CORD BLOOD EVALUATION

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1 CORD BLOOD EVALUATION Principle: When there is incompatibility between a mother s antibodies and an infant s red blood cell antigens, the infant is at risk of developing Hemolytic Disease of the Fetus and Newborn (HDFN). Maternal antibodies coating the infant s red blood cells (RBCs) cause the premature destruction of the coated RBCs. If RBC production does not keep pace with RBC destruction, the infant will become anemic and may suffer cardiac failure and death. Additionally, the accumulation of unconjugated bilirubin in the infant s circulation may lead to kernicterus an accumulation of bilirubin in the fatty tissues of the brain, which causes irreversible brain damage. Those infants at risk of developing HDFN include Group A or B infants born to Group O mothers; Rh Positive infants born to Rh Negative mothers; and infants born to mothers with clinically significant alloantibodies. Purpose: This procedure details the testing to be performed on infants suspected of having HDFN. Scope: This procedure may be performed by all trained Transfusion Service personnel. Specimen: The specimen of choice is a 2.5 ml EDTA specimen collected from the umbilical cord at the time of delivery. Alternatively, 2 ml of venous or capillary blood collected in EDTA may be submitted for testing. The specimen shall be labeled with the following information: 1. Infant s last name, Baby and gender. (e.g. Smith, Baby Girl) 2. Infant s Medical Record Number 3. Collection date and time 4. Identity of person collecting specimen 5. Designation of Cord Blood, if applicable 6. Mother s last name, first name (e.g. Smith, Susan) 7. Mother s Medical Record Number 8. Mother s ABO and Rh type, if known Materials: 12 x 75 test tubes Anti-A Test tube racks Anti-B 0.9% Saline Anti-D Dispo-pipettes Anti-AB Biohazardous waste container Anti-IgG AHG Timer 6% BSA Coombs Control cells CLS 422 Clinical Immunohematology I Page 1 of 5

If RBC production does not keep pace with RBC destruction, the infant will become anemic and may suffer cardiac failure and death.

2 Equipment: Serofuge (set at 3400 rpm) Cell washer Agglutination viewer 37 o C heat block Procedure: 1. Mix sample well. Remove an aliquot of red blood cells (RBCs) and place in test tube labeled with infant s last name and gender (e.g. Smith Girl). 2. Add saline to tube (2/3 ¾ full). Centrifuge for 60 seconds. Decant saline. Resuspend RBCs in residual saline. Repeat wash at least once, for a total of at least 2 washes. 3. Resuspend washed RBCs with saline to a 2-5% suspension. 4. Perform ABO forward grouping on infant s RBCs. a. Label 3 test tubes with patient identification and: Anti-A, Anti-B and anti- AB b. Place one drop of the appropriate reagent in each tube. c. Place one drop of the RBC suspension in each tube. d. Mix and centrifuge for 15 seconds. e. Examine tubes for agglutination. f. Grade reactions and interpret results. Record on worksheet. 5. Perform Rh typing on the infant s RBCs. a. Label a test tube with patient identification and Anti-D. b. Place one drop of anti-d in the tube. c. Place one drop of the RBC suspension in the tube. d. Mix and centrifuge for 15 seconds. e. Examine tubes for agglutination. f. Grade reactions and interpret results. Record on worksheet. g. If the infant types as Rh Negative, and the mother is also Rh Negative (or Rh type is unknown), a weak D test must be performed using the infant s RBCs. If the infant is Rh Negative, but the mother is Rh Positive, no weak D test is required. To perform the weak D test: 1) Following examination of the immediate spin phase, incubate tube with anti-d and infant s RBCs at 37 o C for minutes in heat block. 2) Following incubation, wash RBCs 3-4 times with saline to remove anti-d. Blot tube after final wash to create a dry cell button. 3) Add 2 drops anti-igg AHG to tube. CLS 422 Clinical Immunohematology I Page 2 of 5

Decant saline. Resuspend RBCs in residual saline. Repeat wash at least once, for a total of at least 2 washes. 3. Resuspend washed RBCs with saline to a 2-5% suspension. 4.

3 4) Mix and centrifuge for 15 seconds. 5) Examine for agglutination macro- and microscopically. 6) If no agglutination is detected, add 1 drop of Coombs Control cells to tube. 7) Mix and centrifuge for 15 seconds. 8) Examine tube for agglutination. 9) Grade reactions and interpret results. Record on worksheet. 6. Perform a Direct Antiglobulin Test (DAT) using the infant s RBCs. Reporting Results: a. Label 2 test tubes with patient identification and: Anti-IgG and BSA. b. Place one drop of the RBC suspension in each tube. c. Wash RBCs 3-4 times with saline to remove unbound globulins. Blot tubes after final wash to create a dry cell button. d. Place two drops of the appropriate reagent in each tube. e. Mix and centrifuge for 15 seconds. f. Examine for agglutination macro- and microscopically. g. If no agglutination is detected in the anti-igg tube, add 1 drop of Coombs Control cells to tube. 1) Mix and centrifuge for 15 seconds. 2) Examine tube for agglutination. Agglutination must be observed in order for DAT test results to be valid. h. Grade reactions and interpret results. Record on worksheet. 1. Agglutination indicates antigen and antibody have reacted and is considered a positive result. 2. No agglutination indicates that antigen and antibody did not react and is a negative result. 3. Negative results at the AHG phase must be confirmed using Coombs Control cells. 4. If the BSA control is positive, no interpretation can be made from a positive DAT or positive weak D test. Further investigation is required to determine the source of the agglutination, before reporting results. Consult supervisor for further instructions. 5. All results must be recorded on the appropriate worksheet and in the laboratory computer system. CLS 422 Clinical Immunohematology I Page 3 of 5

Perform a Direct Antiglobulin Test (DAT) using the infant s RBCs. Reporting Results: a. Label 2 test tubes with patient identification and: Anti-IgG and BSA. b.

4 Procedural Notes: 1. ABO reverse grouping is not performed on neonates. Infants do not produce antibodies to antigens of the ABO system until approximately 4 months of age. Confirm results of anti-a and anti-b reagents with anti-ab. 2. Results of a positive weak D test are invalid if the infant has a positive DAT. For purposes of treating the mother with Rh Immune Globulin (RhIG), consider the infant Rh Positive and treat the mother with RhIG. For purposes of transfusing the infant, consider the infant Rh Negative. 3. If the DAT is positive and the BSA control is negative, IgG antibody is coating the infant s RBCs. Perform an elution on the infant s RBCs and identify the antibody. See eluate procedure. 4. If an Rh Positive infant is born to an Rh Negative mother, notify the mother s care giver, and request that an order be placed for an Rh Immune Globulin Investigation on the mother. References: Manufacturers package inserts Roback, J., MD PhD, ed. Technical Manual, 16 th ed., Bethesda, MD: AABB, Author: Greta Tech, MT(ASCP) Date: 8/1/2008 Reviewed and approved by: Bud Bank, MD Date: 8/10/2008 Implemented: 8/18/2008 CLS 422 Clinical Immunohematology I Page 4 of 5

For purposes of treating the mother with Rh Immune Globulin (RhIG), consider the infant Rh Positive and treat the mother with RhIG.

5 After reading the cord blood evaluation procedure, watch the video of a cord blood being tested according to this procedure. The tech does not exactly perform the testing as instructed! There are at least 10 mistakes. Can you identify them? When you are finished, check your answers against the key in BlackBoard. CLS 422 Clinical Immunohematology I Page 5 of 5

There are at least 10 mistakes. Can you identify them? 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. When you are finished, check your answers against the key in BlackBoard.

Direct Antiglobulin Test (DAT)

Exercise 8 Direct Antiglobulin Test (DAT) Objectives: 1. State the purpose for performing the DAT. 2. State what a positive DAT indicates. 3. List the reagents which are used for performing the DAT. 4.