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Report on Unexpected Antibodies Detected Exclusively in the Serum
혈청에서만 동정되는 비예기항체의 보고
Korean J Blood Transfus 2023;34:211−213
Published online December 31, 2023;
© 2023 The Korean Society of Blood Transfusion.

Bo Kyeung Jung, M.D., Yoo Na Chung, M.D., Joowon Park, M.D.

Department of Laboratory Medicine, Dankook University Hospital, Dankook University College of Medicine, Cheonan, Korea
단국대학교병원 진단검사의학과
Joowon Park, M.D.
Department of Laboratory Medicine, Dankook University Hospital, Dankook University College of Medicine, 201 Manghyang-ro, Dongnam-gu, Cheonan 31116, Korea
Tel: 82-41-550-3097, Fax: 82-41-550-6662, E-mail:, ORCID:
Received August 14, 2023; Revised September 1, 2023; Accepted September 4, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In 2023, an 81-year-old woman requested a preliminary red blood cell transfusion during pancreatic cancer surgery, and antibody screening was performed using automated blood typing equipment. The antibody screening test results were negative when plasma was used as the sample but positive when the serum was used instead. The 4℃ test was positive when using the enzyme method but negative at warm temperatures. An unknown antibody was identified using a commercial antibody identification test (Bio-Rad, CA, USA). A clinically insignificant cold antibody was detected only in the serum samples, and the negative blood was ultimately released following the third stage of cross-matching using anti-human globulin.
Keywords : Cold antibody, Antibody screening test, Cross-matching

An 81-year-old woman with a B+ blood type required a blood transfusion for cancer surgery to treat a malignant neoplasm of the ampulla of Vater. In March 2023, a blood transfusion test was performed at the blood bank of the Department of Laboratory Medicine at Dankook University Hospital (Cheonan, South Korea). The patient’s blood transfusion history included red blood cell, platelet, and plasma transfusions in 2021 and 2022, followed by red blood cell transfusions in 2006 and 2014. The unexpected pre-transfusion antibody screening test yielded the following results: negative for ethylenediaminetetraacetic acid (EDTA), positive for plain and serum separating tube (SST), and negative for the direct anti-globulin test (DAT). On the other hand, autoantibodies were detected at a 1+ compatibility level. Unexpected antibody screening was conducted on a plasma sample using an IH-500 instrument (Bio-Rad, CA, USA). The test results were negative, but suitable blood was difficult to find during cross-matching because of the positive autocontrol results. Unex-pected antibody screening was conducted using a serum sample after a round of centrifugation because ZZAP tests cannot be performed at the authors’ laboratory. Two units of red blood cells were supplied to fulfill the request for the least incompatible blood transfusion. This was achieved by preparing blood samples ranging from negative to 1+ compatibility levels. The unexpected pre-transfusion antibody screening test was performed on plasma using the IH-500 instrument. The results were negative with no ABO discrepancy, as shown in Fig. 1 (Table 1).

Fig. 1. Negative antibody screening test when plasma was used as the sample (Biorad IH-500 equipment).

Pre-transfusion tests

Pre-transfusion tests
1) ABO typing B & Rh D (+)
2) Antibody screening test With plasma (–); serum (2+) in I and II
3) Antibody identification test Unknown antibody; Autocontrol (trace, +/–)
4) Enzyme method 4℃ (+); 37℃ (–)
5) Autocontrol and direct antiglobulin test (DAT) Autocontrol (1+) & DAT (–)
6) Cross-matching Released 2 pints of compatible RBCs

During the patient’s first visit in February 2021, an unexpected pre-transfusion antibody screening test conducted using blood bottles supplemented with EDTA yielded negative results, whereas another conducted using plain blood bottles was positive. The enzymatic method yielded negative results for EDTA and plain blood samples. EDTA yielded negative results in an antibody identification test, whereas the plain and SST antibody test results were positive. The enzymatic method yielded positive results when the samples were treated at 4℃ but negative results for the warm-temperature treatment. Therefore, the sample was released using the serum method after high-speed centrifugation. Cross-matching for the blood transfusion was performed until the final anti-human globulin step. Similar results were obtained again in December 2022.

Cold-reactive antibodies mainly consist of the IgM type. Cold alloantibodies exhibited specificity for antigens, such as MNS, Lea and Leb, and P1, whereas cold autoantibodies show specificity for antigens I, I, HI, and Pr [1]. Cold autoantibodies are often associated with conditions, such as cold hemagglutinin disease, typically arising from infections caused by Mycoplasma pneumoniae or Epstein–Barr virus [2]. On the other hand, no tests were performed for these infections in this case. Antibody identification tests were unnecessary for cases that yield negative antibody screening test results, positive immediate cross-matching results, and clinically non-significant findings for suspected cold-reactive antibodies [1]. Alth-ough an antibody identification test or red blood cell antigen phenotyping was not essential for this patient, and chronic transfusion was not required, blood preparation for surgery was needed. A mixed-field reaction or atypical agglutination involving a fibrinogen test was performed after removing fibrin from the plasma to eliminate the possibility of Rouleaux formation. This test also yielded negative results. The presence of warm-reactive autoantibodies was dismissed because the antibody test showed a negative reaction during warm processing and a positive reaction during 4℃ processing when treated using the enzyme method. Antibodies against Lewis, P, and I antigens, which were enhanced after the enzyme treatment, were considered absent. Although anti-MNS, an antibody susceptible to destruction by enzymatic treatment [2], was suspected, an unknown antibody was detected using a commercial antibody identifi-cation test. This paper reports this case due to the detection of cold antibodies exclusively at 4℃. The British Committee for Standards in Hematology suggested that plasma-based tests may miss weak or complement-dependent antibodies, in contrast to serum-based ones. More clinically significant antibodies have been detected in EDTA-treated plasma than in serum [3]. In the present case, clinically insignifi-cant antibodies were found exclusively in serum samples, escaping detection by the general antibody identification test.


2023년 81세 여성이 췌장암 수술을 위해 수술 시 예비용 적혈구 수혈 요청이 있어 혈액형 자동화장비를 이용해 비예기항체 선별검사를 시행하였다. 혈장을 검체로 비예기항체 선별검사 시행 시 결과는 음성이나 혈청을 검체로 시행 시 결과가 양성이었다. 효소법 시행 시 냉장온도 검사에서 양성이었으나 항온 시 결과는 음성이었다. 시행한 상품화된 비예기항체 동정검사(DiaPanel, Bio-Rad, CA, USA)에서는 동정되지 않았다. 이에 혈청 검체에서만 검출되는 임상적으로 중요하지 않은 한랭항체를 가진 것으로 판단되었으며, 교차시험은 3단계 항온 단계까지 하여 최종 음성인 혈액이 불출되었다.


No funding was received for this study.

Conflict of Interest


  1. Ontario Regional Blood Coordinating Network. Document No. NRT.006. Transfusion technical resource manual. [Online] (last visited on 8 August 2023)
  2. Yudin J, Heddle NM. A 13-question approach to resolving serological discrepancies in the tra-nsfusion medicine laboratory. Lab Med 2014;45:193-206.
    Pubmed CrossRef
  3. Blood Transfusion Task Force. Microplate tech-niques in liquid phase blood grouping and anti-body screening. In: Roberts BE; British Committee for Standards in Haematology. Standard haematology practice. Oxford: Blackwell Scientific Publications, 1991: 164-88.


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