Questions for Research
1. Give the purpose of incubation in the saline tube method
For cells suspended in saline, incubation times may vary between 30 and 120 minutes. Most clinically
significant antibodies can be detected after 30 minutes of incubation, and extended incubation times are
usually not necessary. If a LISS or PEG technique is being used, incubation times may be shortened to 10
to 15 minutes. With these shortened times, it is essential that tubes be incubated at a temperature of 37°C.
Extended incubation (i.e., up to 40 minutes) in the LISS technique has been shown to cause antibody to
elute from the RBCs, decreasing the sensitivity of the test.
2. Describe and give the procedure for
a. Stick test - determine the blood type in a fastest and easier manner.
PROCEDURE:
1. Heat the water bath for 40°C-45°C
2. Put the slide for 15 minutes
3. Perform venipuncture
4. Drop blood on the prewarmed slide
5. Add anti-D on test and RAM on control
6. Mix the blood in two different applicator sticks
7. Observe agglutination
b. Modified test tube - determine the presence of variance of D of the blood samples.
PROCEDURE:
1. Prepare 2% of red cell suspension
2. Add one drop of washed RBCs into the tube.
3. Mix cells and antiserum
4. Incubate the tube at 37C heat block for 5 to 10 minutes
5. Centrifuge the tube for 1 minute, making sure to balance the tube with a blank tube.
6. Observe agglutination
3. Describe HDN in detail
Hemolytic Disease of the Newborn (HDN), also known as erythroblastosis fetalis, isoimmunization, or
blood group incompatibility, occurs when fetal red blood cells (RBCs), which possess an antigen that the
mother lacks, cross the placenta into the maternal circulation, where they stimulate antibody production.
The antibodies return to the fetal circulation and result in RBC destruction.
DIFFERENTIAL DIAGNOSIS OF HEMOLYTIC ANEMIA IN A NEWBORN INFANT:
-Isoimmunization
-RBC enzyme disorders (e.g., G6PD, pyruvate kinase deficiency)
-Hemoglobin synthesis disorders (e.g., alpha-thalassemia’s)
-RBC membrane abnormalities (e.g., hereditary spherocytosis, elliptocytosis)
-Hemangiomas (Kasabach Merritt syndrome)
-Acquired conditions, such as sepsis, infections with TORCH or Parvovirus B19 (anemia due to RBC
aplasia) and hemolysis secondary to drugs.
ISOIMMUNIZATION
A. Rh disease (Rh = Rhesus factor)
(1) Genetics: Rh positive (+) denotes presence of D antigen. The number of antigenic sites on RBCs
varies with genotype. Prevalence of genotype varies with the population. Rh negative (d/d) individuals
comprise 15% of Caucasians, 5.5% of African Americans, and <1% of Asians. A sensitized Rh-negative
mother produces anti-Rh IgG antibodies that cross the placenta. Risk factors for antibody production
include 2nd (or later) pregnancies, maternal toxemia, paternal zygosity (D/D rather than D/d),
fetomaternal compatibility in ABO system and antigen load.
(2) Clinical presentation of HDN varies from mild jaundice and anemia to hydrops fetalis (with ascites,
pleural and pericardial effusions). Because the placenta clears bilirubin, the chief risk to the fetus is
anemia. Extramedullary hematopoiesis (due to anemia) results in hepatosplenomegaly. Risks during labor
and delivery include asphyxia and splenic rupture.
Postnatal problems include:
Asphyxia Pulmonary hypertension Jaundice Kernicterus (from
Pallor (due to anemia) Edema hyperbilirubinemia)
(hydrops, due to low serum albumin) Hypoglycemia (due to
Respiratory distress Coagulopathies hyperinsulinemia from islet cell
(↓ platelets & clotting factors) hyperplasia)
(3) Laboratory Findings vary with severity of HDN and include:
Anemia Hyperbilirubinemia + Direct Antiglobulin Test
Reticulocytosis (6 to 40%) Hypoalbuminemia
↑ nucleated RBC count (>10/100 Rh negative blood type
WBCs) Smear: polychromasia, anisocytosis,
Thrombocytopenia Leucopenia no spherocytes
*HDN can occur in 1st pregnancy, but this is uncommon.
*Cord blood bilirubin >4 mg/dL indicates severe isoimmunization.
With severe HDN, high quantities of antibody may block Rh antigen site resulting in a Rh+ infant
typing as Rh- and having a negative Direct Antiglobulin test.
B. Minor Blood Group Incompatibility is uncommon, occurs in ~0.8% of pregnant women and usually
with E, c, Kell, Kidd or Duffy. Clinical presentation is like Rh disease. Anti-Kell disease may be severe
due to hemolysis or erythroid suppression. Lewis antigen stimulates only IgM production, so maternal
antibody screen may be positive, but fetus is not affected.
C. ABO Incompatibility
(1) Genetics: With maternal blood types A and B, isoimmunization does not occur because the naturally
occurring antibodies (anti-A and -B) are IgM, not IgG. In type O mothers, the antibodies are
predominantly IgG, cross the placenta and can cause hemolysis in the fetus. The association of a type A
or B fetus with a type O mother occurs in ~15% of pregnancies. However, HDN occurs in only 3%, is
severe in only 1%, and <1:1,000 require exchange transfusion. The disease is more common and more
severe in African American infants. Unlike Rh, ABO disease can occur in first pregnancies, because anti-
A and anti-B antibodies are found early in life from exposure to A- or B-like antigens present in many
foods and bacteria.
(2) Clinical presentation: generally, less severe than with Rh disease.
(3) Laboratory findings that differ from Rh disease:
Smear: micro spherocytosis
MCV <95, microcytic for a newborn (normal for adult)
Direct Coombs test is often weakly +.
REFERENCES:
Denise M. Harmening, PhD, MT(ASCP) Modern Blood Banking and Transfusion Practices 6th Edition
John W. Ridley (2011) Essentials OF Clinical Laboratory Science
https://www.ucsfbenioffchildrens.org/pdf/manuals/42_Hemol.pdf
