Aberrant Blood Grouping Results in a Patient with Splenomegaly ...

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case study [blood banking/transfusion medicine]
Aberrant Blood Grouping
Results in a Patient
with Splenomegaly
and Thrombocytopenia
Nancy L. Sapanara, MD, 1 Vanlila Swami, MD, 1
Emmanuel C. Besa, MD 2
Departments of 1 Pathology and Laboratory Medicine, and
2 Medicine, Medical College of Pennsylvania Hospital and
Drexel University College of Medicine, Philadelphia, PA
DOI: 10.1309/1637L2KFT6F728ME
Patient
43-year-old male.
Chief Complaint
The patient presented to our outpatient laboratory for
pre-operative blood work prior to splenectomy. He had
persistent thrombocytopenia that was felt to be secondary
to hypersplenism. An appropriately labeled blood
sample was sent and received in the blood bank for type
and cross.
History of Present Illness
The patient had presented 8 months earlier with
anemia, thrombocytopenia, and splenomegaly. He was
diagnosed with kappa light chain-restricted chronic
lymphocytic leukemia (CLL) with high CD38 expression,
Rai stage IV. Complete remission was achieved
after treatment with cyclophosphamide, fludarabine,
and rituximab.
Past Medical History
No significant history other than as above.
Physical Examination
The pertinent positive finding on physical exam was an
enlarged spleen extending to the iliac crest. The patient
was afebrile and without palpable lymphadenopathy.
Principal Laboratory Findings
[T1] and [T2]
Patient’s Forward and Reverse Blood Grouping and Rh Testing Results
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Questions:
1. What is (are) this patient’s most striking laboratory
result(s)?
2. How do you explain this patient’s most striking laboratory
result(s)?
3. What is this patient’s blood group and Rh type?
4. What other tests can be done to confirm this patient’s
blood group and the presence of hypogammaglobulinemia?
5. What are other possible causes of ABO discrepancies?
Possible Answers:
1. The discrepancy between forward (group O) and reverse
(group AB) ABO blood typing results [T1];
decreased globulins; mildly increased WBC count;
markedly decreased platelet count; and modestly
increased blood urea nitrogen (BUN) concentration
[T2]. The failure of expected agglutination reactions to
occur during forward and reverse typing is called an
Forward Grouping Rh Grouping Reverse Grouping
Reaction Conditions Anti-A Anti-B Anti-D D u Rh control A 1 Cells B Cells
Immediate spin 0 0 0 0 0 0 0
Incubation 5 min, RT 0 0
Incubation 10 min, 3°C 0 0
Incubation 60 min, RT vw+ vw+
RT, room temperature; 0, no reaction; vw+, very weakly positive
Principal Laboratory Findings
Test Patient’s Result “Normal”
Reference Range
Hematology
WBC count 12.1 4.0-10.8 x10 3 /µL
Hemoglobin 14.3 14.0-18.0 g/dL
Hematocrit 42.9 42.0-52.0%
MCV 94.8 80-94 fL
Platelet count 33 150-450 x10 3 /µL
Differential
Neutrophils 69 42-75%
Bands 30 0-5%
Monocytes 1 5-10%
Chemistry
Sodium 139 133-145 mEq/L
Potassium 4.2 3.3-5.1 mEq/L
Chloride 102 96-108 mEq/L
Bicarbonate 23 22-29 mmol/L
BUN 20 6-19 mg/dL
Creatinine 0.9 0.5-1.2 mg/dL
Glucose 90 70-110 mg/dL
Total protein 6.1 5.9-8.4 g/dL
Albumin 4.7 3.2-5.2 g/dL
Globulins * 1.4 2.5-3.5 g/dL
Blood Bank
Antibody screen Neg Neg
Direct coombs test Neg Neg
* Calculated from total protein concentration – albumin concentration
WBC, white blood cell; MCV, mean cell volume; BUN, blood urea nitrogen
©
T2
T1

“ABO discrepancy.” Discrepant forward and reverse ABO
grouping results should be brought to the attention of the
blood bank director and reviewed in tandem with the patient’s
clinical history, which in this case, indicated the
diagnosis, chronic lymphocytic leukemia (CLL). To enhance
the reverse group reaction, the tubes were incubated
at room temperature for 5 minutes, and at 3ºC for 10 minutes.
The results in all 4 tubes were still negative [T1]. Repeat
testing provided the same results. While not standard
procedure, the reverse group typing was repeated after a
60 minute incubation at room temperature, and the results
were very weakly positive for both A 1 and B reagent
RBCs [T1].
2. Hypogammaglobulinemia is known to occur in patients
with CLL as a result of both leukemia-related immune
dysfunction and therapy-related immunosuppression. The
deficiency in humoral immunity is largely responsible for
CLL patients’ increased risk of infection-related morbidity
and mortality, especially by encapsulated microorganisms. 1
The overall decrease in antibody production that occurs
during immunosuppression also causes a decrease in ABO
antibody levels. Weak, or missing, ABO antibodies due to
CLL-related hypogammaglobulinemia are the most logical
explanation for the failure of the patient’s serum to react
against the A 1 and B cells at immediate spin. Moreover,
hypogammaglobulinemia following rituximab therapy has
been reported previously. 2-4 In each of these reports, however,
the disease being treated was post-transplant lymphoproliferative
disorder not CLL. Nevertheless, it is
possible that rituximab therapy contributed to the development
of hypogammaglobulinemia in the CLL patient. The
decreased globulin value may also be a manifestation of
the patient’s hypogammaglobulinemia. An elevated WBC
count may be related to a variety of conditions, including
infection, stress (physical or emotional), and hematologic
disorders. Thrombocytopenia may result from decreased
production or increased consumption of platelets caused
by immune- and nonimmune-mediated mechanisms. In
this patient with marked splenomegaly, increased platelet
sequestration by the spleen is the most likely cause of
thrombocytopenia. Changes in intravascular volume due
to hypogammaglobulinemia is the most likely cause of
this patient’s slightly increased blood urea nitrogen (BUN)
concentration.
3. Group O, Rh-negative.
4. To confirm the patient’s blood type, additional cell typing
can be done to rule out the presence of an inhibitory
substance in the patient’s blood that could cause all reactions
to appear negative. When we tested the patient’s
RBCs using anti-C, anti-E, anti-c, and anti-e antisera, his
RBCs were found to have both c and e antigens, thus ruling
out the presence of a global inhibitor in his blood
[T3]. The patient’s Rh phenotype is rr, the most common
phenotype found in Rh-negative white (Caucasian) blood
©
donors. 5 To confirm and characterize this patient’s
hypogammaglobulinemia, serum protein electrophoresis
and/or quantification of serum levels of the 3 principal
serum immunoglobulins, IgA, IgG, and IgM, could be
performed. Quantification of these immunoglobulins in
our patient’s serum indicated a marked decrease in IgA
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C E c e
Patient’s RBCs 0 0 4+ 3+
Positive control 3+ 2+ 4+ 2+
Negative control 0 0 0 0
0, no reaction
T3
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Possible Causes of Aberrant Blood Group Typing Reactions*
Factors Affecting Typing Reaction
Type of Forward Reverse
Aberrant
Result
Positive Acquired B antigen in patients with:
gastric or colon cancers
intestinal obstructions
other conditions (eg, inflammatory bowel disease; UTI with
E. coli O86; pancreatic cancer, primary and metastatic)
Rouleaux formation due to:
elevated globulin
elevated fibrinogen
Wharton’s jelly
dextran/plasma expanders
Polyagglutination:
microbial-associated (ie, T polyagglutination)
nonmicrobial-associated (ie, Tn activation)
Autoagglutination due to:
potent cold autoantibodies
Antibody-coated RBCs due to:
warm autoantibodies
transfusion reaction
Acriflavin antibodies
Negative Weak A or B subgroups causing weak or mixed field
agglutination reactions or no reaction
Excess blood group-specific soluble substances in blood
samples from patients’ with:
gastric cancer
pancreatic cancer
mucinous ovarian cysts
Antigen depression due to disease
*Excluding technical and human error. UTI, urinary tract infection
repeating the test on the same sample suspended in saline
may resolve the discrepancy by washing away the excess
BGSS. 7 Excess BGSS has been associated with carcinoma
of the stomach and pancreas. 7 There is, however, at least 1
case where excess BGSS was identified in the serum, cyst
fluid, and ascites of a woman with mucinous cystadenoma
of the ovary. 8 After surgical resection of the cyst, the
serum BGSS returned to a normal level. Weak antigens
are less common than weak or missing antibodies, but
when encountered, they too may cause aberrant typing
reactions. Weakly reacting antigens that may be present
include subgroups of A and/or B. Group A is subdivided
into A 1 and A 2 phenotypes, which together represent approximately
99% of group A individuals, and several rare
A phenotypes, A 3 , A x , A end , A m , A y , and A el , which comprise
the remaining 1%. The rare A subgroups may yield
negative, weak, or mixed-field agglutination reactions
upon testing with anti-A, putting the patient at risk of
being mistyped as group O. Similarly, B subgroups B 3 ,
B x , B m , and B el demonstrate variable reactivity to anti-B.
The presence of unexpected antibodies may also produce
confusing results. For example, anti-A 1 may be present in
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Unexpected antibody present in blood grouping reagent that
may react with an unknown, unexpected patient antigen
Genetic chimerism (cis-AB) (rare)
Unexpected ABO antibody in patient’s serum such as:
anti-A1 in A subgroups (including A2B)
anti-B in cis-AB individuals
anti-H in A1 and A1B individuals
Rouleaux formation due to:
elevated globulin
elevated fibrinogen
Wharton’s jelly
dextran/plasma expanders
Unexpected alloantibody in the patient’s serum that may
react with an unknown, unexpected reagent RBC
antigen
Cold reactive allo- or auto-antibodies that may agglutinate
reagent red blood cells
Weak or missing antibodies in patients with:
hypogammaglobulinemia
newborn or elderly
immunodeficiency
immunosuppression
congenital deficiency (agammaglobulinemia)
Genetic chimerism (cis-AB) (rare)
up to 8% of blood group A 2 individuals and in 22% to
35% of blood group A 2 B individuals, 6 causing a positive
reaction to A 1 cells during reverse typing. This discrepancy
may be resolved by repeating the forward typing
using anti-A 1 lectin reagent instead of the usual anti-A
reagent. Group A 1 cells will react with both anti-A 1 lectin
and anti-A, but group A 2 cells will only react with anti-A.
A negative reaction with anti-A 1 will confirm the suspicion
that one is most likely working with group A 2 cells.
In addition to blood group A 2 , some of the rare A
subgroups can also produce anti-A 1 . If a positive reaction
to A 1 cells is observed on reverse typing and this finding
is coupled with an absent or weak reaction to anti-A on
forward typing, a patient will erroneously appear to be
group O. Finally, an unusual case has been reported in
which a transfusion-naïve blood group A 1 individual produced
anti-A 1 autoantibodies. 9 Acquired B antigens are
another important, although uncommon, cause of ABO
discrepancies. The B antigen is acquired as a result of
bacterial enzymatic activity that modifies N-acetylgalactosamine,
the immunodominant sugar on A red blood
cells, to D-galactosamine. D-galactosamine is similar
©
T4

enough to D-galactose, the immunodominant sugar of B
red blood cells, to cause reactivity with most anti-B
reagents, but not with autologous anti-B. Acquired B antigens
are most commonly seen in conditions where the
breakdown of normal bowel mucosa allows bacterial enzymes
to enter the systemic circulation. The underlying
disease state is often gastric cancer, 10 colon cancer, 11 or
intestinal obstruction, 7 but the acquired B phenomenon
has been reported in patients with inflammatory bowel
disease, 12 urinary tract infection with E. coli O86, 13 and
pancreatic cancer 14 as well. ABO typing reactions may be
misinterpreted as positive in the presence of rouleaux formation
or polyagglutination. Rouleaux may be attributable
to conditions in which there are elevated levels of
globulin, such as multiple myeloma, elevated levels of fibrinogen,
or in the presence of dextran, other plasma expanders,
or Wharton’s jelly. 7 In case of the latter,
repeating the tests on the same sample after washing the
patient’s RBCs with saline may resolve the discrepancy by
removing the Wharton’s jelly. Polyagglutination is
caused by an alteration in the RBC membrane that enables
agglutination to occur in the presence of most ABO-compatible
sera. Alteration of the RBC membrane may occur
by microbial- or nonmicrobial-associated methods. In either
case, normal carbohydrate residues are removed from
the RBC membrane causing exposure of what are called
hidden, or “cryptantigens.” Cryptantigens then react to
naturally occurring IgM antibodies in the patients’ serum
and cause agglutination. 15 Cold autoantibodies may be
potent enough to cause agglutination of the patient’s
RBCs used in forward grouping, as well as the reagent
cells used in reverse grouping. To resolve the forward
grouping problem, the patient’s RBCs could be incubated
at 37ºC, then washed with saline that has also been
warmed to 37ºC. If warming methods are unsuccessful,
dithiothreitol (DTT) can be used to remove IgM-related
agglutination. 6 For reverse-grouping discrepancies, the
reagent RBCs may need to be warmed to 37ºC before
mixing with the patient’s serum. If IgM-related agglutination
still occurs, adsorption may be required to remove
IgM antibody from the serum. Warm autoantibodies may
also cause ABO discrepancies; however, they typically
yield weaker reactions than those observed with cold antibodies.
When warm autoantibodies are suspected to be the
cause of false-positive results in forward grouping, elution
may be required to remove bound immunoglobulin from
the RBCs. Last, RBCs may become coated with warm antibodies
after a transfusion reaction, secondary to the use
of certain drugs such as alpha-methyldopa, or in patients
with warm autoimmune hemolytic anemia. 7
©
Patient Outcome
Given our patient’s profound hypogammaglobulinemia,
splenectomy was postponed so that he could first receive
replacement doses of intravenous immunoglobulin (IvIg).
Later, he underwent splenectomy with removal of a 1,550gram
spleen, without complication. It showed no
evidence of CLL by histologic examination or flow
cytometry. The patient’s platelet count recovered;
however, he remained hypogammaglobulinemic.
Keywords: ABO discrepancy, hypogammaglobulinemia,
chronic lymphocytic leukemia, forward grouping, reverse
grouping
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