6888_Ch05_103-118 29/10/18 4:46 PM Page 116

116 PART II Blood Groups and Serologic Testing

the medical surgical floor. Because you are confident you is O-positive, and the blood bank technologist performed
can finish before your shift ends, you decide to proceed an antibody screen using the patient serum and a two-cell
with testing. The patient types as an A-positive. The anti- screen using the solid-phase automated platform. One of
body screen results were negative in all phases of testing. the cells in the antibody screen is weakly reactive (1+),
Therefore, check cells were added to all tubes and the so you proceed with an antibody identification using a
reactions after centrifuging were also negative. panel. However, only two cells are weakly reactive (1+)
and show no pattern of reactivity. A second panel is run
1. Can the antibody screen be interpreted as negative?
and yields similar results. You decide to run an antibody
2. What steps must be taken to resolve the problem?
identification panel using the conventional tube testing
3. What is the most likely cause for the discrepant
method with LISS as your enhancement medium. All
results?
reactions are negative using LISS.
4. What are other causes for the results?
1. Can the antibody screen be interpreted as negative?
Case 5-2 2. Provide an explanation for the observed results.
3. Are there additional tests that should be conducted to
A 44-year-old white female is admitted for an exploratory
complete testing on this patient? If so, which tests
laparotomy. A type and screen is ordered prior to her
should be run?
scheduled surgery. ABO and Rh typing reveal the patient

SUMMARY CHART
 The antiglobulin test is used to detect RBCs sensitized  The IAT detects in vitro sensitization of RBCs and can
by IgG alloantibodies, IgG autoantibodies, and com- be applied to compatibility testing, antibody screen,
plement components. antibody identification, RBC phenotyping, and titra-
 AHG reagents containing anti-IgG are needed for the tion studies.
detection of IgG antibodies because the IgG monomeric  A positive DAT is followed by a DAT panel using
structure is too small to directly agglutinate sensitized monospecific anti-IgG and anti-C3d to determine the
RBCs. specific type of protein sensitizing the RBC.
 Polyspecific AHG sera contain antibodies to human  EDTA should be used to collect blood samples for the
IgG and the C3d component of human complement. DAT to avoid in vitro complement attachment associ-
 Monospecific AHG sera contain only one antibody speci- ated with refrigerated, clotted specimens.
ficity: either anti-IgG or antibody to anti–C3b–C3d.  There are multiple sources of error that can be intro-
 Classic AHG sera (polyclonal) are prepared by inject- duced into the AHG procedure.
ing human globulins into rabbits, and an immune  LISS, PEG, polybrene, and albumin can all be used as
stimulus triggers production of antibody to human enhancement media for AHG testing, with each having
serum. its own advantages and disadvantages.
 Hybridoma technology is used to produce monoclonal  Conventional tube testing, gel technology, enzyme-
antiglobulin serum. linked technology, and solid-phase testing are available
 The DAT detects in vivo sensitization of RBCs with methods to use in AHG testing.
IgG or complement components. Clinical conditions  Method-dependent antibodies do exist and should be
that can result in a positive DAT include HDFN, HTR, evaluated on a case-by-case basis.
and AIHA.

2. Polyspecific AHG reagent contains:

Review Questions
a. Anti-IgG and anti-IgA.
1. A description of the antiglobulin test is: b. Anti-IgG and anti-IgM.
c. Anti-IgG and anti-C3d.
a. IgG and C3d are required for RBC sensitization.
d. Anti-IgA and Anti-C3d.
b. Human globulin is completely eluted from RBCs
during saline washings. 3. Monoclonal anti-C3d is:
c. Human globulin is injected into an animal. a. Derived from one clone of plasma cells.
d. AHG reacts with human globulin molecules bound to b. Derived from multiple clones of plasma cells.
RBCs. c. Derived from immunization of rabbits.
d. Reactive with C3b and C3d.
6888_Ch05_103-118 29/10/18 4:46 PM Page 117

Chapter 5 The Antiglobulin Test 117

4. Which of the following is a clinically significant 11. A positive DAT may be found in which of the following
antibody whose detection has been reported in some situations?
instances to be dependent on anticomplement activ- a. A weak D-positive patient
ity in polyspecific AHG? b. A patient with anti-M
a. Anti-Jka c. HDFN
b. Anti-Lea d. An incompatible crossmatch
c. Anti-P1
12. What do Coombs’ check cells consist of?
d. Anti-H
a. Type A-positive cells coated with anti-IgG
5. After the addition of IgG-coated RBCs (check cells) b. Type A-negative cells coated with anti-IgG
to a negative AHG reaction during an antibody c. Type O-positive cells coated with anti-D
screen, a negative result is observed. Which of the d. Type B-negative cells coated with anti-D
following is a correct interpretation based on these
findings? 13. Which of the following IAT methods requires the use of
check cells?
a. The antibody screen is negative.
b. The antibody screen cannot be interpreted. a. Manual tube method with albumin
c. The saline washings were adequate. b. Gel
d. AHG reagent was added. c. Automated solid-phase analyzer
d. Enzyme-linked
6. RBCs must be washed in saline at least three times
before the addition of AHG reagent to: 14. Which uncontrollable factor can affect AHG testing?
a. Wash away any hemolyzed cells. a. Temperature
b. Remove traces of free serum globulins. b. Antibody affinity
c. Neutralize any excess AHG reagent. c. Gravitational force in the centrifuge
d. Increase the antibody binding to antigen. d. Incubation time

7. An in vivo phenomenon associated with a positive 15. Which would be the most efficient method for a labora-
DAT is: tory staffed by medical laboratory technicians?
a. Passive anti-D detected in the maternal sample. a. LISS
b. Positive antibody screen tested by LISS. b. Polybrene
c. Identification of alloantibody specificity using a c. Solid-phase or gel
panel of reagent RBCs. d. Enzyme-linked
d. Maternal antibody coating fetal RBCs. 16. A 27-year-old group O mother has just given birth to a
8. False-positive DAT results are most often associated group A baby. Since the mother has IgG anti-A, anti-B
with: and anti-A, B in her plasma, which of the following
methods and tests would be most effective at detecting
a. Use of refrigerated, clotted blood samples in which
the anti-A on the baby’s RBCs?
complement components coat RBCs in vitro.
b. A recipient of a recent transfusion manifesting an a. DAT using common tube technique
immune response to recently transfused RBCs. b. DAT using gel
c. Presence of antispecies antibodies from adminis- c. IAT using common tube technique
tration of immune globulin (IVIG). d. IAT using gel
d. A positive autocontrol caused by polyagglutination.
References
9. Polyethylene glycol (PEG) enhances antigen-
1. Coombs RRA, et al. A new test for the detection of weak and
antibody reactions by: “incomplete” Rh agglutinins. Br J Exp Pathol. 1945;26:255.
a. Decreasing zeta potential. 2. Coombs RRA, et al. In-vivo isosensitisation of red cells in babies
b. Concentrating antibody by removing water. with haemolytic disease. Lancet. 1946;i:264.
c. Increasing antibody affinity for antigen. 3. McCullough J. Transfusion medicine., 4th ed. Wiley-Blackwell
2016.
d. Increasing antibody specificity for antigen. 4. Moreschi C. Neue Tatsachen über die Blutkorperchen Aggluti-
10. Solid-phase antibody screening is based on: nationen. Zentralbl Bakteriol. 1908;46:49.
5. Coombs RRA, Mourant RA. On certain properties of antisera
a. Adherence. prepared against human serum and its various protein frac-
b. Agglutination. tions; their use in the detection of sensitisation of human red
c. Hemolysis. cells with incomplete Rh antibody, and on the nature of this
d. Precipitation. antibody. J Pathol Bacteriol. 1947;59(1-2):105-11.
6. Dacie JF. Differences in the behaviour of sensitized red cells to
agglutination by antiglobulin sera. Lancet. 1951;ii:954.
7. Dacie JV, et al. “Incomplete” cold antibodies: role of complement
in sensitization to antiglobulin serum by potentially haemolytic
antibodies. Br J Haematol. 1957;3:77.