paternal antigen (anti-Wra, -Kpa, -Jsa), or IVIG / maternal autoantibody.

Rosette, Kleihauer–Betke, and flow

Postpartum RhIG dosing depends on knowing whether the fetomaternal hemorrhage (FMH) exceeded the 30 mL whole blood that one 300 µg vial covers.

Rosette test (fetal bleed screen): a qualitative screen used when the mother is D-neg and the infant is D-pos. Maternal EDTA blood is incubated with anti-D; enzyme-treated D+ indicator cells then form rosettes around any D+ fetal cells.

• Negative rosette → FMH is below the screen’s threshold (on the order of 10 mL) → one 300 µg vial is enough.
• Positive rosette → quantify the bleed. Do not guess vials from rosette strength.

Rosette fails when you cannot use D as the marker:

• Infant is weak D (too few D sites) → skip the rosette, go straight to KB or flow.
• Mother types as weak D or mixed-field D (often a large FMH of D+ cells) → do not call her D-pos; quantify.
• Mother is D-pos (wrong test) or infant is D-neg (nothing to detect).

Kleihauer–Betke (acid elution): peripheral smear, acid buffer, HbF resists elution, adult Hb washes out. Fetal cells stain pink; adult cells are ghosts. Count a large number of cells (classically 2,000) and compute % fetal cells. Limitations: subjective, maternal F cells in pregnancy or HPFH / thalassemia inflate the percentage, and the math assumes a 5,000 mL maternal blood volume unless you know otherwise.

Flow cytometry: anti-HbF and/or anti-D, objective, more precise, preferred when available. Anti-HbF flow works even when D types do not (weak D infant, or a need to measure FMH that is not D-marked). Anti-D flow only works for a D-neg mother / D-pos infant pair.

Exam traps

• Treating 16 as a law of physics. It is a common tube-AHG critical titer, not a gel constant and not the anti-K trigger.
• Repeating titers forever after the critical titer instead of sending the patient for MCA-PSV.
• Skipping weak D on the newborn of a D-neg mother.
• Using the rosette on a weak-D infant.
• Calling a D-neg mother D-pos because delivery type looks weakly D+ — that is often fetal cells, not a new blood type.
• Ruling out ABO HDFN with a negative DAT.
• Titration of Lewis or cold anti-M.