Hematology

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Forward and reverse typing discrepancy and

crossmatch incompatibility of ABO blood groups:
cause analysis and treatment

Hongmei Qiu, Xuechun Wang & Yan Shao

To cite this article: Hongmei Qiu, Xuechun Wang & Yan Shao (2023) Forward and reverse
typing discrepancy and crossmatch incompatibility of ABO blood groups: cause analysis and
treatment, Hematology, 28:1, 2240146, DOI: 10.1080/16078454.2023.2240146

To link to this article: https://doi.org/10.1080/16078454.2023.2240146

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Published online: 03 Aug 2023.

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HEMATOLOGY
2023, VOL. 28, NO. 1, 2240146
https://doi.org/10.1080/16078454.2023.2240146

Forward and reverse typing discrepancy and crossmatch incompatibility of

ABO blood groups: cause analysis and treatment
Hongmei Qiua#, Xuechun Wanga and Yan Shaob#
a
Department of Blood Transfusion, The First Hospital of Jiaxing, Jiaxing, People’s Republic of China; bDepartment of Laboratory, The
Second People’s Hospital of Pinghu, Pinghu, People’s Republic of China

ABSTRACT ARTICLE HISTORY

Objective: Analyze the reasons for the mismatch between forward and reverse typing of ABO Received 21 March 2023
blood types and the mismatch between cross matching. Accepted 19 July 2023
Methods: When the forward and reverse typing do not match, use physiological saline
KEYWORDS
method, polyamine method, anti human globulin method, and anti screening positive Crossmatch incompatibility;
samples are used for antibody identification. discrepant forward and
Results: The factors contributing to discrepancies in blood typing between forward and reverse blood group typing
reverse typing include weakened serum typing, condensation, monoclonal immunoglobulin of ABO blood groups;
influence, bone marrow transplantation, and blood type subtypes. The causes of cross interfering factors
matching incompatibility include homologous antibody, warm Autoantibody, cold
Autoantibody and daretozumab.
Conclusion: Regular red blood cell homologous antibody screening should be conducted
based on disease type, blood transfusion history, and medication history. Antigen matched
red blood cells should be selected for cross matching, and different experimental methods
should be used for testing to ensure the safety of clinical blood transfusion.

1. Introduction group typing discrepancies at the blood transfusion

department of our hospital and 57 instances
Transfusing blood is a crucial therapeutic and life-saving
of crossmatch incompatibility tested at Jiaxing
procedure in the medical field. However, it is crucial to
Central Blood Station. This study was conducted
confirm the blood group of the recipient before a trans-
in accordance with the Declaration of Helsinki and
fusion is given. Typically, crossmatching of blood is con-
approved by the ethics committee of our hospital.
ducted in cases where time is not of the essence.
Written informed consent was obtained from all
Discrepancies in forward and reverse blood group
participants.
typing and/or crossmatch incompatibility are common
clinical occurrences [1]. In such a scenario, the blood
transfusion (blood bank) staff must investigate the root
2.2 Instruments
causes, run numerous tests, rule out potential interfer-
ences, double-check the accuracy of blood group Centrifuge KA-2200, Japan KUBOTA; Electric thermo-
results, and select compatible blood for transfusion. In static water bath S.H.W 21.600S, Shanghai Yuejin
this study, we conducted a retrospective analysis of 23 Medical Device Co., Ltd.
cases of forward and reverse blood group typing discre-
pancy and 57 cases of crossmatch incompatibility, to
determine the causes and treatments that facilitate 2.3 Reagents
accurate blood group typing and crossmatching, and Monoclonal anti-A/B positive setting reagent
thus, ensure timely and safe clinical blood transfusion. 20210608, ABO negative setting reagent 20215349,
irregular antibody screening reagent 20217056, spec-
trum cell 20211129, acid release, anti-spheroid
2. Data and methods human protein reagent, 2-Me, and anti-A1 were all pur-
chased from Shanghai Blood Biomedicine; polybrene
2.1 Specimen source
reagent A1210803 was purchased from Zhuhai Beso;
Between January 2015 and December 2021, we ana- imported spectrum cells 8000459055 were purchased
lyzed 23 instances of forward and reverse blood from Sanquin.

CONTACT Xuechun Wang [email protected]

#
The authors contributed equally to this study.
© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been
published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
2 H. QIU ET AL.

2.4 Methods both, the diffusion solution and the supernatant

of the last cell wash were positive.
We performed positive and negative blood group
typing, antibody screening, and the selection of
Criteria: ABO positive setting /RhD agglutination
ABO and Rh homologous blood for cross matching
degree should meet 4+, and anti-setting agglutination
prior to the transfusions (Flowchart 1). Forward
degree should not be less than 2+ (except subtype). At
and reverse blood group typing and Rh(D) blood
the same time, the agglutination degree of the Oc tube
group identification method: Microlab STAR IVD
and self-tending should be 0. The standard of cross-
Automatic Blood Analyzer. Irregular antibody
matching blood is that there is no agglutination and
screening was carried out using the normal saline
hemolysis on the primary and secondary sides. The
method, coacylamine method, and anti-human glo-
results of antibody identification were interpreted
bulin method. We followed the manufacturer’s
against the matching ’Red Blood Group Antibody
instructions provided in the package inserts and
Identification Cell Response Pattern Table.’
National Clinical Laboratory Procedures [2] for the
Steps of the polybrene test:
other tests, including antiglobulin test, absorption
test, and diffusion test. Saline tube test, microcol-
(1) Add the serum into the labeled test tube.
umn agglutination, and polybrene were adopted
(2) Add +/- control serum to the quality control tube
for crossmatch tests, and we followed the manufac-
of the +/- control, respectively.
turer’s instructions provided in the package inserts.
(3) Add 3% concentration Rh(D) positive erythrocyte
Antibody subtypes were identified with a heat
suspension to the quality control tube and mix
release test, while isoantibodies and autoantibodies
the test tubes.
were detected with an acid release test. The
(4) Add 500∼1000 μL of low ion solution and mix
flowchart about blood crossmatch incompatibility
again.
test was shown in Figure 1.
(5) Incubate at room temperature for 1 min (or longer).
The procedures of the diffusion test are:
(6) Add 2∼4 drops of polycoagulant reagent to each
test tube.
(1) Cool all reagents to 4°C for testing. (7) Centrifuge for 1 min (3100 r/min).
(2) Washing before diffusion: wash the red blood cells (8) Discard the supernatant, shake the test tube
in the whole blood to remove impurities. gently, and observe whether there is strong
(3) Add 1 unit of hematocrit to 1 unit of citric acid agglutination after the red blood cells are re-
diffusion solution and place in a 13 mm × suspended.
100 mm test tube. (9) Add 1 drop of the re-suspension.
(4) Seal and shake the tube upside down for 90 s. (10) Gently shake the test tube and observe and
(5) Unseal and centrifuge for 45 s. record the results within 1 min.
(6) Remove the supernatant into a clean test tube and
add the neutralizing solution into the diffusion sol- Result analysis:
ution until the pH is adjusted to about 7.0. 1. Step (8) above: Observe whether all test tubes
(7) Re-centrifuge to remove impurities and collect the have strong agglutination.
upper layer of the diffusion liquid.
(1) If there is strong agglutination, it can be used for
Diffusion test analysis: the next test.
(2) If there is low or medium mass agglutination, the next
(1) Antigen-positive cells were subjected to a reaction test can also be carried out, but the test results can
with the solution from step (7) and the super- only be negative or positive, not strong or weak.
natant solution from the final cell wash prior to (3) If there is no agglutination, the test failed; need to
diffusion. be tested again.
(2) The diffusion test was successful and the diffusion
solution could be used if the reaction with the 2. Step (10) above: Gently shake the test tube to
diffusion solution was positive and with the super- fully mix the suspension with the clot. Observe the
natant of the final cell wash was negative. results for 1 min; do not shake at this time.
(3) There was either no antibody in the diffusion sol-
ution or it was a drug-dependent antibody if the (1) Observe the quality control tube – negative and
reaction of both, the diffusion solution and the positive control. The weak positive control should
supernatant of the final cell wash, were negative. have small clots that do not disperse within
(4) The diffusion test failed, and the cells need to be 1 min, while all clots should disperse within
washed and diffused again if the reaction of 1 min in the negative control.
 HEMATOLOGY 3

Figure 1. The flowchart about blood crossmatch incompatibility test. DAT: direct antiglobulin test; IAT: indirect antiglobulin test;
BCEIRRS: blood crossmatch by examining the interaction between the recipient’s RBCs and serum.

(2) Observe the results of all test tubes for 1 min, and autoantibody and warm autoantibody, 18 cases with
determine the agglutination intensity according to a combination of alloantibody and autoantibody, 6
the size of the clot. cases of alloantibody, 6 cases of warm autoantibody,
3 cases of cold autoantibody, and 1 case with a combi-
3. Step (10) above: Directly record the results and nation of daratumumab therapy combined with auto-
end the test. antibody. Among these causes, combinations of cold
autoantibodies and warm autoantibodies accounted
for the largest share (40.35%), while combinations of
3. Results alloantibodies and autoantibodies accounted for the
second-largest share (31.58%) (Table 3).
3.1 Cause analysis for discrepant forward and
reverse blood group typing
Different factors contribute to discrepant forward and 3.3 Specificity test results of blood transfusion
reverse blood group typing, the most common of recipients
which is a serotyping weak response or no response. The most common cause of crossmatch incompatibil-
11 cases in total, including 6 instances of cold aggluti- ity among the 24 blood transfusion recipients was an
nin, 2 instances of monoclonal immunoglobulin, 2 irregular antibody of the Rh blood group system,
instances of bone marrow transplant, and 2 instances with 6 cases of anti-E (25.00%), 5 cases of anti-c, E
of blood group subtype (Table 1). Conditions that (20.83%), 2 cases of anti-C, e (8.33%), 1 case of anti-c
cause weakened serotype included multiple (4.17%). The MNS blood group system accounted for
myeloma (MM), pancytopenia, and leukemia che- the second biggest proportion, with 7 cases of anti-M
motherapy. There was 1 case of cold autoantibody in (29.17%) and 1 case of anti-S (4.17%). The Lewis
a patient with hemolytic anemia; 1 case of acute hepa- blood group accounted for the third highest pro-
titis E among the 6 cases of cold agglutinin (high portion, with 1 case of Lea (29.17%) (Table 4).
potency cold agglutinin); 1 case of abnormal mono-
clonal immunoglobulin secretion due to MM; and 1
case of bone marrow transplant in a patient with 3.4 Blood transfusion recipient disease type
acute leukemia (Table 2).
Types of diseases among the 57 blood transfusion reci-
pients: there were 11 cases of immune disorders,
including immune hemolytic anemia, rheumatoid
3.2 Cause of crossmatch incompatibility
arthritis, and immune thrombocytopenic purpura; 32
Among the 57 cases of crossmatch incompatibility, cases of hematologic diseases, including MM, myelo-
there were 23 cases with a combination of cold dysplastic syndrome (MDS), diffuse large B-cell
4 H. QIU ET AL.

Table 1. The forward and reverse blood group typing in our study.
Forward blood group typing Reverse blood group typing
Anti-A Anti-B Anti-AB Ac Bc Oc Autoantibody Blood type
Serotyping weak response or no response. 0 4+ 4+ 0 0 0 0 B
0 0 0 0 4+ 0 0 O
0 0 0 4+ 0 0 0 O
4+ 0 4+ 0 0 0 0 A
0 4+ 4+ 0 0 0 0 B
0 4+ 4+ ± 0 0 0 B
0 0 0 4+ 0 0 0 O
0 4+ 4+ 0 0 0 0 B
4+ 0 4+ 0 0 0 0 A
4+ 0 4+ 0 0 0 0 A
4+ 0 4+ 0 0 0 0 A
Cold agglutinin 4+ 4+ 4+ 4+ 4+ 4+ 4+ O
0 4+ 4+ 4+ 3+ 3+ 3+ B
4+ 0 4+ 2+ 3+ 2+ 2+ A
0 4+ 4+ 3+ 3+ 3+ 3+ B
4+ 0 4+ 2+ 2+ 2+ 2+ A
0 4+ 4+ 3+ 3+ 3+ 3+ B
MM 0 4+ 4+ 3+ 1+ 1+ 1+ B
4+ 0 4+ 1+ 4+ 1+ 1+/± A
Bone marrow transplant 0 0 0 3+ 0 0 0 Positive O negative B
0 4+ 4+ 0 0 0 0 Positive B negative AB
ABO subtype 1+ 4+ 4+ 0 0 0 0 AB subtype
4+ 1+ 4+ 0 0 0 0 AB subtype
MM, multiple myeloma.
已将部分血型结果0修正为O.

Table 2. Cause analysis and treatment of forward and reverse blood group typing discrepancy.
Number of
Cause cases Disease Method
Serotyping weak response or 11 (47.83%) MM, pancytopenia, leukemia chemotherapy, Placement at 4°C for enhancing antigen-antibody
no response. tumor, etc. reaction, absorption, and diffusion test.
Cold agglutinin 6 (26.09%) Hemolytic anemia, acute hepatitis E, anal Wash red blood cells with normal saline at 37°C, 2-ME
papilla hypertrophy, skin mass inactivation.
M protein 2 (8.70%) MM Saline doubling dilution[3]
Bone marrow transplant 2 (8.70%) Acute lymphoblastic leukemia, acute Learn about medical history, etc.
myelogenous leukemia
ABO blood group 2 (8.70%) Atherosclerosis, acute myelogenous leukemia Add special typing reagent, H substance test and
absorption and diffusion test

tumor, B-cell lymphoma, plasma cell lymphoma, acute occur in clinical practice. Diseases, individual patient
myelogenous leukemia, and aplastic anemia; 4 cases of circumstances, and medication administration are
tumors, including pancreatic cancer, prostate cancer, just some of the causes.
and others; and 10 cases of other diseases, such as cir- Among all other causes of discrepant forward and
rhosis, hepatic failure, tuberculous pleurisy, and others. reverse ABO blood group typing, serotyping weak
response or no response accounted for the largest pro-
portion (6 cases of MM, 1 case of whole blood cell
4 Discussion decrease, 1 case of tumor, 2 cases of leukemia che-
motherapy, and 1 case of ectopic pregnancy) with
Safe blood transfusions depend on consistent positive
reverse blood group typing agglutination < 2 + in all
and negative blood group typing and cross-matching
cases. Herein we provide four treatment options for
of blood. Blood group typing and agglutination, as
the exclusion: enhancing the serum volume for the
measured by major and (or) minor crossmatch, can

Table 3. Cause analysis of 57 cases of crossmatch Table 4. Specificity of irregular antibodies in anti-ethmoid
incompatibility. positive patients with cross-matched blood.
Percentage Antibody specificity Percentage (%)
n (%)
Anti-M antibody 7 29.17
Cold autoantibody combined with warm 23 40.35 Anti-E antibody 6 25.00
autoantibody Anti-c, E antibody 5 20.83
Alloantibody combined with autoantibody 18 31.58 Anti-C, e antibody 2 8.33
Alloantibody 6 10.53 Anti-c antibody 1 4.17
Warm autoantibody 6 10.53 Anti-S antibody 1 4.17
Cold autoantibody 3 5.26 Anti-M, E antibody 1 4.17
Retuzumab combined with autoantibodies 1 1.75 Anti-Lea antibody 1 4.17
Total 57 100.00 Total 24 100
 HEMATOLOGY 5

reverse blood group typing in the tube test (serum: red discrepancy; (2) consistent forward and reverse ABO
blood cell = 3:1); centrifuging the specimen after it is blood group typing with forward blood group typing
stored in a refrigerator at 4°C [4];incubating the speci- response intensity ≤ 3 + or reverse blood group
men for 30–60 min at 4°C and submitting it for absorp- typing response intensity ≤ 2, as seen in the two
tion and diffusion test; and enhancing antigen– cases of subtype AB in this study. Anti-H agglutination
antibody response by adding a low ionic solution [5] intensity is the major criterion for defining ABO sub-
to the specimen to lower its red blood cell surface types. From strong to weak, the H antigen of each
potential. blood group is O>A2>B>A1>A2B>A1B. The antigen
MM, a malignant plasma cell dyscrasia, produces a identification method was determined based on the
considerable quantity of aberrant immunoglobulins, results of the absorption and diffusion test in which
the M-protein. Significant amounts of M-protein can the absorptive capacity of subtypes was lower than
invert the ratio of plasma albumin to globulin [6]. An that of normal red blood cells but the diffusion
excessive increase in plasma globulin and plasma glo- capacity was higher [10]. The absorption and
bulin-coated red blood cells decreases negative diffusion tests are typically used to detect ABO sub-
charges on the surface of red blood cells and repulsion types when there are discrepancies between forward
between blood cells, ultimately leading to aberrant and reverse ABO blood group typing, or when
rouleaux agglutination of red blood cells [7]. Herein forward blood group typing is O. This method,
we provide two treatment options. Option 1: Isotonic however, has a few drawbacks. An additional genetic
saline dilution. Add 1 drop of saline onto a glass test based on molecular biology is recommended if
slide; the abnormal rouleaux agglutination of red possible.
blood cells disappears instantly [8]. Option 2: Saline Serum cold agglutinins are typically IgM, which can
replacement. Replace the clear plasma supernatant agglutinate an individual’s red blood cells or isotype
liquid for reverse blood group typing with 100 μl red blood cells at temperatures below 32 °C, and rever-
normal saline and resuspend it. The agglutination dis- sibly disperse agglutinated red blood cells at 37°C [11].
appears instantly [9]. Cold agglutinins are most effective at a temperature of
Blood groups of bone marrow transplant patients 4°C, where their agglutination abilities are at their
more closely resemble those of donors as a result of highest. In addition, cold antibodies are active at temp-
hematopoietic reconstitution of hematopoietic stem eratures lower than 30°C, suggesting that temperature
cells in recipients [1]. Considering that both donor plays a significant role. However, nonspecific aggluti-
and recipient red blood cells can be found in the per- nation, the reaction product of red blood cell and
ipheral blood, forward blood group typing is a hybrid cold agglutinins, can interfere with forward and
technique. Variations in reverse blood group typing reverse ABO blood group typing due to its role in
are to be expected after a transplant. Using forward mycoplasma pneumoniae infection, autoimmune
and reverse blood group typing, we found that a hemolytic anemia, and other diseases. Treatments:
patient with blood group B could successfully receive Forward and reverse blood group typing should be
a bone marrow transplant from a donor who was performed at 37°C. For forward blood group typing,
typed as blood group O and B in forward and the red blood cells of the patient can be repeatedly
reverse blood group typing, respectively. Our study washed with normal saline at 37°C, and the serum of
also included a patient with blood group AB who the patient can be absorbed and reused multiple
received a bone marrow transplant from a donor times for reverse blood group typing. Forward blood
with blood group B. This donor was typed as B and group typing should be performed after specimens
AB in forward and reverse group typing, respectively. have been incubated for 15 min in a water bath at
Within 4 months of their non-homotypic hematopoie- 37°C and the red blood cells have been treated with
tic stem cell transplants, both patients underwent a 2-Mercaptoethanol (2-ME) to prevent red cell suspen-
transformation that rendered ABO blood group sion preparation failure caused by high potency cold
typing difficult. Patients with forward and reverse autoantibodies [1]. The capacity of cold agglutinin to
blood group typing discrepancies should have their agglutinate corresponds with its potency. Patients
medical records reviewed to learn more about their with low potency requiring a blood transfusion
diseases, and both the patient and donor involved in should have a crossmatch conducted in a 37°C water
the bone marrow transplant should be questioned bath. Crossmatching with absorbed serum and
about their blood types. With this information, it is diffused red blood cells should be performed on
possible to confidently determine the blood group of patients with high potency requiring a transfusion.
the patients and provide them with the correct Patients with high potency should get a gradual
blood transfusion. blood transfusion using an infusion warmer while
ABO subtype, also known as ABO variant, is associ- their vital signs are closely monitored [12].
ated with the following blood group test results: (1) Most warm autoantibodies are IgG, while IgM is
forward and reverse ABO blood group typing uncommon. Warm autoantibodies can obfuscate
6 H. QIU ET AL.

clinically relevant alloantibodies and impede blood occur if this antibody is present, making a compatible
transfusion assays. Life-threatening complications can crossmatch more difficult [20]. The Rh blood group
arise from transfusing the wrong type of blood. system accounts for the vast majority of the reported
Treatments: The presence of alloantibodies in instances of significant irregular antibodies, although
serum can be determined by performing a diffusion the MNS blood group system and the Lewis blood
test on red blood cells with positive direct antiglobulin, group system also play a role. This result is consistent
which exposes the antibody binding site, absorbs with prior findings [21].
autoantibodies in the serum, and prevents autoanti- Treatments: Antibody identification should be per-
bodies from disturbing the red blood cells, allowing formed on positive antibody specimens to determine
for accurate blood group typing and crossmatching the type and clinical significance of the antibody.
[13]. (1) In the event of a transfusion emergency, it is
best to use blood from a donor whose ABO and Rh
types are consistent with those of the recipient’s, 5 Conclusion
whose blood has fewer reactive direct antiglobulins Forward and reverse ABO blood group type, as well as
than the recipient’s blood, and whose blood agglutin- crossmatch incompatibility, may occur for a variety of
ates only slightly with the recipient’s serum. (2) For reasons. The study of blood types and crossmatch
blood transfusions under normal circumstances, it is incompatibility is of utmost importance, and those
best to use blood from a donor whose ABO and Rh working in the field of blood transfusions must have
types are consistent with those of the recipient’s, and a comprehensive awareness of all test techniques.
with isotypic MN, Ss, and kidd. The diagnostic process should not, however, prevent
MM has high levels of CD38 expression. When it or postpone an emergency blood transfusion. If a
comes to treating relapsed, refractory multiple blood transfusion facility does not have the necessary
myeloma, daratumumab (DARA), a monoclonal anti- equipment, it should send blood samples to the
body that targets CD38, is both safe and effective regional blood center so that they may be properly
[14,15]. In this study, we found one case of daratumu- typed. This allows for a more precise crossmatch, and
mab therapy combined with autoantibodies. CD38 is hence a safer and more effective blood transfusion.
widely expressed in human cell membranes and is a
type II transmembrane protein. Indirect antiglobulin
tests (IATs) including those used for screening and Disclosure statement
identifying abnormal antibodies, red blood cell No potential conflict of interest was reported by the author(s).
antigen phenotyping analyses, and crossmatch tests
rely on the ability of DARA to agglutinate with the
reagent or donor red blood cells [16,17]. However, Funding
DARA has no effect on crossmatching between ABO The author(s) reported there is no funding associated with
blood groups, RhD blood groups, or saline medium. the work featured in this article.
Consequently, polybrene can be used for blood speci-
mens, antibody screening tests, and crossmatches for
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