Patient's question:
Hello, doctor. I recently have a friend who wants to have a child with her husband, but they don't know the risk of hemolysis. They especially asked me to ask you what the titer of blood type and the risk of hemolysis actually mean.Doctor's answer:
Polyacrylamide Immunodiffusion Method (Basic Principle)Antigens and antibodies diffuse within the same gel, forming specific precipitate lines upon encountering each other.
Antigens and antibodies are separately added to two small wells in the same gel plate, spaced at a certain distance, allowing them to diffuse and meet. When the antigen-antibody concentration ratio is appropriate, they form a white arc-shaped precipitate line.
Advantages and Disadvantages: Simple but with low sensitivity, prone to false-negative results.
Procedure of Immunodiffusion Method
1. Wash the glass plate with water and rinse it with 75% ethanol, then dry it and place it on a flat surface for use.
2. Melt 1% agar and place it in a 56°C water bath.
3. Spread the gel on the glass plate to a thickness of about 1.5 mm, then punch holes (diameter 3 mm) with a spacing of 10 mm.
4. Add antiserum (antibodies are pre-diluted in a series of ratios, e.g., 1:2, 1:4, 1:8, 1:16, 1:32, etc.) to the wells:
- Surrounding wells: 5 μL per well
- Center well: 5 μL of each dilution
5. Place the gel plate in a moist chamber and allow it to diffuse at room temperature for 24 hours.
6. Observe the results.
Other Methods for Antibody Titer Detection
- Ring precipitation test: Requires a large amount of antiserum and is rarely used now.
- Counterimmunoelectrophoresis: More sensitive than agar immunodiffusion, simpler and more practical.
- Enzyme-linked immunosorbent assay (ELISA).
Typically, an antibody titer below 1:128 is acceptable. Some pregnant women may have titers as high as 1:256 or more. It is recommended to check antibody titer once a month, as it may fluctuate.
Hemolytic Disease of the Newborn (HDN)
HDN is an autoimmune disease caused by blood type incompatibility between a pregnant woman and fetus, which can occur in early fetal and neonatal stages. If the dominant antigen inherited from the father is absent in the mother, it can enter the mother’s body during pregnancy or childbirth, stimulating the production of immune antibodies. When these antibodies cross the placenta into the fetal circulation, they can cause red blood cell agglutination and destruction, leading to immune hemolytic anemia in the fetus or newborn. This condition has no effect on the mother but can cause severe anemia, heart failure, or kernicterus (jaundice affecting the brain) in the child, even if they survive. Survivors may suffer from neurological and intellectual developmental issues and motor dysfunction.
Blood type incompatibility mainly includes ABO and Rh types, with other systems like MN being rare causes. ABO incompatibility is more common and usually milder, often overlooked. Rh incompatibility is less common in China but more severe, often leading to fetal death in utero or kernicterus in newborns.
### I. Etiology
In the ABO system, the mother is usually type O, while the father and fetus are A, B, or AB. The fetal A or B antigens act as allergens. The Rh system has six antigens (C, c, D, d, E, e), with D being the most antigenic and clinically significant. Rh-negative mothers may develop antibodies against other antigens (e.g., anti-E or anti-C), leading to incompatibility. Fetal red blood cells normally do not cross the placenta but can enter the mother’s circulation during pregnancy or childbirth if the placenta is damaged. The amount and frequency of fetal red blood cell entry influence antibody production and the severity of hemolysis.
Blood type antibodies are immunoglobulins, including IgG and IgM. IgG (7S-globulin) is an incomplete antibody (colloidal medium antibody or blocking antibody) and can cross the placenta, while IgM (19S-γ-globulin) is a complete antibody (salt-agglutinating antibody) and cannot. IgG is the active form in Rh and ABO incompatibility.
### II. Pathology and Clinical Manifestations
1. Hemosiderin deposition: Increased red blood cell destruction can lead to hemosiderin deposition in reticuloendothelial systems, liver, and kidneys.
2. Compensatory hematopoiesis: Bone marrow and extramedullary hematopoietic tissues may hypertrophy, causing hepatosplenomegaly. Microscopic examination reveals scattered extramedullary hematopoietic foci in liver, spleen, lungs, pancreas, and kidneys.
3. Anemia: Cardiac enlargement, hypoproteinemia, pallor, edema, and hepatosplenomegaly with scattered extramedullary hematopoietic foci in tissues like the chest, abdomen, and pericardium.
4. Hyperbilirubinemia: Can cause generalized jaundice and kernicterus, primarily affecting basal ganglia, hippocampal gyrus, globus pallidus, subthalamic nucleus, caudate nucleus, and dentate nucleus. Neuronal degeneration with cytoplasmic yellowing and nuclear loss occurs. Mild cases may be asymptomatic, while severe hemolysis can lead to fetal edema, abortion, premature birth, or stillbirth. Postnatally, symptoms include anemia, edema, hepatosplenomegaly, jaundice, and kernicterus. Severity depends on antibody levels, neonatal maturity, and compensatory hematopoietic capacity.
### III. Diagnosis
Diagnosis primarily relies on laboratory-specific antibody testing. Women with a history of miscarriage, unexplained stillbirth, blood transfusion, or severe neonatal jaundice should be ruled out for incompatibility. Routine blood typing during pregnancy is recommended:
- If the mother is type O and the father is A, B, or AB, specific antibody testing is necessary. A negative result suggests sensitization.
- Rh incompatibility with antibody titer >1:32 or ABO incompatibility with titer >1:512 indicates severe disease.
- Amniotic fluid analysis can be performed using a spectrophotometer to measure bilirubin absorbance at 450 nm.
- Dangerous value: ΔOD450 > 0.06
- Warning value: 0.03–0.06
- Safe value: <0.03
- After 36 weeks of gestation, amniotic fluid bilirubin >0.2 mg% suggests fetal hemolysis.
### IV. Management
#### (1) Prenatal Management
1. Comprehensive therapy: To enhance fetal resistance and bilirubin metabolism, administer comprehensive treatment at 24, 30, and 33 weeks for 10 days:
- Daily intravenous injection: 40 mg 50% glucose + 100 mg vitamin C
- Oxygen therapy: 2–3 times daily, 15–20 minutes per session
- Vitamin E: 30 mg, 3 times daily
- Phenobarbital: 10–30 mg, 2–3 times daily, starting 2 weeks before delivery, to enhance fetal liver glucuronidation enzyme activity and reduce kernicterus risk.
2. Chinese herbal medicine: Chaihuang decoction (Chaihuang 9g, Da Huang 4.5g, Huangqin 9g, Gancao 6g) is taken daily until delivery.
3. Induction of labor: Consider induction if:
- Antibody titer: Rh >1:32 or ABO >1:512
- History of stillbirth, especially due to hemolytic disease
- Changes in fetal movement or heart rate, indicating unsafe continuation of pregnancy
- Dark yellow amniotic fluid or elevated bilirubin levels
#### (2) Intraoperative Management
- Aim for natural delivery to avoid sedatives or anesthetics, which may increase fetal asphyxia risk.
- Prepare for neonatal resuscitation.
- Clamp the umbilical cord immediately after birth to reduce antibody entry. Retain about 10 cm of the cord for medication or exchange transfusion.
- After birth, inject 10 mL 25% glucose, 100 mg vitamin C, 125 mg nikethamide, and/or 25 mg hydrocortisone via the umbilical vein.
- Send placental-side umbilical cord blood for blood typing, bilirubin, specific antibody testing, and red blood cell, hemoglobin, and nucleated red blood cell counts.
#### (3) Neonatal Management
Focus on three critical periods:
1. First 1–2 days: Monitor jaundice onset, progression, and severity. If severe anemia at birth (hemoglobin <12 g%), consider transfusion.
2. 2–7 days: Prevent kernicterus due to hyperbilirubinemia. Closely monitor indirect bilirubin levels to avoid exceeding the critical threshold (18–20 mg%).
3. First 2 months postpartum: Watch for anemia due to red blood cell regeneration dysfunction and transfuse if necessary.